FIELD

The present description relates to packaging for extending the useful shelf life of a product contained in the packaging and to compositions for forming such packaging and to methods of improving the shelf life of packaged products.

In one form, the present description relates to extended life packaging for food, particularly food susceptible to spoilage from contamination by pathogens during storage, and to compositions for forming such packaging, and to methods of using such packaging to extend the shelf life of foods contained within the package. In one form, the present description relates to packaging in the form of pathogen resistant food containers for forming environments internally within the containers, which environments preserve the quality of foods stored in such containers. The pathogen resistant containers assist in extending the shelf life of the food contained within the packaging whilst preserving the quality of the food for an extended period, and to methods of preserving food using such packaging.

In one form, the packaging of the present description finds particular application as packaging containing anti-spoilage agents to reduce spoilage of foods due to microorganisms contamination when the foods are packed and/or stored in containers manufactured from such packaging, and to compositions having anti-spoilage agents for forming the containers, and to methods of using the containers to preserve food.

Although forms of the packaging will be described with particular reference to specific embodiments, it is to be noted that the scope of protection is not restricted to the described embodiments, but rather the scope of protection is more extensive so as to include other forms and arrangements of the packaging, other forms of the anti- spoilage agent, other compositions having spoilage resistant properties, and to other methods of using the packaging and compositions.

BACKGROUND

Food preservation, including maintaining quality of food substances in packages, and ensuring the safety of packaged food, are considerations for the food industry when packaging food. Such considerations are also relevant to incorrect packaging procedures, or the selection of inappropriate materials from which the packaging is manufactured, or to mistreatment and/or mishandling of products once packaged, so that food is often spoiled to an extent so as to be unusable, even only after a short period of time from when packaged, if not treated correctly prior to, during and after packaging. One meaning of food spoilage is the process of contamination of foods leading to loss of colour, texture, nutritive value, organoleptic properties, consumer appeal, and the like. Usually, the contamination results in the growth of pathogenic microbes, or other micro-organisms, which deteriorate the quality of the food product in the package, often to an extent that the food is unusable and must be discarded as waste. Food contamination can occur as a result of a number of different sources in the overall chain of obtaining the food and its ingredients, through preparing the food for packaging and actually packaging the food, and also during storage of the food after packaging, such as post processing, storage and shipping abuse. Thus, sources of contamination can be derived from the food itself, or from the packaging being contaminated.

Post processing contamination is one of the major causes of food borne illnesses and associated product recalls resulting in the need for destruction of such contaminated food which is both a major public health issue and a huge economic loss for the food producers, including loss of reputation. As post processing contamination of packaged foods is a serious problem within the food processing and storage industries, any developments which result in

improvements to either preserving packaged foods against contamination, particularly pathogenic contamination and/or extending the shelf life of packaged food whilst retaining quality, would be welcomed and can result in large economic gains for the food industry generally, and the food processor in particular, as well as having a major public health benefit.

Accordingly, there is a need for improvements in the packaging of food for preserving the quality of the food to extend the shelf life of the packaged food, and to maintain quality of the food for extended storage periods. Accordingly, it is an aim of the present invention to provide a packaging material having improved preservation properties or characteristics.

Accordingly, it is an aim of the present invention to provide a package for food in which the safety and shelf life of the food is improved or extended.

Accordingly, it is an aim of the present invention to provide a method of extending the shelf life of a packaged food whilst maintaining its quality by using improved packaging. Accordingly, it is an aim of the present invention to improve the safety of packaged foods, particularly for longer periods of time to increase shelf life to reduce the chance of illness or injury to a person consuming spoiled food.

Accordingly, it is an aim of the present invention to provide a composition having a spoilage resistant component for making packaging for preserving food against spoilage.

SUMMARY

According to one form of the present invention, there is provided spoilage resistant packaging suitable for use in preserving quality of an item during storage of the item in the packaging, the item having an extended shelf life when in the packaging, the packaging comprising a container for containing the item during storage and a spoilage resistant agent wherein the spoilage resistant agent interacts with the item contained in the packaging to preserve quality of the item when in the package, and wherein the spoilage resistant agent is present in an amount effective to preserve quality of the item. According to one form of the present invention, there is provided a spoilage resistant composition suitable for use in manufacturing spoilage resistant packaging, the composition comprising at least one spoilage resistant agent substantially

homogeneously dispersed throughout at least a part or a layer of the packaging, wherein the spoilage resistant agent is present in an amount sufficient to provide effective spoilage resistant properties so as to extend the shelf life of the item stored in the packaging.

According to one form of the present invention, there is provided a method of extending the shelf life of a food item stored in packaging whilst maintaining quality of the food item comprising the steps of providing a packaging material suitable for storing the food item to retain the quality of the food item, packaging the food item using the packaging material, and treating the packaging material to form a sealed container containing the food item, wherein the packaging material includes a spoilage resistant agent in an amount effective to preserve quality of the food item against spoilage when in the sealed container so as to extend the useful shelf life of the food item. BRIEF DESCRIPTION OF EMBODIMENTS

One form of the spoilage resistant agent is an anti-spoilage agent. Typically, the anti- spoilage agent is an anti-microbial agent (AMA). However, other forms of the spoilage resistant material and/or the anti-spoilage agent can be used to preserve foods, such as for example, biocides, microbiocides, bioactive substances, pathogen growth inhibitors and the like, typically in combination with the AMA.

Different anti-microbial agents (AMA) have different activities on different pathogenic micro-organisms due to many factors including the physiology of the micro-organisms and how the anti-microbial agent is delivered to the micro-organism and/or absorbed by the microorganism. Other factors of the micro-organisms include cell wall composition, whether the micro-organisms are gram-negative or gram-positive, facultative anaerobes, whether the micro-organisms are aerobic or anaerobic, their acid/osmosis resistance, optimal growth temperatures, whether the micro-organisms are mesophilic, thermophilic or psychotropic, among other factors.

The selection of anti-microbial agent or combinations of two or more anti-microbial agents is dependent in part on the likely pathogens that are expected to be present in the particular food being packaged and subject to particular pathogen spoilage.

Although any suitable anti-microbial agent can be used to produce anti-microbial packaging, it is to be noted that certain anti-microbial agents are more effective than others, and hence, are preferred for use in forming the AMA-containing packaging. This is due not only to the effectiveness of the agent, but also to the physical form of the agent, and to its ease or convenience of use.

One preferred anti-microbial agent is or includes, a silver compound or silver- containing composition or component. More preferably, the anti-microbial agent is a silver inorganic salt, including a complex inorganic salt of silver, particularly combined with a metal. A particularly preferred form of anti-microbial agent is silver sodium hydrogen zirconium phosphate (Ag0.18Na0.57HO.25Zr ₂ (PO4)3). However, other silver containing materials can be used, such as silver zeolite (HEZ), silver zirconium phosphate silicate (AgZrPsi), and silver zirconium phosphate (AgZrP), and the like.

A most preferred AMA is Microban Additive IB20 provided by Microban International Limited, which is silver sodium hydrogen zirconium phosphate

(Ag0.18Na0.57H0.25zR ₂ (P04)3).

Microban Additive IB20 is suitable for use in food contact applications in some countries, most notably United States of America and European Union, if not all countries, particularly for incorporation into polymers (Reference No. 86434, SML 0.05 mg/kg silver in food). The final packaging or article containing the anti-microbial agent may contain from 0.1 % to 2.0% by weight of the anti— microbial agent.

In one form, Microban Additive IB20 is able to withstand high temperatures in the manufacture of materials, such as plastics, films, fibres, polymeric materials and ceramics.

One form of the AMA is a microbicide which contains, as an active ingredient, a specific phosphate containing a metal ion having antibacterial, antifungal or antialgal activity such as silver, copper, zinc, tin, mercury, lead, iron, cobalt, nickel, manganese, arsenic, antimony, bismuth, barium, cadmium or chromium ion, and the microbicide can be used as the anti— microbial composition or in anti-microbial compositions which comprise the microbicide mixed with various binders or as shaped products containing the antimicrobial composition or component, which shaped products comprise the microbicide supported on carriers such as fibres, films, papers, and plastics either as a surface layer or moulded into the shape of the item.

As compared with organic microbicides, inorganic microbicides have the

characteristics that they are higher in safety, have prolonged anti-microbial effect, and are superior in heat resistance, which makes their use as anti-microbial agents convenient and cost effective.

In one form, the AMA is a material which exhibits maximum antifungal, antialgal and antibacterial activities of silver, copper, zinc, tin, mercury, lead, iron, cobalt, nickel, manganese, arsenic, antimony, bismuth, barium, cadmium and chromium ions. It has been found that specific phosphates having metal ions exhibit anti-microbial activity such as silver, copper, zinc, tin, mercury, lead, iron, cobalt, nickel, manganese, arsenic, antimony, bismuth, barium, cadmium and chromium ions, especially containing silver ion. Such compounds and compositions have markedly excellent chemical and physical stability and besides, can exhibit effective antifungal, antialgal and antibacterial activities for a long period of time. Forms of the AMA include a microbicide comprising a phosphate represented by the following general formula:

wherein M ¹ represents at least one element selected from silver, copper, zinc, tin, mercury, lead, iron, cobalt, nickel, manganese, arsenic, antimony, bismuth, barium, cadmium and chromium, M ² represents at least one element selected from tetravalent metal elements, A represents at least one ion selected from hydrogen ion, alkali metal ion, alkaline earth metal ion, and ammonium ion, n is a number which satisfies 0≤n≤6, a and b are positive numbers and satisfy la+mb=1 or la+mb=2, and when a and b satisfy la+mb=1 , c is 2 and d is 3, and when a and b satisfy la+mb=2, c is 1 and d is 2 where 1 is valence of M ¹ and m is valence of A.

Forms of the microbicide can be suitably mixed with other components or formed into a composite with other materials depending on uses. Typically, there is a synergistic effect between the AMA and the other components.

The microbicides exhibit antifungal, antialgal and antibacterial activities for any use against fungi, algae and bacteria on which antimicrobial metal ions such as silver ion effectively act, and can be effectively used. Examples of carrier materials include papers such as wall papers; films such as food-packaging films, medical films, and synthetic leather; paints such as paints for sterilizers, corrosion-resistant paints, and antifungal paints; powders such as agricultural soil; and liquid compositions such as shampoo, paste and gel compositions, such as toothpaste, and other oral health preparations including anti-tooth decay materials and preparations.

Forms of AMA and the compositions containing them have very low solubility in water and hence can exhibit anti-microbial activity for a prolonged period of time, even in contact with water vapour, moisture, wet conditions and similar. Typically, the AMA is effective against the following examples of micro-organisms. Escherichia coli

Pseudomonas aeruginosa

Staphylococcus aureus

Bacillus subtilis

Candida yeast

Saccharmoyces yeast

Aspergillus niger

Gliocladium

Aureobasidium

Cladosporium

Salmonella,

Shigella

Shewanella (rare to find this contamination)

Enterobacteriaceae

Campylobacter

Clostridia (Clostridium botulinum, Clostridium perfringens)

Pseudomonas,

Acinetobacter

Moraxella.

Bacillus

Lactobacillus

Monocytogenes

Carnobacterium

Leuconostoc.

Yersinia (Y. enterocolitica)

Brochothrix (B.thermosphacta)

Another AMA is AlphaSan RC5000 supplied by Milliken Chemical which has a similar composition to Microban Additive IB20.

The anti-microbial agent can be incorporated into any or all of the materials or layers from which the packaging material is manufactured or into only one component of the packaging, such as one layer or part of one layer or into part of the packaging only, such as the base of the packaging, or the lid or closure of the packaging. The antimicrobial agent can be embedded in the material or can be incorporated into one or more layers or can be applied to one or other surface of the materials from which the packaging is manufactured or can be added, embedded, incorporated or applied, typically coated on an insert located within the packaging, such as for example, an insert inside the packaging, such as a sachet, or as part of the absorbent layer or absorbent pad, or absorbent lining often included in packaging to absorb liquids and/or other materials exuded from the food stuff, such as for example, the juices purged from meat.

The packaging can be made from natural materials, synthetic materials, composite materials or combinations of two or more different materials including plastics materials, paper and cardboard materials, textile and fabric materials including non- woven materials, woven materials, cast materials, fibrous materials, cellular materials, aerated materials, or the like.

Materials that can be used to form the packaging, either in part or entirely include the following, either alone, or in combination of two or more.

LLDPE MLDPE

Metallocine LDPE

HDPE

POLYOLEFIN POLYETHYLENE PVdC

PVC EVOH PLA NYLON POLYESTER

POLYPROPYLENE PAPER

COATED PAPER FOILS Forms of the packaging include active packaging, controlled release packaging, delayed action packaging, continuous release packaging, or the like.

Active anti-microbial packaging is used to actively modify the internal environment within the sealed package by continuous interaction with the food contained within the package over the stipulated shelf life of the package. In one form, active packaging can be defined as a system that modifies the environment inside the food package to alter the state of the packaged food system, including the head space within the package to extend the shelf life of the food package, to enhance the sensory and/or organoleptic qualities of the food and to maintain safety of the food for consumption.

In forms of the anti-microbial packaging, the packaging materials can include additives other than the anti-microbial agent such as for example, oxygen scavengers, carbon dioxide scavengers, ethylene absorbing agents, moisture absorbing agents, carbon dioxide or ethanol emitting systems, anti-oxidants or similar. Scavenging systems can absorb deleterious compounds from the surface of the food within the package or from the headspace of the package. Emitting systems release certain compounds which act at the surface of the food or within the headspace.

One form of active packaging is controlled release packaging which is also known as time-release, or slow-release packaging of active substances, such as having controlled release of the anti-microbial agent into the environment inside the packaging, particularly sealed packaging.

The anti-microbial agent can be incorporated into films or coatings for application to the food. One form of the film is edible film coating which incorporate anti-microbial agents or coatings resulting in biodegradable active packaging. Such edible film coatings are particularly useful to reduce and inhibit the growth of micro-organisms on the surface of food products by applying a barrier around the food products. In one form, low density polyethylene packaging film is used with an anti-microbial agent coating. However, other polymers include methylcellulose (MC), hydroxypropyl methylcellulose (HPMC), and the like.

Forms of anti-microbial packaging include packaging in which the anti-microbial agents may be coated, incorporated, immobilised, or surface modified onto the packaging materials and/or the surface or surfaces of the food or packaging.

Forms of the anti-microbial packaging include incorporation of anti-microbial agents into a sachet connected to the package from which the volatile bioactive substance is released during further storage of the package. Another form is direct incorporation of anti-microbial agents into the packaging film or into one or more of the layers from which the container is manufactured. A still further form is a coating of the packaging material with a matrix that acts as a carrier for the anti-microbial agent.

In one form, the anti-microbial agent is a non-volatile anti-microbial substance, in which case the non-volatile anti-microbial agent must contact the surface of the food so that the anti-microbial agent can diffuse into the surface of the food product to effectively suppress contamination by pathogens by attacking any pathogens present in the food. The anti-microbial agent is effective against the pathogen by contact with the cell walls of the pathogen, passing through the cell wall, diffusing through the cells of the pathogen, or the like. Other forms of the packaging rely on diffusion of the anti-microbial agent from the packaging material to the environment within which the food product is stored.

Preferably, the anti-microbial agent is positively charged or has a positive ion.

In one form, the anti-microbial agent is a small molecule.

Small molecule anti-microbial agents have increased diffusion rates due to their low molecular weights while absorption is better achieved by anti-microbial agents having large molecular weight such as when incorporated or embedded into polymers.

Preferably the molecular weight of the polymer having the anti-microbial agent is in the molecular weight range of

Preferably, the polymers in which the anti-microbial agent is incorporated or bound have longer chains since longer chains have higher anti-microbial activity.

In one form, the polymers which are biocidal polymers that typically require contact times of the order of hours to provide substantial reductions in pathogens are less preferred owing to the time taken to destroy the pathogen. Preferably the contact time between the anti— microbial agent and the food for the pathogen to be destroyed by the anti-microbial agent should be as short as possible, typically in the order of seconds, or minutes, at most.

The item can include food, such as a wide range of fresh, cooked or preserved food, and can also include healthcare products generally, such as toothpaste, mouthwash, or other healthcare products that are ingested or topically applied to the body and which have a tendency to spoil over time. Forms of the anti-microbial agent can contain up to about 99.9% by wt. of the active component, typically the silver containing compound, such as for example, silver sodium hydrogen zirconium phosphate.

Forms of the AMA can contain up to about 10% by wt. silver on a weight/weight basis. Typical amounts of the active component incorporated into the anti-microbial agent include amounts of up to about 20% by weight of the total weight of the composition, more typically from about 0.01 % to about 10% by weight, even more typically, about 0.1 % to about 5%, preferably about 0.1 % to 2.0% by weight.

Forms of the AMA can be used in any suitable concentration. Typically, the concentration is in the range of from about 0.1 % to about 2.0% by weight based on the amount of active ingredient/silver.

In forms, the AMA is incorporated into the materials from which the packaging is manufactured during the manufacturing process, such as for example, during vacuum moulding processes, during injection moulding processes, including co-injecting processes when two or more materials are coextruded, extrusion processes, lamination processes, or other processes for forming the materials from which the containers are made or processed for forming the container. Forms of the packaging and/or containers are used for manufacturing, packing, packaging, transporting, handing and/or holding food. Forms of the materials associated with the AMA's include plastics, coatings, films, laminates, barrier fabrics, bottles, cook wear, food and drink containers, food packaging, food storage containers and bags, food trays, food covers, food wrap, coated deli paper, coated meat interleaves, plastic wrap, plastic lids, absorbent pads, inserts, and similar. In one form, the packaging can include an integral or separate insert added to, contained within or formed as part of the packaging. One form of the insert is an absorbent pad, typically an absorbent pad having the dimensions 75 x 94mm. One form of the absorbent pad is identified by product code 7594B of the applicant, whereas another form of the pad is identified by product code 64141W having dimensions of 64 x 141 mm. Other absorbent pads are identified by product codes

150150W having dimensions of 150 x 150mm; 80158 having dimensions of 80 x 158mm; and, 225158BB having dimensions of 225 x 158mm.

The absorbent pads are for use with meats, such as beef, lamb, pork, poultry, such as chicken; seafood, such as fish, crustaceans, and the like. The anti-microbial agent is incorporated into the absorbent pad, together with the super absorbent powder, typically in the form of acrylic acid.

Other examples of packaging include Bone-In Barrier Bags, such as for example, bags having a film of wall thickness in the range from about 60μ ίο about 150μ, typically in the range of from about 70μ to about 135μ. More typically, the bags have a thickness of about 75μ, 86μ, 95μ, 105μ and 125μ, depending upon the application of the bag to the particular food being stored.

One example of packaging includes vacuum barrier shrink bags in which the antimicrobial agent is incorporated into one or more layers of the vacuum barrier shrink bag. Typically, the anti-microbial agent is incorporated into vacuum barrier shrink bags for boneless primals is in which the vacuum barrier shrink bags can range in thickness from about 45 μηι to about 60 μηι or more, typically 60 μηι.

In one form, the packaging is a high barrier packaging, such as for example, a 9 layer structure incorporating Nylon and one or more of the barrier layers being EVOH.

Typical properties of one form of the high barrier packaging include the following:

Puncture Resistance >280 Newton (N)

Free Shrink@86°C 20/20 %MD/TD

OTR(Oxygen transmission rate) <15 cc/m ² /24hr@23°C, 85%RH

Bone-In bags are primarily for preserving meat having bones, such as beef, pork and lamb cuts containing integral bones.

Even though the precise mechanisms of biocidal activity of silver against

microorganisms is not fully understood, it is believed that the proposed antimicrobial mechanisms are first that, silver ions can associate with the cell wall, cell membrane, and cell envelope of microorganisms. The positive charge of a silver ion is thought to be significant for anti-microbial activity, allowing electrostatic attraction between the negative charges of the bacterial cell membrane and positively charged silver particles causing cell membrane rupturing. Low concentrations of silver ions were demonstrated to have the ability to induce a massive proton leakage through the bacterial membrane causing subsequent cell death.

Second, silver ions can react with nucleophilic amino acid residues in proteins, attached to sulfhydryl, amino, imidazole, phosphate and carbonyl groups of membrane or enzyme proteins. A number of oxidative enzymes have been reported to be inhibited by silver ions such as yeast attached dehydrogenase, enzymes associated with the uptake of succinate by membrane vesicles and respiratory chains of bacteria. There resulted in the metabolites efflux, interfering with DNA replication, inactivation of ATP production and inhibition of growth, all for reducing the growth of pathogens. Third, the antimicrobial action of silver particles is suggested to be related to the formation of free radicals and subsequent free radical-induced membrane damage. As a result of the catalytic action of silver ions, oxygen is changed into oxygen radicals by the action of light energy and/or H ₂ 0 in the air or water only at polar surfaces and these oxygen radicals cause structural changes in microorganisms. Embodiments comprising anti-microbial agents will now be described and exemplified with reference to the following examples.

EXAMPLE 1

Evaluation of the activity of anti-microbial agents and material containing the agents is conducted in two ways: microbroth dilution and disc susceptibility testing. This example tests the anti-microbial material using methods akin to each of these approaches. Each method tests up to six anti-microbial material concentrations which are 0.12, 0.18, 0.24, 0.30, 0.50 and 1 .00%, and three bacterial concentrations. A cocktail mixture of E. coli organisms that have been previously used in a range of challenge and storage trials is used in the study. Absorbent pads are used during vacuum packaging of meat products to absorb purge thereby enhancing the appearance of the packs during their shelf life. Forms of an antimicrobial absorbent pad are for use with vacuum packaged meat and their performance evaluated in terms of their ability to extend the shelf life of vacuum packaged lamb products. There are indications from the evaluations that the anti- microbial pad may be useful in extending the storage life of vacuum packaged lamb products, however little is known about the anti-microbial capacity of the anti-microbial material that is incorporated into the antimicrobial pad. Studies have been undertaken to determine the ability of the material to kill (bactericidal) or retard (bacteriostatic) bacterial growth in such meat products.

Tests have been conducted to evaluate the capacity of the antimicrobial material at a range of antimicrobial concentrations and microbial concentrations in order to determine the optimum material concentration for use in a commercial setting.

Anti-microbial susceptibility testing is typically conducted in two main ways: microbroth dilution and disc susceptibility testing. This involved testing the anti-microbial material using methods akin to each of these approaches. Each method tested up to six (6) anti-microbial material concentrations which are 0.12, 0.18, 0.24, 0.30, 0.50 and

1 .00%, and three (3) bacterial concentrations. A cocktail mixture of E. co// ^' organisms that have been previously used in a range of challenge and storage trials was used in the study.

Method 1 : Anti-microbial discs of 90mm diameter are prepared and placed into sterile 92mm x 16mm petri dishes. A cocktail mixture of E. coli organisms are prepared following overnight growth and subsequently diluted in buffered peptone water (BPW) to achieve working concentrations of approximately 6.00, 4.00 and 2.00 log ₁₀ CFU/mL. An aliquot (10ml_) of each dilution is added to a separate petri dish and subsequently stored at - 0.5°C for a period of one (1) or (4) weeks. At the completion of storage the

concentration of E. coli present in each petri dish is determined and compared to the starting value. Each anti-microbial/bacterial combination is tested in triplicate and appropriate controls included.

Method 2: Anti-microbial discs of 50mm diameter are prepared and placed onto petri dishes containing a pre-poured tryptone soya agar base. A cocktail mixture of E. coli organisms is prepared following overnight growth and subsequently added to seven (7) mL of molten tryptone soya agar (top agar) to achieve working concentrations of approximately 6.00, 4.00 and 2.00 log ₁₀ CFU/mL. Each top agar is used to overlay the pre-prepared agar bases containing the anti-microbial discs. After allowing the agar to set the plates are incubated at 25°C for 3 days or 37°C for 1 day. The zone of clearance immediately above and extending from the edges of the anti-microbial discs, are subsequently assessed. Each anti-microbial/bacterial combination is tested in triplicate and appropriate controls included.

Below are 2 tables showing the average reductions for each absorbent pad concentration from both the starting bacterial count (table 1) and from the 0.00% count (table 2). The 6, 4 and 2 in the concentration column refer to estimated starting concentrations. The actual starting values were 6.90, 4.67 and 2.68 logs respectively. It probably makes sense to just focus on the rows in italic as there were readily countable numbers prior and post treatment. The cells highlighted by underlining are done due to there being nothing to count at the end so the actual reduction is almost certainly higher than the number listed.

Reductions from starting count

Cone 0.00% 0.12% 0.18% 0.24% 0.30% 50GSM 100GSM No Pad

6 1.45 5.60 5.26 5.33 5.26 1.12 1.07 2. 10

4 0.97 3.67 3.67 3.67 3.67 1 .1 1 1 .12 1 . 14

2 1 .25 1 .68 1 .68 1 .68 1 .68 1 .00 1 .22 1 . 52

Reductions from 0.00%

Cone 0.00% 0.12% 0.18% 0.24% 0.30% 50GSM 100GSM No Pad

6 0.00 4.15 3.81 3.88 3.81 -0.33 -0.38 0. 65

4 0.00 2.70 2.70 2.70 2.70 0.14 0.15 0. 17

2 0.00 0.43 0.43 0.43 0.43 -0.25 -0.03 0. 27

Some initial observations:

Reductions for 0.12% - 0.30% exceed 5 logs however there appears to be little additional activity associated with an increase in concentration.

It would appear that the material itself has a slight antimicrobial activity of approx. 1 .22 logs meaning at least 3.8 logs of bacterial reduction can be attributed to antimicrobial activity. In reality the 1 .22 log reduction observed with the 0.00% material is probably a combination of cold shock to the bacteria and the material.

The 50 and 100GSM material is ineffective and is directly comparable to the 0.00% result.

These are very encouraging results.

EXAMPLE 2: Shelf-life of vacuum packaged lamb Activity objective: · Determine the shelf-life of vacuum packaged lamb racks stored using a variety of packaging combinations.

Materials and Methods

Samples:

Vacuum packaged lamb racks sourced from a registered Australian establishment were stored under refrigeration (-0.5°C ± 1 °C) at the test facility of the Testing Authority Food and Nutrition Laboratory. Lamb racks were each packed using one of five packaging combinations:

• Pack 1 : Thermasorb bag (printed AI688) with white 125x158 Thermasorb

antimicrobial pad

· Pack 2: Thermasorb bag (printed AI688) with black 125x158 standard Thermasorb absorbent pad

• Pack 3: Thermasorb bag (printed AI688) with no absorbent pad

• Pack 4: Thermasorb high barrier 86μηι bag with no absorbent pad

• Pack 5: Thermasorb high barrier 86μηι bag with white 125x158 Thermasorb

antimicrobial pad

All samples were received by the Testing Authority within ten days of packaging with sampling occurring after the second and third weeks of storage followed by fortnightly sampling from the 4th week of storage through to week 14.

Microbiological analysis Lean meat and adipose tissue collected from each sample were analysed as a composite sample for total viable count (TVC), lactic acid bacteria (LAB), and

Enterobacteriaceae. A 100 mL aliquot of 0.85% saline was added to stomacher bags containing a total of 40cm2 of lean meat and adipose tissue and samples were subsequently stomached for 30s. Decimal dilution series were prepared in 0.85% saline and subsequently plated onto Petrifilm Aerobic and Petrifilm Enterobacteriaceae plates for TVC and Enterobacteriaceae, respectively. The dilutions were also prepared in MRS broth (Oxoid) and plated onto Petrifilm Aerobic according to the Petrifilm method for enumeration of LAB. Petrifilm Aerobic count plates were incubated at 25°C ± 1 °C for 72 ± 3 h; Petrifilm Enterobacteriaceae plates were incubated at 35°C ± 1 °C for 24 ± 2h; LAB plates were incubated anaerobically at 25°C ± 1 °C for 120 ± 3 h. Sampling was conducted in triplicate at each time point. Microbial counts were converted to log10CFU/cm2 for each sample and then the mean count determined. For the purposes of generating the mean counts, samples with counts below the limit of detection (LOD) were arbitrarily assigned a count equal to the limit of detection. The LOD's for TVC, Enterobacteriaceae and LAB were 0.40, 0.40 and 1 .40 logl 0CFU/cm2, respectively. pH measurements

Muscle pH was measured on all lamb samples at each storage time using a digital pH meter (TPS) fitted with a combination electrode (glass body with spear tip) with temperature compensation.

Results and Discussion

Mean TVC of incoming product was <3.00 log ₁₀ CFU/cm ² after two weeks of storage and rose progressively during 14 weeks of storage. Using an arbitrary cut off for acceptability of product of >7.00 log ₁₀ CFU/cm ² , packs 2 and 3 were unacceptable after 10 weeks of storage, packs 1 and 4 were unacceptable after 12 weeks of storage, and pack 5 was considered unacceptable after 14 weeks of storage (see Table 3 and Figure 1 ). Pack types 1 to 3 all utilised the same packaging film but differed in the type of absorbent pad included. Comparison of mean TVC at week 10 determined that packs containing the antimicrobial absorbent pad were on average 1 .27 log ₁₀ CFU/cm ² lower than packs with the standard absorbent pad (pack 2) or with no absorbent pad (pack 3). Similarly the mean TVC at week 12 in packs utilising the high barrier bag was shown to be 1 .07 log ₁₀ CFU/cm ² lower in packs with the antimicrobial pad (pack 5) than those without (pack 4). Despite similar differences being observed in packs containing the antimicrobial absorbent pad only the difference at week 12 between pack 5 and pack 4 was shown to be significant (p<0.05).

Table 3. Mean TVC of vacuum packaged lamb racks stored for 14 weeks at -0.5°C

Mean TVC exceeding 7.00 log ₁₀ CFU/cm ² were considered unacceptable and are shaded in red.

Figure 1 , Mean TVC of vacuum packaged lamb racks stored for 14 weeks at -0.5°C

Good correlation was observed between mean TVC and LAB counts during storage for all pack types (Table 4 and Figure 2). Pack 5 mean LAB counts at week 12 were 6.24 logioCFU/cm ² compared with 7.13 log ₁₀ CFU/cm ² for pack 4. This difference was considered significant (p<0.05) and mirrors the finding observed with respect to mean TVC between pack 5 and pack 4 at week 12. Comparison of mean LAB counts in packs 1 to 3 at week 10 identified a difference of at least 1 .50 log ₁₀ CFU/cm ² , however this was shown not to be significant (p<0.05). Despite the large differences being observed for both TVC and LAB counts, the mean TVC and LAB counts for pack 1 samples are skewed by an individual sample with very low TVC and LAB counts that are not supported by the results of the remaining pack 1 samples at week 10, hence pack 1 samples are not significantly (p<0.05) different from packs 2 or 3.

Table 4. Mean LAB counts of vacuum packaged lamb racks stored for 14 weeks at - 0.5°C

Enterobacteriaceae counts for each pack type during the 14 weeks of storage are shown in Table 4. Counts remained below 3.00 log ₁₀ CFU/cm ² during the first 10 week of storage for all samples with pack types 3 and 4 remaining below that value through 12 weeks of storage. Pack 5 samples marginally exceeded 3.00 log ₁₀ CFU/cm ² at weeks 12 and 14 with counts of 3.18 and 3.03 log ₁₀ CFU/cm ² , respectively. However they were found to not be significantly higher than any other pack type at week 12 or 14. Mean pH values for all samples rose from 5.53 at week 2 to 5.81 at week 14 and appeared generally consistent at each sampling time point across all pack types. Table 5. Mean Enterobacteriaceae counts of vacuum packaged lamb racks stored for 14 weeks at -0.5°C

Conclusions · The use of the antimicrobial absorbent pad in combination with Thermasorb's bag (printed AI688) gave lower mean TVC and LAB counts after 10 weeks of storage than storage using Thermasorb's bag (printed AI688) with or without an absorbent pad. However, these differences were dominated by an individual sample with very low counts and the differences were not deemed significant (p<0.05).

· Comparisons of mean TVC and LAB counts for samples stored in the high barrier bag with or without the antimicrobial absorbent pad determined that counts after 12 weeks of storage were significantly lower in packs containing the antimicrobial absorbent pad. • The results of this study indicate that there are potential gains in the storage life of vacuum packaged lamb racks through the combined use of Thermasorb's high barrier bag and antimicrobial absorbent pad.

EXAMPLE 3

Antimicrobial material evaluation

Activity objective:

• Determination of antimicrobial material effectiveness at differing antimicrobial concentrations

• Ability of antimicrobial material to reduce bacterial concentrations during repeat challenge

• Ability of antimicrobial material to reduce bacterial concentrations in ideal and abuse conditions

Materials and Methods Materials

Thermasorb provided the Testing Authority with six samples of antimicrobial containing material as well as one sample of material containing no antimicrobial (placebo). The concentrations of antimicrobial in the material provided were stated as 0.12%, 0.18%, 0.24%, and 0.30%.

Bacterial strains

A cocktail mixture of E. co/i organisms (EC1604-1608;) were used in the study. E. coli were recovered from storage at -80°C and grown at 37°C for 18-24 h in buffered peptone water (BPW; Oxoid). Bacterial inoculums were subsequently prepared by combining equal volumes of enrichment culture of each E. coli strain followed by serial dilution in 0.85% saline.

Determination of antimicrobial material effectiveness at differing antimicrobial concentrations

Previous studies determined that the rates of bacterial decline for antimicrobial material concentrations of 0.12%, 0.18%, 0.24% and 0.30% were >4.00 log ₁₀ CFU/ml_ when assessed after seven days storage. In order to determine the efficacy of each antimicrobial concentration the rates of reduction were examined over a shorter more intensive timeframe. Antimicrobial squares measuring 6cm x 6cm (36cm ² ) were prepared and placed into sterile 92mmx16mm petri dishes. A cocktail mixture of E. coli organisms was prepared as outlined previously and each concentration of antimicrobial material was challenged with 15 ml_ of >7.00 log ₁₀ CFU/ml_ of E. coli. Each sample was evaluated hourly for eight hours and then daily for up to five days. If differences in the rate of reduction were not observed using these timeframes then a repeat experiment would be conducted with sampling to occur at five minute intervals or at daily intervals for seven days. The concentration of E. coli present in each petri dish at the point of sampling was determined by plating serial dilutions onto tryptic soy agar (TSA, Oxoid) and incubating for 18-24 h at 37°C. The resulting counts were compared to the starting count to determine the nett reduction. A material only sample (i.e no antimicrobial activity) was used as a control. Ability of antimicrobial material to reduce bacterial concentrations during repeated challenge

The capacity of the antimicrobial material to respond to dynamic changes in microflora likely to be encountered during vacuum packaged storage was assessed by challenging each antimicrobial concentration with 15 ml_ of the E. coli cocktail at a starting count of ~5.00 log ₁₀ CFU/mL. Samples were subsequently stored at -0.5°C for either seven or 14 days. At the completion of the initial storage period (either seven or 14 days) the reduction of bacterial numbers was assessed by plating serial dilutions onto tryptic soy agar (TSA, Oxoid) and incubating for 18-24 h at 37°C. Following sampling, the same antimicrobial material was again challenged with ~5.00 log ¹⁰ CFU/ml_ and stored for seven or 14 days. This process was repeated seven times for samples stored for seven days (49 days in total) and four times for samples stored for 14 days (56 days in total). A material only (i.e no antimicrobial activity) and a no material sample (i.e inoculum only) were used as controls. Ability of antimicrobial material to reduce bacterial concentrations in ideal and abuse conditions.

All activities within this project have utilised ideal storage conditions (i.e -0.5°C). The final phase of the antimicrobial material evaluation investigated the capacity of the antimicrobial material to be bacteriostatic or bactericidal when exposed to a range of temperatures and media. The efficacy of the antimicrobial material was assessed at three storage temperatures (-0.5, 4 and 10°C) and in three media (saline, BPW, and tryptone soya broth (TSB); Oxoid). Antimicrobial material concentrations of 0.12%, 0.18%, 0.24% and 0.30% were challenged with 15 mL of -5.00 log ₁₀ CFU/ml_ that had been prepared in the three different media. The samples were then stored at -0.5, 4 or 10°C for up to seven days and assessed daily for changes in bacterial concentrations.

Microbiological assessment was conducted by plating serial dilutions onto tryptic soy agar (TSA, Oxoid) and incubating for 18-24 h at 37°C. A material only (i.e no antimicrobial activity) and a no material sample (i.e inoculum only) were used as controls. Results and Discussion

Determination of antimicrobial material effectiveness at differing antimicrobial concentrations Antimicrobial material of differing concentrations were inoculated with 8.35 log ₁₀ CFU/ml_ of E. coli and their ability to reduce the concentration of E. coli measured over a five day period. Reductions in E. coli concentrations were not observed during the first eight hours of storage and did not exceed 0.76 log ₁₀ CFU/ml_ for any antimicrobial concentration after five days (Figure 3). Interestingly, the largest reduction observed occurred in the 'material only' control and most probably represents an effect of storage at -0.5°C as opposed to an antimicrobial effect caused by the material.

Further evaluation of the antimicrobial material was conducted using a starting inoculum of 7.93 log ₁₀ CFU/ml_ and daily sampling over a seven day storage period resulted in the observation of increased reductions. Reductions for samples containing material with antimicrobial ranged from 2.19 log ₁₀ CFU/ml_ for 0.12% to 2.87 log ₁₀ CFU/ml_ for 0.24% (Figure 4). In comparison, the 'material only' sample had a reduction of 0.90 log ₁₀ CFU/ml_ which suggests the nett effect of the antimicrobial is at least 1 .28 log ₁₀ CFU/ml_ over the seven days. This is less than the >3.81 log ₁₀ CFU/ml_ reductions observed in a previous study (data not shown), however the inoculum levels were much higher in this study (8.35 and 7.93 log ₁₀ CFU/ml_) than previously used (6.90 log ₁₀ CFU/ml_) and suggests there are diminishing benefits of the antimicrobial substance at these levels. It should be noted that the elevated inoculum levels used in this study are concentrations at which product would be considered spoiled and are therefore not the primary focus of this packaging solution. Bactericidal or bacteriostatic effects during the early phases of vacuum packaged storage are of greater benefit to long term shelf life and bacterial concentrations <5.00 log ₁₀ CFU/ml_ are most likely during this stage.

5 Ability of antimicrobial material to reduce bacterial concentrations during repeated challenge

The ability of the antimicrobial material to withstand repeated bacterial challenge with either seven or 14 days challenge are shown in Tables 6 and 7, respectively. All samples were initially inoculated with 4.62 log ₁₀ CFU/ml_ and then repeatedly l o challenged with a minimum of 4.07 log ₁₀ CFU/ml_ E. coli at either day seven or 14. High reproducibility was observed for all antimicrobial concentrations with most samples below the limit of detection at each sampling point. Total reductions for samples challenged every seven or 14 days were at least 3.07 log ₁₀ CFU/mL. There did appear to be an enhanced effect of storage temperature as the trial progressed, however,

15 even when accounted for, reductions still exceeded 2.45 log ₁₀ CFU/ml_ for any sample regardless of antimicrobial concentration. Table 6. E. coli counts of samples repeatedly challenged and stored at -0.5°C for seven days

Table 7. £. coli counts of samples repeatedly challenged and stored at -0.5°C for seven days

Ability of antimicrobial material to reduce bacterial concentrations in ideal and abuse conditions

Cocktails of E. coli were prepared in saline, BPW and TSB and used to challenge the antimicrobial material during seven days storage at -0.5, 4 or 10°C. Each antimicrobial material sample was challenged with a minimum of 4.26 log ₁₀ CFU/mL. A summary of the data is shown in Tables 8 (-0.5°C), 9 (4°C) and 10 (10°C). Table 8. E. coli counts of antimicrobial material samples stored in differing media at - 0.5°C for seven days

Table 9. E. coli counts of antimicrobial material samples stored in differing media at 4°C for seven days

Table 10. E. coli counts of antimicrobial material samples stored in differing media at 10°C for seven days

Regardless of storage temperature used, reductions were observed for all samples prepared in saline. Reductions were a minimum of 3.37 log ₁₀ CFU/ml_ with all samples having counts below the limit of detection by the end of the trial. Interestingly, samples stored at the highest temperature of 10°C had the most rapid decline of E. coli with all samples below the detectable limit by day 3. In contrast, the concentration of E. coli fell below detectable limits at day 5 for samples stored at 4°C and at day 7 for samples stored at -0.5°C. These results suggest that the elevated temperatures aid in the solubilisation and distribution of the antimicrobial throughout the test sample.

Temperatures below 7°C are utilised with food distribution networks and are used to control the growth of spoilage and pathogenic organisms with temperatures exceeding that mark enabling relatively rapid growth of these organisms. Whilst, as mentioned previously, samples prepared in saline and stored at 10°C produced the fastest reductions in E. coli counts, contrasting observations were made for samples stored in BPW and TSB where nutrients for growth are readily available. Samples stored in BPW and TSB at 10°C took four and three days, respectively for all samples to exceed 8.00 log ₁₀ CFU/ml_. Conclusions

• Antimicrobial containing material produced at concentrations of 0.12, 0.18, 0.24 and 0.30% were shown to reduce E. coli concentrations by at least 3.37 log ₁₀ CFU/ml_ when challenged for at least seven days in saline solution, regardless of storage

temperature.

• The bactericidal effect of the antimicrobial was enhanced in saline solutions stored at 10°C compared with 4 or -0.5°C

• The antimicrobial material stored in BPW at 4°C demonstrated nett E. coli reductions of at least 1.07 log ₁₀ CFU/ml_, however similar observations were not made when TSB was used in place of BPW.

ADVANTAGES

Anti-microbial food packaging reduces, limits and retards the growth of pathogenic and/or spoilage microorganisms that may be present on packaged food surfaces due to the release of anti-microbial components. It is to be understood that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art, in Australia or any other country.

In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

It will be understood to persons skilled in the art of the invention that many modifications may be made without departing from the spirit and scope of the invention.