An antimicrobial food package

Description

Introduction

[0001] The invention relates to an antimicrobial package suitable for containing an item susceptible to microbial growth or spoilage. In particular, the invention relates to an antimicrobial package for comestible products such as food and drink products.

[0002] Microbial contamination of food reduces the quality of food, limits the shelf-life of the food, and increases the risk of food-borne illness to the consumer. Contamination occurs primarily on the surface of foods, especially foods such as cheeses and meats. For the food industry, prevention of food spoilage is an important varaible when determining profit. Moreover, prevention of food spoilage can prolong the shelf-life of products and thus extend market boundries, resulting in increased profit. The growth of microorganisms on food products largely occurs post processing during stroage, therefore the packaging of the ffod is extremely important to the quality and shelf life of the food.

[0003] The antimicrobial properties of silver have been recognised for a long time. More recently, silver nanoparticles have been recognised as having antimicrobial properties, and have been suggested for use as an antibacterial agent in surgical masks, wound dressings, and cotton fibres, Use in food packaging has also been described ― see for exampl Appendini et al, Innovative Food Science & Emerging technologies, 2002, Vol. 3, US6942897, US10/726512, WO2004/012998, and US7311933. Use in food packaging, where silver-containing nanoparticles are incorporated into the matrix of polymeric packaging materials, has not gained acceptance due to percieved health risks associated with the silver nanoparticles, and also due to the large amounts of metal required to achieve an antibacterial effect (1-5wt% ).

[0004] It is an object of the invention to overcome at least one of the above-referenced problems.

Statements of Invention

[0005] The Applicant has solved the above-referenced problem by the provision of a package defining an enclosed space suitable for containing an item, in which an interior of the package comprises a surface bearing a nanopattern or nanoarray, ideally an ordered nanopattern or nanoarray, of metal or metal oxide nanostructures.

[0006] The term "nanopattern or nanoarray of metal or metal oxide nanostructures" should be understood to mean an arrangement of metal or metal oxide structures, for example nanodots or nanolines, that are formed on a surface of a substrate, have dimensions in a nanometre range, and are formed by self-assembly from a microphase separated block copolymer in which one of the polymers selectively incorporates a metal ion salt prior to treatment of the block copolymer to oxidise the metal ion salt and remove the polymers. Methods of forming such arrays or patterns of nanostructures is described in the semi-conductor and microelectronics literature, albeit not in the context of antimicrobial packaging - see for example: US2010/102415; Kuemmel et al., J. Sol Gel Sci. technol. 2008, Vol. 48; CN101609743; and US2011/250745. The invention therefore relates to the use of a surface bearing such an array of nanostructures as an antimicrobial surface in a packaging for an item prone to microbiological spoilage or growth.

[0007] Thus, the invention provides a package for an item, typcially an item susceptible to microbial growth or spoilage, ideally a comestible product, in which an interior of the package comprises a surface that bears a nanoarray or nanopattern of metal or metal oxide nanostructures. The provision of a nanoarray of nanostructures, for example nanostructures of silver oxide, significantly reduces the amount of silver required to achieve an antimicrobial effect (<0.001wt%) compared to convention technology where silver-containing nanoparticles are incorporated into the packaging material at 1-5wt%. Further, due to their process of manufacture, the nanostructures are rigidly anchored to the surface on which they are formed, thereby allaying concerns that the nanostructures will be ingested by consumers.

[0008] The invention also provides a package defining an enclosed space containing an item, generally an item susceptible to microbial growth or spoilage, in which an interior of the package comprises a surface bearing a nanopattern or nanoarray of metal or metal oxide nanostructures.

[0009] The invention also relates to a packaging material in the form of a sheet or film, for example a roll of polymeric flim, having a first face and a second face, in which at least a portion of one of the faces of the film comprises a surface bearing a nanopattern or nanoarray of metal or metal oxide nanostructures.

[0010] The invention also relates to the use of a packaging material of the invention as an antimicrobial agent, typically an antimicrobial agent against packaged comestible items such as food products.

[0011] The invention also relates to a method of extending the shelf life of a packaged comestible item, typically a food product, which method employs a packaging material at least part of which comprises a surface bearing a nanoarray of metal or metal oxide nanostructures, the method comprising a step of enclosing the comestible item within the packaging material to form a package such that the surface bearing the nanoarray of metal or metal oxide nanostructures is disposed on an interior of the package. The surface bearing the nanoarray of metal or metal oxide nanostructures may be integrally formed with the packaging material, or may take the form of an insert.

[0012] The invention also relates to a method of preserving a packaged comestible item, typically a packaged food product, which method employs a packaging material at least part of which comprises a surface bearing a nanoarray of metal or metal oxide nanostructures, the method comprising a step of enclosing the comestible item within the packaging material to form a package such that the surface bearing the nanoarray of metal or metal oxide nanostructures is disposed on an interior of the package. The surface bearing the nanoarray of metal or metal oxide nanostructures may be integrally formed with the packaging material, or may take the form of an insert.

[0013] The invention also relates to a method of preventing or inhibiting microbial growth on a comestible item contained within a package, typically a packaged food product, which method employs a packaging material at least part of which comprises a surface bearing a nanoarray of metal or metal oxide nanostructures, the method comprising a step of enclosing the comestible item within the packaging material to form a package such that the surface bearing the nanoarray of metal or metal oxide nanostructures is disposed on an interior of the package. The surface bearing the nanoarray of metal or metal oxide nanostructures may be integrally formed with the packaging material, or may take the form of an insert.

[0014] The invention also relates to the use of a substrate having a surface bearing an array of metal or metal oxide nanostructures as a packaging material for an item susceptible to microbial growth or spoilage, especially a comestible item such as a food product. Typically, the substrate is a material suitable for packaging, such as for example a material formed from a polymeric, paper, foil, material or composites thereof.

Detailed Description of the Invention

[0015] Methods for generating an nanoarray of metal or metal oxide nanostructures on a substrate are known from the literature in the field of mciroelectronics, and are described in many of the documents as a resist for pattern transfer to a substrate via an etch process. The invention is based on the finding that such nanoarrays have antimicrobial properties, and may be provided on packaging materials to confer an antimicrobial property to the packaging material. Items that are packaged in such packaging material are therefore subjected to the antimicrobial effects of the nanoarray of nanostructures. The invention is particularly applicable to the packaging of comestible items that are susceptible to microbial growth or spoilage.

[0016] The metals employed for the package, packaging materials, and methods and uses of the invention, may be selected from the group consisting of:

The surface bearing the nanoarray or nanopattern of nanostructures may be an interior surface of the packaging material itself, or it may be an insert that it inserted into the package. Inserts could be, for example, a planar film or card on which the nanoarray of nanostructures is formed. The packaging material may be polymeric, for example a polymeric bottle, pouch, tray, wrapper, bag, carton or film, with the nanostructures formed on an interior surface of the material, preferably a portion of the material that in use is in contact with at least a portion of the item.

[0017] Typically, the nanoarray or nanopattern of nanostructures has a density on the surface of at least 1 x 10⁶ nanostructures/cm², preferably at least 1 x 10⁷ nanostructures/cm², preferably at least 1 x 10⁸ nanostructures/cm², preferably at least 1 x 10⁹ nanostructures/cm², preferably at least 1 x 10¹⁰ nanostructures/cm². The nanostructures generally have a diameter of from 10-50nm, preferably 15-30nm, more preferably 20-30nm. Suitably, the centre to centre distance of the nanostructures is 30-60nm, preferably 40-50nm. Characterisation techniques such as atomic force microscopy, secondary electron microscopy, and helium ion microscopy, are employed.

[0018] Typically, the nanostructures have a flattened dome shape, such that the edges of the nanodot are not less than one fifth of the height at the centre. However, other types of nanostructures are envisaged, for example lines.

[0019] In one embodiment, the nanoarray or nanopattern of nanostructures is ordered (i.e. in an eqispaced pattern), and ideally periodically ordered. The term "periodically ordered" should be understood to mean that the system exhibits a pattern formed that has both short range and long range order. In this way the local pattern is reproduced so that the spacing of features is uniform in any chosen direction across the substrate.

[0020] The nanoarray of nanostructures may be provided in a pattern, for example having a round, oval, square, rectangular, triangular, or any other shape. Moreover, the surface may bear a plurality of nanoarrays, for example a pattern of dots on the surface in which each dots comprises a nanoarray of nanostructures.

[0021] Typically, the metal is a transition metal (i.e. iron, copper, silver, nickel, aluminium, tungsten, silicon cadmium) or a lanthanide (i.e. cerium), although other metals may be employed in the present invention. Preferably, the metal is silver.

[0022] The term "metal oxide" as employed herein should be understood to mean a chemical compound containing a metal and an anion of oxygen typically in a -2 state. Generally, the metal is selected from a transition metal (i.e. iron, copper, silver, nickel, aluminium, tungsten, silicon cadmium) or a lanthanide (i.e. cerium), although other metals may be employed in the present invention.

[0023] The term "metal ion salt" should be understood to mean an ionic compound comprising a salt-forming metal cation and a salt-forming cation. Examples of salt-forming anions include nitrates, nitrites, phosphates, sulphates, chlorides and carbonates. Suitably, the metal ion salt is a metal nitrate, for example iron (III) nitrate nonahydrate, cerium nitrate hexahydrate, and copper nitrate hemipentahydrate.

[0024] The term "item susceptible to microbial growth or spoilage" should be understood to mean an item that supports microbial growth in a packaged environment. Examples of items are comestible products, such as food and drink products, including red meat, poultry, fish, shellfish, vegetables, fruit, ready-made meals, dairy products, yoghurts, yoghurt drinks, fruit drinks, confectionary products.

[0025] Various forms of packages may incorporate a surface bearing a nanopattern or nanoarray of metal or metal oxide nanostructures, for example:

• film packaging, where the item is wrapped in the film and an interior face of the film provides the surface bearing the metal nanostructures ― the antimicrobial surafce may extend across all, some or only a portion of the interior face of the film;
• film plus tray packaging, where the item sits in a tray and the item and tray are wrapped in a film. In this case, the nanostructures may be formed on the tray, an interior surface of the film, or both, and the antimicrobial surafce may extend across all, some or only a portion of the interior face of the film or the tray;
• TETRAPAK packs, generally for drinks, milk, soups, sauces, yoghurts, in which the nanostructures are generally formed on an inner surface of the pack - the antimicrobial surafce may extend across all, some or only a portion of the interior face of the film;
• plastic wrappers, for example wrappers for chocolate bars or plastic wrapping for snacks, where the nanostructures are generally formed on an inner surface of the pack - ; the antimicrobial surafce may extend across all, some or only a portion of the interior face of the film
• plastic bag type packages, such as those employed to pack potatoes and vegetables, where the nanostructures are generally formed on an inner surface of the pack- the antimicrobial surafce may extend across all, some or only a portion of the interior face of the film;
• paper or paper polymer composite sheets, of the type employed to wrap fish and which is sealed by heat-sealing, where the nanostructures are generally formed on an inner surface of the sheet - the antimicrobial surafce may extend across all, some or only a portion of the interior face of the film;
• plastic bottles or vials, of the type employed to contain beverages or liquid samples, where the nanostructures are generally formed on an inner surface of the bottle, and typically disposed on a part of the inner surface of the bottle that in use is in contact with the item contained within the bottle. The antimicrobial surafce may extend across all, some or only a portion of the interior face of the film;
• glass bottles, where the nanostructures are generally formed on an inner surface of the pack - the antimicrobial surafce may extend across all, some or only a portion of the interior face of the film;
• cardboard boxes or cartons.

[0026] The surface bearing a nanopattern or nanoarray of metal or metal oxide nanostructures may be integral with the packaging material, or may be separate from the packaging material, for example an insert in the form of, for example, a sheet of card or polymer. The insert may be disposed within the package such that in use substantially all or a part of the insert abuts at least a part of the item. The surface bearing the nanopattern or nanoarray is preferably disposed within the packaging such that it abuts the item contained within the package. Thus, substantially all or a portion of the surface bearing the nanoarray of metal or metal oxide nanostructues generally abuts substantially all or a portion of the item contained wirthin the packaging.

[0027] The packages of the invention ideally enclose the packaged item, This means that they fully or partially enclose the item.

[0028] Nanorrays of metal or metal oxide nanostructures are created from a simple block copolymer self-assembly technique. Briefly, a thin film of a microphase separating solution is applied to a substrate (ceramic, metal, glass, polymer and films thereof) which is then treated by heating and/or solvent exposure to induce microphase separation into an ordered nanopattern. The so-formed film is then subject to exposure to a solvent containing metal cations. The solvent is chosen so that it selectively swells one block allowing the metal cations to enter one block. The substrate is removed and dried and placed under a UV/ozone atmosphere for a period of time. During this, the metal cations are oxidized to a solid oxide replication the polymer pattern formed by the microphase separation. The remaining polymer material is also oxidized in the treatment to CO2 so that a surface containing nanodots of metal oxide may be formed. Reduction to metal (from the oxide) can be achieved by exposure to reducing gas. The nanodots formed (oxide or metal) are well-adhered to the surface, robust, thermally stable and of uniform size. Nanodot sizes of 5nm to 100nm in diameter can be formed by choice of block copolymer.

Experimental

Example 1

[0029] A polystyrene-b-poly(ethylene oxide) (PS-b-PEO) diblock copolymer was purchased from Polymer Source and used without further purification (number-average molecular weight, M_n, PS = 42 kg mol^-1, M_n, PEO = 11.5 kg mol^-1, M_w/M_n = 1.07, M_w: weight-average molecular weight). Microscopic glass substrates were cleaned by ultrasonication in ethanol and toluene for 30 min each and dried under a nitrogen stream. PS-b-PEO was dissolved in toluene to yield 0.9 wt% polymer solution at room temperature and this solution was aged for 12 hours. The PS-b-PEO thin film was fabricated by spin coating the polymer solution at 3000 rpm for 30 sec on Si wafer. The film was exposed to toluene/water (50:50, v/v) mixed vapour placed at the bottom of a closed vessel kept at 50°C for 1h to induce mobility and allow microphase separation to occur. Separate reservoirs were used for each solvent to avoid azeotropic effects. The resultant phase separated film was immersed in ethanol at 40°C for 15h. For the fabrication of silver and zinc oxide nanodots silver nitrate (AgNO₃) and zinc nitrate hexahydrate (Zn(NO₃)₂,6H₂O) were used respectively. 0.6wt% and 1wt% concentrations of silver and zinc precursors were dissolved in ethanol respectively and spin coated onto the nanoporous film. UV/Ozone treatment (3h) was used in order to oxidize the precursor as well as to remove polymer residues. The spin coating of the precursors and UV/Ozone treatment were repeated four times in order to increase the concentrations of the resultant nanodots.

Example 2

[0030] The antimicrobial activity of the glass slides containing silver nanoarrays as produced according to Example 1 was carried out by agar diffusion method and viable cell count method. The sensitivity of bacterial strains (Gram-positive and Gram-negative bacteria) commonly present in food products and native microflora from chicken to the glass slides containing silver nanoarrays were determined by the agar diffusion method. Prior to use glass slides contaianing and not containing silver nanoarray were sterilised in a laminar flow using UV. The slides were then aseptically placed on the surface of the inoculated Muller Hinton agar (MHA) with 0.1 ml of inocula containing indicator microorganisms in the range of 10⁶ CFU/ml. The following bacterial strains were used: Escherichia coli (E. coli): (NCIMB 11943), Staphylococcus aureus (S. aureus): (NCIMB 13062), Bacillus cereus (B. cereus): (NCIMB 9373) and Pseudomonas fluorescences (Ps. fluorescences): (NCIMB 9046). A microflora isolated from raw chicken sourced locally was also used. Each strain was subcultured twice in 10 mL of Muller Hinton Broth and incubated at 30 for Ps. Fluorescens and B. cereus or 37°C for E. coli and S. aureus, respectively, for 18 h to reach a final concentration of 10⁹ CFU/mL. After incubation, culture was then diluted in sterile maximum recovery diluent (Oxoid, UK) to obtain a final cell density of ~10⁶ CFU/mL. The sensitivity to the silver nanoarray antimicrobial glass slides was defined by the area of the inhibition zone produced.

[0031] To test the antimicrobial activity of glass slides containing silver nanoarrays using a viable cell count method; glass slides (1.7 cm x 2.5 cm) containing silver nanoarrays were placed in individual sterile flasks to which 30 ml of inoculums, containing indicator microorganisms in the range of 10⁶ CFU/ml, were added. Inoculums of cell suspension in a flask with glass slides without silver nanoarrrays were used as a control. The flasks were incubated using an orbital shaker and rotated at 168 rpm at 30 or 37°C and aliquots of 1.5 ml were taken from the flasks and optical density at 610 nm measure every using a UV-visible spectrophotometer. The changes in the optical density were monitored for up to 36 hr.

[0032] The susceptibility of the native microflora obtained from chicken and pure bacterial strains to the silver nanoarray glass slides as determined by the agar diffusion method are presented in Table 1. The silver nanoarray showed antimicrobial effect against all bacteria tested (Gram (+) and Gram (-) bacteria) including chicken microflora. Inhibition zones were noticed in all bacterial strains tested and the most susceptible microorganism to the silver nanoarrays was Ps. fluorescence followed by S. aureus. A spore forming bacteria (Bacillus cereus) was also inactivated, indicating that the silver nanoarrays are a powerful antimicrobial with a wide spectrum. After measuring the inhibition zone area, plates were stored for up to 7 days and the inhibition zone area was measured again. The area of the inhibition zone did not change after 7 days storage indicating that the bacteria are inactive and possibly death and the effect of the silver nanoarrays is biocide and not only bacterisotatic.

[0033] The antimicrobial activity of the glass slides containing silver nanoarrays against Ps. Fluorescens and S. Aureus using a viable cell growth in liquid media is shown in Table 2. The silver nanoarrays delayed significantly the outgrowth of the bacteria tested compared to control samples.

Table 1: Antimicrobial activity of silver nanoarrays against pure culture and chicken microflora

Bacterial strain	Inhibition zone area* (cm2)
Pseudomonas Fluorescens	7.8
E. coli	5.0
Staphylococcus aureus	7.1
Bacillus cereus	5.7
Chicken microflora	6.6

Initial area of silver nanoarray slides = 4.20 cm2. The results presented are the average of 2 measurements.

Table 2: Antimicrobial activity of glass slides containing silver nanoarrays against Gram (+) and Gram (-) bacteria.

Bacterial strain	OD at 610 nm
	Time (hr)
	0		8		24		36
	C	SN	C	SN	C	SN	C	SN
Ps. fluorescens	0.004	0.004	1.024	0.004	1.631	0.0073	ND	0.011
S. aureus	0.004	0.004	1.12	0.009	1.745	0.012	ND	0.015

C: glass slides without silver nanoarrays SN: glass slides containing silver nanoarrays ND: not determined

Example 3

[0034] A beef steak was packaged in a Styrofoam tray, and the steak and tray were wrapped within a polyvinylidine chloride barrier film (SARANEX 451) bearing a nanoarray of silver chloride nanostructures on an interior surface of the film. The surface bearing the nanoarray of nanostructures covers an area of approximately 5cm², with the nanostructures provided by nanodots having an average diameter of approximately 20nm, and arranged at a density of approximately 1 x 10⁸ nandots/cm². As packaged, the surface bearing the nanoarray of silver chloride nanodots abuts a top surface of the steak. A simlar steak was packaged in similar packaging but without the array of nanodots on the film. Both packages were stored for five days at refridgeration conditions, and the microbial load on the steaks sampled at days 3, 4, and 5.

Example 4

[0035] A beef steak was packaged in a Styrofoam tray, and the steak and tray were wrapped within a polyvinylidine chloride barrier film (SARANEX 451) bearing a nanoarray of silver chloride nanostructures on an interior surface of the film. Prior to packaging within the film, a stiff polymeric insert was placed on a top surface of the meat, the insert bearing a nanoarray of silver chloride nanostructures on a surface of the insert that bears against the meat. The surface bearing the nanoarray of nanostructures covers an area of approximately 5cm², with the nanostructures provided by nanodots having an average diameter of approximately 20nm, and arranged at a density of approximately 1 x 10⁸ nandots/cm². A simlar steak was packaged in similar packaging but without the array of nanodots on the film insert. Both packages were stored for five days at refridgeration conditions, and the microbial load on the steaks sampled at days 3, 4, and 5.

Example 5

[0036] A salmon steak was packaged heat sealable poly-coated foil paper packaging material bearing a nanoarray of silver chloride nanostructures on an interior surface of the material. The surface bearing the nanoarray of nanostructures covers an area of approximately 5cm², with the nanostructures provided by nanodots having an average diameter of approximately 20nm, and arranged at a density of approximately 1 x 10⁸ nandots/cm². As packaged, the surface bearing the nanoarray of silver chloride nanodots abuts a top surface of the salmon steak. A simlar steak was packaged in similar packaging but without the array of nanodots on the film insert. Both packages were stored for five days at refridgeration conditions, and the microbial load on the steaks sampled at days 3, 4, and 5.

[0037] The invention is not limited to the embodiments hereinbefore described which may be varied in construction, detail and process step without departing from the spirit of the invention.

Claims

1. A package comprising packaging material defining an enclosed space suitable for containing an item susceptible to microbial growth or spoilage, an interior of the package comprising at least one antimicrobial surface bearing an ordered nanoarray of metal or metal oxide nanostructures.

2. A package as claimed in any preceding Claim in which the surface bearing a nanoarray of metal or metal oxide nanostructures is part of an interior surface of the packaging material.

3. A package as claimed in any of Claims 1 to 2 in which the nanoarray of nanostructures has a density on the surface of at least 1 x 10⁸ nanostructures/cm².

4. A package as claimed in any of Claims 1 to 3 in which the nanostructures have an average diameter of from 10-50nm.

5. A package as claimed in any preceding Claim in which the antimicrobial surface is formed of a polymeric material.

6. A package as claimed in any preceding Claim and selected from a tray, a carton, a bag, a pouch, a bottle, and a wrapper.

7. A package as claimed in any preceding Claim, in which the antimicrobial surface bearing an array of metal or metal oxide nanostructures has a surface area of at least 5cm².

8. A package as claimed in any preceding Claim containing an item susceptible to microbial growth or spoilage.

9. A package as claimed in Claim 8 in which the item susceptible to microbial growth or spoilage is a comestible product.

10. A package as claimed in any preceding Claim in which at least a portion of the at least one antimicrobial surface is in contact with at least a portion of the item.

11. A packaging material in the form of a sheet or film having a first face and a second face, in which at least a portion of one of the first or second faces of the film comprises a surface bearing a nanoarray of metal or metal oxide nanostructures.

12. A packaging material as claimed in Claim 11 in which: the nanoarray of nanostructures has a density on the surface of at least 1 x 10⁸ nanostructures/cm²; and/or in which the nanostructures have an average diameter of from 10-50nm.

13. A package as claimed in of Claims 1 to 10 or a packaging material as claimed in Claim 11 or 12 in which the metal is silver.

14. Use of a packaging material as claimed in Claim 12, 13 or 14, as an antimicrobial agent.

15. A method of preventing or inhibiting microbial growth on a comestible item contained within a package, which method employs a packaging material at least part of which comprises a surface bearing a nanoarray of metal or metal oxide nanostructures, the method comprising a step of partially or completely enclosing the comestible item within the packaging material to form a package such that the surface bearing the nanoarray of metal or metal oxide nanostructures is disposed on an interior of the package.