The invention relates to a packaging material having antimicrobial properties in form of a layered composite, containing a nanocomposite coating with zerovalent iron nanoparticles. The invention further relates to a method for producing a packaging material and use of a nanocomposite containing zerovalent iron nanoparticles for the production of a coating having antimicrobial properties. The packaging material can be used primarily for packaging of food, but also drugs, cosmetics, medical equipment (especially disposable tools) and electronic components (such as microprocessors).

Basic functions of packagings are closely related to their suitability in ensuring food quality and safety in the supply chain and during food storage by end consumers [Tichoniuk 2019] Commonly used traditional packaging forms primarily protect the packed products, provide information about them, and also allow for their convenient consumption [Vanderoost et al. 2014, Miiller and Schmid 2019] Protection against unfavorable external factors, chemical and/or microbiological contamination results mainly from the barrier properties of packaging materials and the packaging structure as such, but it is realized in a passive manner [Cierpiszewski 2016, Schaefer and Cheung 2018] Active packagings and materials actively contribute to reducing the effects of the above-mentioned unfavorable factors and increase the functionality of traditional packaging systems, most often by releasing the desired or absorbing harmful substances from the packed product and/or its environment [Lisinska-Kusnierz 2010, Schaefer and Cheung 2018] Use of active packaging solutions can significantly extend food shelf life and reduce the costs associated with protecting food against its deterioration, as well as the costs resulting from the management of already spoiled products. Active packaging systems are especially useful for perishable food products, which can thus be protected in the supply chain while reducing the degree of processing of the above-mentioned products, as well as the amount of food preservatives used. The most frequently used active packaging solutions include moisture adsorbers, oxygen absorbers, carbon dioxide emitters, antioxidant or antimicrobial elements [Wyrwa and Barska 2017, Poyatos-Racionero et al. 2018, Yildirim et al. 2018]

Food is a very good environment for the growth of various microorganisms, the existence of which leads to or accelerates changes in the taste, smell, appearance and consistency of food products and thus significantly shortens their shelf life. In some cases, contamination with pathogenic microorganisms may also lead to a threat to the health or life of consumers [Realini and Marcos 2014, Sofi et al. 2017] Antimicrobial active packaging materials are most often designed to extend the adaptive phase (lag phase) and to limit the growth phase of undesired microorganisms. In the case of non-volatile substances, the packaging materials must be in direct contact with the food to be protected, for example by using so-called antimicrobial coatings. In such case antimicrobial substances can be immobilized on the surface of the active package or released from it directly into the protected product [Malhotra et al. 2015] Antimicrobial coatings and similar direct wrappers are particularly effective against the microflora developing on the surface of microbiologically contaminated food products. Among the antimicrobial substances used in antimicrobial active packaging, the most common are natural essential oils [Atares and Chiralt 2016], enzymes and bacteriocins [Barbiroli et al. 2012], polymeric substances having antimicrobial properties [Soysal 2015], organic acids and their derivatives [Jiinior et al. 2015] as well as other substances with nanoparticle sizes [Mihaly Cozmuta et al. 2015, Mlalila et al. 2016] Table 1 below lists exemplary, commercially available antimicrobial active packages [Tichoniuk 2019]

Table 1. Examples of comercially available antimicrobial food packagings [Tichoniuk 2019]

Numerous antimicrobial films have also been developed based on antimicrobial agents or nanometals such as silver, gold and copper nanoparticles [Rzeszutek et al. 2014, Hoseinnejad et al. 2018] Antimicrobial coatings applied on the surface of various materials, including those intended for packaging purposes are also known [Cierpiszewski 2016, Foltynowicz

2017]

For example, antimicrobial packaging properties of poly(lactic acid) (PLA) films are obtained by applying thereon coatings containing bioactive substances against Escherichia coli bacteriae [Karakurt et al., 2019]

Due to the fact that recently an increasing range of so-called "bio" products is available on the market, the products being made of raw materials obtained without the use of chemicals and as such containing no preservatives, it is important to protect these products against microbiological spoilage by using functional packagings. Currently, the products containing no preservatives are used mainly by a more demanding group of consumers, but due to their high pro-health value, these products are becoming more and more popular among the general public. The rapid microbial spoilage of these products can cause losses for producers, which can discourage them from extending their production range. Decreased quality associated with the development of unfavorable microflora can also pose a health risk to consumers.

In reviews on the use of metal nanoparticles having antimicrobial activity in packaging industry, no mention is made of iron nanoparticles. The phrase "iron nanoparticles" is found in titles of many reports, but very often this term includes mainly iron oxides. Iron nanoparticles, especially these at zero oxidation state (zerovalent iron, ZVI), are used as active oxygen scavenger components for packagings [Foltynowicz 2017, Foltynowicz

2018] In PL 227585 a method is disclosed for the production of nano-iron doped with boron in the amount of 0.01-10 wt. % as a result of reduction of iron (III) salt with NaBLL under strictly defined reaction conditions, as well as the use of this nano-iron as an oxygen absorbing material placed in sachets made of paper or plastic permeable to oxygen, the sachets being intended to be placed inside the packagings as oxygen scavengers. Another method for producing nano-iron is disclosed in PL 227096, also with the indication that the material thus obtained can be used in a particulate form as an oxygen absorber in sachets to be placed in the packagings.

Patent application no. P.397499 discloses a nanocomposite oxygen scavenger containing a polymer in combination with nano-iron or zero-valent iron boron-doped. In this composition, the ZVI nanoparticles constitute an active filler, dispersed throughout the entire volume of the polymer matrix. Preferably the nano-iron is the iron obtained by the method disclosed in PL 227096, and the zero-valent iron doped with boron is preferably the product of the process described in PL 227585. Preferred polymers mentioned in P.397499 are silicone rubbers, polysiloxanes, modified cellulose acetate butyrate, polyamides, poly(vinyl alcohol), polyethylene, polyethylene terephthalate), cellulose derivatives, modified starch, biodegradable polymers [such as poly(lactic acid), poly(hydroxybutyrobutylate) and poly(oxymethylene)], and mixtures thereof.

In the course of research on the possibilities of using ZVI nanoparticles in packaging materials, it has been unexpectedly found that these nanoparticles introduced into the polymer matrix and in the form of a thin coating applied to a packaging support layer (plastic film as well as paper or other material) not only show oxygen absorption properties, but also antimicrobial properties.

In its first aspect the invention therefore relates in a first aspect to a packaging material having antimicrobial properties in form of a layered composite having a support layer with an outer surface and an inner surface intended for contacting the package contents, wherein at least a part of the inner surface of the support layer is provided with a nanocomposite coating comprising zerovalent iron nanoparticles dispersed in a polymer matrix. Such a solution not only allows to limit the growth of microorganisms on the surface of products packed in the packaging material according to the invention, but also to optimally use the activity of ZVI nanoparticles in packaging materials produced on a large scale, and to easily modify the existing technological lines for the production of packaging materials according to the invention. Preferably the support layer is made of a natural or synthetic polymer, paper, impregnated paper, metal or a composite material. In particular, the support layer is formed as a polymer film, preferably a polyolefin film.

Preferably, the polymer matrix of the nanocomposite coating is made of a biodegradable polymer, preferably poly(lactic acid) (PLA) or polyhydroxybutyrate (PHB). The possibility of using biodegradable polymers to obtain the coating in combination with the use of environmentally friendly materials for the support layer (biodegradable polymers as well as paper) is an attractive alternative to conventional packagings made of synthetic plastics, especially in food industry.

In one preferred embodiment, the zerovalent iron nanoparticles are obtained in a reaction of an iron salt with sodium tetraborohydride NaBTL _t , wherein:

- molar ratio of the iron salt to NaBTLt is 1 : 3 ± 5%,

- the iron (III) or (II) salt concentration ranges from 0.01 to 0.05 M, and NaBTL _t concentration ranges from 0.04 to 0.2 M,

- NaBTL _t solution is added dropwise to the iron salt solution at a rate corresponding to 0.6 to 1.1 parts by weight per 100 Te gram-atoms per minute,

- the reaction is carried out in an atmosphere of deoxygenated inert gas,

- the reaction is carried out at room temperature, and once all the NaBTLt has been added dropwise, it is heated to a temperature from 70 °C to 90 °C and then cooled.

In another preferred embodiment, the zerovalent iron nanoparticles are obtained in a reaction of an iron (III) salt with sodium tetraborohydride NaBTL _t , wherein:

- molar ratio of the iron (III) salt to NaBTLt is 1 : 3 with a stoichiometric excess of NaBTLt to the iron (III) salt of 30 to 50%,

- the iron (III) salt concentration ranges from 0.01 to 0.05 M, and NaBTL _t concentration ranges from 0.05 to 0.2 M,

- NaBTL _t solution is added dropwise to the iron (III) salt solution at a rate corresponding to 3.5 to 5.5 parts by weight per 100 Te gram-atoms per minute,

- the reaction is carried out in an atmosphere of deoxygenated inert gas,

- the reaction is carried out at room temperature, and once all NaBH4 has been added dropwise, it is heated to the temperature from 70 °C to 90 °C, and then cooled. Preferably, the packaging material according to the invention is formed as a sheet in a shape selected from a rectangle, square, rectangle with rounded corners, triangle and circle, but any other forms are possible depending on the characteristics of the packaged product.

In a further aspect the invention relates to a method for producing a packaging material according to the invention, wherein zerovalent iron nanoparticles are stored for at least 3 hours in 70% ethanol containing 0.01% by weight of a-lipoic acid, and then after evaporation of ethanol, the nanoparticles are introduced into a solution of a matrix polymer in an organic solvent, and from the suspension thus obtained a nanocomposite coating is formed on a support layer. The storage of the ZVI nanoparticles (preferably directly upon obtaining thereof) in 70% ethanol with the addition of a-lipoic acid protects them against oxidation, and thus allows to maintain their activity after they are embedded into the structure of the packaging material according to the invention.

In one preferred embodiment of the method according to the invention, the nanocomposite coating on the support layer is formed by pouring the zerovalent iron nanoparticles suspension in the matrix polymer solution directly onto the support layer.

In another preferred embodiment of the method according to the invention, the nanocomposite coating on the support layer is formed by producing a nanocomposite film followed by applying the film to the support layer by lamination.

Preferably, the organic solvent used in the method according to the invention is chloroform or an ionic liquid, preferably didecyldimethylammonium nitrate. Chloroform allows to obtain solutions of poorly soluble biodegradable polymers, such as PLA. In turn, ionic liquids, apart from their usefulness as solvents of such polymers, are compounds that are safe to use and meet the criteria of agents used in the so-called "green chemistry".

In a further aspect, the invention relates to the use of a nanocomposite containing zerovalent iron nanoparticles dispersed in a polymer matrix for producing a coating having antimicrobial properties on an inner surface of a support layer of a packaging material in form of a layered composite.

The subject of the invention is in examples of embodiment is illustrated in the attached drawing, in which Fig. 1 shows the packaging material according to the invention in form of a rectangular sheet, in Fig. 2 - in form of a square sheet, in Fig. 3 - in form of a rectangular sheet with rounded corners, in Fig. 4 in form of a triangle and in fig. 5 - in form of a circle.

Examples of invention embodiments The ZVI nanoparticles used in the examples below were obtained according to the methodology disclosed in WO 2012/091587, the scope of which included the content of the solutions covered by the patents PL 227585 and PL 227585 and the application P.397499.

Example 1: Preparation of Z VI/PL A foil

The PLA film containing ZVI nanoparticles was obtained by pouring a mixture of PLA and ZVI nanoparticles in chloroform onto a polished steel plate, and then distributing the liquid using a steel plate with a slot milled along the base. The slit was 0.4 mm high and 100 mm long (see Fig. 1). Once the liquid was distributed, it was allowed to dry. The films were removed from the polished steel plate 48 hours after its application. A starting solution of PLA in chloroform was obtained by mixing 10 g of PLA and 135 g of chloroform in a beaker. The ingredients were mixed with a magnetic stirrer until the PLA granules were completely dissolved and a homogeneous solution was formed. The beaker was covered with a watch glass to prevent chloroform evaporation. Mixing is carried out at room temperature for about 3 hours. After the PLA was completely dissolved, the magnetic stir bar was removed from the beaker and an appropriate test portion of ZVI nanoparticles was added. Amounts of 0.10, 0.20; 0.30 or 0.50 g of the additive per 10 g of PLA were introduced. Finally, the films were obtained containing the addition of ZVI nanoparticles in the amount of 1; 2; 3 and 5% by weight. The mixture was then stirred for 10 minutes in an ice-water bath using an ultrasonic homogenizer VCX130, Sonics, USA (frequency: 20 kHz, vibration amplitude 50%).

Example 2: Preparation of a ZVI/PLA coating applied to polyolefin films

To prepare the ZVI/PLA suspension, the procedure described in the example 1 was followed. The suspension was then spread over a polyolefin film placed on a polished steel plate. Chloroform was allowed to evaporate for 24 hours.

Coatings made of PLA without addition and with the addition of 1%, 3% or 5% by weight of ZVI nanoparticles were tested.

Example 3: Antimicrobial activity of the ZVI/PLA film against standard microbial strains

In order to evaluate the antimicrobial activity of the ZVI/PLA film obtained according to the procedure described in the example 1, the following procedure was followed: 1) A ZVI/PLA film containing the addition of ZVI in an amount of 3% by weight was immersed in 70% ethanol for 15 minutes, then transferred to sterile plates and left to allow ethanol to evaporate.

2) Inoculation was carried out on a medium: nutrient agar (bacteriae) and Sabouraud's medium (fungi: yeasts and molds).

3) Inoculation using the surface method: solidified medium was inoculated with 100 pi of the microorganism suspension having a density of 1.5 x 10 ⁷ cells per 1 ml (bacteriae) and 1 ^c 10 ⁶ - (fungi), and then the films previously dipped in the microorganism suspension were applied to the substrate.

4) The plates were incubated at 37 °C in case of bacteriae or at room temperature in case of fungi.

The results of determination of antimicrobial activity of the ZVI/PLA film against the standard microbial strains are presented in Table 2.

Table 2. Inhibition of growth of the standard bacteriae strains by ZVI formulations „+” - growth inhibition on the foil surface, - no growth inhibition on the foil surface

Example 4: Antimicrobial activity of ZVI/PLA coating applied to polyolefin films

The antimicrobial activity of the ZVI/PLA coating, applied to the polyolefin films according to the procedure described in example 2, was determined by testing the stability of the product, which was goat cream (bio) curd cheese with a unit weight of 150 g, packed in films covered with the ZVI/PLA coating containing the addition of ZVI nanoparticles in the amount of 3% by weight. During the product storage, it turned out that the use of the PLA coating containing ZVI nanoparticles increased the shelf life of the cheese to 6 weeks. During this time, no growth of microorganisms on the package surface (wrapping made of the tested polyolefin film covered with the ZVI formulation) and the product surface was observed. The packaging containing the ZVI nanoparticles did not inhibit the development of the natural (desired) microflora present in the cheese mass. In order to assess the antimicrobial properties of the functional packaging, the following media were inoculated: - PCA - (plate count agar) (assessment of the total amount of psychrophilic microorganisms - incubation at 20°C ± 2°C; 72 h)

- Sabouraud with chloramphenicol (assessment of the total amount of fungi - incubation at 20°C ± 2°C; 5-7 days)

The results of the determination of the antimicrobial activity of the ZVI/PLA nanoparticle coating applied on polyolefin films are presented in Table 3.

Table 3. Results of microbiological tests of goat cheese packed in packages with ZVI/PLA nanoparticle coating (inoculation of the package surface, product surface and product mass)

„+” - visible growth of microorganisms on the plates, - no growth of microorganisms on the plates, the control was polyethylene film covered with PLA coating

Examples 3 and 4 show the possibility of industrial application of the coatings having antimicrobial activity based on ZVI nanoparticles, e.g. in the packaging industry.

Example 5: Shapes of packaging material sheets

Depending on the characteristics of a specific product to be packed, the packaging material is cut to appropriate shapes. As shown in the figures of drawings, exemplary shapes of sheets of the packaging material according to the invention are: a rectangle having sides a and b (where a ¹ b) (Fig. 1), a square with side a (Fig. 2), a rectangle with rounded corners (Fig. 3), a triangle (fig. 4) and a circle (fig. 5).

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