Coating composition for food packaging with antimicrobial properties

Abstract

The present invention relates to an antimicrobial coating of food products comprising the application of an active and flexible packaging; and more particularly to a substrate material coated with a permanently positively charged natural polymer selected from chitosan and gelatin, wherein the substrate material surface is oxygen or corona plasma pre-treated prior to coat, and the method of preparation thereof.

Description

[0001] Polymer coatings for antimicrobial surfaces for food preservation

[0002] The present invention relates to ah antimicrobial coating of food products comprising the application of an active and flexible packaging: and more particularly to a substrate materia! coated with a permanently positively charged natural polymer selected from chitosan and gelatin, wherein the Substrate material surface is oxygen or corona piasma pre-treated prior to coat, and the method of preparation thereof.

[0003] BACKGROUND ART

[0004] Food products need to be protected from mold, rot and water damage. Coatings may provide this kind of protection and may also improve their appearance and. hence, their marketability. Common coatings now in use employ proteins, gums, resins hydrocollgids. waxes, and oils either alone or in combination, to achieve certain desired objectives. Enhancing appearance, slowing moisture loss.: affecting the respiration or ripening process of fruits, nuts, and vegetables, are several of the goals of many modern food coatings.

[0005] One of the most well-known coatings used is chitosan, a natural polysaccharide derived from the deacetylation of chitin, that has a great potential for a wide range of applications. As an edible coating material, chitosan has been reported to be highly effective in extending the post-harvest life of many crops, while reducing moisture loss, respiration, ethylene production, ripening, softening, controlling the decay, and maintaining fruit quality, along with storability. Chitosan exhibits intrinsic antimicrobial activity against both Gram-positive and Gram-negative bacteria, fungi, and mold, due to the presence of primary amine side groups in its structure. However, chitosan is soluble in dilute acidic solutions only and its reported antimicrobial activity is limited to acidic conditions, where its amine groups are positively charged, which limits its practical applications.

[0006] On the other hand, polypropylene (PR) and polyethylene (PE) are among the most widely used today conventional packaging material in a variety of food packaging applications. Also, PP arid PE are the standard coating material in use today in board and paper-based packaging. Additional benefits of using PP and PE packaging for

[0007] SUBSTITUTE SHEET (RULE 26) food and beverages include their puncture resistance, sterility, light-weighting capability, and versatility for a wide range of package sizes or types.

[0008] The aim of the current invention is to provide improved antimicrobial coatings, which will have more favourable application properties, especially in food packaging and preservation, without an impairment of the organoleptic or aesthetic properties.

[0009] SUMMARY OF THE INVENTION

[0010] The present invention discloses a protective coating on the surface of the food product comprising a conventional packaging such as PE, PP, bio-based polyesters (PLA, PCL), glass, or paper, coated with a modified natural polymer such as chitosan, and a crosslinker, wherein the said coating composition forming a film in contact with the food product has antimicrobial properties.

[0011] In a first aspect, the present invention is related to a substrate material coated with a permanently positively charged natural polymer selected from chitosan and gelatin, characterised in that the substrate material surface is oxygen or corona plasma pretreated prior to coat, and the coating is cross-linked and bound with the surface of the substrate material by a cross-linker.

[0012] In a preferred embodiment, the material is selected from polyethylene, polypropylene, polyethylene terephthalate), poly(vinyl chloride), polystyrene, copolymer poly(ethylene terephthalate glycol) (PETG), glass, paper, paperboard, cellulose fibers, cellulose acetate butyrate, cellulose acetate propionate, poly(lactic acid), or other recycling plastic packaging. In a more preferred embodiment, the packaging

[0013] In another preferred embodiment, the crosslinker is selected from ammonium zirconium carbonate and dialdehyde. In a more preferred embodiment, the crosslinker is selected from a dialdehyde, glutaraldehyde, oxidised sucrose and glyoxal; and in an even more preferred embodiment the dialdehyde is glyoxal.

[0014] In another preferred embodiment, wherein the permanently positively charged natural polymer is quaternized chitosan or gelatin with quaternization degrees between 20% and 70% of the chitosan repeat units and between 5 % to 30 % of the gelatin repeat units.

[0015] On the other hand, the present invention regards to the method for preparing the substrate material as described before, which comprises the chemical modification of natural polymers such chitosan and gelatin to bear permanently positively charged quaternary amine groups along their chains. In particular, chitosan (CS) can be modified through the reaction of its amine groups (CS) with (2,3-epoxypropyl)- trimethylammonium chloride (GTMAC) to form the quaternized derivatives of chitosan QCS, according to the following scheme:

[0016] Through this chemical modification, quaternized chitosan derivatives (QCS) show significantly enhanced biocidal activity regardless of the solution pH and at the same time they become water-soluble, as a result, acids are not required for their solubilization to prepare the coating solution.

[0017] Following to that modification, in case the substrate material is polyethylene, the polyethylene surface is also treated using oxygen plasma. Polyethylene is a hydrophobic polymer that does not contain functional groups, so it has a low affinity for hydrophilic natural polymers such as chitosan. For this reason, the polyethylene developed to avoid dewetting of the coating.

[0018] Finally, for the development of stable coatings onto the polyethylene substrates, aqueous solutions of the modified chitosan in the presence of a cross-linker were deposited on the oxygen plasma pretreated polyethylene surfaces. This crosslinker stabilizes the coating by forming a crosslinked film of modified chitosan chemically bound onto the packaging surface. The crosslinker remains on the packaging during use in food. Therefore, another aspect of the invention regards to the method for preparing a substrate material as described above, which comprises the following steps: a) chemical modification of the natural polymer selected from chitosan and gelatin to bear permanent quaternary ammonium salt groups; b) pre-treatment of a material surfaces with oxygen or corona plasma; c) coating of an aqueous solution of the chemically modified natural polymer from step (a) in the presence of a crosslinker on the pretreated substrate material surface from step (b), at pH values between 3.5 and 6.

[0019] In a preferred embodiment, the natural polymer is selected from chitosan and gelatin bearing quaternary ammonium salt groups. In a more preferred embodiment, the material of the invention is based on quaternized chitosan. In an even more preferred embodiment, the quaternized chitosan or gelatin is prepared by reaction of chitosan with a quaternization agent based on an epoxy trimethylammonium salt such as (2,3- epoxypropyl)-trimethylammonium chloride, on a chloro- or bromo-trimethylammonium salt such as (3-chloro-2-hydroxy-propyl)trimethyl ammonium chloride and (3- bromopropyl)trimethylammonium bromide to provide quaternary ammonium salt groups on the polymer repeat units.

[0020] In another preferred embodiment the degree of quaternization of the chitosan in step (a) ranges between 20% and 70%; even more preferred is between 40% and 60%.

[0021] In another preferred embodiment the degree of quaternization of the gelatin in step (a) ranges between 5 and 30%, more preferably between 10% and 20%. polyethylene, polypropylene, polyethylene terephthalate), poly(vinyl chloride), polystyrene, copolymer poly(ethylene terephthalate glycol), glass, paper, paperboard, cellulose fibers, cellulose acetate butyrate, cellulose acetate propionate, polylactic acid; more preferably the material is polyethylene. In a more preferred embodiment, the packaging material is polyethylene.

[0022] In another preferred embodiment the crosslinker in step (c) is selected from ammonium zirconium carbonate and dialdehyde. In a more preferred embodiment, the crosslinker is selected from a dialdehyde, glutaraldehyde, oxidised sucrose and glyoxal; and in an even more preferred embodiment the dialdehyde is glyoxal.

[0023] In another preferred embodiment, the crosslinker in step (c) is added at a crosslinker / natural polymer repeat units molar ratio that ranges between 0.3 and 0.6; more preferred between 0.3 and 0.4; and even more preferred the crosslinker in step (c) is added at a 0.35 crosslinker / natural polymer units molar ratio.

[0024] In another preferred embodiment, the coating step (c) is applied by a roll-to-roll process, rotogravure, flexography, dipping, spraying or inkjet printing, followed by heating above 40 °C, more preferably between 40 °C and 100 °C, depending on the substrate tolerance to heat.

[0025] Another aspect of the invention is the use of the coated substrate material as described above as antimicrobial film, preferably for packaging.

[0026] In a preferred embodiment, the coated substrate material of the invention is used for packaging of food products and more preferably for dairy products; and even more preferably for packaging cheese and / or cheese derivatives including a soft, semi-soft, hard, and extra hard cheese.

[0027] In the present invention the term “food products” refers to all types of edible products, such as fruit, vegetables, ornamentals, dairy products, sausage and ham products, eggs, dairy or bakery products. Preferred food products are cheese and / or cheese derivatives including a soft, semi-soft, hard, and extra hard cheese. Preferred cheeses include feta cheese.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skilled in the art to which this invention belongs. Methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. Throughout the description and claims the word "comprise" and its variations are not intended to exclude other technical features, additives, components, or steps. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples are provided by way of illustration and are not intended to be limiting the present invention.

[0029] DESCRIPTION OF THE DRAWINGS

[0030] Fig.1 : Cross-section FE-SEM images of PE coated with QCS55.

[0031] Fig. 2: Population decrease of L. monocytogenes in QCS55 at 37 °C.

[0032] Fig. 3: S. aureus population decrease in QCS55 at 37 °C.

[0033] Fig. 4: Population decrease of L. monocytogenes in QCS55 in escalating concentration at 20 °C.

[0034] Fig. 5: Population decrease of L. monocytogenes in QCS55 in escalating concentration at 4 °C.

[0035] Fig. 6: S. aureus population reduction after inoculation onto glass slides bearing the modified biopolymer coatings.

[0036] Fig. 7: S. aureus population reduction after inoculation onto the glass slides bearing the modified biopolymer coatings.

[0037] Fig. 8: E. coli population reduction after inoculation onto the glass slides bearing the modified biopolymer coatings.

[0038] Fig. 9: L. monocytogenes population reduction, inoculation on slides bearing modified biopolymers.

[0039] Fig. 10: Reduction of S. aureus after inoculation onto the control (PE), non-modified CS and quaternized chitosan (PEQCS55) coatings.

[0040] Fig. 11 : E. coli population reduction after inoculation onto the control (PE), nonmodified CS and quaternized chitosan (PEQCS55) coatings.

[0041] Fig. 12: L. monocytogenes population reduction after inoculation onto the control (PE), non-modified CS and quaternized chitosan (PEQCS55) coatings.

[0042] EXAMPLES

[0043] Example 1. Chemical modification of Chitosan

[0044] For the modification of chitosan with quaternary ammonium salt groups, chitosan with Mn = 30K and with a degree of deacetylation (DD) -90% w / w was used, and the following synthetic procedure was followed. First, chitosan was dissolved in an aqueous solution containing acetic acid (1%v / v) and then GTMAC was added in two different molar ratios (GTMAC / CS building units) (3 / 1 , 6 / 1 ). The reaction was allowed to proceed at 70 °C for 24 h. The reaction product was purified by precipitation in acetone. Then, the product was diluted in water and lyophilized for 24 h. The chemical structure of the modified chitosan was characterized by nuclear magnetic resonance (NMR) and attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopies. In addition, the percentage of the quaternary ammonium salt groups in the structural units of chitosan was calculated by1H NMR spectroscopy, by rationing the integrals of the peaks at 2.2 ppm, assigned to the methyl groups of chitosan, to the peak at 3.9 ppm assigned to the methyl protons of the quaternary ammonium groups. Thermal stability of the materials was determined by thermogravimetric analysis. Two different quaternized chitosan derivatives (QCS) with quaternization degrees of 25% and 55% were prepared by the above process. The degree of quaternization is crucial for achieving the desired antimicrobial properties and adhesion of the coating on the PE. In particular, a balance between the biocidal quaternary ammonium salt groups that provide the antibacterial properties and the free amine groups which serve as cross-linking and anchoring points of the film to the PE substrate is necessary, to ensure high coating bioactivity and stability at the same time. For example, when the degree of quaternization was greater than 70% stable coatings for prolonged time immersed in water could not be prepared.

[0045] Example 2. Oxygen plasma treatment of the polyethylene surface

[0046] For the oxygen plasma treatment, the polyethylene membranes were first cut into 10 cm*20 cm dimensions, cleaned with ethanol, and then irradiated with a 89-watt oxygen plasma for 3.5 sec, keeping the pressure constant at 0.5 mbar. Oxygen plasma treatment of polyethylene surfaces is expected to change their hydrophobicity and roughness. For this reason, the polyethylene (PE) membranes before and after the oxygen plasma treatment were characterized for their hydrophilicity by water

[0047] SEM in order to examine any changes in the surface roughness of PE after the oxygen plasma treatment, while the changes in the chemical structure of the PE surfaces were studied by (ATR-FTIR). The ATR-FTIR spectrum of oxygen plasma-treated PE shows the characteristic peaks of PE at 2912, 2834 cm-1, 1456 cm-1and 710 cm'1assigned to the asymmetric and symmetric stretching vibrations of C-H, the bending C-H, and the rocking C-H vibration, respectively and two additional peaks at 1680 erm1and 1070 cm-1attributed to the C=O and C-0 stretching vibrations, indicating the presence of polar groups on the PE surface. Furthermore, the induced change in hydrophilicity of PE after oxygen plasma treatment was examined by water contact angle measurements. Oxygen plasma treatment decreased the contact angle of water on the PE surface from 93 ± 2° to 55 ± 2°, indicating an increased hydrophilicity of PE as polar functional groups were formed on the surface after treatment, which was in agreement with the ATR-FTIR analysis.

[0048] Example 3. Coating of quaternized Chitosan on the plasma pretreated polyethylene surface

[0049] For the fabrication of the QCS coatings, an aqueous solution of QCS 0.05 g / ml was prepared. The pH of the solution was adjusted at 5 with the addition of acetic acid (0.25 v / v % in water). Next, glyoxal was added at a 0.35 crosslinker / chitosan molar ratio. In order to deposit the solution on the polyethylene surface, either specially engraved rollers (Mayer rods), for the small-scale experiments, or rotogravure printing, for large-scale experiments, were used. The large-scale experiments were carried out in a roll-to-roll process on a normal production line (Nordmeccanica machine) with a production speed of 40 m / min and varnish drying temperatures of 60 - 80 °C. Furthermore, in the large-scale experiments, the QCS aqueous solution applied, contained 5 to 20% v / v ethanol relative to water, facilitating the rapid drying of the ink and making it feasible to apply it in the roll-to-roll process. The coatings obtained with Mayer rods were allowed to dry in air overnight. Several parameters are crucial for the preparation of stable QCS coatings on the PE surfaces. First of all, the pH value of the QCS / cross-linker solution is essential for the coating preparation since it can affect the material’s adherence to the PE surface. At pH values lower than 3,5, no uniform coatings were formed, indicating low adhesion of the polymer to the PE surface. In addition, at pH values higher than 6, the amine groups of QCS react with the aldehyde groups of the cross-linker agents at a faster rate, which results in link the polymer and thus become less available to anchor the QCS to the PE surface. Furthermore, the cross-linker content has a high impact on the stability of the coatings. In particular, at a cross-linker / QCS molar ratio below 0.30, the coatings were not stable after prolonged immersion in water.

[0050] The prepared coatings were characterized by infrared spectroscopy and FE-SEM, while the wettability of the coatings was examined by water contact angle measurements. The ATR-FTIR spectrum of coated PE packaging surfaces shows the characteristic vibrations derived from PE (715, 1471 and 2910-2850 cm-1), as well as the chitosan-derived bond vibrations. In particular, the characteristic peaks of chitosan at 1642 cm-1and 1150 cm-1, are observed which correspond to the stretching vibrations of the C=O bond of the acetyl group of CS, and to the asymmetric stretching vibration of the C-O-C bonds, respectively. The thickness of the QCS / GL coatings on the PE substrates was determined by FE-SEM cross-section images and was found to be about 3.0 pm (Figure 1 ). In addition, the thermal properties of the QOS coated PE membranes were studied by thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) measurements. The mechanical properties of the PE membranes before and after the development of the QCS coatings were studied. The stability of the coatings was evaluated by submerging them in water for a predetermined amount of time, and ATR-FTIR and SEM were employed to examine the presence of the coating on the PE surface.

[0051] Example 4. Antimicrobial Activity

[0052] Description-Measurement of the Antimicrobial activity of the Developed Coatings

[0053] The antimicrobial activity of the coatings was determined according to the ISO22196 / 2011 standard, “Measurement of antibacterial activity on plastics and other non-porous surfaces”. The coatings were exposed to an initial microbial population and were incubated for 24 h at 37 °C. Staphylococcus aureus ATTC6538P and Escherichia coli ATTC8739 strains were used, as the standard dictates. In addition, the experiments were repeated with the foodborne pathogen Listeria monocytogenes (isolated from food products at the Unit of Food, Water and Environmental Microbiology, School of Medicine, University of Crete). The cell population that formed the microbial inoculum had a size of 2.5*10Λ5 to 10*10Λ5 cells / ml, with a target concentration of 6 *10Λ5 cells / ml.

[0054] For each microorganism in question, a minimum of 3 valid experiments was perfom^rTeFTneasurem^C'aTTe^TT^oSingFwreneste^r^’^ntrorTF (substrate without coating), 3 PECS (coatings of unmodified chitosan) and 3 PEQCS (coatings bearing 55% modified chitosan). On the first day of the experiment, the microbial inoculum was created, and all the coatings were inoculated. Regarding the coatings belonging to the control group (PE), in half of them, the inoculum was recovered immediately after exposure to the coating, cultured in PCA (Plate Count Agar) and incubated for 24 h. All the other coatings were incubated (while still inoculated). On the second day, the inoculum of all the other coatings was recovered and cultured similarly. The antimicrobial activity of the coatings was calculated after the validity of the measurement was confirmed, by comparing the microbial load of the coatings to the load of the controls.

[0055] Determination of the Antimicrobial Activity of quatemized biopolymers-survival / death rates

[0056] The unmodified and modified biopolymers that were used to create the coatings were first studied and compared in terms of their antimicrobial activity in solution. The aim of these experiments was to determine which one of the quatemized biopolymers displayed the most potent antibacterial activity. Specifically, quatemized chitosan (QCS) of 55% and 26% were prepared by the synthesis group and were studied, with the percentages demonstrating the proportion of modified amine groups of CS to bear cationic charge. Liquid solutions / broths containing the polymers in different concentrations were inoculated with the aforementioned bacteria. For each time point, a sample of the broths was cultured, and the surviving microbial load was determined. The experimental procedure took place at three different temperatures (4 °C, 20 °C and 37 °C). The following tables show the experimental conditions as well as the concentrations of the biopolymer solutions used.

[0057] Table 1. Concentrations of biopolymer Table 2. Incubation temperatures and test organisms for QCS55, QCS26

[0058] We found that both biopolymers (QCS26 and QCS55) displayed potent antimicrobial activity against L monocytogenes and S. aureus bacteria. Below are the results for QCS55.

[0059] Biopolymer QCS55 showed a more intense antimicrobial activity compared to QCS26 against L. monocytogenes, which can be attributed to the higher fraction of cationic quaternary ammonium salt groups. In particular the entire microbial population even at the lowest concentration, 0.01 % w / w was completely killed in the first 24 h of the test (Figure 2). Concentrations greater than 0.1% w / w are not shown (complete killing as in the case of 0.01 % w / w and 0.1 % w / w).

[0060] Similarly, in the case of inoculation of S. aureus in QCS55, in the control samples (liquid broth with no biopolymers -0% concentration of biopolymers) a slight increase in population is detected, which is not surprising (Figure 3).

[0061] Figure 4 shows the population decrease of Listeria monocytogenes in escalating concentrations of the QCS55 polymer at 20 °C. A sharp drop in population size was observed in the first hour of the test, with the reduction ranging from 2Log (99% kill of the initial population) at the lowest concentration of 0.01 %w / w to 3Log (99.9% kill) at the highest concentration of 0.1 % w / w. At time t = 6 h the population exposed to the highest concentration of antimicrobial agent was completely killed, the same happens in the next measurement for the immediately lower concentration of 0.05 %w / w.

[0062] Accordingly (Figure 5), when incubated at 4 °C, the population decreased abruptly in lowest concentration of 0.01 %w / w and 2.5Log at the highest concentration of 0.1 %w / w. The population continued to decrease and at time t = 8 h it was reduced to zero for the 0.1 % concentration. It appears that the low temperature by some mechanism protected the cells from the biocidal effect of the polymer, possibly because chitosan acts on the cell membrane of the cells, which at low temperatures has less fluidity, preventing penetration by the substance. The remarkable difference in population reduction between temperatures is of particular interest, because the modified coatings covering the cheeses will are preserved at low temperatures.

[0063] The above results led to the conclusion that the modified coatings shall be prepared using the QCS55 polymer.

[0064] Determination of the Antimicrobial Activity displayed by the quaternized biopolymers / fixation onto glass slides.

[0065] Before the modified biopolymers could be formed as coatings on PE packaging, the protocol described in section 1 was applied to silanized glass slides coated with the biopolymers. The coatings on the pre-modified glass slides with (3- aminopropyl)triethoxysilane (APTES) were prepared by dipping them in biopolymer solutions of different concentrations. The purpose of these preliminary experiments was to evaluate the optimal concentration of the polymers to be used for the formation of the coatings onto the food packaging substrates. Antimicrobial activity was not quantified with the given samples due to the non-acceptable (based on the standard) dimensions of the glass slides.

[0066] Table 3: Sample description

[0067] The results are presented in Figure 6. It is observed a 1 Log population reduction was observed in the control samples AP01 (glass slides that did not carry a biopolymer with antimicrobial properties). One interpretation of this involves a lack of nutrients for the bacteria, that were injured and starved. A decrease by one log was recorded for samples APCS and APQCS26. APQCS55 showed a greater microbial death.

[0068] Figures 7, 8 and 9 show the reduction of the initial microbial population for the different microorganisms after 24 h of exposure to the quaternized CS coatings on glass slides. In the case of L. monocytogenes and E. coli, all bacterial cells were killed (the population is shown as one Log cells = 10 live cells / ml of suspension). In the case of S. aureus, a 2.5Log reduction was measured compared to the control. In all cases, a significant difference was also observed between the control samples after 24 h, which was attributed to the lack of nutrients on the slide, and thus cell death due to starvation. In all cases, we note Control Oh, an untreated glass slide, Control 24h a glass slide with CS and QCS55, a glass slide coated with APQCS55. In the case of the Control 24h, and QCS55, the bacterial inoculum was exposed to the material for 24h, then was recovered, cultured, incubated and the bacterial population was calculated.

[0069] These results were encouraging for the formation of the modified CS coatings, using QCS55, onto the PE food packaging.

[0070] In vitro evaluation of the antimicrobial activity of the modified CS coatings on the PE

[0071] As mentioned above, the samples were PE (polyethylene control), PECS (polyethylene coated with unmodified chitosan) and PEQCS55 (polyethylene coated with quaternized chitosan). The results were based on at least 3 experimental replicates, in each of which 3 coatings of each group were inoculated (6 for control), and two internal replicates per sample were performed during cultivation.

[0072] Regarding the antimicrobial activity of QCS55, Figure 10 shows the reduction of the S. aureus population after inoculation onto the three surfaces described above. The population is reduced by 1 Log in the control samples after 24 h of incubation and 1.5Log in the presence of unmodified chitosan (PECS). Importantly, complete death of bacteria was observed in all modified CS coatings (PEQCS). We examined whether the results were statistically significant (p < 0.5). In all of the following cases, the microbial populations were found to be significantly altered in the presence of the antimicrobial coating of modified CS.

[0073] With all test validity criteria in place, the antimicrobial activity of the coatings was calculated as an average of the individual test results.

[0074] The results found are summarized below:

[0075] Table 4: Antimicrobial activity R, of PEQCS55 against S. aureus

[0076] Table 5: Antimicrobial activity R, of PECS (non quaternized chitosan) against S. aureus.

[0077] In general, the results were considered consistent regarding the study of the antimicrobial activity between the novel coatings comprising the modified CS biopolymer and those comprising unmodified CS.

[0078] Regarding E. coli (Figure 11 ), the population was reduced by less than 1 Log in the control samples (PE) after 24 h of incubation and in the presence of the unmodified chitosan (PECS). Some coatings displayed complete population death, while in others viable cells were detected, hence the standard deviation increased. To confirm the validity of the tests, the experiments were repeated more than 3 times. The antimicrobial activity was calculated and is shown in the tables below:

[0079] Table 6: Antimicrobial activity R of PEQCS55 against E. coli

[0080] Table 7: Antimicrobial activity R of PECS against E. coli.

[0081] The antimicrobial capacity against E. coli is lower compared to that against S. aureus. This is related to the cellular structure of the microorganism, which reduces the biocidal action of the coating, and agrees with the literature which reports a lower activity of cationic polymer coatings for gram negative bacteria. The population decfine was in any case remarkable.

[0082] L. monocutogenes is a foodborne pathogen of particular interest for the food industry, for this reason the effect of the modified CS coatings on this species was studied (Figure 12). Indicative tests were carried out according to the ISO:22196 / 2011 standard. After 24 h of contact of the microbes with the coatings, the population was reduced by 1.5Log units (approximately 95% death). This result was promising regarding the antimicrobial activity of the modified packages, when they are in contact with dairy products such as cheeses. Table 8: Antimicrobial activity R of PEQCS55 against L. monocytogenes.

[0083] Table 9: Antimicrobial activity R of PECS against L. monocytogenes.

[0084] The antimicrobial activity of the coatings bearing the modified CS (PEQCS55) coating was notably higher than that of the PECS coating, which demonstrates that the presence of the cationic groups on the coatings affect the survival of the pathogens.

Claims

CLAIMS1. A substrate material coated with a permanently positive charged natural polymer selected from chitosan or gelatin, characterised in that the substrate material surface is oxygen or corona plasma pre-treated prior to coat, and the coating is cross-linked and bound with the surface of the substrate material by a cross-linker.

2. The substrate material according to claim 1 wherein the substrate material is selected from polyethylene, polypropylene, poly(ethylene terephthalate), poly(vinyl chloride), polystyrene, copolymer polyethylene terephthalate glycol) (PETG), glass, paper, paperboard, cellulose fibers, cellulose acetate butyrate, cellulose acetate propionate, poly(lactic acid); and more preferably the material is polyethylene.

3. The substrate material according to any preceding claim wherein the crosslinker is selected from a dialdehyde, glutaraldehyde, oxidised sucrose and glyoxal; and more preferably the crosslinker is glyoxal.

4. The substrate material according to any preceding claim, wherein the permanently positive charged natural polymer is quaternized chitosan or gelatin with quaternization degrees between 20% and 70% of the chitosan repeat units and between 5% and 30 % of the gelatin repeat units.

5. A method for preparing the coated substrate material according to any of the claims1 to 4, which comprises the following steps: a) chemical modification of the natural polymer selected from chitosan or gelatinb) pre-treatment of a substrate material surface with oxygen or corona plasma; c) coating of an aqueous solution of the chemically modified natural polymer from step (a) in the presence of a crosslinker on the pretreated substrate material surface from step (b), at pH values between 3.5 and 6.

6. The method according to claim 5, wherein the natural polymer is chitosan and the quaternized chitosan is prepared by reaction of chitosan with a quaternization agentbased on an epoxy trimethylammonium salt such as (2,3-epoxypropyl)- trimethylammonium chloride, on a chloro or bromo trimethylammonium salt such as (3-chloro-2-hydroxy-propyl)trimethyl ammonium chloride and (3- bromopropyl)trimethylammonium bromide to provide quaternary ammonium salt groups on the polymer repeat units.

7. The method according to any of the claims 5 or 6, wherein the degree of quaternization of chitosan in step (a) ranges between 20 and 70%, more preferably between 40% and 60%.

8. The method according to claim 5, wherein the natural polymer is gelatin and the quaternized natural polymer is prepared by reaction of gelatin with a quaternization agent based on an epoxy trimethylammonium salt such as (2,3-epoxypropyl)- trimethylammonium chloride, or a chloro- or bromo-trimethylammonium salt such as (3-chloro-2-hydroxy-propyl)trimethyl ammonium chloride and (3- bromopropyl)trimethylammonium bromide to provide quaternary ammonium salt groups on the polymer repeat units.

9. The method according to any of the claims 5 or 8, wherein the degree of quaternization of the gelatin in step (a) ranges between 5 and 30%, more preferably between 10% and 20%.

10. The method according to any of claims 5 to 9, wherein the substrate material in step (b) is selected from polyethylene, polypropylene, poly(ethylene terephthalate), poly( vinyl chloride), polystyrene, copolymer poly(ethylene terephthalate glycol), glass, paper, paperboard, cellulose fibers, cellulose acetate butyrate, cellulose acetate11 . The method according to any of claims 5 to 10, wherein the crosslinker in step (c) is selected from a dialdehyde, glutaraldehyde, oxidised sucrose and glyoxal; more preferably the crosslinker is glyoxal.

12. The method according to any of claims 5 to 11 , wherein the crosslinker in step (c) is added at a crosslinker / natural polymer repeat units molar ratio that ranges between 0.3 and 0.6.

13. The method according to any of the claims 5 to 12, wherein the coating is applied by a roll-to-roll process, rotogravure, flexography, dipping, spraying or inkjet printing, followed by heating above 40 °C, more preferably between 40 °C and 100 °C.

14. The use of the coated substrate material according to any of claims 1 to 4 as antimicrobial film , preferably for packaging.

15. Use according to claim 14, wherein the antimicrobial film is used for packaging of food products and more preferably for dairy products; and even more preferably for packaging cheese and / or cheese derivatives including a soft, semi-soft, hard, and extra hard cheese.

Images