PACKAGING MATERIALS HAVING A DISCONTINUOUS CHITOSAN COATING THEREON, METHODS OF MANUFACTURE AND USES THEREOF

Cross-Reference to Related Applications

[0001] The present application claims priority to U.S. Provisional Patent Application No. 62/775,966, filed December 6, 2018, which is incorporated herein by reference in its entirety.

Technical Field

[0002] The embodiments disclosed herein relate to coatings, and, in particular to methods and apparatuses for forming discontinuous chitosan coatings on packaging materials.

Background

[0003] Microbial growth is of a concern for public health as it can cause foodborne diseases and food spoilage. The development of antimicrobial food packaging is therefore a promising research area [1 ] Chitosan is a biopolymer extracted from crustacean shells or fungi that has the potential to be used as antibacterial biomaterial given its intrinsic antimicrobial properties.

[0004] Chitosan is generally purchased as powder or flakes and can be processed into different physical forms through solubilisation in acidic media or by using an adequate solvent. Forms such as films [2-6], fibers [7-12], micro- and nanobeads [13-26], hydrogels [27-29], membranes [30] and sponge (foams) [31 ] of chitosan alone or in blends [30, 32- 34] are produced by several technological processes for applications in different fields [35] For instance, chitosan film is commonly studied as for use as packaging material given its results regarding the antibacterial activity [36, 37] However, neat chitosan films have brittle mechanical properties and poor water barrier performance, and therefore need to be blended with another polymer for the improvement of these properties [38, 39] Chitosan films are generally produced by the solution casting/solvent evaporation method [1 , 40], or less often by blending with a thermoplastic polymer via twin-screw extrusion [41 -43] [0005] Chitosan films prepared by solution casting require the dissolution of chitosan in aqueous solutions of hydrochloric, formic, acetic, lactic propionic, or citric acids, among others [44, 45] Solvent evaporation proceeds at room or high temperature, after pouring the solution into a thin layer. Processing of chitosan by extrusion generally requires to be blended with a low melting point thermoplastic polymer such as polycaprolactone (PCL), polyethylene oxide (PEO) or low-density polyethylene (LDPE). An addition of a cross-linker, an acid or plasticizer, is often required [41 -43] After processing, films are generally subjected to a neutralization step by immersion in sodium hydroxide for the determination of the antimicrobial effect [41 , 46] Various blends containing chitosan have been reported [45]: chitosan/corn starch [36, 47-49], chitosan/potato starch [36], chitosan enriched with essential oils including thymol, oregano, carvacrol and cinnamaldehyde among others [36, 49, 50], chitosan/oleic acid [36], chitosan/amylose [36], chitosan/sorbitol [43, 51 , 52], chitosan/glycerol [43], chitosan/protein [48], chitosan/cellulose [53], chitosan/acetic acid [44, 46, 52, 54], chitosan/propionic acid [54], chitosan/metal complexes (Ag, Zn) [48, 55, 56] or blended with a polymer in solution (PVP, PEO) [57, 58] or by extrusion (LDPE, mPE, PP) [41 , 42, 59-61 ]

[0006] Several chitosan-based films have been reported to prevent bacteria development and growth because of antibacterial and antifungal activities, as shown in Table 1 [45] For instance, chitosan/PVP, chitosan/PEO and chitosan/PP blends reported the higher antibacterial effect [57, 59] with a reduction of more than 2 logs CFU/mL in bacterial population, which indicates that at least 99 % of bacterial density was killed by the action of chitosan [45]

Table 1. Antibacterial and antifungal activity of chitosan films [45]

Composition Bacteria Antibacterial/antifungal effect

Enterobacteriaceae

I 90% in 12 h of contact

Chitosan/acid acetic (G-)

0% in 12 h of contact

Escherichia coli (G ^~ ) † inhibition zone

Staphylococcus

Chitosan/starch [47] † inhibition zone

aureus (G ⁺ )

Bacillus subtilis (G ⁺ ) † inhibition zone Chitosan/sorbitol Aspergillus niger 27.08 % of Fungistatic index

G511 after 72 hours

75, 68 and 55% of viable cells

Chitosan/cellulose Staphylococcus

after 24 hours (2, 3, and 5%

[53] aureus (G ⁺ )

chitosan, respectively)

Escherichia coli (G ) I 2.82, 2.48 and 2.15 log

Chitosan/PVP [57] (CFU/mL) after 6 h of contact

(ratio 75/25, 50/50 and 25/75)

Escherichia coli (G ) I 3.42, 3.75 and 2.93 log

Chitosan/PEO [57] (CFU/mL) after 6 h of contact

(ratio 75/25, 50/50 and 25/75)

Escherichia coli (G-) I 1 .5 log (CFU/mL) after 4 h of

Chitosan/mPE [41 ]

contact

Clavibacter I 5 log (CFU/mL) after 20 h of michiganensis contact

Chitosan/PP [59]

Pseudomonas I 5 log (CFU/mL) after 20 h of solanacearum contact

[0007] The use of edible coatings containing chitosan has been demonstrated to be useful for decreasing food spoilage as well as foodborne pathogen risk. The potential of chitosan to act as a food preservative of natural origin has also been widely reported based on in vitro trials as well as through direct application on real complex matrix foods [62-68] Chitosan was added to food during processing and used as natural preservative. Chitosan dipping solutions have shown capacity to inhibit bacterial growth in strawberries [69], tomatoes and cucumbers [70] Chitosan is also an excellent film forming material and chitosan films have a selective permeability to gasses (CO2 and O2), but brittle mechanical properties with a very brittle character. The fact that chitosan films are highly permeable to water vapor limits their use since an effective control of moisture transfer is a desirable property for most foods. To this end, several researchers proposed adding different components to improve both mechanical and barrier properties of chitosan- based coatings such as essential oils, clays, metallic particles, proteins, etc. [45, 69, 71 - 77] [0008] Cellulose, starch, alginate, carageenan, xanthan, are available as edible coatings, however, no chitosan-based coating is commercially available yet, even though many studies have succeeded in developing such materials for food applications [45, 48, 62, 67, 78-80]

[0009] A market study conducted during Technopreneur program (Polytechnique Montreal, Quebec, Canada, September 2016-May 2017) validated that there is a need of agri-food industry for the extension in the shelf life of food (vegetables, fruits, meat, milk, prepared meals and salads).

[0010] Accordingly, there is a need for new methods and apparatuses for forming discontinuous chitosan coatings to extend food shelf life.

Summary

[0011] According to some embodiments, apparatus and methods of forming a discontinuous coating of chitosan on a packaging material are disclosed herein. The discontinuous layer of chitosan on the packaging material may be used as a form of active food packaging with antimicrobial activity. In some embodiments, the layer of chitosan is a discontinuous layer to facilitate formation of the layer into a piece of packaging. In other embodiments, the layers of chitosan described herein are formed on a previously treated polymer surface. In some embodiments, the polymer surface is treated by an ultraviolet (UV), plasma or Corona treatment.

[0012] According to an aspect, a method of forming a discontinuous chitosan coating on a packaging material is disclosed herein The method includes applying an obstacle to the packaging material to cover a portion of the packaging material underlying the obstacle; dissolving chitosan in an acid to form a chitosan solution; depositing the chitosan solution onto the packaging material to form the chitosan coating on the packaging material; drying the packaging material to remove the acid from the packaging material; and removing the obstacle from the packaging material to reveal a chitosan-free region of the packaging material interrupting the chitosan coating.

[0013] According to another aspect, a packaging material is disclosed herein. The packaging material includes a body having a discontinuous chitosan coating disposed on a surface of the body, the discontinuous chitosan coating defining a chitosan-free region of the packaging material interrupting the chitosan coating.

[0014] According to a further aspect, a method of inhibiting bacterial growth in food is disclosed herein. The method includes contacting a food to be packaged onto a discontinuous chitosan coating of a packaging material disclosed herein.

[0015] According to yet another aspect, a method of inhibiting bacterial growth in food is disclosed herein. The method includes contacting a food to be packaged onto a discontinuous chitosan coating of a packaging material disclosed herein when the packaging material is in a first configuration; and reconfiguring the packaging material into a second configuration to form a pocket by coupling the chitosan-free regions of the packaging material, the food to be packaged resting in the pocket when the packaging material is in the second configuration.

[0016] According to another aspect, a method of inhibiting bacterial growth in food is disclosed herein. The method includes disposing a food to be preserved onto a discontinuous chitosan coating of a first packaging disclosed herein when the packaging material is in a first configuration; and sealing the first packaging material with a second packaging material disclosed herein to form a pocket by coupling the chitosan-free regions of the first and second packaging materials, the food to be preserved resting in the pocket when the packaging materials are sealed.

[0017] According to one aspect, a method of preserving food is disclosed herein.

The method includes contacting the food to be packaged onto a discontinuous chitosan coating of a packaging material disclosed herein.

[0018] According to a further aspect, a method of preserving food is disclosed herein. The method includes contacting a food to be packaged onto a discontinuous chitosan coating of a packaging material disclosed herein when the packaging material is in a first configuration; and reconfiguring the packaging material into a second configuration to form a pocket by coupling the chitosan-free regions of the packaging material, the food to be packaged resting in the pocket when the packaging material is in the second configuration. [0019] According to yet a further aspect, a method of preserving food is disclosed herein. The method includes disposing a food to be preserved onto a discontinuous chitosan coating of a first packaging disclosed herein when the packaging material is in a first configuration; and sealing the first packaging material with a second packaging material disclosed herein to form a pocket by coupling the chitosan-free regions of the first and second packaging materials, the food to be preserved resting in the pocket when the packaging materials are sealed.

[0020] Other aspects and features will become apparent, to those ordinarily skilled in the art, upon review of the following description of some exemplary embodiments.

Brief Description of the Drawings

[0021] The drawings included herewith are for illustrating various examples of apparatuses and methods of the present specification. In the drawings:

[0022] FIG. 1 is a schematic representation of a discontinuous layer of chitosan on a polymer, according to one embodiment;

[0023] FIG. 2 is schematic representation of an industrial solvent casting film system, according to one embodiment;

[0024] FIG. 3 is a series of bar graphs showing the bacterial population (CFU/mL) of Paenibacillus at different initial inoculum size at different contact time with and without chitosan film;

[0025] FIG. 4 is a graph showing the evolution of the total bacteria flora of milk in the presence and in the absence of chitosan-based films at 7 °C;

[0026] FIG. 5A is a graph showing in situ antibacterial activity of chitosan nanofiber-based packaging (CNFP) against E. coli after 7 days storage at 4 °C;

[0027] FIG. 5B is a picture showing the appearance of packed red meat with and without CNFP, before and after grinding;

[0028] FIG. 6 is an image of a Corona treatment setup used for the surface activation of PE films; [0029] FIG. 7 is a bar graph showing the contact angle measurements of untreated and corona-treated PE films as a function of time;

[0030] FIG. 8 is an image showing a discontinuous coating process using the Phantom Proofer assembly;

[0031] FIG. 9A is an image showing the coating efficacy of non-corona treated PE films and FIG. 9B is an image showing the coating efficacy of corona treated PE films;

[0032] FIG. 10 is a graph showing thickness distribution of the chitosan (CS) coating layer;

[0033] FIG. 1 1 is an image showing the FosiTest Pull-Off Adhesion Tester;

[0034] FIG. 12 shows a FTIR analysis spectra of the surface of chitosan/PE films inside and outside of the dolly; and

[0035] FIG. 13 is an image of a milk pouch prototype produced according to a method described herein.

Detailed Description

[0036] Various apparatuses will be described below to provide an example of each claimed embodiment. No embodiment described below limits any claimed embodiment and any claimed embodiment may cover apparatuses that differ from those described below. The claimed embodiments are not limited to apparatuses having all of the features of any one apparatus described below or to features common to multiple or all of the apparatuses described below.

[0037] Terms of degree such as“about” and“approximately” as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% or at least ±10% of the modified term if this deviation would not negate the meaning of the word it modifies.

[0038] According to an aspect, a method of forming a discontinuous chitosan coating on a packaging material is disclosed herein The method includes applying an obstacle to the packaging material to cover a portion of the packaging material underlying the obstacle; dissolving chitosan in an acid to form a chitosan solution; depositing the chitosan solution onto the packaging material to form the chitosan coating on the packaging material; drying the packaging material to remove the acid from the packaging material; and removing the obstacle from the packaging material to reveal a chitosan-free region of the packaging material interrupting the chitosan coating.

[0039] According to some embodiments, prior to depositing the chitosan solution on to the packaging material, the packaging material is subjected to a treatment chosen form a ultraviolet (UV) treatment, a plasma treatment, a Corona treatment and mixtures thereof.

[0040] According to some embodiments, the treatment encourages sealability of the packaging material.

[0041] According to some embodiments, the dissolving of the chitosan in the acid takes place at a temperature in a range of about 20 °C to about 40 °C.

[0042] According to some embodiments, the dissolving of the chitosan in the acid takes place for a period of time in a range of about 1 hours to about 3 days.

[0043] According to some embodiments, the chitosan has a molecular weight of about 50 to about 200 kDa. According to some embodiments, the chitosan has a molecular weight of about 100 to about 180 kDa. According to some embodiments, the chitosan has a molecular weight of about 120 to about 150 kDa.

[0044] According to some embodiments, the chitosan is a chitosan nanofiber, said fiber having an average diameter of about 60 to 100 nm. According to some embodiments, the chitosan is a chitosan nanofiber, said fiber having an average diameter of about 70 to 90 nm.

[0045] According to some embodiments, the packaging material comprises a polyolefin polymer.

[0046] According to some embodiments, the polyolefin polymer comprises polymerized ethylene monomers, polymerized propylene monomers or a mixture thereof.

[0047] According to some embodiments, the packaging material comprises a polyethylene-based polymer. [0048] According to some embodiments, the packaging material comprises a low density polyethylene-based polymer or a high density polyethylene-based polymer.

[0049] According to some embodiments, the packaging material comprises one or more of polycaprolactone (PCL), polylactic acid (PLA), ethylene vinyl alcohol (EVOH), ethylene vinyl acetate (EVA) and mixtures thereof.

[0050] According to some embodiments, the acid is an aqueous organic acid solution.

[0051] According to some embodiments, the acid is chosen from acetic acid, citric acid, lactic acid and mixtures thereof.

[0052] According to some embodiments, the acid comprises at least one of citric acid and lactic acid.

[0053] According to some embodiments, the acid is a solution that comprises about 1 % to about 3% (v/v) of citric acid.

[0054] According to some embodiments, the acid is a solution that comprises about 4% to about 6% (v/v) of lactic acid.

[0055] According to some embodiments, the acid is a solution that comprises about 0.5% to about 1.5% (v/v) of acetic acid and about 0.5% to about 1.5% (v/v) of lactic acid.

[0056] According to some embodiments, the acid is a solution that comprises about 0.5% to about 1.5% (v/v) of acetic acid.

[0057] According to some embodiments, the acid is a solution that comprises about 0.5% about 1.5% (v/v) of acetic acid and about 0.5% to about 1.5% (v/v) of lactic acid.

[0058] According to some embodiments, the acid is a solution that comprises about 0.5% to about 1.5% (v/v) of acetic acid, about 0.5% to about 1.5% (v/v) of lactic acid and about 0.5% to about 1.5% (v/v) of citric acid.

[0059] According to some embodiments, the chitosan solution is an aqueous solution having a range of about 0.5 to about 10% (w/v) chitosan.

[0060] According to some embodiments, the chitosan solution is an aqueous solution having a range of about 0.5 to about 4% (w/v) chitosan. [0061] According to some embodiments, the depositing the chitosan solution is by spraying the chitosan solution onto the packaging material.

[0062] According to some embodiments, the depositing the chitosan solution is by pouring the chitosan solution onto the packaging material.

[0063] According to some embodiments, the depositing the chitosan solution is by hand proofing the chitosan solution onto the packaging material.

[0064] According to some embodiments, the discontinuous chitosan coating allows for inactivation of Paenibacillus spores disposed on top of the discontinuous chitosan coating.

[0065] According to some embodiments, the discontinuous chitosan coating is effective for about a 2 log reduction of Paenibacillus spores disposed on top of the discontinuous chitosan coating.

[0066] According to some embodiments, the discontinuous chitosan coating is effective for about at least a 1 log reduction of Paenibacillus spores disposed on top of the discontinuous chitosan coating. According to some embodiments, the discontinuous chitosan coating is effective for about at least a 1.5 log reduction of Paenibacillus spores disposed on top of the discontinuous chitosan coating. According to some embodiments, the discontinuous chitosan coating is effective for about at least a 2 log reduction of Paenibacillus spores disposed on top of the discontinuous chitosan coating.

[0067] According to some embodiments, the discontinuous chitosan coating allows for inactivation of Pseudomonas fluorescens spores disposed on top of the discontinuous chitosan coating.

[0068] According to some embodiments, the discontinuous chitosan coating is effective for about a 2 log reduction of Pseudomonas fluorescens spores disposed on top of the discontinuous chitosan coating.

[0069] According to some embodiments, the discontinuous chitosan coating is effective for about at least a 1 log reduction of Pseudomonas fluorescens spores disposed on top of the discontinuous chitosan coating. According to some embodiments, the discontinuous chitosan coating is effective for about at least a 1.5 log reduction of Pseudomonas fluorescens spores disposed on top of the discontinuous chitosan coating. According to some embodiments, the discontinuous chitosan coating is effective for about at least a 2 log reduction of Pseudomonas fluorescens spores disposed on top of the discontinuous chitosan coating.

[0070] According to some embodiments, the depositing the chitosan solution is by solution casting or by hand proofing (e.g. Phantom Proofer assembly or Doctor Blade).

[0071] According to some embodiments, the thickness of the chitosan coating is about 20 to about 200 pm, about 30 to about 180 pm or about 40 to about 160 pm. According to some embodiments, the thickness of the chitosan coating is about 1 to about 200 pm, about 1 to about 180 pm or about 1 to about 160 pm. According to some embodiments, the thickness of the chitosan coating is about 1 to about 20 pm, about 1 to about 10 pm or about 1 to about 5 pm.

[0072] According to another aspect, a packaging material is disclosed herein. The packaging material includes a body having a discontinuous chitosan coating disposed on a surface of the body, the discontinuous chitosan coating defining a chitosan-free region of the packaging material interrupting the chitosan coating.

[0073] According to some embodiments, the discontinuous chitosan coating forms a central region of the packaging material surrounded by the chitosan-free region.

[0074] According to some embodiments, the packaging material has four edges and the central region is central to the packaging material.

[0075] According to some embodiments, the packaging material has a general shape of a square or rectangle and the central region is central to the square or rectangle.

[0076] According to some embodiments, the central region has a general shape of a square or rectangle.

[0077] According to some embodiments, the packaging material has a general shape of a circle and the central region is central to the circle.

[0078] According to some embodiments, the central region has a general shape of a circle. [0079] According to some embodiments, the chitosan-free region defines an outermost portion of the packaging material.

[0080] According to some embodiments, the chitosan-free region is sandwiched between two discontinuous chitosan regions of the packaging material.

[0081] According to some embodiments, the discontinuous chitosan coating is formed by dissolving chitosan in an acid to form a chitosan solution and depositing the solution onto the body, wherein prior to depositing the chitosan solution onto the body, the packaging material is subjected to a treatment chosen from a UV treatment, a plasma treatment, a Corona treatment and mixtures thereof.

[0082] According to some embodiments, the treatment encourages sealability of the packaging material.

[0083] According to some embodiments, the treated packaging material has a contact angle of about 40° to about 80°. According to some embodiments, the treated packaging material has a contact angle of about 50° to about 70°. According to some embodiments, the treated packaging material has a contact angle of about 60°.

[0084] According to some embodiments, the contact angle is maintained for at least 6 months.

[0085] According to some embodiments, the body comprises a polyolefin polymer.

[0086] According to some embodiments, the polyolefin polymer comprises polymerized ethylene monomers, polymerized propylene monomers or a mixture thereof.

[0087] According to some embodiments, the body comprises a polyethylene- based polymer.

[0088] According to some embodiments, the body comprises a low density polyethylene-based polymer or a high density polyethylene-based polymer.

[0089] According to some embodiments, the packaging material comprises one or more of polycaprolactone (PCL), polylactic acid (PLA), ethylene vinyl alcohol (EVOH), ethylene vinyl acetate (EVA) and mixtures thereof. [0090] According to some embodiments, the discontinuous chitosan coating is formed by dissolving chitosan in an acid to form a chitosan solution and depositing the solution onto the body, wherein the acid is an aqueous organic acid solution.

[0091] According to some embodiments, the acid is chosen from acetic acid, citric acid, lactic acid and mixtures thereof.

[0092] According to some embodiments, the acid comprises at least one of lactic acid and citric acid.

[0093] According to some embodiments, the acid is a solution that comprises about 1 % to about 3% (v/v) of citric acid.

[0094] According to some embodiments, the acid is a solution that comprises about 4% to about 6% (v/v) of lactic acid.

[0095] According to some embodiments, the acid is a solution that comprises about 0.5% to about 1.5% (v/v) of acetic acid and about 0.5% to about 1.5% (v/v) of lactic acid.

[0096] According to some embodiments, the acid is a solution that comprises about 0.5% to about 1.5% (v/v) of acetic acid.

[0097] According to some embodiments, the acid is a solution that comprises about 0.5% about 1.5% (v/v) of acetic acid and about 0.5% to about 1.5% (v/v) of lactic acid.

[0098] According to some embodiments, the acid is a solution that comprises about 0.5% to about 1.5% (v/v) of acetic acid, about 0.5% to about 1.5% (v/v) of lactic acid and about 0.5% to about 1.5% (v/v) of citric acid.

[0099] According to some embodiments, the chitosan solution is an aqueous solution having a range of about 0.5 to about 10% (w/v) chitosan.

[0100] According to some embodiments, the chitosan solution is an aqueous solution having a range of about 0.5 to about 4% (w/v) chitosan.

[0101] According to some embodiments, the depositing the chitosan solution is by spraying the chitosan solution onto the packaging material.

[0102] According to some embodiments, the depositing the chitosan solution is by pouring the chitosan solution onto the packaging material. [0103] According to some embodiments, the depositing the chitosan solution is by hand proofing the chitosan solution onto the packaging material.

[0104] According to some embodiments, the discontinuous chitosan coating allows for inactivation of Paenibacillus spores disposed on top of the discontinuous chitosan coating.

[0105] According to some embodiments, the discontinuous chitosan coating is effective for about a 2 log reduction of Paenibacillus spores disposed on top of the discontinuous chitosan coating.

[0106] According to some embodiments, the discontinuous chitosan coating is effective for about at least a 1 log reduction of Paenibacillus spores disposed on top of the discontinuous chitosan coating. According to some embodiments, the discontinuous chitosan coating is effective for about at least a 1 .5 log reduction of Paenibacillus spores disposed on top of the discontinuous chitosan coating. According to some embodiments, the discontinuous chitosan coating is effective for about at least a 2 log reduction of Paenibacillus spores disposed on top of the discontinuous chitosan coating.

[0107] According to some embodiments, the discontinuous chitosan coating allows for inactivation of Pseudomonas fluorescens spores disposed on top of the discontinuous chitosan coating.

[0108] According to some embodiments, the discontinuous chitosan coating is effective for about a 2 log reduction of Pseudomonas fluorescens spores disposed on top of the discontinuous chitosan coating.

[0109] According to some embodiments, the discontinuous chitosan coating is effective for about at least a 1 log reduction of Pseudomonas fluorescens spores disposed on top of the discontinuous chitosan coating. According to some embodiments, the discontinuous chitosan coating is effective for about at least a 1 .5 log reduction of Pseudomonas fluorescens spores disposed on top of the discontinuous chitosan coating. According to some embodiments, the discontinuous chitosan coating is effective for about at least a 2 log reduction of Pseudomonas fluorescens spores disposed on top of the discontinuous chitosan coating. [0110] According to some embodiments, the depositing the chitosan solution is by solution casting.

[0111] According to some embodiments, the thickness of the chitosan coating is about 20 to about 200 pm, about 30 to about 180 pm or about 40 to about 160 pm. According to some embodiments, the thickness of the chitosan coating is about 1 to about 200 pm, about 1 to about 180 pm or about 1 to about 160 pm. According to some embodiments, the thickness of the chitosan coating is about 1 to about 20 pm, about 1 to about 10 pm or about 1 to about 5 pm.

[0112] Referring to FIG. 1 , illustrated therein is packaging material 100 with a coating of chitosan 102. Chitosan 102 can form a continuous coating (e.g. can be formed without any interruptions) or can form a discontinuous coating (e.g. with interruptions) on top of packaging material 100.

[0113] In embodiments where chitosan 102 forms a continuous coating on packaging material 100, chitosan 102 forms a surface 103 that is continuous across packaging material 100. Chitosan 102 has generally a consistent thickness in a range of about 47 to about 157 pm.

[0114] In embodiments where chitosan 102 forms a discontinuous coating on packaging material 100, surface 103 is not continuous across packaging material 100. Rather, surface 103 of chitosan 102 defines at least one opening 104 where surface 103 is interrupted and at least a portion of packaging material 100 is exposed through opening 104. Opening 104 can be any appropriate size or shape to provide for exposure of packaging material 100 through surface 103. In some embodiments, more than one opening 104 can be formed in surface 103 to of chitosan 102.

[0115] In the embodiment shown in FIG. 1 , chitosan 102 is shown as a discontinuous coating with an opening 104 spaced from and adjacent to an edge 106 surrounding the packaging material 100. Opening 104 substantially surrounds a central portion 108 and is surrounded by a perimeter portion 1 10. Opening 104 generally forms a square shape around central portion 108 and has a consistent width W. [0116] Opening 104 is sized and shaped to facilitate sealing of packaging material 100 to itself upon folding packaging material 100. For example, in the embodiment shown in FIG. 1 , folding packaging material 100 in half would result in opening 104 being folded onto itself such that uncoated portions of packaging material 100 would rest upon uncoated portions of packaging material 100.

[0117] In some embodiments, the coating of chitosan 102 can define a series of openings 104 where portions of packaging material 100 are exposed through chitosan 102. In these embodiments, openings 104 are generally sized and shaped such that folding packaging material 100 upon itself provides for openings 104 to align with each other and results in exposed portions of polymer film 100 resting upon exposed portions of packaging material 100 to facilitate sealing of the packaging material 100 upon itself.

[0118] It should be noted that the dimensions of the active films can be adjusted by simply changing the location of the obstacles (uncoated parts of the commercial film). Given the poor sealability of chitosan films, the obstacles are desirable for the sealing of the activated packaging.

[0119] Packaging material 100 can comprise a polyolefin polymer. The polyolefin polymer can comprise polymerized ethylene monomers, polymerized propylene monomers, or mixtures thereof. The packaging material 100 can comprise a high density polyethylene-based polymer, or a low density polyethylene-based polymer. It can also comprise a polypropylene-based polymer, an ethylene-propylene-based polymer, an ethylene-propylene copolymer, or mixtures thereof. Other types of polymers may also be suitable, such as but not limited to polycaprolactone (PCL), polylactic acid (PLA), ethylene vinyl alcohol (EVOFI), ethylene vinyl acetate (EVA) and mixtures thereof.

[0120] Chitosan is the common name for poly-[1 -4]-p-D-glucosamine. Chitosan is chemically derived from chitin which is a poly-[1 -4]-p-N-acetyl-D-glucosamine which, in turn, is derived from the cell walls of fungi, the shells of insects and, especially, crustaceans. Chitin is treated with strong alkalis to remove acetyl groups producing chitosan. Depending on the specific treatment of chitin, chitosan may vary in the degree of deacetylation (DDA). Chitosan is generally insoluble in water, but dissolves in dilute solutions of organic acids such as acetic, formic, tartaric, valeric, and propionic acids. Preparations of unusually short chitosan polymers of low molecular weight, that is less than about 10,000 Daltons, are soluble in water. This type of preparation is uncommon and not used in the present disclosure. Typical chitosan preparations with varying molecular weights of individual species are used in the present disclosure.

[0121] In some embodiments, chitosan 102 may have a degree of deacetylation (DDA) in a range of about 95% to about 97%, average molecular weights ( _w ) of about 130 to about 150 kDa and polydispersity indices of 1 .6 and 2 such as may be obtained from Marinard Biotech (Riviere au Renard, Quebec, Canada) or Primex (Iceland).

[0122] In one exemplary embodiment, the coating of chitosan 102 is formed by a solution casting and/or solvent evaporation method disclosed herein.

[0123] During the solution casting and/or solvent evaporation method, chitosan is dissolved in an acidic solution (i.e. solvent). In some embodiments, the acidic solution can be an aqueous organic acid solution such as but not limited to an acetic acid solution, a lactic acid solution, a citric acid solution or combinations of different organic acid solutions. Acetic acid concentrations can vary from about 0.25 to about 2% (v/v). Chitosan concentration can be about 0.5 to about 4% (w/v) depending on solvent type. Chitosan concentration can also reach about 10% (w/v) for medium and low molecular weight chitosans.

[0124] The solvent may also be a mixture of more than one acid solution, such as but not limited to a mixture of two or more of an acetic acid solution, a lactic acid solution and a citric acid solution. Depending on solvent type, acid concentration can vary from about 0.5 to about 5% (v/v) and depending on the chitosan grade (low, medium or high molecular weight), the concentration of chitosan can be about 0.5 to about 10% (w/v). In some embodiments, the solvents can be acetic acid (glacial, 99.7%), lactic acid (90% w/w), citric acid (anhydrous), and mixtures thereof.

[0125] In some embodiments, to form a mixed solution of solvent, mixing can occur under magnetic agitation overnight and then be used immediately either for the preparation of neat chitosan films or for the coating process. [0126] Following this, the chitosan dissolved in the acidic solution is deposited over the packaging material 100 to form chitosan coating 102.

[0127] In some embodiments, the deposition can be by pouring the chitosan solution over the packaging material 100. In other embodiments, the deposition can be by spraying the chitosan solution of over the packaging material 100. For instance, chitosan solution can be deposited onto the packaging material 100 via spraying using a vaporizer/sprayer or by pouring and spreading the chitosan solutions using a Doctor Blade (or tape casting) coating device.

[0128] In some embodiments, chitosan films can be prepared by a solution casting method where the solvent solution was poured onto a flat surface and solvent evaporation proceeded at a temperature in a range from about 15 °C to about 50 °C. In other embodiments chitosan films can be prepared by a solution casting method where the solvent solution was poured onto a flat surface and solvent evaporation proceeded at a temperature in a range from about room temperature (e.g. ~20 °C) to about 40 °C.

[0129] In some embodiments, the thickness of the chitosan coating can be in a range from about 20 pm to about 200 pm. In other embodiments, the thickness of the chitosan coating can be about 30 to about 180 pm. In other embodiments, the thickness of the chitosan coating can be about 40 to about 160 pm.

[0130] It will be understood that the method used to prepare the films will impact the thickness of the chitosan coating. For example, when preparing the chitosan films using a Phantom proofer assembly, the chitosan may have a decreased thickness. In some embodiments, the thickness of the chitosan coating can be about 1 to about 200 pm, about 1 to about 180 pm or about 1 to about 160 pm. In other embodiments, the thickness of the chitosan coating can be about 1 to about 20 pm, about 1 to about 10 pm or about 1 to about 5 pm.

[0131] After the chitosan solution is deposited onto the packaging material 100, the chitosan-coated packaging material can be dried by any method commonly known in the art, for example, by vacuum, evaporation (ambient air drying), and air forced drying, each with or without heat, and by oven drying. [0132] In some embodiments, the chitosan deposited on top of the packaging material 100 dries by evaporation. For example, evaporation may proceed in a range from about room temperature (approximately 20 °C) to about 40 °C. Depending on the drying conditions, the solvent evaporation may take about 1 to about 24 hours.

[0133] In some embodiments, packaging material 100 can be treated prior to forming chitosan coating 102 on top of packaging material 100. In one embodiment, packaging material 100 can be treated by Corona treatment prior to forming chitosan film 102 on top of polymer layer 100. In some embodiments, Corona treatment of packaging material 100 prior to being coated with chitosan facilitates sealability (i.e. adhesion) of the chitosan-coated packaging material to itself for packaging.

[0134] Corona treatment (sometimes referred to as air plasma) is a surface modification technique that uses low temperature corona discharge plasma to impart changes in the properties of a surface. Corona plasma is generally generated by application of high voltage to an electrode having a sharp tip; the Corona plasma generally forms at the tip of the electrode. A linear array of electrodes can be used to create a curtain of corona plasma.

[0135] Materials such as plastics, cloth, or paper may be passed through the Corona plasma curtain to change the surface energy of the material. Surface treatment systems are available for virtually any surface format including dimensional objects, sheets and roll goods that are handled in a web format. Corona treatment is a widely used surface treatment method in the plastic film, extrusion, and converting industries.

[0136] In some embodiments, the coatings can be discontinuously formed by placing one or more obstacles (not shown) on the top surface of the packaging material 100 prior to depositing the chitosan solution. The obstacles mask portions of the packaging material 100 from the chitosan solution to inhibit chitosan solution from contacting the portions of the packaging material 100 underlying the obstacles. Following deposition of the chitosan solution, the obstacles are removed from the packaging material 100 to reveal openings 104 formed in the chitosan coating 102. [0137] The antibacterial efficacy of chitosan coatings 102 may be tested and validated against several bacterial strains frequently involved in the alteration of the microbiological quality of food.

[0138] In one specific example described further below, in one embodiment, chitosan coating 102 can be applied either by spraying or spreading the chitosan solution (dissolved either in acetic acid, lactic acid, citric acid, or a mixture of two or more of acetic acid, lactic acid, and citric acid) on the top surface of a Corona-treated polyethylene- based film for packaging food. The coated films were left for solvent evaporation overnight at room temperature under a conventional hood or for 1 hour at 40 °C in the oven. The coating was done discontinuously. The discontinuity was achieved by placing physical obstacles adjacent to the edges of the polyethylene-based films (as shown in FIG. 1 ), thus providing for the sealing of the activated packaging at the uncoated regions.

[0139] Methods of forming a packaging material 100 according to the embodiments disclosed herein are also disclosed herein

[0140] Methods of inhibiting bacterial growth in food are disclosed herein.

[0141] In one embodiment, the method comprises contacting food to be packaged onto a discontinuous chitosan coating of packaging materials disclosed herein.

[0142] In another embodiment, the method includes contacting the food to be packaged onto the discontinuous chitosan coating of a packaging material disclosed herein when the packaging material is in a first configuration; and reconfiguring the packaging material into a second configuration to form a pocket by coupling the chitosan- free regions of the packaging material, the food to be packaged resting in the pocket when the packaging material is in the second configuration.

[0143] In another embodiment, the method includes disposing a food to be preserved onto the discontinuous chitosan coating of a packaging material disclosed herein when the packaging material is in a first configuration; and sealing the first packaging material with a second packaging material disclosed herein to form a pocket by coupling the chitosan-free regions of the first and second packaging materials, the food to be preserved resting in the pocket when the packaging materials are sealed. [0144] In another embodiment, a method of preserving food is disclosed herein, the method includes contacting the food to be packaged onto the discontinuous chitosan coating of the packaging material disclosed herein.

[0145] In another embodiment, a method of preserving food is disclosed herein, the method includes contacting the food to be packaged onto the discontinuous chitosan coating of the packaging material disclosed here when the packaging material is in a first configuration; and reconfiguring the packaging material into a second configuration to form a pocket by coupling the chitosan-free regions of the packaging material, the food to be packaged resting in the pocket when the packaging material is in the second configuration.

[0146] In another embodiment, a method of preserving food is disclosed herein, the method including disposing a food to be preserved onto the discontinuous chitosan coating of a first packaging material disclosed herein when the packaging material is in a first configuration; and sealing the first packaging material with a second packaging material disclosed here to form a pocket by coupling the chitosan-free regions of the first and second packaging materials, the food to be preserved resting in the pocket when the packaging materials are sealed.

[0147] In another embodiment, the food is chosen from vegetables, fruits, meat, milk, dairy products, prepared meals and salads.

[0148] In another embodiment, the method is effective for extending the shelf life of meat for at least one week.

Examples

Example 1 : Preparation of chitosan -based packaging

Lab scale prototype - proof of concept

[0149] Chitosan films were prepared by a solution casting/solvent evaporation method. This method describes dissolving chitosan in aqueous acids (such as acetic, lactic, citric, etc.) and pouring the solution in a thin layer, leading to solvent evaporation. In one example, chitosan solutions were discontinuously deposited on top of a conventional packaging of which surface was previously Corona-treated, to provide for sealability of the final activated packaging. The coating of the commercial polyethylene (PE)-based films was done by spraying using a vaporizer/sprayer or by pouring and spreading the chitosan solutions using a Doctor Blade coating device. The antibacterial efficacy of these chitosan films was tested and validated against several bacterial strains frequently involved in the alteration of the microbiological quality of food.

[0150] Phase 1 . Preparation of chitosan-based films. Films were prepared by solution casting method. Chitosan was dissolved in acetic acid (other acids such as lactic acid, citric acid and mixtures thereof were also considered) and solvent evaporation proceeded in a range from about room temperature (e.g. about 20 °C) to about 40 °C. Preliminary tests showed that the blend of acetic, citric and lactic acid exhibited better performance in terms of mechanical and adhesion properties (results not shown).

[0151] Phase 2. Incorporation of chitosan-based films into packaging. PE-based multilayer films were coated by the chitosan casted film active layer. The coating was done discontinuously to allow the sealing of the activated packaging films. To improve the adhesion of chitosan on top of the PE film, surface treatment (corona) of the conventional packaging was performed (2 min, 16 kV).

[0152] Phase 3. Characterization of chitosan-based films. Films were characterized for their antibacterial, mechanical, optical, thermal, barrier, adhesive and sealing properties. Also, composition at the surface was analyzed. The physicochemical characterization was conducted using the following techniques: thermal gravimetric analysis (TGA), differential scanning calorimetry (DSC), Flaze, water vapor transmission rate (WVTR), oxygen transmission rate (OTR), rheology (for the precursors solutions), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), transmission electron microscopy (TEM) and mechanical tests. For assessing the antibacterial effect, in vitro and in situ tests were performed using different pathogenic and non-pathogenetic bacteria commonly found in milk and meat spoilage.

Industrial scale processing of activated chitosan-based packaging

[0153] At this step, the preparation of chitosan-based films at the industrial level was optimized and scaled-up to test the antibacterial activity in situ against different pathogenic and non-pathogenetic bacteria (including Pseudomonas, Bacillus, Paenibacillus, Streptococcus, Clostridium, Listeria, Salmonella, Escherichia coli, Staphylococcus) commonly found in animal source food spoilage such as milk and meat. Also, the study of the mechanical, optical, adhesive, thermal, barrier and sealing properties may be addressed to ensure that the activated films meet the requirements of food packaging in terms of mechanical and barrier properties. Finally, the compatibility between chitosan films and conventional packaging such as PE films may be studied to maximize the adhesion of the active layer on the PE layer. FIG. 2 is one example of a commercial device currently used in the packaging industry for the preparation of solvent casted films. Knowing that packaging manufacturers are equipped with devices for spraying additives on top of plastic films and also with coating systems such as industrial Doctor Blades, the discontinuous coating device described herein could be transferable on an industrial and commercial scale [81 ]

Materials and methods

Materials

[0154] Polymers. Two chitosan (CS) grades with 97% and 95% degree of deacetylation (DDA), average molecular weights ( _w ) of about 130 to about 150 kDa and polydispersity indices of 1 .6 and 2 were obtained from Marinard Biotech (Riviere au Renard, Quebec, Canada) and Primex (Iceland), respectively.

[0155] Solvents. Acetic acid (AcOFI, glacial, 99.7%), lactic acid (LA, 90 % w/w) and citric acid (CA, anhydrous) were purchased from Fisher Scientific (Saint-Laurent QC, Canada). All materials were of analytical grade and used as received.

Methods

Preparation of chitosan-based solutions and films

[0156] CS solutions were prepared by dissolution of the polymer powder in aqueous acetic acid (AcOH), lactic acid (LA), citric acid (CA) solutions and mixtures thereof at various polymer concentrations, as indicated in Table 2. The solutions were mixed under magnetic agitation overnight and then immediately used either for the preparation of neat CS films or for the coating process. CS films were prepared by solution casting method where, in brief, the appropriate volume/weight of polymer solution was poured onto a flat surface and solvent evaporation proceeded at a temperature in a range from about room temperature to about 40 °C. The thickness of the neat CS films was also measured and found to be about 47 to about 157 pm.

Table 2. Formulations of CS solutions.

Preparation of active chitosan-based packaging

[0157] Chitosan-based packaging were prepared by the impregnation of poly(ethylene) (PE)-based films by depositing and spreading CS solutions on the PE- based layer. Prior to the coating process, the surface of the latter underwent a corona treatment to improve the adhesion between the CS layer and the PE one. As mentioned, the coating was done discontinuously to allow the sealing of the activated CS-based packaging.

Surface treatment of PE-based conventional packaging

[0158] Corona treatment was applied to the surface of PE-based films to enhance their compatibility with the CS layer. Uniform and symmetrical films were cut and used for the surface treatment. A voltage of 16 kV was determined as an optimum value, above which breakdown voltage occurred. Surface treatment was applied for 30 sec, 1 min and 2 min. surface treated PE-based films were then coated with CS in order to confer the desired antibacterial properties to the final packaging.

Discontinuous coating process

[0159] Three different PE-based films were activated with a CS layer by the discontinuous coating process using the setup shown in FIG. 1 . In this process, the coating was applied either by spraying or spreading the CS solution (dissolved either in AcOH, LA, CA or mixtures thereof) on the top surface of the Corona-treated PE-based conventional film. The coated films were left for solvent evaporation overnight at room temperature under the hood or for 1 hour at 40 °C in the oven. The coating was done discontinuously. The discontinuity was achieved by placing physical obstacles adjacent to the edges of the PE-based films (FIG. 1 ), thus allowing the sealing of the activated packaging at the uncoated regions. Three PE films differing in the structure and composition and labeled as film 1 , film2 and film3, as described in Table 3 below, were activated by the discontinuous coating process.

Characterization of the activated packaging

Adhesion

[0160] The adhesion of the CS layer onto the PE-based film revealed to be affected by the type of solvent employed to dissolve CS (AcOH, LA, CA and tmixtures thereof). Table 3 presents the results of adhesion between the CS and PE layer before and after corona treatment for the three different PE-based films (film 1 , film2, film3) and as a function of the formulation and solvent type. The results showed that citric acid (CA) and lactic acid (LA) improved both the elasticity of CS films (data not shown) as well as their adhesion to the surface of the PE layer. When the adhesion was not optimal or only partial (+/-), corona treatment was necessary to improve the compatibility between the two layers.

Table 3. Adhesion between the CS and PE layer before and after corona treatment for PE-based films, film 1 , film2, film3 as a function of the formulation and solvent type.

Polymer 3 % C S in Adhesion Adhesion Adhesion

(before (after corona (after corona corona treatment)* treatment)** treatment)

Filml 1 % AcOH - +

2% LA +

5% CA +

1% AcOH + 1 % LA - +

1% AcOH + 1% LA + 1% CA - rid ~

Film2 1 % AcOH - +/- +

2% LA +

5% CA +

1% AcOH + 1 % LA - +/- +

1% AcOH + 1% LA + 1% CA - +/- +

Film3 1 % AcOH - +/-

2% LA +

5% CA +

1% AcOH + 1 % LA + +

* 16 kV, 1 min; ** 16 kV, 2 min; + means adhesion, - means no adhesion, nd: not determined.

Antibacterial properties

[0161] The antibacterial efficacy of chitosan-based packaging was tested against various strains including Paenibacillus and Pseudomonas fluorescens which are commonly associated with altering the microbiological (milk unsuitable for consumption) and organoleptic properties (unpleasant taste) of milk and indeed economical losses to milk industry. FIG. 3 shows the antibacterial activity of chitosan-based packaging vs control packaging at 7 °C. The results reveal that at different initial bacterial populations, chitosan films significantly inhibited bacterial growth of Paenibacillus. FIG. 4 shows the evolution of the total bacterial flora of milk in the presence and in the absence of chitosan films at 7 °C. The results indicate a reduction of 2-logs magnitude of bacterial population of milk in the presence of chitosan-based films, which is promising for chitosan-based active packaging in the extension of the shelf life of milk.

[0162] In previous research [12, 82-86], the antibacterial activity of different physical forms of chitosan, namely chitosan flakes, powders, solutions, nanoparticles and nanofibers was investigated. In vitro and in situ tests proved that chitosan nanofibers (CNFs) were highly efficient against E. coli, Staphylococcus, Salmonella and Listeria which are frequently involved in the spoilage of meat products [84-86] FIG. 5A shows the antibacterial activity of CNF-based packaging (CNFP) against E. coli (10 ⁴ CFU/mL) at 4 °C, in comparison with the neat packaging (NP) without chitosan and the control group (meat stored uncovered), both at 4 °C. The results revealed that when contaminated meat was packed in a CNFP, bacterial viability was reduced by 95 %, a bactericidal effect that allowed the extension of the shelf life of the tested meat by at least a week, while ensuring food safety and therefore the health of the consumer. FIG. 5B is a picture showing the appearance of packed red meat with (CNFP) and without CNFP (NP), before and after grinding.

[0163] In the best cases where plasticized chitosan/PE blends were successfully extruded [41 , 42], the antibacterial activity of the obtained CS-based films was negatively affected (as opposed to melt-batch mixing) since the functional groups of CS were no longer exposed at the surface of the films in direct contact with the food, as a result of the improved dispersion and distribution of chitosan in PE.

Example 2: Additional preparation of chitosan-based packaging

Solution preparation

[0164] Chitosan: A chitosan grade having a molecular weight (M _w ) of 147 kDa and a degree of deacetylation (DDA) of 97% was used. Chitosan solutions used during the coating process were prepared by dissolving chitosan powder at concentrations of 3% (w/v), in 1 % (v/v) acetic acid (AcOH). Solutions were stirred using a magnetic stirrer for 24h at room temperature to ensure complete dissolution of the polymer chains.

[0165] Poly(ethylene) (PE): PE films, provided by a food packaging manufacturer (Saint-Leonard, Montreal, Canada) were treated by corona treatment prior to use and further characterization. The PE films have the following structure and composition:

• Three layers

• 2.25 mm total thickness

• Interior layer: mLLDPE + Melt LDPE + additives (ratio: 30%)

• Center layer: oct-LLDPE + PP (ratio: 40%)

• Exterior layer: mLLDPE + Melt LDPE + additives (ratio: 30%)

Corona treatment

[0166] The surface treatment of the poly(ethylene) (PE) is desirable for the plastic films because, by creating functional groups, the surface tension of these materials is increased to permit good wetting. In this case, this treatment aims to improve the adhesion between the chitosan coating and the PE films used to make food packaging (e.g. milk pouches).

[0167] The surface of PE films was treated by a corona setup (CoronaFlex 28eluxe from Enercon) (FIG. 6). The corona treatment provides reliable adhesion promoting surface treatment and increases the wettability of the treated films. The operating conditions of the corona treatment are mentioned in Table 4.

Table 4. Operating conditions of corona treatment process.

PE Power (kW) Speed (mpm*) Pressure

Multilayer PE- 3Ϊ 2.1 -1 .8 Atm

based film _

^* mpm: meter per minute.

Contact angle measurements

[0168] The contact angle of corona-treated PE films was measured in order to evaluate the efficacy of the surface modification. Contact angle is the angle conventionally measured using the sessile drop technique, where a liquid-vapor interface meets a solid surface. It quantifies the wettability of a solid surface by a liquid by using the Young’s equation.

[0169] The results presented in FIG. 7 show that after corona treatment (parameters shown in Table 4), the contact angle was decreased from 90° (untreated PE) to 60°, immediately after treatment. The same films were analyzed and the contact angle was measured every month (results not shown) during six months (FIG. 7). The results indicate no significant change in the contact angle. This indicates that the corona treatment remains stable up to six months with contact angle values of 60°. Without wishing to be bound to this theory, it is believe that such contact angle values are desirable to ensure good adhesion between chitosan and PE in the next step of the coating process.

Coating process

[0170] The multilayer PE films were coated with a mono-active layer of chitosan. This coating step was realized discontinuously by the batch casting process using a “Phantom proofer assembly” (FIG. 8). In this process, the coating is carried out in a discontinuous manner to allow adequate sealing of the packaging. After solvent evaporation, the activated chitosan-based packaging films were kept at 21 °C and 10-30 % relative humidity for further characterization. The conditions of the coating process are presented in Table 5.

Table 5. Conditions of the chitosan coating process.

PE Surface area Formulation Volume (ml_)

_ (cm ² ) _

Multilayer PE film 10 x 7 3 % CS, 1 % AcOH 0.3

[0171] FIG. 9A and FIG. 9B show the adhesion of the chitosan layer on the top surface of untreated (FIG. 9A) and corona-treated PE (FIG. 9B). In the case of non-treated PE films (FIG. 9A), the chitosan solution retracted and was not able to spread on the surface of the films. On the other hand, in the case of treated PE films (FIG. 9B), corona treatment appears to help the creation of functional groups that favor the interactions between chitosan and PE, usually thermodynamically incompatible. Consequently, the corona treatment significantly improved the wettability of the films and therefore the adhesion of the chitosan coating on the surface of PE films. The coating chitosan solution was stained using methylene blue (see droplets on left side in FIG. 9A and evenly dispersed chitosan solution in bottom portion in FIG. 9B in order to better visualize the coating efficacy.

Coating thickness

[0172] FIG. 10 shows the thickness distribution of the chitosan coating layer, estimated using ProGage 00089-2007 Thickness Tester manufactured by Thwing-Albert, using different independent samples. Results shown in FIG. 10 reveal that the chitosan coating layer have an average thickness of about 1 pm.

Analysis of the adhesion force of the coating

[0173] In order to evaluate the adhesion force of the chitosan coating layer on the top surface of PE films, the following adhesion test was performed using a PosiTest Pull- Off Adhesion Tester, as shown in FIG. 1 1 . The test measures the forces required to pull a specified test diameter of coating away from its substrate using a hydraulic pressure pump. This pressure represents the coating strength of adhesion to the substrate. It evaluates the adhesion of a coating by the determination of the greatest tensile pull-off force that it can bear before detaching. For comparative purpose, the same test was performed for coated films obtained by both Phantom proofer assembly and solvent casting method. Results of the pull-off adhesion test are presented in Table 6.

Table 6. Results of the pull-off tests

Adhesion forces (Mpa) Adhesion forces (Mpa)

Corona-treated untreated samples

_ samples _

Phantom proofer _ 1 .39 _ No adhesion _

Solvent casting _ 0A _ No adhesion _

[0174] The results presented in Table 6 show that the adhesion forces between the chitosan coating layer and the surface of PE films obtained using the Phantom proofer assembly is three times higher than that obtained by solvent casting method. In comparison, as expected, in the absence of corona treatment, the adhesion was not possible and the test could not be performed. Without wishing to be bound by such theory, it is believed that increased adhesion forces is obtained by the lower contact angle reached after corona treatment and corroborates with the higher wettability and improved spreading of the chitosan solution on top of PE film.

[0175] To confirm that chitosan coating was not pulled-off after the adhesion test, the inside and outside surface of the dollies (pouches) were analyzed by Fourier transformed infrared spectroscopy (FTIR), as shown in FIG. 12. The FTIR spectra indicate that there is no significant difference in the surface composition of both spots of the CS/PE films (inside dolly and outside dolly). This result confirms that the applied coating was not pulled-off by the equipment, which is an indication of the coating strength.

X-ray photoelectron spectroscopy (XPS) Analysis

[0176] XPS is a surface-sensitive quantitative spectroscopic technique that measures the elemental composition, the chemical and electronic state of the elements that exist within a material. More simply, XPS is a useful measurement technique because it not only shows what elements are within a film but also what other elements they are bonded to. Table 7 presents the XPS analysis of the surface composition of untreated and corona-treated PE films. The results presented in Table 7 indicate that the surface treatment of PE films by corona allowed the creation of mainly oxygen and more specifically carbonyl groups, thus improving the interactions and the compatibility between PE films and chitosan coating and consequently enhancing the wettability of the former and the adhesion of the latter.

Table 7. XPS analysis of the surface composition of untreated and corona-treated PE films.

Element _ Be (ev) Untreated PE Treated PE _

Cls _ 285.0 91 .6 _ 76Ό _

N1S _ 399.7 3.3 _ 16 _

01s 531 .9 5.1 22.3 (C=0, 0=C-N: 6.1 ;

C-O: 11 .0; Q ^* -C=0: 4.6)

Conception of a prototype for food packaging, specifically milk pouches

[0177] As shown in FIG. 13, a milk pouch prototype comprising chitosan-activated PE films was manufactured and tested for its antibacterial efficacy in extending the shelf life of milk. As the coating process was performed in a discontinuous way that allows the sealability of the resulting food packaging, milk pouches were designed after sealing the chitosan/PE prepared films. The dimensions of the prototype and real size milk pouches are shown in Table 8. For practical reasons, the size of the developed prototype is half the size of a real milk pouch.

Table 8. Prototyping: resizing of milk pouches.

Container Dimension Volume Internal surface

_ (cm ^* cm) _ (mL) _ (cm ² )

Milk pouch (real 28 ^* 15 1300 840

size) _

Milk pouch 14 ^* 15 650 420

(prototype)

Validation of the antibacterial efficacy of chitosan-based packaging in the extension of the shelf life of food

[0178] The antibacterial efficacy of the chitosan-based packaging prototype was tested against various strains including Paenibacillus and Pseudomonas fluorescens as described in Example 1 and similar results were found, thus demonstrating that the ability of the chitosan films to inhibit bacterial growth and to extend the shelf life of milk. References

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