WO2015079419A1

WO2015079419A1 - Antimicrobically active packaging, antimicrobically active membrane for packaging and related uses

Info

Publication number: WO2015079419A1
Application number: PCT/IB2014/066424
Authority: WO
Inventors: Ernesto Reverchon; Lucia BALDINO; Stefano Cardea
Original assignee: Ernesto Reverchon; Baldino Lucia; Stefano Cardea
Priority date: 2013-11-28
Filing date: 2014-11-28
Publication date: 2015-06-04
Also published as: ITVI20130285A1

Abstract

An antimicrobically active packaging comprising thereinside at least one antimicrobically active membrane having an open-cell structure in a polymeric material loaded with an active antimicrobial agent and in the absence of solvents of the polymeric material of said structure. An antimicrobically active membrane suitable for the production of an antimicrobically active packaging and having an open-cell structure of a polymeric material having pores loaded with an active antimicrobial agent and in the absence of solvents of the polymeric material of said structure. Use of the packaging and of the membrane for the preservation of a food or perishable product of natural origin.

Description

ANTIMICROB IC ALLY ACTIVE PACKAGING, ANTIMICROBICALLY ACTIVE MEMBRANE FOR PACKAGING AND RELATED USES

Description

Technical Field

The present invention finds relates to the technical field of disinfection systems and has for object an antimicrobically active packaging and an antimicrobically active membrane suitable to be used in packaging, in particular for food use or with perishable products for the preservation of food or natural products by reducing the bacterial load. The invention also relates to a membrane with antimicrobial properties for the preservation of products or for use on the human body and a specific use of the packaging and of the membrane.

State of the art

Active packaging with antimicrobial properties are known to have the function of extending the shelf-life of a food by reducing, inhibiting or retarding the growth of microorganisms that may be present in the food or in the material of the packaging [1]. Such packaging avoids the use of traditional techniques involving the addition of antimicrobial active agents directly in the food and that generally cause a reduction in the concentration of the antimicrobial agent on the surface of the food for the diffusion thereof inside of the food .

Therefore, the minimum concentration required for inhibiting the microbial growth may not be achieved, making ineffective the action of the antimicrobial agent [2]. In addition, the mixing of the active agent in the food could lead to the neutralization of the same for possible interactions with food components [1].

For these reasons, the most important property that an active packaging has to posses is the capability of controlling the release of the antimicrobial agent towards the surface of the food [3].

Some authors have tried to control the release of the active agent using a film with three layers. The first layer is the outer barrier, whose function is to prevent the migration of the active agent to the environment; the second layer contains the active agent; the third layer controls the release of the active agent towards the food [4].

Mastromatteo et al. [5] and Buonocore et al. [6] have developed multilayer film constituted by two outer layers, which have the function of controlling the release, and an inner layer which contains the active agent. Other authors have tried to control the kinetics of release of the active agent by changing the degree of crosslinking of the polymer [7].

Gemili et al. [8] have produced films of cellulose acetate, porous and asymmetrical, by means of phase inversion, for applications in active packaging. The authors have tried to control the rate of release of lysozyme, used as an antimicrobial agent, changing the degree of asymmetry and porosity of the film.

However, the above cited techniques are significantly limited by the fact that the release time of the active agent is short (about one day) even when it is adopted the configuration as multilayer film.

Furthermore, the crosslinking of the polymer affects the kinetics of release of the active agent and the amount of active agent released at equilibrium. This phenomenon can be explained by considering that during the crosslinking reaction also part of the antimicrobial agent loaded in the film is chemically bound to the polymer [6].

From CN1723803 it is known the use of an antimicrobial film formed from an aqueous solution containing a mixture of anhydrous acetic acid and chitosan combined with collagen, and subsequently added with antimicrobial agents such as nisin, between 0.025% and 0,05% of the final solution , polyphenols, with a percentage between 0.5% and 1.0%, and lysozyme as a percentage between 0.5% and 1.0%.

The solution so obtained is sprayed onto the product to be treated so as to define an outer protection layer, then wrapped with a coating film.

It is clear that this solution, although according its authors allows to increase the shelf- life of the product up to 35 days, is not easily feasible since it requires that the solution is applied directly to the product.

Moreover, the application of particular components, in particular acetic acid, as legally permitted in determining quantity, however, is not recommended because it will alter the organoleptic features of the food product.

CN102772828 disclose in turn a slow-release membrane made of cellulose loaded with a drug wherein the membrane comprises a biodegradable polymer, polyethylene glycol with CNC structure and an active agent. However there is no evidence for this membrane in terms of increasing the shelf-life of the products treated with the same. CN102363534 discloses a method for treating a biological membrane designed to line pipes for the passage of liquid foodstuffs, which method provides for treating a membrane with a liposome containing lysozyme treated with a process of evaporation of fluids at low pressure. However this solution does not allow to control the release of the biocide agent (lysozyme) and is therefore not suitable to be used as packaging for foods.

A further solution which provides the use of a chemical composition containing enzymes having function of biocide agent to be spread on a membrane designed to be used as a coating in a multiplicity of technical fields is also known from US2010233146.

Porous membranes with open cells loaded with antibiotic, in particular amoxicillin, are also known [20], which membranes being obtained from a solution of a polymeric material and a solvent to which the antibiotic is added in solution or suspension. The mixture thus obtained is treated by means of a dense gas to remove the solvent and obtain the membrane.

A similar process is also disclosed in WO2006/095 372 wherein the active ingredients are selected from antibiotics, anti-inflammatories and other pharmaceuticals.

Therefore is felt the need of a new product that can be used as packaging, particularly for food products, coating or as an element to be applied to any object or also of parts of the human body that guarantees a slow and controlled release of the antimicrobial agent. In particular, in the case of a membrane for the preservation of foods it is required that the same membrane can significantly increase the shelf-life of the treated product without changing appreciably the organoleptic properties.

Scope of the invention

The object of the present invention is to overcome the above drawbacks by providing an antimicrobically active packaging, in particular for food or perishable products, which is characterized by the release of one or more active antimicrobial in a slow and controlled manner.

A particular object is to provide an antimicrobically active packaging that allows to increase significantly the average shelf-life of food or perishable products stored inside. Still another object is to provide an antimicrobically active packaging that does not contain solvents thereinside.

Still another object of the present invention is to provide a membrane loaded with antimicrobial principles that allows a slow and controlled as well as substantially uniform release thereof and which may be used in contact with food or perishable products for prolonging the average shelf-life thereof.

Still another object of the present invention is to provide a membrane loaded with antimicrobial principles that allows a slow and controlled as well as substantially uniform release and that can be used also in contact with the human body for medical and/or therapeutic purposes.

A further object is to provide a porous membrane loaded with antimicrobials principles whose morphology can be controlled during realization simply varying the operating conditions of the process.

Still another object is to provide a porous membrane loaded with antimicrobials principles that can be obtained by processes which enable the uniform distribution of the antimicrobial principle in the polymeric structure of the membrane.

These objects, as well as others which will appear more clearly hereinafter, are achieved by an antimicrobically active packaging that, according to claim 1, comprises thereinside at least one antimicrobically active membrane having a open cell structure in polymeric material loaded with an active antimicrobial agent and in the absence of solvents of the polymeric material of said structure.

This particular embodiment of the packaging, and in particular of the membrane, will allow its safe use even in contact with food or perishable products, in particular of natural origin, increasing the average shelf-life of the product thanks to the slow and controlled release of the antimicrobial agent that will counteract the bacteria which are cause of the biological decay of this kind of products.

Advantageously, the antimicrobial agent may be an enzyme, in particular lysozyme, to have an active agent having both high antimicrobial properties that high biocompatibility also with man.

Alternatively, the antimicrobial principle may be selected among the food preservatives.

Furthermore, the antimicrobial agent may be selected from lysine, nisin, essential oils, organic acids, bacteriocins, fungicides, metals, proteins and may be effective against

Gram + or against Gram -, depending on the uses.

Suitably, the packaging may comprise an outer casing enclosing said membrane in such position to be in contact with the product to be contained and exert its function in a particularly effective manner.

According to a further aspect of the invention there is provided an antimicrobically active membrane that, according with claim 18, is constituted by a open-cell structure in a polymeric material loaded with an active antimicrobial agent and in the absence of solvents of the polymeric material of said structure.

The membrane may be used for the realization of a packaging designed to contain food or perishable products, preferably of natural origin, for the preservation thereof, according to claim 20, or for further medical or pharmaceutical uses. Operatively, regardless of the use to which it is designed, the membrane may be obtained starting from a solution containing one or more solvents and one or more polymers with predetermined respective weight percentages.

The solution can be prepared on the spot at the time of the execution of the process, or may be ready for use or provided in any other way.

Subsequently at least one active ingredient having antimicrobial properties will be added to the solution to obtain a mixture containing at least one polymer and at least one active agent.

The active agent may be soluble in the specific used solvent, or not soluble in order to remain in suspension in the polymer- solvent solution.

The mixture is then treated with a working fluid or with a fluid mixture, containing or consisting of a dense gas, an expanded fluid or a fluid under supercritical conditions, with controlled pressure and temperature.

The treatment step will be performed in such a manner to eliminate at least partially the solvents and more preferably substantially completely, unless possible residual traces in not appreciable amounts.

The possibility of removing the solvents in an almost complete manner, thanks to the use of these fluids, will lead to a relatively short process but mainly to obtain a membrane having an open cell structure loaded with one or more active agents having antimicrobial properties.

The particular embodiment of the membrane will allow the same to contain the active agent in a substantially uniform manner and to release it in a slow and controlled manner, allowing to significantly increase the shelf-life of the food product treated with the membrane.

Furthermore, the ability to control and adjust the parameters of temperature and pressure of the working fluid will also allow to control the morphology and properties of the membrane.

The mixture may be placed in a container for high pressure in which the fluid or mixture of working fluids at high pressure will be subsequently added.

By way of example, the working fluid may be introduced into the container through a suitable pump or other suitable pumping means, without particular limitations.

Suitably, the mixture with the active agent in solution or suspension may be subjected to one or more steps of cooling/freezing to a temperature between -4 ⁰ C and -40 ⁰ C, for a time between lh and 48 h. Preferably, the fluid or mixture of working fluids may be introduced into the container at a pressure between 60bar and 350bar and at a temperature between 25°C and 70°C. Moreover, the overall time of the whole process, which may be either continuous or discontinuous, may be between 5min and 500min.

In particular, by controlling the time of the process, and more specifically by controlling the time of action of the fluid, it will also be possible to control the symmetry of the membrane which will be greater the smaller will be the process time.

The fluid is preferably but not exclusively selected from carbon dioxide (C02) under supercritical conditions, water vapor, ethanol, expanded liquids.

The latter will be preferably mixtures of C02 with one or more organic solvents or the like, such as acetone, water, dichloromethane, dimethylsulfoxide, chloroform, N- methylpyrrolidone, dichloroethane, dioxane, trichloroethane, tetrahydrofuran, methanol, propanol, ethanol, butanol.

The solvents will be selected preferably but not exclusively from the group comprising acetone, water, dichloromethane, dimethylsulfoxide, chloroform, N-methylpyrrolidone, dichloroethane, dioxane, trichloroethane, tetrahydrofuran, methanol, propanol, ethanol, butanol and the like.

The polymers of the solution will be selected preferably but not exclusively between nylon, polymethyl methacrylate, ethyl cellulose, cellulose acetate, polylactide, polyvinyl alcohol, polysulfone, polyethylene glycol, polycaprolactone, dextran, chitosan, inulin, polivinildifluoride, polyvinylpyrrolidone, polyethersulfone, polycarbonate, polyacrylonitrile, polyamide, polyethylene , polyetherimide, alginates, gelatin, and the like.

The mixture may comprise a total percentage of polymer between 1% and 60% of its total weight.

The overall proportion of solvent will preferably be between 20% and 99% of the total weight of the mixture while the total percentage of active agent may be between 0.001% and 50% of the total weight of the mixture.

The membrane has a porous structure, symmetrical or asymmetrical, with porous or non-porous surfaces, with internal morphology of the finger-like type, leafy-like type, nanofiber type with fiber having sizes between 20nm and 5μιη, or with cell morphology with cells having a diameter between 0.1 μιη and 500 mM.

Advantageous forms of the invention are obtained according to the dependent claims

Best modes of carrying out the invention In way of illustrative but not limiting example, herebelow three examples of carrying out a process for the production of a membrane according to the present invention are disclosed.

Exemple 1

Production of a membrane having an open pore structure of cellulose acetate (AC) loaded with lysozyme as active agent (effective against Gram +).

The starting solution is prepared using acetone as a solvent in which only the AC is soluble. It take start, therefore, from a solution containing from 5% to 50% by weight of AC in acetone, preferably between 10% and 30% by weight in AC. An intermediate solution of lysozyme in water, varying between 0,1% and 20% by weight of lysozyme is dissolved thereinto to obtain the final mixture which will also be a solution.

Preferably, the intermediate solution will be introduced in the first solution with a weight percentage between 1% and 15% of the total weight of the final mixture, which will define a second solution.

The final mixture or second solution is loaded in the container for high pressures and put into contact with a dense gas, in the specific case with supercritical C02, at a temperature between 30°C and 60°C and a pressure between 80bar and 300bar, for a total time between 30min and 300min.

At the end of the process, it has been obtained an open-pore structure of AC, containing lysozyme, with symmetrical morphology of cell type and with an average diameter of the cells between 3μιη and ΙΟΟμιη. Lysozyme is homogeneously distributed within the polymer matrix.

Release tests have been conduced, which showed that the kinetics of release of lysozyme is regular during time and the duration of release is greater in the case of polymeric structures generated from more concentrated solutions.

Finally, tests were made of antimicrobial activity that have demonstrated the effectiveness of the structures generated against a target microorganism (Micrococcus lysodeikticus).

Exemple 2

Production of a membrane having an open pore structure of polyvinyl alcohol (PVA) loaded with silver as active agent (effective against Gram -).

The starting solution is prepared using water as solvent in which only the PVA is soluble. It take start, therefore, from a first solution containing from 5% to 45% by weight of PVA in water, preferably between 7% and 35% by weight in PVA. An amount of silver variable between 0.01% and 15% by weight of the amount of the starting polymer is suspended thereinto.

This suspension undergoes to a freezing stage at a temperature between -4°C and -30°C for a time from 2h and 24h and subsequently loaded into the container for high pressures and put into contact with an expanded liquid, in the specifc case consisting of a mixture of supercritical C02 and ethanol, at a temperature between 30°C and 50°C and a pressure between 90 and 350bar, for a total time between 50min and 420min. At the end of the process, it has been obtained an open-pore structure of PVA containing silver, with asymmetric morphology of finger-like type and with an average cell diameter of between Ιμιη and 80μιη. The silver is homogeneously distributed within the polymer matrix.

Tests were performed of the antimicrobial activity that have shown the effectiveness of the generated structures against a target microorganism (Escherichia coli), monitoring the kinetics of the death of the organism.

Exemple 3

Formation of a membrane having open pore structure consisting of two polymers, polylactide (PLA) and polyethylene glycol (PEG), loaded with nisin.

The first solution is prepared by dissolving PLA and PEG in dichloromethane (DCM), and subsequently it is joined to it an intermediate solution of nisin dissolved in water to obtain the second solution.

The latter will have a concentration of between 40% and 90% by weight of DCM, preferably between 60% and 85% by weight, PLA between 40 and 90% by weight, PEG between 2% and 30% and Nisin between 5% and 25% by weight.

This second solution is then loaded into the container for high pressures and put into contact with a dense gas, in the specific case supercritical C02, at a temperature between 35°C and 60°C, pressure between 90bar and 250 bar and a total time of the process between 25min and 250min.

It has been obtained a porous structure with homogeneous morphology of nanofiber tyoe, with average size of the fibers between 200nm and 500nm, wherein the bioactive substance is homogeneously incorporated.

Release test were conducted that showed that the kinetics of release of nisin is regular during time. Finally, tests were made of antimicrobial activity that have demonstrated the effectiveness of the generated structures against a target microorganism (Micrococcus lysodeikticus). Bibliography

[I] P. Appendini, J.H. Hotchkiss, Review of antimicrobial food packaging, Innovative Food Sci. Emerg. Technol. 3 (2002) 113-126.

[2] S. Min, J.M. Krochta, Inhibition of penicillium commune by edible whey protein films incorporating lactoferrin, lactoferrin hydrosylate and lactoperoxidase systems, J. Food Sci. 70 (2005) 87-94.

[3] M. Uz, S.A. Altinkaya, Development of mono and multilayer antimicrobial food packaging materials for controlled release of potassium sorbate, LWT - Food Sci. Technol. 44 (2011) 2302-2309.

[4] J.H. Han, J.D. Floros, Simulating diffusion model and determining diffusivity of potassium sorbate through plastics to develop antimicrobial packaging films, J. Food. Process Pres. 22 (1998) 107-1220.

[5] M. Mastromatteo, G. Barbuzzi, A. Conte, M.A. Del Nobile, Controlled release of thymol from zein based film, Innovative Food Sci. Emerg. Technol. 10 (2009) 222-227.

[6] G.G. Buonocore, A. Conte, M.R. Corbo, M. Sinigaglia, M.A. Del Nobile, Mono and multilayer active films containing lysozyme as antimcirobial agent, Innovative Food Sci. Emerg. Technol. 6 (2005) 459-464.

[7] G.G. Buonocore, M.A. Del Nobile, A. Panizza, M.R. Corbo, L. Nicolais, A general approach to describe the antimicrobial agent release from highly swellable films intended for food packaging applications, J. Controlled Release 90 (2003) 97-107.

[8] S. Gemili, A. Yemenicioglu, S.A. Altinkaya, Development of cellulose acetate based antimicrobial food packaging materials for controlled release of lysozyme, J. Food Eng. 90 (2009) 453-462.

[9] S. Liparoti, R. Adami, E. Reverchon, PEG micronization by supercritical assisted atomization, operated under reduced pressure, J. Supercrit. Fluids 72 (2012) 46-51.

[10] I. De Marco, E. Reverchon, Supercritical carbon dioxide + ethanol mixtures for the antisolvent micronization of hydro soluble materials, Chem. Eng. J. 187 (2012) 401-409.

[I I] I. De Marco, O. Knauer, F. Cice, A. Brauer, A. Leipertz, E. Reverchon, Interactions of phase equilibria, jet fluid dynamics and mass transfer during supercritical antisolvent micronization: the influence of solvents, Chem. Eng. J. 203 (2012) 71-80.

[12] E. Reverchon, G. Caputo, S. Correra, P. Cesti, Synthesis of titanium hydroxide nanoparticles in supercritical carbon dioxide on the pilot scale, J. Supercrit. Fluids 26 (2003) 253-261.

[13] G. Delia Porta, R. Campardelli, E. Reverchon, Monodisperse biopolymer nano and micro particles produced by Supercritical Emulsion Extraction technology, J. Supercrit. Fluids 76 (2013) 67-73.

[14] I. De Marco, E. Reverchon, Cellulose acetate nanofibers and nanostructured microfibers produced by supercritical antisolvent precipitation, J. Supercrit. Fluids 55 (2011) 1095-1103.

[15] R. Campardelli, G. Delia Porta, E. Reverchon, Solvent elimination from polymer nanoparticle suspensions by continuous supercritical extraction, J. Supercrit. Fluids 70 (2012) 100-105.

[16] E. Reverchon, S. Cardea, Formation of cellulose acetate membranes using a supercritical fluid assisted process, J. Membr. Sci. 240 (2004) 187-195.

[17] E. Reverchon, S. Cardea, Formation of polysulfone membranes by supercritical C02, J. Supercrit. Fluids 35 (2005) 140-146.

[18] E. Reverchon, E. Schiavo Rappo, S. Cardea, A Flexible Supercritical C02 Assisted process for PMMA structures formation, Polym. Eng. Sci. 46 (2006) 188-197.

[19] E. Reverchon, S. Cardea, PVDF-HFP Membranes Formation by Supercritical C02 Processing: Elucidation of Formation Mechanisms, Ind. Eng. Chem. Res., 45 (2006) 8939-8945

[20] E. Reverchon, S. Cardea, E. Schiavo Rappo, Production of loaded PMMA structures using the supercritical C02 phases inversion process, J. Membr. Sci., 273 (2006) 97-105

Claims

1. An antimicrobically active packaging characterized by comprising thereinside at least one antimicrobically active membrane having an open cell structure in a polymeric material loaded with an active antimicrobial agent and in the absence of solvents of the polymeric material of said structure.

2. Packaging according to claim 1, characterized in that said antimicrobial agent is an enzyme.

3. Packaging according to claim 2, characterized in that said enzyme is lysozyme.

4. Packaging according to claim 1, characterized in that said principle antimicrobial agent is selected from the group consisting of food preservatives.

5. Packing according to claim 4, characterized in that said antimicrobial agent is nisin.

6. Packing according to claim 1, characterized in that said antimicrobial agent is selected from essential oils, organic acids, bacteriocins, fungicides, metals, proteins.

7. Packaging according to claim 6, characterized in that said antimicrobial agent is effective against Gram +.

8. Packaging according to claim 6, characterized in that said antimicrobial agent is effective against Gram -.

9. Packaging according to any one of the preceding claims, characterized in that said membrane is obtained starting from a mixture composed by a solution containing one or more solvents and one or more polymers with the respective predetermined weight percentages and one or more of said active antimicrobial agents in solution or suspension in said solution and subsequent treatment of said mixture with a working fluid adapted to remove said one or more solvents.

10. Packaging according to claim 8, characterized in that said mixture has a percentage of polymers between 1% and 60% of the total weight of said mixture, a total percentage of solvent between 20% and 99% of the total weight of said mixture and a percentage of antimicrobial agents between 0.001% and 50% of the total weight of said mixture.

11. Packaging according to claim 9 or 10, characterized in that said working fluid is a dense gas, an expanded fluid or a fluid in supercritical conditions at a pressure between 60 and 350 bar and at a temperature between 25 and 70 ⁰ C for a time between 20min and 500min.

12. Packaging according to claim 11, characterized in that said working fluid is a dense gas selected from carbon dioxide in supercritical conditions, water vapor, ethanol, mixture of C02 with one or more organic solvents selected from acetone, water, dichloromethane, dimethylsulfoxide, chloroform, N-methylpyrrolidone, dichloroethane, dioxane, trichloroethane, tetrahydrofuran, methanol, propanol, ethanol, butanol.

13. Packaging according to any one of claims 9 to 12, characterized in that said solvents are selected from acetone, water, dichloromethane, dimethylsulfoxide, chloroform, N-methylpyrrolidone, dichloroethane, dioxane, trichloroethane, tetrahydrofuran, methanol, propanol, ethanol, butanol and the like, and in that said one or more polymers of said first solution are selected from the group comprising nylon, polymethyl methacrylate, ethyl cellulose, cellulose acetate, polylactide, polyvinyl alcohol, polysulfone, polyethylene glycol, polycaprolactone, dextran, chitosan, inulin, polivinildifluoride, polyvinylpyrrolidone , polyethersulfone, polycarbonate, polyacrylonitrile, polyamide, polyethylene, polyetherimide, alginates, gelatin.

14. Packaging according to any one of the preceding claims, characterized in that said open-pore structure of said membrane has cells of diameter between 0.1 μιη and 500 μιη.

15. Packaging according to any of claims 1 to 14, characterized in that the open-pore structure of said membrane has internal morphology of the finger-like type, leafy-typelike or nanofiber type with fibers having sizes between 20nm and 5μιη.

16. Packaging according to any one of the preceding claims, characterized by comprising an outer casing enclosing said membrane in such a position as to be in contact with the product to be contained.

17. Use of an antimicrobically active packaging according to one or more of the preceding claims to contain thereinside at least one food or perishable product in contact with said membrane to increase the shelf-life of the product.

18. An antimicrobically active membrane suitable for the production of a packaging according to one or more of the preceding claims and having an open-cell structure of a polymeric material having pores loaded with an active antimicrobial agent and in the absence of solvents of the polymeric material of said structure.

19. Membrane according to claim 18, characterized in that said antimicrobial agent is selected from the group consisting of enzymes, such as lysozyme, food preservatives, amino acids, such as lysine and nisin, essential oils, organic acids, bacteriocins, fungicides, metals, proteins.

20. Use of a membrane according to claim 18 or 19 for the preservation of a food or perishable product.