DESCRIPTION

TECHNICAL FIELD OF THE INVENTION

The present invention relates to compositions for use in packaging of products sensitive to oxygen such as for example food, a drug or a cosmetic product, comprising or consisting of a matrix comprising one or more enzymes capable of catalysing the oxygen reduction. The present invention further relates to a packaging material including said compositions and the methods for the production thereof.

STATE OF PRIOR ART

Several products are subjected to deterioration and/or oxidation when they come in contact with the atmospheric oxygen. Examples of such products include food such as beer, wine, fruit juice, bread, meat, drugs and cosmetic products.

In order to prevent such oxidation, the oxygen has to be removed from the container or it must not penetrate the container wherein the products are preserved. In the state of art several solutions were proposed to avoid or reduce the presence of oxygen in packaging. The most widespread used approach is based upon the attempt at making the used material oxygen-repellent, for example by inserting a barrier layer preventing the passage of oxygen or as described in the patent U.S. Nr. 5.143.763 the insertion of compositions having an oxygen-absorbing material. The disadvantages associated to the known techniques are both complexity and difficulty in removing completely the oxygen from the product. Then the problem is very felt of providing new methods, compositions and materials for the packaging of perishable products such as food, drugs and cosmetic products which are capable of removing oxygen and which do not have the disadvantages of the solutions described in the prior art.

SUMMARY OF THE INVENTION

The good preservation of drugs, food, cosmetic products is directly correlated to the quantity of oxygen thereto they are exposed during the preservation period. In the present invention an active barrier was implemented active in the removal of the molecular oxygen which can permeate at a first layer of packaging ( packaging ), due to the not perfect insulation to the oxygen of the materials. The molecules which are produced as result of the oxygen removal, for example water, are not toxic. The use of an active barrier in the oxygen removal constitutes an effective strategy in the limitation/removal of oxygen inside the packaging. The present invention firstly relates to a composition for use in packaging of products sensitive to oxygen, such as for example food, drugs and cosmetic products, comprising or consisting of a matrix comprising one or more enzymes capable of catalysing the reduction of oxygen.

The present invention secondly relates to the use of such compositions in the preparation of a packaging material such as for example films or bags to contain perishable products.

The present invention also relates to all materials for packaging food, drugs and cosmetic products comprising such compositions and the methods for the production thereof.

BRIEF DESCRIPTION OF FIGURES

Figure 1 : In figure 1 an embodiment of the present invention and the operation thereof is represented schematically.

Figure 2: In figure 2 a second preferred embodiment of the present invention and the operation thereof is represented schematically.

Figure 3: Figure 3 shows an embodiment of the present invention in packaging a liquid.

Figure 4. Figure 4 shows an embodiment of the present invention in packaging solid food.

Figure 5. Figure 5 shows a graph with the concentration of molecular oxygen measured by means of scanning electrochemical microscopy (SECM) in a layer of 1 mm above the barrier. In figure the barrier is placed at first at a distance of 1 mm.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to compositions for use in packaging of products sensitive to oxygen comprising or consisting of a matrix comprising one or more functional enzymes capable of reducing the molecular oxygen without the formation of toxic products.

In the present description under the term“compositions for use in packaging of products sensitive to oxygen” compositions are meant suitable to be used in packaging perishable products in presence of oxygen such as for example food, drugs and cosmetic products.

In the present description under the term“matrix” and“polymeric matrix” composites of hydrogel, matrigel, polymers capable of englobing or absorbing/adsorbing the active enzymes are meant, under“matrixes” substances are further meant which are capable of binding firmly the enzymes thereto so that they could exert their enzymatic function. Under the term‘matrixes’ the composition resulting from these substances with the enzymes themselves then are meant. The term“matrix” is used in case gels (for example protein-based gels) or substances are used capable of absorbing/adsorbing/binding covalently the enzymes,“polymeric matrixes” in case said matrixes are constituted by polymeric structures.

The matrix can be a protein-based matrix, wherein the enzymes are entrapped by crosslinking (for example by using GDA) and by using another sacrificial protein and not active at higher concentrations (for example the bovine serum albumin: BSA) for the formulation of the protein matrix wherein the active enzymes are to be intercalated via crosslinking. The protein constituting the matrix base could be any protein available and at low cost since its function is that of creating a biocompatible protein-based matrix, but it is not necessary that any functionality thereof is retained in the matrix. Proteins such as those of milk, keratin, collagen, and both animal and vegetable proteins and even proteins derived from manufacturing scraps could also be used. Such protein matrix has the shape of a hydrogel and it has the capability of retaining water at good concentrations. Such hydrogel then is also suitable to the solubilization of the substrates of the enzyme.

The enzymes included in the matrixes of the compositions and of the herein described hydrogels could be enzymes consuming 0 ₂ without forming toxic products, the herein described matrixes could include even a second enzyme which removes/consumes the unwished or toxic products in case produced by the first enzyme which catalyses the oxygen reduction. The enzymes in the matrix should be active preferably for a period of time sufficient to the product preservation for example some months or years, in other words the enzymes should not be in denatured form inside the hydrogel or the matrix, but in their catalytically active form.

The matrix could be prepared based upon different interaction principles between the enzymes and the matrix, for example crosslinking with the glutaraldehyde, entrapping in polymeric matrix, adsorption by physical or electrostatic interactions. The first two are the methods most used to immobilize an enzyme. The crosslinking is a process which is based upon the formation of covalent bonds between two or more molecules whereas the entrapment in polymeric matrix is based upon purely mechanical and electrostatic principles. Examples of agents for the cross-linking are for example the glutaraldehyde (GDA), Bis(sulfosuccinimidyl) suberate BS3, N- hydroxysuccinimide, formaldehyde, use of photoreactive agents in combination with UV.

According to a preferred embodiment the used composition is in form of hydrogel, for example for the entrapment of the enzymes silicone hydrogel, polyacrylamides, cellulose, cellulose derivatives, collagen, carboxymethylcellulose, alginate, chitosan, agar, polymacon, hyaluronic acid, polymethylmethacrylate, hydrogel peptide- mimetics could be used, wherein the enzymes are associated to the matrix with crosslinking agents or o mechanically entrapped after the matrix crosslinking.

Examples of enzymes which catalyze the oxygen reduction are the Glucose oxidase (GOx), the Laccases, NADPH oxidase, xanthine oxidase, lactate oxidase, cytochrome oxidase, any oxidase and oxidoreductase using oxygen as substrate. According to an embodiment the composition, preferably in form of hydrogel, will include the Glucose oxidase and the catalase enzyme. The Glucose oxidase -GOx- is an enzyme of the family of oxidoreductase which catalyses the following reaction:

The hydrogen peroxide is a very reactive molecule, toxic for the cells, for this reason the above reaction was coupled to that of the Catalase -CAT-, another very important oxidoreductase in the biological systems which transforms the reactive oxygen species - ROS - produced in water. Net of both reactions two oxygen molecules are transformed into two molecules of water plus one of oxygen, which returns in the cycle of the Glucose oxidase. The hydrogel consumes oxygen in presence of glucose. This preferred embodiment then has several advantages. The Glucose oxidase for example will be the one purified by the fungus Aspergilus Niger or by other sources, whereas the Catalase could be for example purified by beef liver. In the same way other peroxidase enzymes can be used to remove catalytically the hydrogen peroxide produced in the reaction of the oxygen reduction, for example the horseradish peroxidase (Horseradish peroxidase -HRP) can be used. According to an embodiment the hydrogel could further include the Bovine Serum Albumin (BSA). In the gel formation the use of a solution of Bovine Serum Albumin (BSA -bovine serum albumin-) has resulted extremely advantageous as protective matrix to avoid denaturation of the enzymes during gelification. Moreover BSA existing in huge concentrations enters the network of hydrogel, by constituting it and it creates many cross-links between the molecules of BSA, and between molecules of BSA and molecules of enzyme: in this way the concentration of the enzymes can be reduced or increased to the required amounts without the risk that the enzyme is not fixed in the gel, if present at very low concentrations; moreover the presence of BSA decreases the probability of intermolecular cross-links in the enzymes which can denature them by inhibiting the activity thereof. The composition acting as active barrier for the oxygen could include the substrates required to the enzymatic reaction used in the oxygen removal, for example glucose, sucrose, corn syrup and other molasses, in particular when the Glucose oxidase is used or other oxidases using these substrates. It is to be noted that these compounds are approved by FDA for food use and they are available at very low cost. Such aspect is particularly interesting for its applications since the reaction substrate is the only sacrificial component of the barrier and it can be used at very high concentrations, by increasing the effectiveness and allowing very long operating time of the system. Example of oxidoreductase and respective substrates: Glucose oxidase/b -D glucose (sucrose, molasses and corn syrups etc, fructose and hexokinase ATP, etc); Hexose Oxidase/Hexose; Cholesterol oxidase/cholesterol; galactose oxidase/ D-galactose; lactate oxidase/ lactate; pyruvate oxidase/ pyruvate; aminoacid oxidase/ aminoacids). As above said the composition acting as active barrier can be prepared by exploiting different principles of interaction between the enzymes and the matrix, for example crosslinking with the glutaraldehyde, by means of entrapping in polymeric matrix, adsorption by physical or electrostatic interactions. The high viscosity of high concentrations of substrate, such as for example high aqueous concentrations of sucrose and glucose, could allow the use of the solution-substrate itself as matrix for the dissolution of the enzymes themselves. Such matrix preferably will be arranged then between layers of material for passive packaging.

The present invention further relates to a material for the packaging of an oxygen sensitive product, for example food, a drug or a cosmetic product, comprising a composition as herein described. Such material for example will be in form of film, bag or other form suitable to include the product to be preserved.

According to an embodiment the packaging material will include a material layer suitable to the food contact whereon the composition according to any one of the herein described embodiments is deposited. Such layer for example could be a plastic film, preferably polyethylene terephthalate (PET). According to an embodiment the packaging material will include three layers, a first and a second layer preferably made of plastic, such as PET or other materials and a third layer positioned between the other two comprising or constituted by the composition according to any one of the herein described embodiments. In figure 1 a schematic representation of an embodiment of this type is shown.

The present invention also relates to a method for the preparation of a multi-layer material as herein described. According to an embodiment such method provides a first passage wherein a composition is prepared comprising or consisting of a matrix comprising one or more enzymes capable of catalysing the reduction of oxygen according to anyone of the herein described embodiments and a second passage wherein the composition is deposited between two layers of plastic, such as for example films of polyethylene terephthalate (PET) or other polyesters and thermoplastic resins suitable to the food contact. The so-formed multilayer for example could be sold in rolls to be used for packaging the products. The thickness of the layers made of plastic or other suitable materials preferably will be comprised between 10 and 100 micrometres, whereas the thickness comprising the composition acting as active barrier will be preferably comprised between 10 and 1000 micrometres.

According to an alternative embodiment the material will be constituted by a double two-component film to be joined at time of packaging, one of these layers comprising a film for packaging plus the composition with the enzyme and the other one a film for packaging comprising the enzyme substrate. The two rolls could be joined in one single product directly during the final packaging in a machine for packaging.

According to an embodiment in the first step for preparing the composition a hydrogel will be prepared, for example according to the following process:

a) preparing a solution comprising one or more enzymes apt to catalyze the oxygen reduction;

b) adding a cross-linker agent;

c) gelling the solution. Preferably the process will include the addition of BSA followed by the addition of a cross-linker agent such as for example the glutaraldehyde (GDA). The enzymes and BSA preferably will be mixed in biocompatible buffers such as for example PBS. For example, at first a mixture comprising Glucose oxidase and Catalase will be prepared, thereto at first BSA and then the cross-linking agent, for example la GDA, is added. EXAMPLES

Embodiment example:

After having prepared the solutions of Catalase (32 mg/ml_) and Glucose oxidase (16 mg/ml_) they are mixed in a ratio 1 :1 in a solution including the BSA (62 mg/ml_), an amount of glutaraldehyde (25% in water) equal to 1.38% of the total final volume is added. The Glucose oxidase (type X) of the fungus Aspergillus Niger whereas the Catalase of beef liver. All used compounds were purchased by Sigma Aldrich. As sacrificial substrate b-D-glucose 1 mM is added. The matrix is then placed on a PET film or other film used for packaging. In order to determine the capacity of the so-prepared material with the active barrier in the oxygen removal the scanning electrochemical microscopy (SECM) was used.

SECM instruments used for measurements:

The instrument used for the measurements of the oxygen gradient is the probe scanning electrochemical microscope "CH Instrument Texas" model CHI B910. SECM uses an ultramicroelectrode (UME) for detecting electrochemical processes such as the oxygen reduction and then its concentration. The probe (UME) is connected to a system of three engines and three piezoelectrical elements, and then can be moved and positioned in the three cartesian axes X, Y and Z. Such system allows to solve in space the process measured at UME and then it allows to display the chemical concentration of redox species (for example oxygen) depending upon the specific position of the probe. The distance between the probe and the substrate made of plastic in the specific case is controlled through a feedback mechanism. In this way we can evaluate the oxygen concentration depending upon the distance from our active barrier for the oxygen removal and we can measure the functionality thereof in time. Through this particular probe scanning technique, the concentration of molecular oxygen was measured near the protective film. In figure 5 the oxygen concentration in a layer of one millimetre in proximity of the barrier is shown. It is to be noted that the oxygen concentration falls to zero completely near the barrier film.