WO2007063097A1

WO2007063097A1 - Process for production of active matrices with antimicrobial activity

Info

Publication number: WO2007063097A1
Application number: PCT/EP2006/069131
Authority: WO
Inventors: Matteo Alessandro Del Nobile; Milena Sinigaglia; Amalia Conte; Giovanna Giuliana Buonocore
Original assignee: Universita' Degli Studi Di Foggia
Priority date: 2005-12-01
Filing date: 2006-11-30
Publication date: 2007-06-07
Also published as: ITMI20052301A1

Abstract

The invention relates to a process for lysozyme immobilization on a polymeric matrix containing hydroxyl groups. The characteristics of this process enable covalent binding of lysozyme to a matrix without loss of antimicrobial activity. According to a preferred embodiment of this process, lysozyme and other necessary reagents are sprayed on a matrix which is already reticulated and moist. This invention also relates to products resulting from the process which are endowed with antimicrobial properties. Among possible applications, the product is specifically intended for use in coating or packaging or as part of a coating and/or packaging for foodstuffs.

Description

Process for production of active matrices with antimicrobial activity FIELD OF THE INVENTION

The present invention relates to the field of immobilization of enzymes with antimicrobial activity on polymeric reticulated and organic matrices for preparation of coatings or packaging films for alimentary use. STATE OF THE ART

Studies on "active coating or packaging" focus on investigation and preparation of matrices, generally in the form of films, capable of slowing down the process of deterioration of manufactured food or drinks. Recently, various procedures for immobilization of active antimicrobial principles on matrices have been set up, however the final product obtained not always meets the requirements. These are:

• minimum release of the active principle after immobilization. As the final product is a commercial product, the occurrence of even a minimum release can cause the concern of the nearest or remote buyer about quality of the coating and of the final product, β the antimicrobial material should be of organic nature even though it is not released in the product, since it comes in contact with food, thus the immobilization of xenobiotic products should be avoided,

• the material used should conform to European or National regulations, for instance it should have a low environmental impact. Moreover, it is appropriate that such materials are commercially convenient and widely available,

• immobilization must not inactivate the active principle,

• linkage between active principle and matrix should be durable and resistant, and should be mostly concentrated on the surface of the matrix. Finding materials that meet all these requirements is still a problem in the art, even though similar attempts based on matrix immobilization of an enzyme, the naringinase, have been published for instance in Naringinase Immobilization in Packaging Films for Reducing Naringin Concentration in Grapefruit Juice, NFF Soares e JH Hotchkiss, Vol. 63, No1. Journal of Food Science, 1998. Authors compared the effects of naringinase on two matrices and found that the quality of the final product involved a compromise: a stronger and more resistant linkage of naringinase to the matrix resulted in lower enzymatic activity. In fact the authors of this study privileged the linkage resistance over naringinase activity for a commercial product.

Other attempts to immobilize lysozyme on various types of matrices have been published in Immobilization of Lysozyme on Food Contact Polymers as Potential Antimicrobial Films, (1997) P. Appendini and J. H. Hotchkiss, Packag. Technol.

Sci. Vol. 10: 271-279. The results obtained well illustrate the actual difficulty to immobilize proteins with enzymatic activity, such as lysozyme.

Therefore, devising a process for immobilization of a product, that ensures that the active principle durably retains its properties, is still an issue in the art. SUMMARY OF THE INVENTION

The invention relates to a process for immobilization of lysozyme on a polymeric matrix containing hydroxyl groups, involving the following steps: a) preparation of the polymeric matrix by addition of a reticulating agent to a polymer containing hydroxyl groups b) lysozyme activation by addition of a dialdehyde linker to an aqueous solution of lysozyme c) combination of the solution obtained in step (b) with the reticulated matrix obtained in step (a).

A preferred embodiment of this process consists in partial exsiccation of the matrix after step (a) and addition, by spraying, of the solution prepared in step (b) onto the surface of the partially exsiccated matrix.

The invention includes also the use of products obtained according to this process.

The linkage formed between a linker and the lysozyme makes possible to retain lysozyme in the polymeric matrix durably without impairing its effectiveness. Among possible applications, the product is specifically intended for use in coating or packaging or as part of a coating and/or packaging for foodstuffs.

DESCRIPTION OF THE FIGURES

Figure 1: Release of lysozyme from films prepared by bulk and spray methods Graph representing the release of lysozyme over time from films prepared by the bulk method and by the spray method, containing various amounts of lysozyme and glutaraldehyde, after washing in 4,5L of distilled water. (D) Film Bb: bulk method carried out with 500 mg of lysozyme and addition of

0.025 mi (50% w/v) glutaraldehyde.

(o) Film Cb: bulk method carried out with 100 mg of lysozyme and addition of

0.005 ml (50% w/v) glutaraldehyde. (Δ) Film Db: bulk method carried out with 50 mg of lysozyme and addition of 0.005 ml (50% w/v) glutaraldehyde.

(■) Film Bs: spray method carried out with 500 mg of lysozyme and addition of

0.025 ml (50% w/v) glutaraldehyde.

(•) Film Cs: spray method carried out with 100 mg of lysozyme and addition of 0.005 ml (50% w/v) glutaraldehyde.

(A) Film Ds: spray method carried out with 50 mg of lysozyme and addition of

0.005 ml (50% w/v) glutaraldehyde.

Figure 2: Antimicrobial activity of various lysozyme containing films on bacterial suspensions Panel A) Graphical representation of the antimicrobial activity of various films produced by binding different amounts of lysozyme by glutaraldehyde, using the bulk method, as assessed on a suspension of M. lysodeikticus cells by monitoring the absorbance at 450nm of the microbial suspension.

(D) Film A: matrix without lysozyme, with only 0.025 ml (50% w/v) glutaraldehyde. (0) Film B: matrix obtained with 500 mg of lysozyme and 0.025 ml (50% w/v) glutaraldehyde.

(■) Film C: matrix obtained with 100 mg of lysozyme and 0.005 ml (50% w/v) glutaraldehyde.

(o) Film D: matrix obtained with 50 mg of lysozyme and 0.005 ml (50% w/v) glutaraldehyde.

(•) Film E: matrix obtained with 20 mg of lysozyme and 0.005 ml (50% w/v) glutaraldehyde.

Panel B) Graphical representation of the antimicrobial activity of various films produced by binding various amounts of lysozyme by glutaraldehyde, using the spray method, assessed on a suspension of M. lysodeikticus cells by monitoring the absorbance at 450nm of the microbial suspension.

(o) Film A: matrix without lysozyme, with only 0.025 ml (50% w/v) glutaraldehyde. (T) Film B: matrix obtained with 500 mg of lysozyme and 0.025 ml (50% w/v) glutaraldehyde.

( β ) Film C: matrix obtained with 100 mg of lysozyme and 0.005 ml (50% w/v) glutaraldehyde. (Δ) Film D: matrix obtained with 50 mg of lysozyme and 0.005 ml (50% w/v) glutaraldehyde.

(Δ) Film E: matrix obtained with 20 mg of lysozyme and 0.005 ml (50% w/v) glutaraldehyde.

(0) Control sample: Buffer solution containing microorganisms without film Figure 3: Comparison of the effectiveness of lysozyme immobilized by spray and bulk methods

Graphical representation of different antimicrobial activities (μ_maχ) of films bearing lysozyme immobilized by spray or bulk method, in relation to the amount of immobilized lysozyme. (B) data extrapolated from results with films obtained by the spray method

(D) data extrapolated from results with films obtained by the bulk method

Figure 4: Duration of the antimicrobial activity of lysozyme-containing films on a single strain or on a mixture of five A. acidoterrestris strains

Graphical representation of the antimicrobial activity over time of matrices bearing surface-immobilized lysozyme, assayed on an individual strain (Fig 4A) or on a mixture of 5 A. acidoterrestris strains (Fig 4B) in 600ml of acidified MEB. The bacterial population is evaluated by measuring the CFU/ml value, after taking 1ml of sample from the culture broth.

( ^ ) film A (active film - lysozyme present) ( ^Ώ ) film B (inactive film - lysozyme absent)

( ° ) film C (control without film)

Figure 5: Antimicrobial activity of lysozyme-containing films on A. acidoterrestris spores in acid solutions at various pH

Antimicrobial activity of lysozyme containing matrices on a suspension of A. acidoterrestris spores in 500ml of acidified MEB or in 50OmI of apple juice. The cell population is evaluated by measuring the CFU/ml value of samples taken from the culture.

(0) Film A (active film - lysozyme present) in acidified MEB

(D) Film B (inactive film - lysozyme absent) in acidified MEB (o) Film C (control without film) in acidified MEB

(♦) Film A (active film - lysozyme present) in apple juice

(a) Film B (inactive film - lysozyme absent) in apple juice

(®) Film C (control without film) in apple juice

DETAILED DESCRIPTION OF THE INVENTION In the present description, terms herein reported have the following meaning:

Reticulating agent - A compound that produces reticulation through a chemical bond. In this specific case, the reticulating agent is able to form inter- or intramolecular covalent bonds.

Linker - A compound that enables formation of a chemical bond, either by as interposed bridge or by direct bond, between chemical groups of two different molecules, for instance between the hydroxyl group of a matrix and the side chain of an amino acid in a protein and/or enzyme.

Active matrix - a polymeric matrix derivatized with lysozyme, thereby acquiring antimicrobial properties. Immobilization - generation of a chemical bond between an active principle and a matrix. Object of the present invention is a process for production of a matrix of organic nature, to which lysozyme is immobilized via a resistant and durable chemical bond.

Exsiccation - process based on total or partial evaporation of water from the polymeric matrix.

Foodstuffs- any material that is a (solid or liquid) matrix for alimentary use.

This invention relates to a process for immobilization of lysozyme, a protein having antimicrobial activity, on a polymeric matrix containing hydroxyl groups, comprising the following steps: a) preparation of the polymeric matrix by addition of a reticulating agent to a polymer containing hydroxyl groups b) lysozyme activation by addition of a dialdehyde linker to an aqueous lysozyme solution c) combination of the solution obtained in step (b) with the reticulated matrix obtained in step (a). The enzyme lysozyme has antimicrobial activity since it catalyzes the hydrolysis of the β(1-4) bond between N-acetylglucosamine and N-acetylmu ramie acid; such bond is present in the saccharidic structures of the prokaryotic cell wall. The polymer used for preparation of the matrix is a natural polymer containing hydroxy! groups. Preferred polymers are those bearing alcoholic groups, even more preferred are those polymers in which the alcoholic groups are secondary and are exposed on the matrix surface.

Most preferred polymers are polyvinylalcohol (PVA) homopolymers. Generally, aqueous solutions of PVA homopolymers with molecular weight of 70,000-100,000 Da (Sigma-Aldrich S.r.l., Milano, Italia) are used at concentrations comprised between 5 and 20% w/v, preferably at concentrations comprised between 10 and 15% w/v and even more preferably between 12 and 14% w/v. Polymers may also comprise copolymers, at least one of which containing hydroxyl groups. Other usable polymers should contain hydroxyl groups and meet the other requirements outlined above, such polymers are for instance chitosan, cellulose, starch, alginate, carrageen and agar.

The reticulating agent allows inter- and intra-molecuiar chemical reticulation of polymeric chains. Reticulation prevents possible solubilization of the matrix in water, making the matrix resistant to extreme chemical conditions, as for instance acidic pH. Polymer reticulation (step (a)), lysozyme mixing (step (b)) and the combination of these two steps (step (c)) preferably occur in an acidic environment, preferably at pH below 5, even more preferably below 3, and even more preferably at pH values comprised between 2 and 1. In a preferred embodiment, hydrochloric acid is used to obtain the acidic pH in step (a) and acetic acid is used to obtain the acidic pH in step (b). Among the reticulating agents known in the art dialdehydes are preferred; particularly preferred is glyoxal as reticulating agent in step (a). According to an embodiment, in step (a) 5 μl_ of glyoxal (40% w/v) are mixed with a 13% w/v PVA solution in 25 ml distilled water at room temperature.

In step (b), the linker is a dialdehyde, preferably glutaraldehyde. According to a preferred embodiment, lysozyme and glutaraldehyde are mixed in step (b) at acidic pH, preferably at pH between 1 and 2, at room temperature. The amount of dialdehyde linker added depends on the amount of added lysozyme. If the lysozyme solution obtained is used according to the spray method, it is preferable to use during step (b) a molar ratio between the starting amount of linker and lysozyme comprised between 1 and 50, even more preferably comprised between 2.5 and 35, even more preferably comprised between 4 and 20 and even more preferably comprised between 16 and 18; if the lysozyme solution obtained is used according to the bulk method, the molar ratio is preferably comprised between 12 and 15 and the solution is added to the polymeric matrix obtained in step (a). To obtain a given antimicrobial effectiveness of the film, it is preferable to use a high rather than a low molar ratio between the starting amounts of linker and of lysozyme, for costs reasons.

In step (c) it is preferable to use a weight ratio between the starting amount of PVA and lysozyme, comprised between 0.5 and 250, even more preferably comprised between 1 and 150 and even more preferably comprised between 5 and 15. These ratios are calculated based on laboratory amounts in the order of 10^"6-10^"5 moles, but they are still valid for industrial amounts.

The process may optionally comprise further steps, in between steps (a) and (b), in between (b) and (c), or after (c), such as homogenization and/or exsiccation. These steps can be carried out according to methods well known in the art. In a preferred embodiment, the exsiccation process takes place after step (a) and/or

(C).

According to a preferred embodiment of the process, reagent mixing by homogenization is carried out after step (a) and/or step (b) and/or step (c), in order to obtain better results with respect to reticulation and binding of lysozyme to the matrix

The reticulated matrix obtained after step (a), or after addition of activated lysozyme in step (c), is poured in suitable shapes and amounts, and left to exsiccate. In a preferred embodiment of the process, the matrix solution and the lysozyme are left to exsiccate on a slab for at least 20 hours, preferably 24 hours, at room temperature in order to form a film that preferably has an initial thickness comprised between 150 and 500μm, more preferably comprised between 200 and 400μm and even more preferably comprised between 280 and 320μm.

By use of techniques well known in the art, the process can be modified in order to obtain consistency and shape of the matrix suitable to the needs. For instance, the matrix can be modeled in a gel or in a suitable shape or can be a granulate or can be adsorbed to another material, as for instance material for medications like gauzes.

According to a preferred embodiment, the activated lysozyme solution can be combined with the matrix according to a step (c) by spraying (spray method) carried out by well known techniques. In fact, according to a preferred aspect of the process, the solution in step (b) is sprayed together with an acid onto the matrix prepared in step (a) which is already spread on a slab and partially exsiccated (still moist). This process concentrates lysozyme only on one side of the matrix (for instance, if the matrix is a film used as "active coating"), providing clear economic advantages because lysozyme is only present where needed, thus minimizing reagent waste. A further embodiment of the process consists in adding all the reagents together, by performing the three steps (a), (b) and (c) simultaneously, followed by exsiccation. This embodiment of the process is advantageously easy and simple to perform. According to this embodiment, it is preferable to use glutaraldehyde as both linker and reticulating agent. In one of the preferred embodiments, the matrix obtained in step (c) is then washed in distilled water bath for removal of all unreacted reagents. The efficiency of removal of these free reagents depends on duration of the washing step in distilled water and on the starting concentration of reagents. It is preferable to shake the matrix in the water bath, so that the removal of free reagents occurs more rapidly.

Moreover, it is also possible to carry out only steps (b) and (c) using a preformed polymeric matrix , for instance a preformed polymeric film. The invention relates also to the product obtainable by the described process. The resulting product is a polymeric matrix that is derivatized with lysozyme, thereby acquiring antimicrobial properties. The antimicrobial activity of the so produced matrix can be evaluated by techniques well known in the art, for instance by measuring the cfu/ml (colony forming units/ml: bacterial units producing separate colonies/ml) of a bacterial suspension in presence or in absence of the active matrix. The number of bacterial cfu/ml is always decreased in presence of the active matrix according to the invention. Another way of evaluating the antimicrobial activity of the product of the invention is to measure the change over time of the absorbance at 450nm of a bacterial culture, in presence or in absence of the active matrix. The absorbance at 450nm is proportional to the density of the bacterial population, hence to the antimicrobial properties of the matrix. Moreover, the characteristics of the active matrix can be assessed quantitatively by using the Gompertz equation. In the present invention, the Gompertz equation has been used as modified by Zwietering (Modeling of the bacterial Growth Curve, MH Zwietering et al., Appl. Environ. Microbiol., June 1990, p.1875-1881). According to Zwietering et al., by "fitting" the equation to the experimental data (e.g. from an assay measuring absorbance at 450nm), it is possible to determine the μ_maχ value that represents the maximum rate of decrease of the microbial population in presence of the product. In a sigmoidal curve of decrease of a bacterial population, μ_maχ is graphically represented as the angular coefficient of the tangent at the curve flex point. The matrices produced according to the invention have different μ_maχ values depending on factors such as starting amount of lysozyme, efficiency of the process that varies with the method of lysozyme immobilization to the matrix (e.g. whether lysozyme is immobilized by the spray or the bulk method) and assay specifications (e.g. ratio between the volume of microbial suspension and the active surface of the matrix). Thus μ_max is a value proportional to the antimicrobial activity of the product obtained from the process. Moreover, the product is characterized by stable binding of lysozyme to the matrix, as measured under laboratory conditions involving incubation in aqueous solutions for at least 200 hours. Binding persists even after repeated rinsing or exposure to various substances, as for instance foodstuffs. As seen above, the immobilization by the spray method makes possible to immobilize lysozyme only on one side of the film, and this represents a further advantage of the process, since it makes use of less lysozyme per surface area of polymeric matrix. One of the preferred products among those obtainable according to the process is in the form of a film. By casting, it is possible to produce a film with a thickness comprised between 10 and 200μm, more preferably comprised between 50 and 150μm and even more preferably comprised between 80 and 120μm. This film can be adapted to several coatings for the purpose of preventing bacterial growth. Therefore, its main use is as coating or packaging film for foodstuffs as well as bandage for lesions or wounds. EXPERIMENTAL PART

Example 1 - Preparation of films according to the bulk method Films have been prepared by use of the following reagents: polyvinylalcohol (PVA) with molecular weight 70,000-100,000 Da (Sigma-Aldrich S.rl, Milano, Italia) as polymeric matrix, lysozyme with molecular weight 14,000 Da (Sigma-Aldrich S.r.l., Milano, Italia) as active antimicrobial principle, glyoxal (40% w/v Riedel de Haen, Sigma-Aldrich S.r.l., Milano, Italia) as reticulating agent and glutaraldehyde (50% Aldrich, Sigma-Aldrich S.r.l., Milano, Italia) as linker. Aqueous solutions of 25 ml volume, containing 13% w/v PVA, have been autoclaved for 15 minutes at 121 °C and then cooled at room temperature. After dissolving the PVA in water, 5μL of glyoxal (40%p/v) have been added as reticulating agent and 0.2ml of hydrochloric acid have been added as acid catalyst for polymer reticulation. These mixtures have been subsequently homogenized at a speed of 150 rpm for 15 min, followed by addition of various amounts of lysozyme and glutaraldehyde, as shown in table 1 , together with 2ml of glacial acetic acid as catalyst. Table 1 - Amounts of lysozyme and linker

The resulting solutions have been again homogenized at a speed of 150 rpm for 15 min. After homogenization, solutions have been poured on plexiglass® slabs and left to exsiccate at room temperature. After evaporation, matrices were left to exsiccate for an additional day under vacuum in order to evaporate the excess of acetic acid. The so obtained films had an average thickness of 100μm. Example 2 - Stability of the polymeric matrix in distilled water Samples with a surface of 20x11cm have been prepared as outlined in Table 1 in example 1 and immersed in 4,5L of distilled water at room temperature. To monitor the release of lysozyme from the matrix, a sample of water has been withdrawn periodically until equilibrium was reached. To ascertain the presence of lysozyme, each water sample has been analysed by HPLC (Agilent Mod. 1100), using a C18 reverse phase column (250x4 mm, 5 μm) that has been eluted with acetonitrile and 0.1%TFA, with a 1ml/min elution gradient. The lysozyme calibration curve in the HPLC has been obtained using standard solutions of the substance at concentrations comprised between 6 and 300 ppm. Tests were repeated on 5 samples every time. Results are depicted in figure 1 and shown in table 2. E matrices (see table 1) have not been reported in the figure because no lysozyme was released from these films. Table 2 - Data reporting the release of lysozyme from the matrix during the washing process

The results show that a higher release of (not chemically immobilized) lysozyme occurs from matrices to which higher amounts of lysozyme were initially added.

Presumably, the release of lysozyme reflects the ratio between the amounts of lysozyme and glutaraldehyde - more glutaraldehyde binds more lysozyme in a more stable manner, thereby resulting in lower lysozyme release. Another factor may be the fraction of glutaraldehyde acting as reticulating agent rather than as a linker. However, it is clear that the release is reduced at a minimum with 50mg or lower amounts of lysozyme (100% immobilized).

Example 3 - Antimicrobial activity of the matrix and control of lysozyme release

As outlined in table 2, this example involves the use of films subjected to a washing step as described in example 2. B films, for instance, have reached equilibrium in 4,5L distilled water after 30 hours, whereas D films have not released any lysozyme. Thus, these samples contain only the lysozyme that is chemically bound to the matrix and therefore immobilized.

To measure the antimicrobial activity of these films, freeze-dried Micrococcus lysodeikticus (Sigma-Aldrich S.r.l., Milano, Italia), resuspended at room temperature in 61OmL of 0.1 M phophate buffer pH 6.8 at a cell concentration of

10⁷microorganisms/mL (as determined by adsorbance at 450nm), has been used.

Films containing various amounts of immobilized lysozyme were incubated in presence of their respective microbial suspension in phosphate buffer. As with the determination of the time required to reach equilibrium during washing, the method to determine antimicrobial effectiveness of films involved withdrawal of a sample from the various solutions at defined time intervals, and measurement of the absorbance at 450nm in order to assess the amount of bacteria still present, until reaching a steady bacterial content. These withdrawals have been repeated in tests with three identical films for each active sample studied. In this way, the dynamics of the antimicrobial activity of the film in solution have been measured and evaluated. The control consisted in the same buffer solution containing bacteria without film.

In order to prove that the antimicrobial activity detected was due to fully immobilized lysozyme, after washing, the various active films have been returned to water and any lysozyme release has been again monitored by HPLC. The fact that films have not released any further amount of active substance confirms that the washing step separates sharply and rapidly non-immobilized from immobilized lysozyme. This was confirmed throughout the whole evaluation period (24 hours). Figure 2 shows the effectiveness of the various films in decreasing the population of M. lysodeikticus bacteria in solution. Using the experimental absorbance data, it has been possible to obtain sigmoidal curves of decrease kinetics of the bacterial population in presence of the active film. To evaluate the decrease of the population, the Gompertz formula (equation) has been used as modified by Zwietering (Modeling of the bacterial Growth Curve, MH Zwietering et al., Appl. Environ. Microbiol., June 1990, p.1875-1881) . From the fitting of the Gompertz equation to the experimental data, it was possible to assign values to the variables of the Gompertz equation. Relevant parameters are shown in table 3. E% is the value used to assess the precision of the fitting to the experimental data, and this percentage represents the variation between the results obtained. Therefore, higher E% values indicate less precise fitting of the experimental data to the curve. Table 3 - Gompertz formula parameters drawn from the results shown in Figure 2 for matrices prepared by the bulk method. Sample A is the microbial suspension in phosphate buffer, in presence of film without lysozyme.

In their study, Zwietering et al. point out that the most significant value for evaluation of growth/decrease of a bacterial population is the μ_max value because the mutation rate is maximal in a population (last paragraph of the first column at page 1875 of Appl. and Environ. Microbiol., June 1990, p.1875-1881). In figure 2, μ_max is represented as the angular coefficient of the sigmoidal curve straight line. From data reported in Figure 2 and Table 3, it is possible to observe that a considerably higher elimination of bacteria, therefore a decrease of the population, occurs with lysozyme-containing films compared to the control. A minor killing of M. lysodeikticus can be noted also with the control film, but this is relatively low compared to the loss observed with lysozyme-containing films (μ_max values are e^"6 in comparison to e^"5 for the films with lysozyme). As can be seen in the data in table 3, the equation successfully approximates the experimental data. To assess the importance of lysozyme content relative to the level of activity, μ_max values have been evaluated as a function of the amount of immobilized lysozyme (that is, the lysozyme retained after washing). This assessment is presented in figure 3, showing that the rate of decrease of the bacterial population is proportional to the amount of lysozyme that is present in the film. Example 4 - Comparison between lysozyme immobilization methods on a PVA matrix by measuring lysozyme release during washing. In a comparative experiment, the results of immobilization performed with two different methods have been evaluated. The immobilization method termed "bulk" and the immobilization method termed "spray" have been compared, and the results of such comparison are described in example 1-3. The spray method makes use of the same reagents as the bulk method. The initial matrix preparation is identical: an aqueous solution of 13% (w/v) PVA dissolved in 25ml distilled water is subjected to autoclaving for 15 minutes and subsequently cooled at room temperature. Five μl_ of glyoxal (40%w/v) have been then added as reticulating agent and 0.2ml of hydrochloric acid have been added as acid catalyst. Then the mixture has been homogenized at a speed of 150 rpm for 15 min and, unlike the bulk method, has been allowed to exsiccate in order to form a film. Glutaraldehyde (0.025 and 0.005 ml) and lysozyme (500, 100, 50 and 20 mg) have been then dissolved in 10 ml distilled water and uniformly sprayed on the surface of matrices that were still moist, according to the spray method. Immediately after, glacial acetic acid has been added by spraying in order to favour lysozyme immobilization. Both bulk and spray film types have been exsiccated under the same conditions. By using this method, 4 films (termed B(s), C(s), D(s) and E(s)) have been prepared and compared with those prepared in example 3 (termed (b)), as shown in tables 5, 6 and 7. Furthermore a control has been prepared, consisting in matrix without lysozyme, termed A(s). The main composition of spray and bulk films are shown in table 4. Table 4 - composition of active principles for bulk and spray films. The films termed (b) are obtained by the bulk method and those termed (s) are obtained by the spray method.

Each film, having an area of 20x11cm, has been prepared and washed with 4.5 L distilled water, as described in example 2, and analysed by HPLC for lysozyme release. Also in this case tests have been carried out in triplicate and under the same conditions as those described in example 2.

The results of the washes applied to films obtained with the bulk and spray techniques are described in Figure 1 and in Table 5.

Table 5 - Release of lysozyme from films after washing

In figure 1 and table 5, it can be observed that lysozyme release during washing is similar for both processes: the amount of released lysozyme correlates with the amount of starting lysozyme. It can also be observed that, at lower lysozyme concentrations, the release of lysozyme also occurs for films prepared with the spray method (e.g. at 50mg). It can also be noticed that an equilibrium between released lysozyme and matrix-immobilized lysozyme is reached earlier for spray films than bulk films. To better illustrate the situation at the molecular level, table 6 summarizes the amount of both reagents (glutaraldehyde and lysozyme) in moles:

Table 6 - Amount of immobilized lysozyme and of glutaraldehyde used, and their respective molar ratios.

Glutaraldehyde is always used in molar excess. A higher amount of lysozyme is immobilized by the bulk method (film B(b), C(b) and D(b)) compared to the spray method (film B(s), C(s) and D(s)). In fact, in the surface immobilization technique (spray method), lysozyme adheres only to one side of the matrix. After washing films prepared by the spray method, it is found that more or less the same amount of lysozyme is immobilized for a given amount of linker, while the amount of immobilized lysozyme increases proportionally with the increase of glutaraldehyde. Example 5 - Comparison between the activity of lysozyme immobilized on the surface of the matrix (spray method) and of lysozyme immobilized in the polymeric network of the matrix (bulk method).

Exactly the same methodology as in example 3 has been applied to spray matrices washed in example 4, in order to obtain results comparable to the results obtained for the bulk version in example 3.

In the experimental data shown in Figure 2, fitting of the Gompertz equation to death kinetics of the test microorganism has also been used to evaluate film effectiveness, as for bulk films. Table 7 shows for each film the relevant parameters obtained from fitting the equation.

Table 7 - Gompertz formula parameters drawn from the results shown in Figure 2 for matrices prepared by the spray method. The control consists in a microbial suspension in buffer without film.

Film Mmax E%

Controllo 3 .2057 e-⁶ 0 .2612

A(s) 5 .8580 e-⁶ 0 .3442

B(s) 2 .0871 e^"4 1 .7663

C(s) 8 .6439 e-⁵ 1 .9924

D(s) 3 .7026 e-⁵ 3 .8324

E(s) 4 .1010 e ⁵ 2 .3688

As mentioned in example 3, the μm_ax data are most representative of the antimicrobial activity of the film. As with systems termed bulk, the inactive film (A(s)) and the control sample show a minor and relatively low antibacterial activity. Also in this case, it can be observed that the antibacterial activity is correlated with the amount of lysozyme present: the activity is higher in presence of a higher amount of lysozyme. The validity of fitting is given by E% values. E% values are higher for spray versions of the films, however they remain statistically significant. Figure 3 shows μ_maχ values as a function of the amount of lysozyme present, for both bulk and spray films. As previously observed in both systems, film activity increases with the amount of immobilized lysozyme. It is interesting to note a comparison of the antibacterial activity between the two types of film, in figure 3. The bulk system retains more lysozyme, however it produces a μ_maχ value comparable to or lower than spray films. The difference in film activity is clearly observed at the lowest concentrations, where the μ_max value for the spray film is higher compared to the bulk film. The reason for this difference in effectiveness relates to the fact that lysozyme acts as antimicrobial agent by direct contact with the substrate (the β(1-4) bond between N-acetylglucosamine and N-acetylmuramic acido) in the bacterium. The lower activity observed in the bulk system may be due to the fact that the lysozyme present in the matrix does not necessarily come into contact with bacteria, because it is not immobilized on the outer surface of the matrix, but is rather immobilized inside the film. In fact, Figure 3 shows that the activity or amount of lysozyme present in bulk films is less than half compared to spray films: this is partly due to the fact that all lysozyme in the spray film is immobilized on one side, whereas the lysozyme in the bulk film is immobilized inside the matrix, thus resulting in two active surfaces contacting the bacterial substrate. A test for release of lysozyme from active films has been carried out also for spray films, after washing in water bath, in order to evaluate if the active substance was partly released, thus challenging the results on antimicrobial efficacy obtained above. These further washing tests have shown that no lysozyme was released from the film and therefore the antimicrobial activity exhibited by active films, in the various buffer solutions used, should be ascribed to the immobilized lysozyme. All the following examples have made use of films in which lysozyme was immobilized by the spray method. Example 6 - Evaluation of the effectiveness of immobilized lysozyme on Alicyclobacillus acidoterrestris

A 25ml PVA solution (13% w/v) has been reticulated by addition of 5 μl_ of glyoxal and 0.2ml of hydrochloric acid as catalyst. After homogenization, the solution has been poured onto a slab and left to exsiccate partially in order to form a film. A 10ml aqueous solution containing 2% lysozyme, mixed with 0.05ml of 50% (w/v) glutaraldehyde, has been prepared and sprayed on the moist film. Immediately after, 2 ml of glacial acetic acid have been sprayed to favour the reaction. Subsequently the matrix has been left to exsiccate completely, resulting in a film with a thickness of 100μm. An identical film lacking lysozyme has been prepared and used as control.

After appropriate soaking in distilled water, as seen in example 2, these films have been immersed in microbial culture suspensions (samples A). Also control films, after soaking in distilled water as in example 2, have been immersed in microbial culture suspensions (samples B). A second control has been used consisting of microbial culture suspensions without film (sample C). To evaluate the effectiveness against Alicyclobacillus acidoterrestris microorganisms, a suspension of an individual A. acidoterrestris strain has been used in one test and a mixture of 5 A. acidoterrestris strains has been used in another test. These strains have been grown separately in acidified (pH 4.5) malt extract broth (MEB, Oxoid, Milano, Italy) for two days at 44⁰C. To inoculate the individual A. acidoterrestris strain in suspension, a cell suspension was withdrawn from MEB and diluted in sterile saline solution (0.9% NaCI) in order to obtain a concentration of about 10⁸ cfu/ml. An amount of 1 ml of this suspension, at a concentration 10⁸ cfu/ml, has been inoculated in MEB pH 4.5, respectively in 150ml, 300 ml and 600ml volumes.

To inoculate the five A. acidoterrestris strains, a suspension of identical volume has been withdrawn from the respective 5 cultures and diluted in sterile saline solution (0.9% NaCI) in order to reach a total concentration of about 10⁸ cfu/ml. As with the example involving the inoculum of an individual strain, 1 ml of this solution has been inoculated in 3 different volumes of MEB: 150ml, 300ml and 600ml. Once A. acidoterrestris cells have been inoculated in these various volumes of MEB, the active versions of films with lysozyme (version A) or without lysozyme (version B) have been added, except for the control consisting in the sample without film (version C). All experimental solutions have been maintained at 44 ⁰C, under continuous shaking. Periodically, 0.1ml samples have been withdrawn from the solutions, diluted in saline and cfu/ml have been counted on acidified malt extract (MEA) in order to evaluate the concentration of A. acidoterrestris . These experiments have been carried out in duplicate, thus obtaining the mean and the standard deviation of the results.

Figure 4 shows the results relative to the activity of films on A. acidoterrestris populations. Table 8 shows the difference in cfu/ml between the initial microbial load present in the broth and the load measured after monitoring for 200 hours (Δ₂₀₀). Since the microbial load relates to the activity of immobilized lysozyme, Δ₂₀₀ can be considered an index of antimicrobial effectiveness of the active matrix.

Table 8 - Δ200 values (representing film effectiveness) obtained by fitting the equation 3 to the experimental data, together with confidence intervals (square brackets).

The results show that the active film effectively prevents growth of both the individual strain and the mixture of 5 strains. In fact, values and confidence intervals for active samples do not overlap with those of their respective controls, therefore, the results can be considered statistically significant. Moreover, the same values in table 8 show that the effectiveness of lysozyme on the film does not depend on the volume in which the film is placed.

Example 7 - Measure of film effectiveness on A. acidoterrestris spores in a liquid food (apple juice) compared to MEB. As seen in example 6, the effectiveness of lysozyme (represented by the Δ₂oo parameter) does not depend on solution volume. Therefore, the effectiveness of films on the spores has been evaluated only in a solution volume of 500ml. Films have been prepared as in example 6. Spore solutions were prepared according to the method of Sinigaglia et al. (ref. Sinigaglia M., M. R. Corbo, C. Altieri, D. Campaniello, D. D'Amato, and A. Bevilacqua. 2003. Combined effects of temperature, water activity, and pH on Alicyclobacillus acidoterrestris spores. J. Food Prot. 66: 2216-2221). For this, 10⁶ spores/ml have been inoculated in 500 ml of acidified MEB, as in example 6, and in 500 ml of a commercial apple juice. As in example 6, the antimicrobial activity of the active film has been assessed (version A) by comparison with two controls (version B, inactive film, and version C, without film).

Prior to addition of films (time=0), a sample of the solution (0.1 ml) has been withdrawn, diluted in saline and cfu/ml have been counted on acidified malt extract (MEA) in order to determine the initial bacterial load of samples. After addition of films, counts have been monitored at defined time intervals until 200 hours. As in example 6, experiments have been carried out in duplicate, and means and standard deviations have been calculated. The results shown in figure 5 demonstrate that A. acidoterrestris spores can survive and grow in acidified MEB in presence of a control film or in a solution without film (version B and C), whereas the microbial population decreases in presence of the active film (with lysozyme). Figure 5 shows that the active film has a similar noxious effect on A. acidoterrestris spores in a fruit juice solution. In the same figure, it is noted that the effectiveness of the active film on microorganisms depends on the type of environment: Lysozyme acts rapidly in acidified MEB but more slowly in fruit juice.

Claims

I. Process for immobilization of lysozyme on a polymeric matrix containing hydroxyl groups, comprising the following steps: a) preparation of the polymeric matrix by addition of a reticulating agent to a polymer containing hydroxyl groups b) lysozyme activation by addition of a dialdehyde linker to an aqueous lysozyme solution c) combination of the solution obtained in step (b) with the reticulated matrix obtained in step (a).

2. Process according to claim 1 , wherein the polymeric matrix to be reticulated exposes alcoholic groups.

3. Process according to claim 2, wherein said exposed alcoholic groups are secondary.

4. Process according to claim 3, wherein the polymeric matrix polymer is polyvinil alcohol.

5. Process according to claims 1-4, wherein a homogenization follows step (a) and/or (b) and/or (c).

6. Process according to claims 1-5, wherein step (a) and/or (b) and/or (c) is carried out at pH lower than 5.

7. Process according to claim 6, wherein step (a) and/or (b) and/or (c) is carried out at a pH comprised between 1 and 2.

8. Process according to claims 1-7, wherein the starting concentration of polyvinil alcohol is comprised between 5% and 20% (w/v).

9. Process according to claim 8, wherein said concentration is comprised between 10% and 15% (w/v).

10. Process according to claim 9, wherein said concentration is comprised between 12% and 14% (w/v).

I I . Process according to claims 1-10, wherein in step (b) the molar ratio between linker and lysozyme is comprised from 1 to 50.

12. Process according to claim 11 , wherein said molar ratio is comprised from 2.5 to 35.

13. Process according to claim 12, wherein said molar ratio is comprised between 4 and 20.

14. Process according to claims 4-13, wherein the weight ratio between polyvinilalcohol and lysozyme is comprised between 0.5 and 250.

15. Process according to claim 14, wherein said ratio is comprised between 1 and 150.

16. Process according to claim 15, wherein said ratio is comprised between 5 and 15.

17. Process according to claims 1-16, comprising the following step: d) Exsiccation of the solution obtained in step (c) poured on a suitable support.

18. Process according to claim 17, wherein the exsiccation of step (d) lasts at least 24 hours.

19. Process according to claims 17-18, wherein the exsiccation of step (d) is carried out under vacuum.

20. Process according to claims 17-19, comprising a washing step in distilled water after step (d).

21. Process according to claims 17-20, wherein in step (b) the molar ratio between linker and lysozyme is comprised between 12 and 15.

22. Process according to claims 1-16, wherein the reticulated matrix obtained in step (a) is poured on a support and the addition of activated lysozyme obtained in step (b) according to step (c) is made by spraying.

23. Process according to claim 22, wherein the poured matrix is partially or totally exsiccated before the combination according to step (c).

24. Process according to claims 22-23, comprising a subsequent exsiccation step.

25. Process according to claim 24, wherein exsiccation is carried out under vacuum.

26. Process according to claims 22-25, wherein the molar ratio between linker and lysozyme in step (b) is comprised between 16 and 18.

27. Process according to claims 22-26, comprising a subsequent washing step in distilled water.

28. Process according to claims 1-27, wherein the reticulating agent and/or linker is a dialdehyde.

29. Process according to claim 28, wherein the reticulating agent and/or linker is glutaraldehyde.

30. Process according to claim 29, wherein the reticulating agent is glyoxal.

31. Process according to claims 1-16, wherein steps (a) and/or (b) and/or (c) occur simultaneously.

32. Process according to claim 31 , comprising the following further steps: d) exsiccation of the matrix for at least 24 hours, optionally under vacuum e) washing in distilled water.

33. Process according to claims 31-32, wherein the polymer used is polyvinilalcohol and glutaraldehyde is both the linker and the reticulating agent.

34. Matrix derivatized with lysozyme obtainable by the process according to claims 1-33.

35. Matrix derivatized with lysozyme obtainable by the process according to claims 22-27.

36. Matrix according to claim 35, wherein lysozyme is present on at least one side of the matrix.

37. Matrix according to claims 1-36, wherein the matrix is a film with thickness comprised between 10 and 200 micrometers.

38. Matrix according to claim 35, wherein said thickness is comprised between 50 and 150 micrometers.

39. Use of the product according to claims 34-38 for packaging and/or coating products.

40. Use of the product according to claim 39 for the coating and/or packing of foodstuffs.