This application claims the benefit of European Patent Application EP17382264.4 filed May 1 1 , 2017.

Technical field

The present invention relates to active packages for foodstuff, particularity for fresh meat, more particularly for fresh broiler meet.

Background art

In the last EU summary report on zoonoses, zoonotic agents and food-borne outbreaks issued by the European Food Safety Authority (EFSA) and the European Centre for Disease Prevention and Control (cf. "The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2014", EFSA Journal 2015; 13(12): 4329, 190 pp. doi:10.2903/j.efsa.2015.4329), it was found that in 2014 Campylobacter continued to be the most commonly reported gastrointestinal bacterial pathogen in humans in the European Union (EU). The number of reported confirmed cases of human campylobacteriosis was 236,851. However, the actual number of cases is believed to be around 9 million each year, according to EFSA.

The cost of campylobacteriosis to public health systems and to lost productivity in the EU is estimated by EFSA to be around EUR 2.4 billion a year.

Although broiler meat is considered to be the most important source of human campylobacteriosis (only in 2014, 38.4% of the 6,703 samples of fresh broiler meat were found to be Campylobacter positive), there are other foodstuffs that can contain Campylobacter. Campylobacter can live in the intestines of healthy animals and can be transferred to the meat at slaughter, cutting up and handling. Despite all the measures followed during these processes, Campylobacter is still found in fresh meat. A lot of effort has been devoted by consumer societies and regulatory agencies to provide guidelines and recommendation to combat Campylobacter, including pre- slaughter measures, meat production measures, at-home safe-handling, and cooking measures amongst others. However, further measures are needed to reduce the prevalence of Campylobacter and to mitigate the risk of food poisoning.

Therefore, despite the different measures allowing minimizing the incidence of campylobacteriosis, there is a need in the art to find new active packages providing higher antimicrobial potencies against Campylobacter, thus allowing reducing the contamination of packaged raw chicken meat by this microorganism.

Summary of the invention The present inventors, in an attempt for developing an improved active package, have found that the combination of a specific modified atmosphere (MA) and cinnamaldehyde contained in and release by a polymeric matrix provides an unexpected bactericidal effect on Campylobacter, particularly on Campylobacter jejuni, in a short period of time. This has an improved effect on the preservation of raw meat, particularly of raw chicken meat (e.g. broiler meat), and as a consequence on minimizing the incidence of campylobacteriosis.

Although it is known the antimicrobial activity of cinnamaldehyde against, amongst others, Campylobacter (cf. S. Ravishankar ei a/., "Plant-derived compounds inactivate antibiotic-resistant Campylobacter jejuni strains", J Food Prot., 2008, Vol. 71 , pp. 1 145-9) and also the effect of modified atmosphere packaging on

Campylobacter survival (cf. J. Byrd et al., "Effect of selected modified atmosphere packaging on Campylobacter survival in raw poultry", Poultry Science, 201 1 , vol. 90, pp. 1324-1328), the combined use of these two elements, particularly wherein cinnamaldehyde is incorporated in a packaging material used to manufacture an active package, has not been disclosed. Inventors have surprisingly found that an active package comprising cinnamaldehyde incorporated in a polymeric matrix and provided with a modified atmosphere with a high content of 0 ₂ , particularly an atmosphere comprising from 30 to 100% of 0 ₂ , has an improved bactericidal effect on Campylobacter and, as a consequence, on the preservation of raw chicken meat. As can be seen from the examples, this effect is higher than expected. Thus, the combination of cinnamaldehyde and a modified atmosphere with a high content of 0 ₂ provides a synergic effect on the preservation of raw meat susceptible of being contaminated by Campylobacter, particularly of raw poultry meat such as raw chicken meat. Particularly, as can be seen from the Examples,

Thus, a first aspect of the invention relates to the use of an active formulation comprising a polymeric matrix and cinnamaldehyde in combination with a modified atmosphere comprising from 30 to 100% of 0 ₂ for the eradication of Campylobacter in a foodstuff. The potency of this antimicrobial combination guarantees that smaller amounts of the antimicrobial compound can be used in active packaging materials, which ensures: i) the active packaging materials are more convenient in terms of overall manufacturing costs; ii) the accumulation of volatile compounds in the packaged meat is minimized; thus potential undesirable flavours are also minimized, as the effective antimicrobial concentration used in the films is lower due to the synergistic antimicrobial activity of the mentioned combination.

This aspect can also be formulated as the use of the antimicrobial combination of the active formulation and the modified atmosphere as defined above for the preservation of raw meat susceptible of being contaminated by Campylobacter, particularly of raw poultry meat such as raw chicken meat.

A second aspect of the invention relates to a package for the preservation of poultry meat comprising:

- an active formulation comprising a polymeric matrix and cinnamaldehyde and - a modified atmosphere comprising from 30 to 100% of 0 ₂ .

The use of such an active package allows reducing the time needed to eradicate Campylobacter jejuni, i.e. to kill the pathogen below the limit of detection of the culture method used in the examples (i.e. <0.48 log cfu gram ^"1 ). Particularly,

Campylobacter is eradicated from foodstuffs in 48 hours (or 2 days), mitigating the risk of consumers getting Campylobacteriosis and therefore reducing the prevalence of Campylobacter in humans. Thus, it also forms part of the invention a method for the preservation of raw meat, particularly of raw chicken meat such as raw broiler meat, comprising the packaging of the mentioned foodstuff with the active package defined above.

Brief description of drawings

Figure 1 shows some of the processes that can be used for manufacturing an active package in form of film, sheet or tray/container. Figure 2 shows the enumeration of Campylobacter jejuni in chicken breast fillets under the specific MA (Table 6) in combination with different compounds, the control and the reference. Detailed description of the Invention

All terms as used herein in this application, unless otherwise stated, shall be understood in their ordinary meaning as known in the art. Other more specific definitions terms as used in the present application are as set forth below and are intended to apply uniformly throughout the specification and claims unless an otherwise expressly set out definition provides a broader definition.

As used herein the term, "modified atmosphere (MA)" means an environment for food storage and transportation that has been modified from ambient conditions in terms of percentage of oxygen (0 ₂ ) and/or percentage of carbon dioxide (C0 ₂ ) or of nitrogen (N ₂ ).

The term "modified atmosphere packaging (MAP)" is a technology that substitutes the atmospheric air inside a package with a modified atmosphere.

As used herein, the term "active formulation" refers to a combination comprising the antimicrobial substance (i.e. cinnamaldehyde) and a polymer, the polymer forming a polymeric matrix wherein cinnamaldehyde is interspersed. The term "interspersed in a polymeric matrix", as used herein, means that the compound to which it relates (i.e. cinnamaldehyde) is at least in part distributed and scattered inside the polymeric matrix, so that it is released to the cavity where the packaged foodstuff is kept. The active formulation can comprise one or more additives such as plasticisers, and antifog compounds. The use of such an active package allows reducing the time needed to eradicate Campylobacter jejuni, i.e. to kill the pathogen below the limit of detection of the culture method used in the examples (i.e. <0.48 log cfu gram ^"1 ).

As used herein, the term "eradication" referred to Campylobacter, means that the microorganism is eliminated at least to a level below the limit of detection of the culture method used by the inventors. Particularly, in the culture method used by the inventors the limit of detection is 0.48 log cfu gram ^"1 . The method used is as follows: a sample of raw chicken meat (25 g) is homogenised with neutralizing broth (50 mL) and, then, an aliquot of the homogeneous broth (1 mL) is added to peptone water (9 mL, 0.1 % weight/volume). Subsequently, serial decimal dilutions are carried out by taking 1 mL of the previous dilution and 9 mL of peptone water. 50 microlitres of each dilution is plated on charcoal cefoperazone deoxycholate (CCD) agar using an automatic spiral plater. Then, the plates are placed into an anaerobic jar and are incubated under 10% C0 ₂ and 3-5% 0 ₂ at 42 °C for 48 hours. Finally, the plates are read after the incubation time to estimate the number of cfu per gram of chicken meat. Valid enumerations are considered when ranged between 30 - 300 colonies per plate.

The term "corona treatment" as used herein refers to a surface modification technique that uses low temperature corona discharge plasma to impart changes in the properties of a surface, in this case the surface of a packaging material.

The term "solvent suitable for food contact" as used herein refers to any solvent that can be used safely in the manufacture of plastics and other materials which come into contact with food for human consumption. The solvent must be harmless and devoid of any toxic properties.

The term "plasticiser" as used herein refers to a product that makes the polymer less brittle, enhancing the flexibility and mobility of the polymeric chain. A list of plasticisers suitable for carrying out the invention follows: phthalate-based

plasticizers, adipate-based plasticizers, benzoates, terephthalates, epoxidized vegetable oils, alkyl sulphonic acid phenyl ester (ASE), sulfonamides,

organophosphates and glycols/polyethers. Among these, preferred plasticisers are tert-butyl citrate, polyadipate or glycerol.

The term "antifog" as used herein refers to a chemical that prevents the condensation of water in the form of small droplets on a surface. A list of antifog agents suitable for carrying out the invention follows: non-ionic surfactants like polyhydric alcohol fatty acid esters, higher fatty acid amines, higher fatty acid amides, polyoxyethylene ethers of higher fatty alcohols, polyoxyethylene glycols of higher fatty acids and ethylene oxide adducts of higher fatty acid, amines, or amides and their admixtures are meant. Among these, preferred antifog agents are polyhydric alcohol fatty acid esters, especially sorbitan derivatives, polyoxyethylene glycols of higher fatty acids and glycerin fatty acid esters and their admixtures. As used herein, the term "wt%" or "percentage by weight" of a component refers to the amount of the single component relative to the total weight of the composition or, if specifically mentioned, of other component.

As stated above, a first aspect of the invention relates to the use of an active formulation comprising a polymer and cinnamaldehyde, wherein the polymer is forming a polymeric matrix wherein cinnamaldehyde is interspersed, in combination with a modified atmosphere comprising from 30 to 100% of 0 ₂ for the eradication of Campylobacter in a foodstuff, particularly in raw meat, more particularly in raw poultry meat. In a particular embodiment, the modified atmosphere comprises from 50 to 100% of 0 ₂ .

The polymeric matrix is capable of progressively delivering the cinnamaldehyde. The polymeric matrix acts as a delivery system limiting the rapid consumption of cinnamaldehyde or its loss due to the significant volatility of cinnamaldehyde. Thus, cinnamaldehyde is released in an amount suitable to exert the sought antibacterial activity in combination with the mentioned modified atmosphere. Thus, this combination of the active formulation and the modified atmosphere defined above can be used either in packaging of foodstuff susceptible of being contaminated by Campylobacter such as raw poultry meat or in a pre-treatment process of the foodstuff before being packaged. In a particular embodiment, optionally in combination with one or more features of the particular embodiments defined above, the amount of cinnamaldehyde in the active formulation is higher than 0.7 wt%, particularly higher than 4 wt%, more particularly from 4.3 to 10 wt% or from 4.3 to 7 wt%, with respect to the total amount of cinnamaldehyde and polymeric matrix. The amount of cinnamaldehyde in the active formulation is as_determined by thermogravimetric analysis, particularly by using a TGA Q5000IR (TA Instruments) wherein the sample is subjected to the following temperature profile: isothermal at room temperature for 1 minute, then ramping at 20°C/min to 900°C and finally isothermal at 900 °C for 3 min (see Analysis of the content of trans-cinnamaldehyde incorporated into the films section in the examples).

In a particular embodiment, optionally in combination with one or more features of the particular embodiments defined above, the polymeric matrix is made of a polymer selected from the group consisting of polyethylene terephthalate (PET), polyethylene (PE), ethylene vinyl alcohol (EVOH), polilactic acid (PLA), polystyrene (PS), ethylene vinyl acetate (EVA), polyamide, and polypropylene (PP), a polyamide (PA), a polycarbonate (PC), or polyvinyl chloride) (PVC). Particularly, the polymer is polyethylene, more particularly low density polyethylene (LDPE).

As commented above, another aspect relates to an active package for the preservation of foodstuff, particularly of raw meat, more particularly of raw poultry meat, and even more particularly raw chicken meat, comprising the active formulation and the modified atmosphere as defined above. Particularly, the modified

atmosphere comprises from 50 to 100% of 0 ₂ .

In a particular embodiment, the active package comprises the foodstuff to be preserved.

In a particular embodiment, optionally in combination with one or more features of the particular embodiments defined above or below, the amount of cinnamaldehyde in the package is from 0.02 to 2 milligrams for every gram of food stuff, particularly of raw meat.

In another particular embodiment of the package as defined above, optionally in combination with one or more features of the particular embodiments defined above, the active formulation is forming a layer having a thickness from 1 to 200 μηη. The active package of the invention can be of any type, either flexible, rigid, or both, and in any form or size, such as in the form of a bag, tray covered by a film, or container.

Manufacture of a cinnamaldehyde and MA active package

The active package can have several packaging structural compositions:

- Composition 1 . Based in one single layer (i.e. layer A) that contains

cinnamaldehyde incorporated into a polymer within the group of the thermoplastics (e.g. PE, PP, EVOH, EVA, PS, PLA, PA, PC, PET, or PVC). The thickness of the layer can be from 2 to 200 microns. - Composition 2. It corresponds to a multilayer structure where layer A as defined in composition 1 is combined with a layer B. Layer B can either be a monolayer or a multilayer material. Layer B as a monolayer can be made of a thermoplastic polymer (e.g. PET, PE, EVOH, PLA, PS, EVA, PP, PA, PC, or PVC) or another material such as paper or aluminium foil. Layer B as multilayer material can be made of whatever combination of the mentioned thermoplastic polymers and/or paper and/or aluminium. Layer A is in direct contact with the food or the medium surrounding the food.

Optionally, between layer A and B, an adhesive could be used to bond both substrates.

- Composition 3. It corresponds to a multilayer structure where layer A as described in composition 1 is between a layer B as described in composition 2 and a layer C. Layer C can either be a monolayer or multilayer material. Layer C as a monolayer can be made of a thermoplastic polymer (e.g. PE, PP, EVA, EVOH, PS, PLA, PA, PC, PET, or PVC) or another material such as paper. Layer B as a multilayer material is made of whatever combination of the mentioned thermoplastic polymers and/or paper. Layer C is in direct contact with the food or the medium surrounding the food. Optionally, between layers A and B, and A and C, an adhesive could be used to bond the substrates.

The active agent cinnamaldehyde can be incorporated to the polymeric matrix by a melting extrusion process or by a coating process in order to obtain a packaging material. This packaging material containing cinnamaldehyde can be used to obtain a final monolayer package (made of composition 1 ) or can take part of multilayer packaging structures as mentioned above (compositions 2 and 3). These packaging compositions can be transformed into a film, sheet or tray/container. To obtain the different active packaging compositions, several processes can be used (see Fig. 1 ).

The manufacture of the active package starts with a compounding process in order to incorporate cinnamaldehyde into a polymeric matrix. Optionally, cinnamaldehyde could be also directly incorporated into the polymeric matrix in the subsequent processes (i.e. by extrusion, co-extrusion or extrusion coating) by means of a feeder connected to the extrusion line when obtaining the film or sheet. Finally, another option to incorporate cinnamaldehyde into a polymeric matrix is by a coating process, wherein a coating composition comprising the thermoplastic polymer and

cinnamaldehyde is applied onto a substrate. The thickness of the layer of polymeric matrix containing cinnamaldehyde depends on the process used for the manufacture of the layer.

Thus, in a particular embodiment, optionally in combination with one or more features of the particular embodiments defined above or below, the package comprises an extruded layer, i.e. a layer obtained by incorporating cinnamaldehyde to a polymeric matrix by an extrusion process, wherein the thickness of the extruded layer is from 5 to 200 μηη, or from 10 to 125, and more particularly from 25 to 75 μηη. In another particular embodiment, optionally in combination with one or more features of the particular embodiments defined above or below, the package comprises a coating layer, i.e. a layer obtained by incorporating cinnamaldehyde to a polymeric matrix by a coating process, wherein the thickness of the coating layer in the packaging material as defined above is from 1 to 10 μηη, or 4 to 9 μηη, or 5 to 6 μηη, particularly 3 μηι.

Optionally, the manufacturing process goes on with a lamination processes (if needed to obtain more complex multilayer structures) and with a thermoforming process for the cases in which a tray/container is desired to be the final package.

During the preparation of the active formulation, i.e. the formulation comprising a polymeric matrix and cinnamaldehyde, part or the cinnamaldehyde dosed in the process can be lost by evaporation, particularly due to the temperature at which the different processes are carried out. Thus, the actual content of cinnamaldehyde in the final active formulation can be lower than the dosed cinnamaldehyde. As mentioned above and as shown in the example, the amount of cinnamaldehyde in the active formulation can be_determined by thermogravimetric analysis.

The following sections explain in more detail the processes referenced above:

1 . Compounding

The compounding process consists of preparing an active compound by blending a polymer or polymers in a molten state with the active ingredient (cinnamaldehyde in this case) and optionally other additives.

Compounding can be done by extrusion. The polymer and the cinnamaldehyde (and optionally other additives) are fed into the extruder through a hopper. The hopper connects with the beginning of a screw that transports the blend towards the end of the extruder. The screw can be confined in a barrel that has different zones that can be heated according to the properties of the polymer. The melted compound exits the extruder in strands, which are cooled and cut into pellets.

2. Extrusion to form a monolayer material

Pellets comprising cinnamaldehyde obtained by the abovementioned compounding process can be used in a film extrusion process. Alternatively, cinnamaldehyde could be directly incorporated in the polymer by means of a gravimetric feeder for liquids.

In this process, the material is gradually melted by the mechanical energy generated by turning screws and by heaters arranged along the barrel. The molten polymer and the additives are then forced into a flat die to obtain the active film. In this way, a monolayer film containing cinnamaldehyde is obtained as defined by composition 1.

3. Co-extrusion to form a multilayer material Pellets incorporating cinnamaldehyde obtained by the abovementioned compounding process are used into a co-extrusion process. Alternatively, cinnamaldehyde could be directly incorporated to the polymer in this process by means of a gravimetric feeder for liquids. Co-extrusion is the extrusion of multiple layers of material simultaneously; the process employs two or more extruders to melt and deliver a volumetric throughput of different materials to a single extrusion head or die. The result is a multilayer film or sheet as defined by compositions 2 and 3. 4. Extrusion coating to form a multilayer material

Pellets incorporating cinnamaldehyde by the abovementioned compounding process are used into an extrusion coating process. Alternatively, cinnamaldehyde could be directly incorporated to the polymer in this process by means of a gravimetric feeder for liquids.

In the extrusion coating process an extruder forces a melted blend of the polymer and the cinnamaldehyde through a horizontal slot-die onto a substrate (i.e. film or sheet). In a co-extrusion coating process, there are two or more extruders to melt and deliver a volumetric throughput of different materials to a single extrusion head or die onto the moving substrate (i.e. film or sheet). The resulting product is a permanently coated web structure as described in compositions 2 and 3.

5. Coating process

The coating process comprises the following steps:

a) preparing the coating composition by

providing a thermoplastic polymer, such as the ones mentioned above, and, optionally, dissolving it in at least one organic solvent, such as ethanol, isopropanol, or ethyl acetate, or in water, or in a mixture thereof; optionally, incorporating one or more additives (plasticisers, antifog compounds, etc.);

incorporating the active ingredient (i.e. cinnamaldehyde);

b) optionally, treating (such as with a corona treatment) the surface of the

substrate to be coated;

c) applying the coating composition to the substrate by casting, impression, dipping, or spraying; and

d) optionally, drying the coated substrate.

The resulting product is a permanently coated web structure as described in composition 2.

In addition, the packaging material obtained by a coating process may be subjected to an additional coating, extrusion coating or lamination process to obtain materials as defined by composition 3. 6. Lamination to form a multilayer material

The packaging material obtained by extrusion, co-extrusion, extrusion coating or coating can be incorporated to other films or sheets to manufacture a final multilayer structure as defined by compositions 2 and 3 by means of lamination processes. The lamination process is an operation where two or more layers of material are permanently joined together. Several lamination processes can be applied including hot roll or thermal lamination, extrusion coating, or adhesive lamination. Thermal lamination

In this process, one of the two materials of the laminate is melted onto the other material under increased temperature and pressure to secure a good bond.

Extrusion lamination

In this process a melted blend of polymer, cinnamaldehyde, and optionally and adhesive or bonding agent, is extruded or co-extruded between two substrates in the form of films or sheets without cinnamaldehyde. The resulting product is a permanently coated web structure as defined by composition 3.

In an alternative extrusion lamination process, a melted blend of polymer and optionally and adhesive or bonding agent is extruded or co-extruded between two substrates, one of the substrates being a film or sheet with cinnamaldehyde in it. The resulting product is a structure as defined by composition 2.

Adhesive lamination Unless one of the substrates has adhesive properties, in certain conditions an additional medium is necessary to secure the bonding between the layers. Wet lamination and dry lamination can be used.

7. Thermoforming to form an active tray

The sheets obtained through any of the abovementioned processes can be transformed into a 3D package such as a container, a tray or a cup by means of a thermoforming process. Thermoforming involves heating the sheets and forming it over a male or female mould. Thus, two basic types of thermoforming processes can be used: vacuum forming and pressure forming.

As can be understood by any skilled in the art, the MA is incorporated in the package during the packaging process, namely after packaging the foodstuff and before the package is sealed.

Throughout the description and claims the word "comprise" and variations of the word, are not intended to exclude other technical features, additives, components, or steps. Furthermore, the word "comprise" encompasses the case of "consisting of. The following examples and drawings are provided by way of illustration, and they are not intended to be limiting of the present invention. Furthermore, the present invention covers all possible combinations of particular and preferred embodiments described herein.

Examples

Materials

Low density polyethylene, LDPE, (ALCUDIA® PE017) from Repsol YPF.

Trans-cinnamaldehyde (CAS number 14371 -10-9), 99% purity from Sigma-Aldrich Co. LLC. Thymol (CAS number 89-83-8),≥99% purity from Sigma-Aldrich Co. LLC. Carvacrol (CAS number 499-75-2),≥98% purity from Sigma-Aldrich Co. LLC.

Example 1. Preparation of an active formulation

Active packaging films were obtained by extrusion processes using a twin screw extruder (DSE 20-40D, Brabender, Germany), equipped with a flat sheet die, obtaining samples of approximately 10 cm wide and 50 μηη thickness. The temperature profile from the first barrel zone to the die of the extruder was set at 170- 175-180-185-190-195°C working at a screw speed of 90 rpm.

Trans-cinnamaldehyde was dosed at 7 wt% versus polyethylene by means of a gravimetric feeder.

The actual content of trans-cinnamaldehyde incorporated to the LDPE films, i.e. the amount of trans-cinnamaldehyde in the active formulation, was determined by thermogravimetric analysis (TGA) using a TGA Q5000IR (TA Instruments). To this end, about 8 mg of the active film was weighted and put into a pan. The sample is then subjected to the following temperature profile; isothermal at room temperature for 1 minute, then ramping at 20°C/min to 900°C and finally isothermal at 900 °C for 3 min. The actual content of cinnamaldehyde incorporated to the active films was of a 4.3 wt%. Example 2. Antimicrobial activity of packages comprising different MAs, and an active formulation (cinnamaldehyde in a polymeric matrix)

The antimicrobial activity in chicken breast inoculated with Campylobacter of films containing cinnamaldehyde in combination with different modified atmosphere (MA) compositions was evaluated. In addition, the antimicrobial activity in chicken breasts inoculated with Campylobacter of packages comprising different MAs (and without cinnamaldehyde) was evaluated. In both cases, seven different modified

atmospheres were evaluated.

Table 1 shows the specific modified atmospheres tested.

Table 1. Modified atmospheres (MA)

- Inoculation of chicken breasts

Samples of 25 grams of chicken breast were inoculated with 0.25 mL of a suspension containing Campylobacter jejuni at a concentration of approximately 10 ⁵ colony forming units (cfu) mL ^"1 . Thus, each chicken breast was inoculated at a concentration of 10 ³ cfu gram ^"1 .

- Procedure followed for the analysis of Campylobacter jejuni in the chicken breasts

The 25 grams of chicken breast was taken from each package and it was

homogenised with 50 mL of neutralizing broth in a Stomacher® homogenizer for 1 minute. Then, 1 mL of the homogeneous broth was added to 9 mL of peptone water (0.1 % weight/volume). After this, serial decimal dilutions were carried out by taking 1 mL of the previous dilution and 9 mL of peptone water. 50 microlitres of each dilution was plated on charcoal cefoperazone deoxycholate (CCD) agar using an automatic spiral plater (easySpiral Pro®, Interscience). Then, the plates were placed into an anaerobic jar and were incubated under 10% C0 ₂ and 3-5% 0 ₂ at 42 °C for 48 hours. Finally, the plates were read after the incubation time to estimate the number of cfu per gram of chicken breast. Valid enumerations were considered when ranged between 30 - 300 colonies per plate.

- Antimicrobial effectiveness of packages containing different contents of modified atmospheres (and without trans-cinnamaldehyde)

First, the antimicrobial effectiveness of different modified atmospheres in chicken breasts inoculated with Campylobacter jejuni was evaluated.

The inoculated chicken breast was packed under each specific MA (Table 1 ) into a PP/EVOH/PP tray (dimensions 170 x 130 x 35 mm. Three packages were packed for each MA condition and day of analysis.

The enumeration of Campylobacter jejuni in chicken breast along the first three days of storage gave the following results (see Table 2):

Table 2. Enumeration of Campylobacter jejuni in chicken breast fillets under MA

- Antimicrobial effectiveness of packages containing different contents of modified atmospheres and an active film containing trans-cinnamaldehyde

The inoculated chicken breast was packed under each specific MA (Table 1 ) into a PP/EVOH/PP tray (dimensions 170 x 130 x 35 mm). A piece of the active film (dimensions 8 x 12 cm and thickness of 50 microns) of PE + 7 wt% dosed cinnamaldehyde (actual amount of cinnamaldehyde incorporated in the active film: 4.3 wt%) as described in Example 1 was attached to a film of PA PP (60 microns thickness) that was thermosealed to the tray using a packing machine SMART 300 (ULMA). The film of PE and cinnamaldehyde was placed in direct contact with the inner headspace of the package in order to release the active agent in vapour phase.

Three packages were packed for each MA condition and day of analysis.

Table 3 below shows the antimicrobial effectiveness in chicken breasts inoculated with Campylobacter jejuni of films containing 7 wt% of dosed cinnamaldehyde (actual content 4.3 wt%) in combination with different modified atmospheres. The

enumeration of Campylobacter jejuni in chicken breast along the first 3 days of storage gave the following results: Table 3. Enumeration of Campylobacter jejuni in chicken breast fillets under MA and active film

The use of the active materials containing cinnamaldehyde increases the

effectiveness of the MA on Campylobacter jejuni in all the cases (Table 3). There are some specific MA compositions that interact with the active film enhancing the antimicrobial effect of the combined system thus reducing the time needed to kill Campylobacter jejuni below the limit of detection of the technique (< 0.48 log cfu gram ^"1 ). This is the case of MA3, MA4 and MA5 at day 2 after packing and MA6 at day 3 after packing.

The common factor among these systems is that the presence of oxygen in the MA is higher than 30%. Therefore, the combination of a specific oxygen containing MA and the active film of cinnamaldehyde enhances the antimicrobial effect against Campylobacter jejuni in comparison to other MA compositions using less than 30% of oxygen.

Particularly, MA compositions in terms of oxygen, carbon dioxide, and nitrogen ranged as follows:

Table 4. MA compositions combined with films containing trans-cinnamaldehyde providing the best results The results show that an active package comprising cinnamaldehyde incorporated in a polymeric matrix in an amount of 4.3 wt% and provided with a modified atmosphere with a high content of 0 ₂ , particularly an atmosphere comprising from 30 to 100% of 0 ₂ , has an improved bactericidal effect on Campylobacter and, as a consequence, on the preservation of raw chicken meat. As can be seen from the obtained results, this effect is higher than expected.

Example 3. Preparation of an active formulation

Active packaging films were prepared following the process of Examplel , but for the dosing of a 12 wt% of trans-cinnamaldehyde instead of a 7 wt%.

The actual content of trans-cinnamaldehyde incorporated to the active films, determined by TGA as disclosed in Example 1 , was of a 6.9 wt%. Comparative Example 1 - Preparation of comparative formulations

Comparative packaging films were prepared following the process of Examplel , but for the dosing of thymol or carvacrol instead of trans-cinnamaldehyde. Thymol and carvacrol were dosed at the wt% indicated in Table 5 (2 ^nd column). The actual content of thymol or carvacrol incorporated to the LDPE films, determined by TGA as disclosed in Example 1 , is indicated in Table 5 (3 ^rd column).

Table 5. wt. % of comparative compounds in the comparative films

Compound | Dosed amount (wt.%) | Actual content (wt. %) Comparative film 1 Thymol 10 3.0

Comparative film 2 Carvacrol 12 8.5

Preparation of reference formulation

A PE film as a "reference sample" was also obtained by the extrusion process described above, but without the addition of cinnamaldehyde nor comparative compounds.

Comparative Example 2. Antimicrobial activity of packages comprising a 50/50 Op/CO? MA and active formulation of Example 3, comparative formulations, reference formulation, or control

The antimicrobial activity in chicken breast inoculated with Campylobacter of films containing active formulation of Example 3 and formulations of Comparative Example 2 in combination with a modified atmosphere (MA) of 50/50 0 ₂ /C0 ₂ was evaluated. In addition, the antimicrobial activity in chicken breasts inoculated with Campylobacter of packages comprising a MA of 50/50 0 ₂ /C0 ₂ (without cinnamaldehyde, thymol or carvacrol) was also evaluated. A "control sample" (package without the PE film and without cinnamaldehyde, thymol or carvacrol) and a "reference sample" (package with the PE film but without cinnamaldehyde or comparative compounds) were prepared.

The procedure followed for the inoculation of the chicken breasts and the analysis of Campylobacter jejuni in the chicken breasts was as disclosed in Example 2 - Antimicrobial effectiveness of packages containing a MA of 50/50 Q?/CO? either alone or with an active packaging films comprising cinnamaldehyde, thymol, carvacrol, the control, the reference

The antimicrobial effectiveness of cinnamaldehyde, thymol or carvacrol in

combination with a MA of 50/50 0 _? /CO _? in chicken breasts inoculated with

Campylobacter jejuni was evaluated. The samples "control" and "reference", which are also under the MA but do not contain cinamaldehyde, thymol or carvacrol, were also evaluated in the same way.

The inoculated chicken breasts were packed under the specific MA into a

PP/EVOH/PP tray (dimensions 170 x 130 x 35 mm). - Packaging with Cinnamaldehyde (active formulation from Example 3)

A piece of the active film (dimensions 8 x 12 cm and thickness of 50 microns) of PE + 12wt% dosed cinnamaldehyde (actual amount of cinnamaldehyde incorporated in the active film: 6.9 wt%) as obtained in Example 3 was attached to a film of PA PP (60 microns thickness) that was thermosealed to the tray using a packing machine SMART 300 (ULMA). The film of PE and cinnamaldehyde was placed in direct contact with the inner headspace of the package in order to release the active agent in vapour phase.

- Packaging with thymol (comparative film 1 from Comparative Example 1 )

A piece of the comparative film 1 (dimensions 8 x 12 cm and thickness of 50 microns) of PE + 10 wt% dosed thymol (actual amount of thymol incorporated in the film: 3.0 wt%) as obtained in Comparative Example 1 was attached to a film of PA PP (60 microns thickness) that was thermosealed to the tray using a packing machine SMART 300 (ULMA). The film of PE and thymol was placed in direct contact with the inner headspace of the package in order to release the thymol in vapour phase.

- Packaging with carvacrol (comparative film 2 from Comparative Example 1 )

A piece of the comparative film 2 (dimensions 8 x 12 cm and thickness of 50 microns) of PE + 12 wt% dosed carvacrol (actual amount of carvacrol incorporated in the film: 8.5 wt%) as obtained in comparative example 1 was attached to a film of PA/PP (60 microns thickness) that was thermosealed to the tray using a packing machine SMART 300 (ULMA). The film of PE and carvacrol was placed in direct contact with the inner headspace of the package in order to release the carvacrol in vapour phase.

- Packaging with the control

A film of PA/PP (60 microns thickness) was thermosealed to the tray using a packing machine SMART 300 (ULMA).

- Packaging with the reference (reference formulation) A piece of the reference film (dimensions 8 x 12 cm and thickness of 50 microns) of PE as described above was attached to a film of PA PP (60 microns thickness) that was thermosealed to the tray using a packing machine SMART 300 (ULMA). The film of PE was placed in direct contact with the inner headspace of the package.

Three packages were packed for each formulation and day of analysis.

The enumeration of Campylobacter jejuni in chicken breast along the first, third, and fifth day of storage gave the following results (see Table 6 and Figure 2):

Table 6.

As can be seen in the results, at day 3 and 5, the enumeration of Campylobacter jenuni in chicken packaged in active packages whose film contained thymol or carvacrol was equivalent to that of the control and the reference samples. Thus, showing that carvacrol and thymol do not enhance the antibacterial activity of packages with a MA having a high content of 0 ₂ .

The sample with cinnamaldehyde, in contrast, showed less Campylobacter than the control and the reference at day 3 and no detectable Campylobacter at day 5.

Therefore, these results provide evidence that not all the compounds that have a proven activity against Campylobacter see their activity enhanced when combined in a package with a MA having a high content of 0 ₂ . Citation List

Non Patent Literature

1 . The European Union summary report on trends and sources of zoonoses, zoonotic agents and food-borne outbreaks in 2014", EFSA Journal 2015; 13(12): 4329, 190 pp. doi: 10.2903/j.efsa.2015.4329.

2. S. Ravishankar et al, "Plant-derived compounds inactivate antibiotic-resistant Campylobacter jejuni strains", J Food P rot., 2008, Vol. 71 , pp. 1 145-9.

3. J. Byrd et al., "Effect of selected modified atmosphere packaging on

Campylobacter survival in raw poultry", Poultry Science, 201 1 , vol. 90, pp. 1324- 1328.