WO2022027152A1

WO2022027152A1 - Polymer coating comprising carvacrol as a natural volatile antimicrobial active agent carried in a polymer vehicle dissolved in organic volatile solvent, and flexible polymer film internally comprising the coating, useful for extending the shelf life of meat products

Info

Publication number: WO2022027152A1
Application number: PCT/CL2021/050074
Authority: WO
Inventors: Francisco Javier RODRÍGUEZ MERCADO; María José Alicia GALOTTO LÓPEZ; Abel GUARDA MORAGA; Julio Elías BRUNA BUGUEÑO; Verónica Alejandra GARCÍA MENA
Original assignee: Universidad De Santiago De Chile
Priority date: 2020-08-06
Filing date: 2021-08-05
Publication date: 2022-02-10
Also published as: CL2020002046A1

Abstract

The present invention relates to a flexible polymer film with antimicrobial properties, which allows the shelf life of packaged meat products to be extended. The film comprises a substrate selected from polystyrene, polylactic acid, polyethylene vinyl alcohol, polyamides, polyolefins, celluloses, waxes, paraffins or a combination of same, preferably single-layer or multilayer low-density polyethylene (LDPE), with an internal coating comprising a polymer vehicle dissolved in volatile organic solvent that carries a natural volatile antimicrobial active agent. According to the invention, the polymer vehicle dissolved in volatile organic solvent is preferably selected from a synthetic or natural varnish, preferably selected from a heat-seal lacquer for dairy products; the natural volatile antimicrobial active agent is carvacrol; the polymer vehicle dissolved in volatile organic solvent is dispersed on the internal faces of the substrate or surface that makes contact with the food; and the meat product is selected from chopped or sliced chicken, pork or turkey meat.

Description

POLYMERIC COATING INCLUDING CARVACROL AS A NATURAL VOLATILE ANTIMICROBIAL ACTIVE AGENT CARRIED IN A POLYMERIC VEHICLE DISSOLVED IN VOLATILE ORGANIC SOLVENT AND A FLEXIBLE POLYMERIC FILM INTERNALLY INCLUDING SAID COATING, USEFUL TO PROLONG THE SHELF LIFE OF MEAT PRODUCTS.

FIELD OF THE INVENTION

The present invention is related to the transforming industry of polymeric containers for food. In particular, it refers to a flexible polymeric film with antimicrobial properties that allows the shelf life of packaged meat products to be extended, and comprises a substrate selected from polystyrene, polylactic acid, polyethylene-vinyl-alcohol, polyamides, polyolefins, celluloses, waxes, paraffins. or a combination thereof, preferably, monolayer or multilayer low-density polyethylene (LDPE), with an inner coating that comprises a polymeric vehicle dissolved in a volatile organic solvent that carries a natural antimicrobial volatile active agent, where said polymeric vehicle dissolved in volatile organic solvent is preferably selected from a synthetic or natural varnish, preferably selected from a heat-sealing lacquer for dairy products, and where said natural antimicrobial volatile active agent is a volatile essential oil, specifically, it is carvacrol, and where said dissolved polymeric vehicle in volatile organic solvent dispersed on the internal faces of the substrate or surface that contacts the food, and said meat product is selected from chopped or cut chicken, pork or turkey meat.

BACKGROUND

An active agent with antimicrobial capacity has the ability to control the growth of microorganisms that cause the deterioration of food products. The mechanism that dominates the food preservation process depends on the physicochemical characteristics of the active agent (food surface contact and container head space). In flexible polymeric food packaging, the main mechanism associated with the action of a volatile active agent is the headspace of the packaging.

Among the patent documents related to the invention, W02015107089A1 (Technological Institute of Packaging, Transport and Logistics ITENE) can be mentioned, which reveals an antimicrobial composition for packaging organic products, comprising the combination of carvacrol, thymol and salicylaldehyde and/or other components and /or excipients with antimicrobial activity; or thymol and salicylaldehyde and/or other components and/or excipients with antimicrobial activity; or carvacrol and salicylaldehyde and/or other components and/or excipients with antimicrobial activity, the concentration of cavacrol being 5-50%; thymol 3-20%; and salicylaldehyde 28-90% by weight based on the total concentration and the organic product being a food. The active formulation for packaging organic product comprises a polymeric matrix and the previous antimicrobial composition, where the polymeric matrix comprises polystyrene, polylactic acid, polyethylene-vinyl-alcohol, polyamides, polyolefins, celluloses, waxes, paraffins or a combination thereof, being the antimicrobial composition interdispersed in the polymeric matrix or is an active cover for the food packaging, the food packaging being a tray, film, bag, pad, among others. A process for preparing said composition is also disclosed, comprising dissolving the polymeric matrix in at least one solvent; optionally incorporate at least one plasticizer and/or an anti-fog compound; and incorporate the antimicrobial composition described above, which may be the plasticizer tert-butyl citrate, polyadipate or glycerol, and antifog lauryl compound, which may be sodium sulfate, glycerol or an ethoxy-amine. The surface of the organic product packaging can be covered with the active composition with a corona treatment; or be applied by coating, printing, dipping or spraying and drying, the coating may be applied by roller printing, flexography, inkjet printing or rotogravure. The composition is deposited on the surface of the substrate totally or partially, it extends the useful life of the product susceptible to being colonized by microbes, the product being able to be a meat-based product.

CL3698-2015 (Universidad de Santiago de Chile) discloses a degradable film for packaging fruit and vegetables that comprises a polymeric matrix based on polyolefin, which incorporates an antimicrobial active agent (biocide or fungicide) of essential oil or said essential oil , and further incorporates degrading agents, and microencapsulation process of said antimicrobial active agent of essential oil, and method of preparation of the film, where said essential oil is selected from the group consisting of carvacrol, cinnaldehyde, cineol, sabinene, thujaplicin or a mixture thereof or incorporates said essential oil selected from the group consisting of: cinnamon oil, oregano oil, eucalyptus oil, nutmeg oil, honokitiol oil or a mixture thereof, and where said polyolefin-based polymeric matrix is selected from the group consisting of polyethylene (PE), polypropylene (PP), polystyrene (PS) and ethyl vinyl acetate (EVA), and where said polymeric matrix also incorporates a degrading agent selected from calcium nanocarbonate, calcium carbonate, starch, cellulose or a mixture thereof, and where the calcium nanocarbonate is also a reinforcing agent, where said essential oil microbial active agent or said essential oil is microencapsulated; process for microencapsulating an essential oil antimicrobial active agent; and process for preparing film.

W02001049121A (Meat product packaging) discloses a meat product container comprising a sheet substrate with a beneficial substance attached, where the substrate of sheet is a shrinkable or clingable plastic wrap film, including the use of a bonding agent. The beneficial substance is an antimicrobial agent, preferably against mold or bacteria. The antimicrobial agent can be a natural antimicrobial agent, preferably an essential oil, which can be selected from linalool, tugineol, eugenol, thymol, citral or carvacrol. The antimicrobial agent may also be an ester of 4-hydroxybenzoic acid or a compound capable of releasing an antimicrobial gas. The antimicrobial agent may be a coating on the sheet. The binding agent may be a lacquer, preferably an acrylic lacquer, or may comprise a polymer, preferably a hydrophilic or lipophilic polymer, where the polymer swells on contact with a release agent, thereby releasing the beneficial substance. The foil may be in contact with the meat product.

W02016140781A (Dow Global Technologies LLC) discloses a packaging material, comprising: a substrate and an antimicrobial composition comprising an antimicrobial active agent and a vehicle, said composition being a hydrogel at temperatures between 2°C and 12°C, and the carrier can be water and at least one polymer of cellulose ether, gelatin, pectin, xanthan gum, guar gum and combinations thereof, in particular it can be water and methylcellulose or water and gelatin. The antimicrobial agent comprises at least one amino acid derivative, an organic acid, a peptide, a quaternary ammonium salt, an amino acid derivative, or combinations thereof, in particular, is selected from cetylpyridinium chloride, lauric arginate, and dimethyloctadecyl[3-(trimethoxysilyl)propyl]ammonium chloride. Said antimicrobial agent may also comprise a bacteriophage and at least one additional antimicrobial agent, and further optionally an antioxidant, a surfactant, a stabilizer, a buffer, a scavenger, and combinations thereof. The substrate can be a polymeric film. The antimicrobial composition is applied to the surface of the substrate prior to packaging, preferably the interior surface of the substrate. The antimicrobial composition is in contact with the product, preferably a meat product.

ES2639914T3 (Universidad de Santiago de Chile) discloses a process for obtaining a film that includes the incorporation of antimicrobial agents of natural origin in a polymeric structure through a double extrusion process to the polymeric material, useful in the development of containers intended for to increase the useful life of refrigerated meat, preferably fresh refrigerated salmon, where said process comprises the following stages: (a) mixing the antimicrobial active agent with powdered low-density polyethylene in a first extrusion, to obtain a pellet, (b ) carry out a second extrusion to obtain a film incorporating the pellet obtained in stage (a) in a proportion of 10% on pelletized polyethylene, (c) carry out a three-layer coextrusion for the development of the film on which the agent was incorporated antimicrobial, wherein the active antimicrobial agent is incorporated into the layer of the film that is in direct contact with the salmon, where the intermediate layer and the outer layer provide the structure requirements of the film, without the incorporation of the active agent.

CN109688834A (Univ, of Guelph) discloses a volatile compound controlled release composition comprising at least one poly(ethylene glycol) (PEG) polymer and at least one or more volatile compounds selected from a combination of antimicrobials, and further optionally comprising acid polylactide (PLA). The volatile compound may be present in a percentage of about 0.01% w/w to about 50% w/w, and is selected from allyl isothiocyanate (AITC), diacetyl, cinnamic acid, thymol, carvacrol and their combinations, preferably a mixture of diacetyl and AITC from 10:1 to 1:1, or a mixture of diacetyl, AITC and cinnamic acid in a 1:4:60 ratio. The volatile compounds are arranged in a vehicle, which can be a fiber spun with electricity . The composition comprises a mixture of polylactic acid (PLA) and poly(epoxy secondary alkane) (PEO), in a PLA to PEO ratio of 7:3. The composition further includes cellulose, which can be selected from ethylcellulose and cellulose acetate. The composition is useful for preserving stored foods selected from fruit and vegetable waters, bakery, bakery, fresh pasta and fresh meat.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 Antimicrobial activity of each compound against Escherichia coli after 24 h of incubation at 35°C. Control A: without compounds, Control B: with 95% ethanol, TX-18: allyl isothiocyanate, TX-09: carvacrol, TX-07: eugenol, TO-08: rosemary extract, TX-08: 2-nonanone, TX -14: cinnamaldehyde, TX-06: thymol, TO-07: clove extract.

Figure 2 Antimicrobial activity of each compound against Staphylococcus aureus after 24 h of incubation at 35°C. TO-08: rosemary extract, TX-18: allyl isothiocyanate, TX-06: thymol, TX-14: cinnamaldehyde, TO-07: : clove extract, TX-09: carvacrol, TX-08: 2- nonanone, TX-07: eugenol.

Figure 3 Comparative analysis of the in vitro antimicrobial capacity of naturally occurring compounds for Escherichia coli and Staphylococcus aureus.

Figures 4A-4G Non-volatile compound C-2 MIC images against Escherichia coli and Staphylococcus aureus. Figure 4A (control, Escherichia coli), Figure 4B (control, Staphylococcus aureus), Figure 4C (10 ppm, Escherichia coli), Figure 4D (10 ppm, Staphylococcus aureus), Figure 4E (20 ppm, Escherichia coli), Figure 4F (20 ppm, Staphylococcus aureus), Figure 4G (30 ppm, Escherichia coli), Figure 4H (30 ppm, Staphylococcus aureus), Figure 4F (40 ppm, Escherichia coli), and Figure 4G (40 ppm, Staphylococcus aureus)

Figure 5 RAM microbiological results expressed in CFU/g.

Figure 6 Graph of useful life test follow-up with modifications. Figure 7 Cross-sectional photograph of LDPE film/dairy product heat sealant lacquer/TX-09 (carvacrol)

Figure 8 Evolution analysis of microbiological quality of chicken samples packaged in active system under refrigeration conditions (4°C). Black: LDPE control; Dark Grey: LDPE/BLSC and Light Grey: LDPE/BLSC/TX-09

Figure 9. Antimicrobial effect of active films based on BLSC and volatile active agent. Figure 9A (4%, Escherichia coli), Figure 9B (4%, Staphylococcus aureus), Figure 8C (6%, Escherichia coli), Figure 9D (6%, Staphylococcus aureus), Figure 9E (8%, Escherichia coli), Figure 4F (8%, Staphylococcus aureus).

DETAILED DESCRIPTION OF THE INVENTION

The present flexible polymeric film with antimicrobial properties that allows to extend the useful life of packaged meat products, and comprises a substrate selected from polystyrene, polylactic acid, polyethylene-vinyl-alcohol, polyamides, polyolefins, cellulose, waxes, paraffins or a combination of the same, preferably, low-density polyethylene (LDPE), monolayer or multilayer, with an inner coating that comprises a polymeric vehicle dissolved in a volatile organic solvent that carries a natural antimicrobial volatile active agent, where said polymeric vehicle dissolved in a volatile solvent is selected of a synthetic or natural varnish, preferably selected from a heat sealing lacquer for dairy products, and where said natural antimicrobial volatile active agent is selected from a volatile essential oil, specifically, carvacrol, and where said polymeric vehicle dissolved in a volatile solvent is scattered on the inner faces of the su stratum or surface that contacts the food, and said meat product is selected from chopped or cut chicken, pork or turkey meat. In this way, said natural antimicrobial volatile active agent interacts both with the surface of the food and in the environment where it is beneficially maintained in the headspace of the package. The coating is applied by means of a standard method of surface treatment of the substrate ("coating"), preferably selected from lamination or surface printing, and in this way, it does not require special implementations by the packaging transforming industry, it can be applied without modifying its processes. classic industrial. The present active packaging showed, in in vivo tests, to extend the shelf life of packaged chicken meat, under traditional refrigeration conditions for this type of food, that is, 4°C.

The active agent (carvacrol) is found in the synthetic varnish (BLSC) in a concentration of 2% to 10% p/p, preferably in concentrations of 2%, 4% and 6% p/p Volatile active agents of both natural origin and synthetic active agents recognized for their antimicrobial capacity were tested. The agent TX-09 (carvacrol) demonstrated a greater antibacterial capacity against bacteria of different types, under in vitro conditions, and compared to a commercial preservative of synthetic origin (C-2, Ethyl lauryl arginate, LAE).

The active agents TX-09 and C-2 were used to functionalize a commercial film of low-density polyethylene (LDPE), which is the type of material that is preferably used in the packaging of chicken meat. The incorporation of the active agents was carried out through the coating technique, and using a vehicle selected from a varnish type heat sealing lacquer (BLSC) for dairy products, preferably, and an acrylic resin (AR), preferably, for the dispersion and fixation of the active agent on the LDPE substrate, each varnish having a particular chemical affinity for the active agent it carries. Thus, TX-09 was used with a lacquer-type varnish, while acrylic resin was used for C-2.

BLSC/TX-09 and RA/C-2 films showed in vitro antibacterial activity against model microorganisms (E. coli and S. aeurus). Figure 7 shows a side section of the LDPE/BLSC/TX-09 film where it is possible to see the presence of the varnish on the LDPE substrate. Due to the excellent antibacterial behavior, its behavior was evaluated in in vivo tests. For this, chicken samples were packed in containers coated inside with both active films. For the evaluation of the antimicrobial capacity under in vivo conditions, a safety limit was considered for a mesophilic aerobic count (RAM) of 1 x10 ⁷ CFU/g of food. This indicator was used to determine the microbiological shelf life of packaged chicken samples. All the tests were carried out under refrigeration conditions (4°C) for at least 10 days in containers made from the developed materials and the respective controls. It should be noted that, in the case of the active material, LDPE/RA/C-2 coating, it was not possible to detect an effect on the shelf life of packaged chicken, since the molecular weight of the active agent hindered migration from the film to the food. . On the contrary, the containers made with the active film LDPE/BLSC/TX-09 showed a clear effect of the active agent in the reduction of AMR during the storage time. Figure 8 shows the evolution of AMR during 10 days for chicken samples stored at 4°C in control containers (LDPE and LDPE/BLSC) and in the active container made with LDPE/BLSC/TX-09. As Figure 8 shows, it is possible to establish that the active packaging allows the shelf life of the packaged samples to be increased by 2 days, going from a shelf life of 6 days for the control packaging to 8 days for the active package. Without being limited to a theory, the generation of antimicrobial activity could be given by the nature of the active compound, which is characterized by volatilizing, a situation that would favor its action in the head space of the container, thus being able to act on a large part of the surface of the product.

The results showed that the film of the invention increases the shelf life of packaged chicken by 33% and that it differs from known antimicrobial films and packages and many proposed solutions that show antimicrobial activity in in vitro tests but that against a food do not show effectiveness, as happened with the LDPE/RA/C-2 film.

Example 1: Efficacy of the natural volatile antimicrobial active agent

The antimicrobial activity of 8 natural volatile antimicrobial active agents allowed as substances or additives to be used in food contact packaging was tested. In vitro radial inhibition against Escherichia coli and Staphylococcus aureus was tested in triplicate by the agar diffusion method (Elgayyar, M., Draughon, F., Goldem, D. and Mount, J. (2001). Antimicrobial Activity of Essential Oils from Plants against Selected Pathogenic and Saprophytic Microorganisms. Journal of Food Protection 64: 1019-1024). Specifically, at the base of a 9 cm diameter Petri dish, 30 mL of sterile Tryptein Soy Agar (TSA) was poured. Once the agar gelled, 100 pL of saturated culture of each microorganism was seeded, in cell concentrations that fluctuate between 1 x 10 ⁸ to 1 x 10 ⁹ (CFU/mL). Subsequently, it is homogenized throughout the surface of the agar by raking. Then, a 6 mm diameter filter paper disc is adhered to which 5 pL of each natural volatile antimicrobial active agent is added, in order to allow its diffusion in the agar. All this procedure was performed in a NuAire NU laminar flow hood, model 425-400E, Massachusetts, USA. Finally, the plates were incubated for 24 h at 35°C, conditions considered optimal for bacterial growth. Fulfilling the incubation time, the inhibition halo associated with each natural volatile antimicrobial active agent in which the halos were present was measured, their diameters were measured in sextuplicate to subsequently calculate a final inhibition area. In this way, the diffusion capacity of the natural volatile antimicrobial active agent through the agar, the release effect of the same compound and the activity against the microorganism were measured (Figure 1 and 2).

Example 2: Minimum Inhibitory Concentration of the control Ethyl Lauryl Arginate (LAE, C-2)

The minimum inhibitory concentration (MIC) defined as the minimum concentration of active compound at which growth inhibition is observed and the minimum bactericidal concentration (MBC), where there is no growth of microorganisms, were tested. The MIC and CMB of the non-volatile compound were determined in TSB liquid medium against two microorganisms: S. aureus as a model of Gram-positive bacteria and E. coli as a model of Gram-negative bacteria. For this, a stock solution of 1000 ppm in Milli-Q water was initially prepared. Subsequently, serial dilutions were made in sterile tubes with 10 mL of TSB, obtaining a range of concentrations between 10-40 ppm. Sterile Milli Q water was used as a control. On the other hand, 100 pL of a miniculture were taken and inoculated in 10 mL of previously tempered TSB, vigorously shaken and introduced into a 37°C stirring bath to bring the microorganism to the exponential phase of growth. Using a visible UV spectrophotometer, the absorbance was measured at 595 nm until reaching a value of 0.2, corresponding to the exponential phase of growth (10 ⁵ CFU/mL). Once the exponential phase is reached, 100 pL of each microorganism are inoculated into the tubes previously prepared with the different concentrations of active. The tubes are incubated in an oven at 37°C for 24 h. Turbidity is an indicator of microbial growth and depending on it, different dilutions were made, 100 pL of the last cloudy tube and the first transparent one were seeded in TSA Petri dishes. They were incubated for 24 h at 37°C in an oven and the colony-forming units (CFU/mL) were counted. All experiments are performed in triplicate. Table 1 and Table 2 show the results obtained for E. coli and S. aureus.

Table 1 Inhibition of the non-volatile compound against S. aureus

Table 2 Inhibition of the non-volatile compound against E. coli

The results show a high antimicrobial action of the non-volatile commercial active compound C-2, both for Gram-positive and Gram-negative bacteria. So the concentration of 10 ppm is capable of inhibiting 5 logarithmic cycles of S. aureus with respect to the control, while higher concentrations manage to completely inhibit its growth. In the case of E. coli, a slightly lower power is observed, but quite acceptable, since the minimum concentration reduces almost 5 logarithmic cycles with respect to the control, achieving total inhibition at 40 ppm. Figure 4 shows images of the in vitro tests for the determination of the MIC of the non-volatile compound C-2 in its different concentrations against E. coli and S. Aureus.

Example 3: Preparation of Coatings

BLSC solutions were prepared for application on polyethylene (LDPE) previously subjected to corona treatment. The solutions were applied on commercial LDPE films, which is currently the most widely used in the food packaging industry to wrap chicken meat trays or make bags in bag format. The corona treatment resulted in a minimum surface energy of 40 dynes/cm, which improved the adhesion of the coating. The coatings were prepared using RK Print K303 laboratory equipment. Materials with different concentrations (2%, 4%, 6% w/w) of volatile active agent (carvacrol) were prepared. The BLSC coating was prepared at 35% w/v. Regarding the content of the active compound, this is expressed based on the mass percentage of the polymer present in the coating.

Table 3 summarizes the homogeneity and adhesion properties of the coating on the LDPE base film. For these purposes, an evaluation scale was used where Value 1 (+) corresponds to the final product with a whitish opaque and heterogeneous coating, while Value 5 (+++++) corresponds to a uniform, homogeneous and transparent coating. total. The results show a good adhesion and homogeneity for the coating with RA with and without active.

Table 3. Properties of the coatings produced

In addition, a test was carried out with different rods to determine the appropriate coating thickness. Table 4 shows the type of rod used and the coating thickness obtained. Table 4. Result of the study of rod and thickness

Rod No. 2 confers an adequate thickness, only increasing the thickness by 3.5 pm with respect to the base material.

Example 4: Characterization of materials

Antimicrobial capacity of coatings

Antimicrobial activity by contact

The method used allowed evaluating the antimicrobial activity on the surface of different materials such as textile, plastic, metallic or ceramic products. E. coli and S. aureus were used as model strains. Control and tested samples were cut into small square pieces (5 x 5 cm). All samples were inoculated in triplicate, with three additional replicates of the control films, with a suspension of microorganisms ca. 10 ⁵ CFU/ml standardized by dilution in a nutrient broth. Next, the inoculum was covered with a thin, sterile polypropylene film (4 x 4 cm) to prevent evaporation of the sample and loss of viability of the microorganisms.

Samples were incubated in a humid environment for +24 hours. After incubation, the surfaces were carefully transferred to a stomacher, from where serial dilutions were made and subsequent seeding in Peths plates, which were incubated for 24 hours. The antimicrobial effect was measured by comparing the survival of the bacteria in the active material with that obtained in a control material.

The value of the antimicrobial activity of the analyzed samples was determined with the following formula (Equation 1):

R = (Ut - U0) - (At - U0) = Ut - At (Equation 1) where

R = is the antibacterial activity;

U0 = is the average common logarithm of the number of viable bacteria, in cells/cm ² , recovered from untreated test samples immediately after inoculation; Ut = is the average common logarithm of the number of viable bacteria, in cells/cm ² , recovered from untreated test samples after 24 h;

At = is the average common logarithm of the number of viable bacteria, in cells/cm ² , recovered from the treated test samples after 24 h; when R > 2.0, the sample is considered to have biocidal properties

Table 5 shows the results obtained after the analysis. It is possible to appreciate a high antimicrobial capacity against E. coli and S. aureus for the active films with different content of non-volatile active agent C-2.

Table 5. Effect of the content of commercial active agent C-2 on the growth of E. coli and S. aureus. contact method.

Antimicrobial activity in the vapor phase.

First, the bacterial strains to be used were recovered. In this case E. coli and S. aureus were selected as models of Gram negative and Gram positive bacteria, respectively. For this, a miniculture is initially prepared, inoculating a small sample extracted with the loop of Henle from the inclined agar tube in a tube with 10 mL of TSB and incubating for 18 h at 37 ^° C.

Subsequently, 100 pL of microorganisms are inoculated at a concentration of 10 ⁵ CFU/mL on TSA plates, then the developed active materials are gently placed on the cover of the Petñ plate ^and incubated at 37 °C for 24 h. After this time, the growth of the bacteria was visually analyzed.

Table 6 shows the effect of BLSC-based films and volatile active agent on the growth of E. coli and S. aureus in the vapor phase. An important effect of the active ingredient is observed on concentrations of 6%. Table 6. Effect of volatile active agent content on the growth of E. coli and S. aureus.

Vapor phase method. See Fig. 9

Figure 9. Shows the antimicrobial effect of active films based on BLSC and volatile active agent.

Antimicrobial activity in liquid medium

The activity of the coatings in liquid medium against the two model bacteria E. coli and S. aureus was evaluated. For this, 100 pL of each microorganism in exponential phase were inoculated in the tubes previously prepared with 10 mL of TSB, to which 0.2 g of each of the active films developed were added. Subsequently, they were incubated at 37°C for 24 h. Depending on the turbidity of the tubes, signed dilutions were made with peptone water. A volume of 100 pL of each dilution was seeded in Petñ plates of approximately 15 mL of TSA culture medium, which were subsequently incubated for 24 h. Finally, the colony-forming units (CFU/mL) were counted. The experiments were carried out in triplicate.

Table 7 summarizes the results obtained for the active films developed. Unlike the test carried out in the previous section, the films with 2% active compound did not show antimicrobial activity in liquid medium against E. coli, which could be due to the fact that the amount released into the medium was less than the MIC, and in this matter it is necessary to mention that for an antimicrobial to be effective by this methodology, it is essential that there is migration of the agent towards the liquid culture medium. From 4%, an important effect is observed in the growth of E. coli with reductions over 5 logarithmic cycles. On the other hand, in the case of S. aureus, only the percentage of 4% was evaluated, a concentration that showed a reduction of more than 2 logarithmic cycles in the growth of this bacterium. Table 7. Effect of the content of commercial active agent C-2 on the growth of E. coli and S. aureus. Method in liquid medium

Subsequent tests are carried out with the film with 4% commercial active ingredients (C-2) for in vivo tests with chicken under refrigeration conditions.

Physical characterization of active films

Mechanical properties - tensile/strain

Tests were carried out using a Zwick Roell model BDO-OC 5HT universal tensile testing machine where the values of Young's Modulus, Tensile Strength, and Percentage of Elongation at Break were determined through a tensile deformation test. The results obtained are shown in Table 8. These results show that the incorporation of the varnish on the base polyethylene film does not significantly affect its mechanical properties.

Table 8. Mechanical properties of coated films and control.

Optical properties

Color

The possible change in coloration after the application of the coating with and without the active agent was evaluated using a Konica minolta CR-410 model colorimeter. Starting at CIELAB coordinates measured L* corresponding to luminosity (black (0) - white (100)), a* (red (+) - green (-)) and b* (yellow (+) - blue (-)). Specimens of 6 x 6 cm were cut for this.

Table 9 shows the color coordinates L*, a*, b*, of the control without varnish (LDPE), with coating with and without active C-2. The values of L* give an idea of the high transparency of the materials, while the low values of a* and b*, close to zero and very similar to white. These results are indicative that the application of varnish does not affect the coloration of the base material.

Table 9. Colorimetric coordinates of the developed films.

Opacity

Additionally, the opacity of the developed material was evaluated by calculating the quotient of the absorbance measured at 600 nm and the thickness of the material. The results of this analysis are presented below. As in the case of coloration, it is evident that the application of the varnish, either with or without active ingredients, on polyethylene does not affect this optical property.

Table 10. Opacity of developed and control films.

Grammage

The basis weight of the RA-coated container with and without the commercial active compound C-2 was determined. For this, 15 samples of 6 cm ² were cut from each condition. Subsequently, the grams of material per square meter were calculated. The results of this analysis are presented in Table 1 1 . Table 1 1 . Determination of LDPE control grammage and active packaging material.

An increase in grammage of approximately 4 g/m ² can be observed due to the application of the coating either with or without active agent C-2 and of 9 g/m ² in the case with TX-09. This result is consistent with the thickness obtained for the films coated with the VT7691 material.

heat sealability

Table 12 reports the results of the LDPE seal strength test at a seal time of 1.5 s. This time has been reported as achieving a proper seal of LDPE without corona treatment. Above this value, the seal strength does not change significantly when comparisons are made at the same seal pressure and temperature conditions. Average Seal Strength (Fav) was measured as the average of the force applied during the peel path of the seal. This value has a representative meaning for the cases where there are wide conventional seals. It can also be expressed per unit width (b) of the specimen (Fav/b). On the other hand, the maximum seal resistance (Fmax) is the maximum force that the seal resists, and its value was significantly higher in the tests, where there is a very narrow thread-type seal produced with the MULTIVAC vacuum packaging machine (Fmax) . It can also be expressed per unit width (b) of the specimen (Fmax/b).

Table 12 Seal Parameters

Polyethylene is recognized for being a thermoplastic material with excellent characteristics in terms of its heat sealability. Due to the above, in many materials manufactured with multilayer structure, this material is included to favor the sealing of the container. Therefore, it is important to note that the application of the varnish generates a significant loss in the heat-sealability of the material. Although this could indicate a disadvantage in the applicability of the material, the use of register printing turns out to be an option to make the application of the active varnish on polyethylene feasible.

oxygen permeability

The determination of the oxygen permeability of the film samples was carried out with an OXTRAN 2/20 MOCON equipment. This equipment allows to measure the speed at which oxygen passes through a polymeric material at a pressure of 760 mmHg and 0% RH. The parameter delivered by the equipment is the OTR (Oxygen Transmission Rate) or the oxygen transmission rate. The results of this analysis are presented in Table 13.

Table 13. Oxygen permeability of the developed films.

Example 4: Evaluation of the microbiological evolution of chicken fillets packed in bags made from the active film LDPE/BLSC/TX-09 and LDPE/RA/C-2. Bag Manufacturing

From the LDPE/BLSC/TX-9 and LDPE/RA/C-2 films, bags with dimensions of 15 x 15 cm were made. For the manufacture, the films were heat sealed with a Multivac heat sealer. It should be noted that in both cases the varnish with the active agents remained on the inside of the bag to allow contact with the food.

The aim is to confirm compliance with the safety limit of 1x10 ⁷ CFU/g of food established in title V of the microbiological criteria, paragraph III, 10.1 of the Food Sanitary Regulations (RSA). A representative sample was taken that was massed (10, 25 or 50 g) and diluted with peptone water (TPA) at 0.1% (90, 225 or 450 mL) inside a digester bag that was homogenized for a maximum of 1 minute in stomacher. Subsequently, serial dilutions are made in sterile tubes with 9 mL of 0.1% APT. Two samples per condition were analyzed and they were seeded in duplicate in each dilution ^and incubated at 37°C for 48 h, reading plates that had between 25-250 colonies. Chicken meat (chicken breast fillets) marketed under refrigeration conditions was used, and later, frozen. For the preliminary evaluation of the microbiological load of commercial samples of chicken meat, a mesophilic aerobic count (-RAM-) of chilled chicken meat was carried out, from 3 different samples (M1, M2 and M3), which were analyzed the day of purchase (DO) and 2 days after purchase or until they were outside the safety limit range of 1x10 ⁷ CFU/g of food. The samples were stored in their original container under refrigeration at 4°C. The results of this study are shown in Table 14.

Table 14 Count of mesophilic aerobes (RAM) in commercial samples of chicken meat.

AMR determination

The antimicrobial efficacy of the active films was determined based on monitoring the count of mesophilic aerobic microorganisms. The proposed method was carried out inside a LABCONCO model 3970421 flow chamber, analyzing separately the three chicken fillets per bag with technical duplicate for each dilution. In turn, the plates were incubated in an Arquimed model ZFD-5090 incubator at 35°C for 7 days or until they were not acceptable from a microbiological or sensory point of view. The concentrations of microorganisms were expressed in CFU/g from the quantification of colonies in a SUNTEX model 570 counter. Shelf life was determined by the acceptability limit for raw poultry meat samples, which is 10 ⁷ CFU/g.

A study was carried out to evaluate the effectiveness of the developed coating LDPE/BLSC/TX-09. For this, 4 systems were analyzed, which included 3 bags of 15x15 cm corresponding to the LDPE control, the LDPE/BLSC control (without the active compound ) and active bag LDPE/BLSC/TX-09. Additionally, a sealed tray was kept maintaining the same conditions as the other samples to be analyzed on the expiration date indicated on the container. The results expressed in colony-forming units per gram of food (CFU/g) are summarized in Table 15. Table 15. Microbiological results of AMR in chicken fillets test with bags (percentage active volatile to vehicle, 8% w/w)

When plotting the results (Figure 5), it is possible to appreciate a slight antimicrobial effect of the developed active bag compared to the other conditions evaluated. However, from a sensory point of view, the sample is unacceptable, with a strong odor of decomposition, a situation that shows that the product is not suitable for consumption. After multiple repetitions, totally heterogeneous results were obtained, where it was not possible to observe antimicrobial activity.

The variability of results was a problem that was already anticipated, the problem of not getting fresh chicken meat to carry out the tests, added to the lack of knowledge of how the food has been treated before it is put up for sale in supermarkets, among other aspects. , further complicate obtaining acceptable and reproducible results over time. Due to the above, different measures were taken in order to reduce external factors that could affect the study, such as the application of UV radiation to the bags prior to packaging the chicken. However, as will be indicated below, starting the study with frozen chicken samples significantly helped to carry out the experiments.

Frozen Chicken Evaluation

Table 16 and Figure 6 show the results obtained after the aforementioned modifications. It is possible to see how the active packaging manages, from a microbiological point of view, to increase the shelf life of chicken meat, at least 3 days more than the other conditions. In addition, from the sensory point of view, the samples were in conditions for sale and consumption, but not the other samples analyzed. Table 16 AMR microbiological results, modified assay

Figure 9 shows the evolution of AMR, expressed as CFU/g, during 10 days for chicken samples stored at 4 °C in control containers (LDPE and LDPE/BLSC) and in the active container made with LDPE/BLSC/TX- 09. As the figure shows, it is possible to establish that the active packaging allows the shelf life of the packaged samples to be increased by 2 days, going from a shelf life of 6 days for the control packaging to 8 days for the active package. The generation of the antimicrobial activity would be given by the nature of the active compound, which is characterized by volatilizing, a situation that would favor its action in the head space of the container, thus being able to act in a large part of the product surface.

Claims

one . Active polymeric container with antimicrobial properties that allows to extend the useful life of meat products selected from chicken, turkey or pork meat, characterized in that it comprises a selected substrate of polystyrene, polylactic acid, polyethylene-vinyl-alcohol, polyamides, polyolefins, celluloses, waxes , paraffins or a combination thereof, monolayer or multilayer, with an inner coating comprising a polymeric vehicle dissolved in a volatile organic solvent selected from a synthetic or natural varnish, which carries a natural antimicrobial volatile active agent, selected from carvacrol

2. The container of claim 1 characterized in that said substrate is selected from low-density polyethylene (LDPE).

3. The container of claim 1, characterized in that said polymeric vehicle dissolved in a volatile organic solvent is dispersed on the internal faces of the substrate or surface that contacts the food.

4. The container of claim 3, characterized in that said synthetic varnish is a heat-sealing lacquer for dairy products.

5. The container of claim 1, characterized in that said meat product is selected from chopped or cut chicken, pork or turkey meat.

6. The container of claim 1 characterized in that the percentage ratio of the volatile active agent to the vehicle is in the range of 2 to 10%.

7. The container of claim 6 characterized in that the percentage ratio of said volatile active agent to said polymeric vehicle dissolved in volatile organic solvent is 4%.