Nanocompuestos materials with antimicrobial activity and the procedure for their obtaination (Machine-translation by Google Translate, not legally binding)

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DESCRIPTION

Nanocomposite materials with antimicrobial activity and the procedure to obtain them.

Technical field of the invention

The present invention relates to clay-based nanocomposites with antimicrobial activity. Said activity is obtained through the formulation of a specific type of nanoadditives based on sheets of natural and / or synthetic clays that are interspersed with metals with antimicrobial capacity and / or salts thereof and / or with other organic, inorganic or other compounds. combination thereof.

Furthermore, the formulation of nanocomposite materials based on the incorporation of the aforementioned nanoadditives in a plastic or ceramic matrix is ​​described, by any method of manufacturing or processing of plastics or preparation and. ceramic powder processing. Thus, nanoadditives are incorporated into plastic matrices by solvent deposition and evaporation methods (eg coatings and lamination), application of the monomeric solution followed by polymerization and curing or crosslinking or vulcanization, operations typically used during the formulation of thermosets and elastomers, by melt mixing processes (eg extrusion, injection, blowing) and / or methods of in-situ polymerization.

Nanocomposites with plastic matrix can be prepared by different procedures typically used in the processing and manufacturing of plastics, such as casting and / or laminating techniques (dissolution and evaporation of the solvent), melt mixing, thermosetting and elastomer formulation and polymerization in-situ, for its advantageous application both in the packaging of products of interest for food and antimicrobial plastics, in surgical equipment, as well as for applications in other sectors.

In the case of nanocomposites with ceramic matrix, these are incorporated during the preparation of powders typically used in the manufacture of ceramic products that involve grinding, atomization, pressing or extrusion, enamelling in the case of enameled products, and firing.

Furthermore, the present invention relates to the use of said materials for multisectoral applications.

Background of the invention

In the field of polymers, one of the areas that is generating the greatest interest is the development of composite materials, and more specifically nanocomposites. There are different techniques for preparing nanocomposites, both by the casting method (Ogata N, Jimenez G, Kawai H, Ogihara T; J Polym Sci Part B: Polym Phys 1997; 35: 389-396, Chen GX, Hao GJ, Guo TY, Song MD, Zhang BH; J Appl Polym Sci 2004; 93: 655-61, Jimenez G, Ogata N, Kawai H, Ogihara T; J Appl Polym Sci 1997; 64: 2211-20), as by the method of melt mixed (Sinha Ray S, Yamada K, Okamoto M, Ueda K. Nano Lett 2002; 2: 1093-6, MD Sanchez-Garcia, E. Gimenez and JM Lagaron (J. App. Pol. Sci., DOI 10.1002 / app), Di Y, lannace S, Maio ED, Nicolais L. J Polym Sci Part B: Polym Phys 2003; 41: 670-8) and by the in-situ polymerization method (Messersmith PB, Giannelis EP. Chem Mater 1993; 5: 1064-6, Knani D, Gutman AL, Kohn DH. JPolym Sci. Part A: Polym Chem 1993 ; 31: 1221-32). Furthermore, these new nanocomposites and their processing techniques are described in US patents Nos. 5747560; 4618528; 4528235; 4874728; 6391449; 6486253; 6376591 and 6156835; WO 95/14733; WO 93/04117 and more specifically with regard to the present invention in WO2007074184A1. In this pCt patent application, a new manufacturing route for nanocomposites is described that may or may not be biodegradable, with antimicrobial properties based on natural products and / or with the capacity for controlled release of other active or bioactive substances. These nanocomposites based on synthetic laminar double hydroxides and / or phyllosilicates are interspersed with different organic modifiers, and once incorporated into thermoplastic and / or thermosetting matrices, they are capable of improving their gas and vapor barrier properties. The documents cited above are some examples of patents and literature on polymer-clay nanocomposites prepared from modified clays. These documents describe a nanocomposite material as an exfoliated or intercalated plate, with a tactid structure of nanometric dimensions, comprising intercalated clay dispersed in a polymer matrix, such as an oligomer, a polymer, or a mixture thereof.

For example, US patent 4739007 describes the preparation of Nylon-6-clay nanocomposites from montmorillonites treated with alkylammonium salts by the melt mixing method.

Protection against the action of microorganisms is a basic requirement for many current applications of plastics, such as preserving the quality of packaged foods, ensuring aseptic conditions in biomedical applications, helping to limit the growth of microorganisms on exposed surfaces and work, among other applications. Inventions have been found related to the manufacture of antimicrobial systems for use in the textile, pharmaceutical and food industries. More specifically, patents US 6841244 and US 7232777 describe the manufacture of fibers with antimicrobial properties that contain silver. Patents KR20030038586, US 6224898 and US 7306777 refer to the use of metallic silver and polyurethane nanocomposites, and metallic silver in dendrimeric polymers, respectively, with antimicrobial properties. Patents US 7306777 and DE202005020859U describe the use of germicidal materials based on silver nanoparticles applied in containers and packaging. Patent application 200703101 covers the manufacture of materials and packaging

ES 2 331 284 A1 passive, (bio) active and intelligent with antibacterial properties due to the incorporation of electrospun nanofibers containing silver nanoparticles. However, until now no specific design has been published describing the manufacturing process of nanocomposites for protection applications against the action of microorganisms.

Microorganisms, and specifically bacteria, are the main cause of diseases caused by the consumption of contaminated food. These can survive the heat treatment required for canning or contaminate the food after such treatment due to sutures or leakage from the package. In addition to its potential danger to health, the proliferation of microorganisms can cause alterations in foods that in turn lead to changes in their physical, chemical and organoleptic properties. Some of the traditional preservation methods such as heat treatments, irradiation, modified atmosphere packaging or the addition of salts, cannot be applied to certain types of food such as vegetables, fruits and fresh meats or ready-to-eat products. On the other hand, the direct application of antibacterial substances on food has limited effects since they are neutralized and quickly diffuse into the food. Considering the above aspects, active packaging constitutes a viable and advantageous way to limit and control bacterial growth in food, since antimicrobial agents slowly migrate from the material to the surface of the product. Migration can be as extensive as required, so that it covers the time of transport, storage and is guaranteed until consumption. In the case of the silver antimicrobial nanoadditives described in the present invention, once incorporated into the containers, they can control microbial contamination by inactivating the enzymatic metabolism of microorganisms.

The effect of microorganisms is also undesirable in other sectors. In the field of medicine, it is essential to eliminate the risks of contagion in invasive treatments, open wounds, as well as in routine treatments. Examples of such treatments include coatings with antimicrobial films of catheters and stethoscopes, and the manufacture of fabrics in fibers pretreated with silver nitrate or with broad spectrum antibiotics for wound and burn treatments. In the textile industry with regard to fashion and work clothing, for example, the use of fibers pre-treated with antibacterial agents limits the proliferation of microorganisms in the face of sweat, humidity and high temperatures, reducing bad body odors and risks of contagion. Fouling is known as the accumulation and deposit of biological material on surfaces exposed to various environmental conditions, such as painted boats, objects or systems exposed to high humidity conditions or other surfaces exposed to active, aggressive or environmentally adverse media. In the case of boats, fuel consumption can increase up to 50% due to the hydrodynamic resistance offered by the accumulation of biological material in the hull. Antimicrobial systems can act as antifouling if applied in layers to the surface of the boat, making fuel consumption optimal, and cleaning and maintenance less frequent. In the case of water containers and tanks, coating the interior with a film of antimicrobial compounds significantly reduces the growth of algae and the generation of bad odors, so the quality of the water contained is guaranteed for a longer time. Coating with films of antimicrobial compounds or manufacturing with these the work surfaces of laboratories (clinical, microbiological, water analysis, food), of shops where fresh food is handled (butchers, fishmongers, etc.), of The pavilions of hospitals and health centers, to mention just a few examples, guarantee adequate hygiene conditions for the development of work and eliminate the risk of contamination and infections. Plastic materials with antimicrobial properties can also be used in the manufacture of cranks, handlebars, handles and armrests of public transport elements, in handrails and support points in high-traffic places, in the manufacture of sanitary parts for public and mass use, as well as in headphones and microphones of telephones and audio systems of public places; kitchen and food transport tools, all in order to reduce the risk of spreading infections and diseases. It is also of emerging interest to manufacture ceramic pieces that inhibit the proliferation of microorganisms on ceramic products, for example, the proliferation of fungi and molds on surfaces covered with ceramic tiles or on their junction points.

In the field of ceramic materials, there are patents that describe the production of antibacterial ceramic compounds with Ag₂WO₄ (silver tungstate) for use in sanitary parts (CN101062786); ceramic compounds with antimicrobial, fungicidal and deodorizing properties containing dolomite and amphiphilic compounds (JP2007169109); vitreous and ceramic materials with incorporated silver as an antimicrobial agent (US2007172661, EP1711060); antimicrobial ceramic composed of mixtures of metal oxides (Ag₂O, Faith₂OR₃, MnO₂, etc.) with which low-density polyethylene antimicrobial nanocomposites can be prepared for use in sheets for food use (KR20010083418); antimicrobial vitreous ceramic compounds for dental applications (US2005142077). The previous examples show some of the applications of antimicrobial ceramic systems that eliminate or reduce the risk of spreading infections and contamination in potentially infectious environments (sanitary parts of public services, for example), in environments where the control of microbial growth is essential. for the proper development of activities (tiles for floors and walls of operating rooms, clinical and toxicological laboratories, childcare centers, for example); in formulations for preparation and / or repair of mobile or fixed replacement teeth (dentistry), among other potential applications.

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Description of the invention

As has been stated, up to now the manufacture of nanocomposite materials with properties both barrier to gases and vapors, flame retardancy, mechanical and thermal properties improved with respect to the pure polymer, has not been described. with the additional ability to block electromagnetic radiation (UV-Vis) and to allow the fixation and / or controlled release of active and / or bioactive substances and additionally has antimicrobial capacity due to the incorporation of antimicrobial metal nanoparticles that they confer sufficient thermal resistance to allow manufacturing and processing of plastics and ceramic firing processes.

However, and as previously seen, the PCT patent application (WO2007074184A1) is the closest work to that developed in the present invention, since it describes the manufacture of nanocomposites with active (eg antimicrobial) and bioactive properties based on compounds mainly of natural origin. The difference between this work and that of the present invention is that the antimicrobial properties are conferred or reinforced by the incorporation of substances based on silver or other metals with biocidal capacity in its structure. The incorporation of metallic biocides in clays is not only interesting for the manufacture of nanocomposites based on the addition of such nanoadditives to plastics, but also, because metallic biocides resist heat treatments, which may also be necessary to favor the reduction of metal salts biocidal to their corresponding metals, They can also be used in the ceramic industry for the manufacture of ceramic and porcelain products with antimicrobial properties. The availability in the ceramic industry of antimicrobial nanoadditives allows increasing the effectiveness of these products, due to the great dispersion exhibited by biocides in these matrices. Excellent results are thus obtained with lower proportions of nanoadditives, and therefore a significant reduction in costs.

The above examples allow to define the field of application of the new nanocomposite materials with antimicrobial properties based on metals, whose procedures for obtaining are detailed in the present patent. . These antimicrobial metal nanocomposites prevent the development, growth and proliferation of microorganisms, as well as the spread of infections, in a wide variety of applications such as packaging (food, drugs and medicines), in fibers and textile fabrics, in materials medical-surgical, in antifouling systems and in the manufacture of plastic parts for public places and for ceramic products.

Therefore, the present invention refers to nanocomposite materials with antimicrobial activity, obtained by the introduction of laminar nanoadditives with or without prior modification of antimicrobial quaternary ammonium salts and / or chitosan and / or derivatives of this antimicrobial that contain nanoparticles of metals and / or its organic and inorganic salts, in plastic or ceramic matrices, with advantageous application in the sectors of coatings, medicine, construction, anti-odor textiles and packaging.

Consequently, a first essential aspect of the present invention refers to nanocomposite materials that have a plastic or ceramic matrix and are constituted from the incorporation of laminar type clay nanoadditives.

Plastic matrices are selected without limiting sense from the group consisting of thermoplastics, thermosets and elastomers such as polyolefins, polyesters, polyamides, polyimides, polyketones, polyisocyanates, polysulfones, styrenic plastics, phenolic resins, amide resins, urea resins, melamine resins, resins made of polyester, epoxy resins, polycarbonates, polyvinylpyrrolidones, epoxy resins, polyacrylates, rubbers and rubbers, polyurethanes, silicones, aramides, polybutadiene, polyisoprenes, polyacrylonitriles, PVDF, PVA, PVOH, EVOH, PVC, PVDC or biomass derivatives and biodegradable materials such as proteins, polysaccharides, lipids and biopolyesters or mixtures of all these and can contain all kinds of additives typically added to plastics to improve its manufacture and / or processing or its properties. Furthermore, said type of matrix is ​​in a proportion from 5% to 99.99%; preferably from 20% to 99.99%, and more preferably from 90% to 99.99%.

Ceramic matrices include, and are not limited to, water, clays (preferably kaolinites and occasionally montmorillonites), deflocculants, feldspars, feldspathic sands and occasionally kaolin, carbonates and zirconium. Ceramic matrices of the enamel type and other types of ceramic coatings include, and are not limited to, kaolin or a kaolinite (5%) or montmorillonite (1%) clay, feldspars, frits, silica and silica sands. Furthermore, said type of matrix is ​​in a proportion from 5% to 99.99%; preferably from 20% to 99.99%, and more preferably from 65% to 99.99%.

According to a preferred embodiment, the plastic or ceramic type matrices can contain agents with barrier properties to electromagnetic radiation and resistance to fire and other active or bioactive substances in addition to nanoadditives, selected from the group consisting of antimicrobial organic and inorganic metal salts. (preferably silver, copper, nickel or cobalt), substances of low molecular weight that have an active or bioactive character selected from ethanol, or ethylene, or of the essential oil type (preferably thymol, carvacrol, linalol and mixtures), or natural or genetically modified antimicrobial peptides (preferably bacteriocins) (preferably nisins, enterocins, lacticins and lysozyme), or natural antioxidants synthetics (preferably polyphenols, preferably flavonoids, rosemary or other plant extract and vitamins, preferably ascorbic acid or vitamin C), or drugs, or enzymes or bioavailable calcium compounds, or prebiotics (indigestible fiber).

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The nanoadditives are selected from the group consisting of layered silicates and / or layered double hydroxides. These above are selected without limiting sense from the group consisting of clays of the montmorillonite, kaolinite, bentonite, smectite, hectorite, sepiolite, gibsite, dicktite, nacritite, saponite, halloisite, vermiculite, mica, and / or mixtures thereof or with other phyllosilicates, mainly, with or without prior organic and / or inorganic surface modification. These materials are characterized in that they are introduced as laminar-type fillers with sizes in the nanometer range in at least the thickness of the particle, in plastic matrices and in ceramic matrices to form antimicrobial nanocomposites.

In plastic matrices, nanoadditives are in a proportion from 0.01% to 95%, preferably from 0.01% to 80% and more preferably from 0.01 to 10%.

In ceramic matrices, nanoadditives are in a proportion from 0.01 to 95%, by weight, preferably between 0.01% and 80% and more preferably from 0.01 to 35%.

In ceramic matrices of the glaze type, the nanoadditives are in a proportion from 0.01% to 50%, preferably from 0.01% to 20% and more preferably from 0.01 to 15%.

The surface modification of the clay nanoadditives when applied allows, in addition to introducing or accentuating the antimicrobial activity, increasing the compatibility between the clay and the matrix to achieve better exfoliation of the clay. Thus, a good morphology is achieved to improve the dispersion and surface exposure of an antimicrobial active substance, which are substances based on metals such as silver, copper, nickel, cobalt, iron, zinc and / or combinations thereof and / or salts. inorganic, organic, organic compounds, preferably salts allowed for food contact (that is, they are included in the lists of monomers and other starting substances authorized by the legislation to be used in the manufacture of plastic materials and objects) such as and without limitation hexadecyltrimethylammonium bromide, esters of polyethylene glycol with aliphatic monocarboxylic acids (C6-C22) and their ammonium and sodium sulfates, perfluorooctanoic acid and its ammonium salt, copolymers of N-methacryloyloxyethyl-N, N-dimethyl-N-carboxymethylammonium chloride, bis (2-hydroxyethyl) -2-hydroxypropyl3- (dodecyloxy) methylammonium chloride; and chitosan and its derivatives, and / or combinations thereof. The inorganic salts are selected from the group consisting of simple salts of the nitrate, acetate, chloride, sulfate and inorganic complexes that include water and the nitrate, acetate, amino and chloride groups.

In the case of plastic materials, they present antimicrobial activity and improvements in their barrier properties and other physical properties, fire resistance and allow blocking electromagnetic radiation, as well as allowing the controlled release of substances with antioxidant and / or bioactive properties with respect to of pure material. In the case of ceramic materials, more effective antimicrobial properties are obtained due to nanoparticulation.

These nanocomposite materials are prepared in the case of plastics by lamination or coating techniques (casting of the solution), by applying the monomeric solution followed by polymerization and curing, operations typically used during the formulation of thermosets, by the previous procedure but followed by crosslinking or vulcanization, operations typically used in the manufacture of elastomers, by melt mixing or by in-situ polymerization.

In the case of their application in ceramics, these are incorporated without limiting sense during the preparation of powders typically used in the manufacture of ceramic products that involve grinding, atomization, pressing or extrusion, enamelling in the case of enameled and fired products.

Plastic nanocomposite materials are of particular interest in the food packaging industry, since these active packages allow the product to be protected from the action of microorganisms and / or the controlled release of active substances and additionally, significantly improve the barrier properties. to gases and vapors, UV barrier mechanical properties and others. In other fields of application, plastic and ceramic nanocomposites reinforced with nanoclays with antimicrobial properties are useful in the medical-surgical, biomedical and pharmaceutical areas, for the manufacture and coating of equipment and materials used in routine and invasive treatments. In “antifouling” applications to avoid the formation of biofilms on submerged surfaces exposed to water and humidity, and in general, for all applications in which a piece, accessory and / or coating of an antimicrobial compound is required to prevent proliferation of microbes and risks of infections.

A second essential aspect of the present invention relates to the process for the manufacture of the nanocomposite materials described in the present invention, which can be based on structures such as lamellar phyllosilicates, including clays (eg montmorillonite, kaolinite, bentonite, smectite , hectorite, sepiolite, saponite, halloisite, vermiculite, mica) or synthetic or natural lamellar double hydroxides with laminar structure comprises the following stages:

1) Reduction of the size of the lamellar particles by mechanical action for example by means of milling technologies. This process is carried out until obtaining a particle size below 30 microns in the D90.

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2) Classification in vibro-screen, centrifuge, filter press or any other dry or wet filtration system up to a range between 0.1 to 100 microns, preferably a reduction in particle size is achieved below 25 microns and more preferably below 3 microns in the so-called D90 (no more than 10% of the material is above that value).

3) Elimination of organic matter by and without limiting sense by means of decantation techniques, collection of supernatant or by chemical reaction with oxidizing substances such as peroxides.

4) Elimination of crystalline oxides and hard particles not subject to modification either by means of centrifugation and / or gravimetric processes in solution or by turbo-dryers, preferably by a centrifugation process either wet or dry followed or not by a process of atomization with controlled depression or by any other industrial drying process including lyophilization.

5) Obtaining laminar fines either in liquid suspension or by subsequent drying by the methods described in step 4) in powder form. These systems, both in liquid suspension and in powder, are considered as the starting product of the present invention.

6) Pre-treatment of the laminar structures in one or in several steps, by using precursors of the expander type as shown in Table 1.

TABLE 1

<td>Modifier</td><td>^ MODIFIER (nm)</td><td>Modifier</td><td>^ MODIFIER (nm)</td>

<td>Unmodified kaolinite</td><td> 0.72</td><td>Montmorillonite without Modify</td><td> 0.98</td>

<td>Dimethyl sulfoxide (DMSO)</td><td> 1.11</td><td>Polyethylene oxide</td><td> 1.12</td>

<td>Silver nitrate</td><td> 0.74</td><td>Silver nitrate</td><td> 0.99</td>

<td>Silver acetate</td><td> 0.74</td><td>Silver acetate</td><td> 0.99</td>

<td>Nickel chlorure</td><td> 0.75</td><td>Nickel chlorure</td><td> 0.99</td>

<td>Cobalt chloride</td><td> 0.76</td><td>Cobalt chloride</td><td> 0.99</td>

<td>Copper nitrate</td><td> 0.76</td><td>Copper nitrate</td><td> 1.00</td>

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<td>N ~ methyl formamide (NMF)</td><td> 1.02</td><td>Cellulose acetobutyrate</td><td> 1.13</td>

<td>Hydrazine hydrated</td><td> 1.03</td><td>Calcium butyrate</td><td>0.92 l</td>

<td>Water</td><td> 0.78</td><td>Acetoisobutyrate saccharose</td><td> 1.08</td>

<td>Alcohols</td><td> 1.10</td><td>Manganese butyrate</td><td> 0.95</td>

<td>Anhydrous hydrazine</td><td> 0.96</td><td>Carboxymethyl starch</td><td> &gt;3</td>

<td>Acetamide</td><td> 1.09</td><td>Starch</td><td> 1.21</td>

<td>DMSO + Methanol (MeOH)</td><td> 1.12</td><td>Hydroxyethyl starch</td><td> 1.15 '</td>

<td>Hexanoic acid</td><td> 1.23</td><td>Hydroxypropyl starch</td><td> 1.14</td>

<td>Achlamidas</td><td> 1.44</td><td>Adonitol</td><td> 1.04</td>

<td>Glucose</td><td> 1.25</td><td>Sorbitol</td><td>1.19 i</td>

<td>Archilamide</td><td> 1.14</td><td>Dibenzylidene sorbitol</td><td> 1.16</td>

<td>Salicidal acid</td><td> 1.07</td><td>Ethylene glycol</td><td> 0.95</td>

<td>Manganese acetate</td><td> 1.41</td><td>Polypropylene glycol</td><td> 1.01</td>

<td>Caprolactam</td><td> 1.18</td><td>Propylene glycol</td><td> 1.01</td>

<td>Vinyl acetate</td><td> 1.21</td><td>Glycolic Acid</td><td> 1.06 '</td>

<td>Potassium acetate</td><td> 1.39</td><td>Triethylene glycol</td><td> 1.08 ,</td>

<td>Tannic acid</td><td> 1.09</td><td>Tetraethylene glycol</td><td> 1.06</td>

<td>í Maleic acid</td><td> 1.20</td><td>Glycerol</td><td> 1.02</td>

<td>Maleic anhydnd</td><td> 1.20</td><td>1,2-Propanediol</td><td> 1.09</td>

<td>Lactic acid</td><td> 1.08</td><td>1,3-Propanediol</td><td> 0.98</td>

<td>Adipic acid</td><td> 1.03</td><td>Polyethylene glycol M_w=1000</td><td> 1.11</td>

<td>Acetic acid</td><td> 1.10</td><td>Polyethylene glycol M_w=3400</td><td> 1.12</td>

<td>Acetaldehyde.</td><td> 0.91</td><td>Sorbitan</td><td> 1.09</td>

<td>Butyric acid</td><td> 1.01</td><td>Dipropylene glycol</td><td> 1.03</td>

<td>Tetra fluoroethylene</td><td> 0.98</td><td>Diethylene glycol</td><td> 1.04</td>

<td>Chlorotrifluoroethylene</td><td> 1.05</td><td>Vinylpyrrolidone</td><td> 1.23</td>

<td>Hexamethylene</td><td> 1.02</td><td>Vinyl Versatate</td><td> 1.11</td>

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Preferably the expanders are selected from the group consisting of DMSO, alcohols, acetates, or water and a mixture of the foregoing, and silver, copper, nickel or cobalt metal salts, which activate the fines through an initial increase in the basal spacing of the sheets and they modify the surface characteristics of the clay. The penetration of the precursors will be accelerated without limitation through the use of temperature, a homogenizer of turbulent regime, ultrasound, supercritical fluids, pressure or a mixture of the above. The drying of these can be carried out by evaporation in an oven, lyophilization, centrifugation and / or gravimetric processes in solution or turbo-dryers or by atomization. According to another preferred embodiment of the present invention, the solution of the intercalated precursor can be used, without a previous drying process, as a starting medium for the next stage of incorporation of the modifier.

7) Additionally, or optionally, in an aqueous base or with polar solvents, inorganic, organic or hybrid substances can be intercalated in the laminar structure. In this same sense, the compounds to be intercalated are selected and without limiting sense from the group formed by PVOH, EVOH and derivatives of the same family, and / or biopolymers such as peptides and natural or synthetic proteins via chemical or genetic modification of microorganisms or plants. and natural or synthetic polysaccharides via chemical or genetic modification of microorganisms or plants and polypeptides, lipids, nucleic acids and synthetic nucleic acid polymers obtained via chemical or genetic modification of microorganisms or plants, and biodegradable polyesters such as polylactic acid, polylacticglycolic acid, polycaprolactone, adipic acid and derivatives and polyhydroxyalkanoates, preferably polyhydroxybutyrate and its copolymers with valeriates, materials biomedicals such as hydroxyapatites and organic salt phosphates. Quaternary ammonium salts can also be intercalated - preferably salts allowed for food contact (that is, they are included in the lists of monomers and other starting substances authorized by the legislation to be used in the manufacture of plastic materials and objects) such as and without limitation hexadecyltrimethylammonium bromide, polyethylene glycol esters with aliphatic monocarboxylic acids (C6-C22) and their ammonium and sodium sulfates, perfluorooctanoic acid and its ammonium salt, N-methacryloyloxyethyl-N, N-dimethyl-N-carboxymethylammonium chloride copolymers, bis (2-hydroxyethyl) -2-hydroxypropyl-3- (dodecyloxy) methylammonium chloride; and chitosan and its derivatives, and / or combinations thereof and more preferably hexadecyltrimethylammonium bromide -, silver, copper, nickel or their organic or inorganic salts, and other particles or nanoparticles with antimicrobial properties.

When the inorganic material to be inserted is based on metals such as silver or organic and / or inorganic salts of silver, copper, cobalt, nickel or other metals with antimicrobial power, a physical or chemical treatment can be subsequently applied to change the state of oxidation of the intercalated metal center, totally or partially. These treatments include but are not limited to: annealing at high temperatures (250-1200 ° C), UV radiation, infrared radiation, microwave radiation, chemical reduction by ethanol and / or NaBH₄ and / or other chemical reducing agents. At the end of any of these treatments, the degree of oxidation of the metal center will have been totally or partially modified (silver, copper, nickel, zinc, cobalt, or other metal used), giving the material more or less intense antimicrobial properties.

When the organic material that is intercalated is EVOH or any material of the same family with molar contents of ethylene preferably less than 48%, and more preferably less than 29%, these are brought to saturation in aqueous medium or in solvents. specific alcoholic type and mixtures of alcohols and water, more preferably of water and isopropanol in proportions by volume of water greater than 50%.

On the other hand, biopolymers with or without plasticizers, with or without crosslinkers and with or without emulsifiers or surfactants or other types of nanoadditives, are from the group formed by synthetic and natural polysaccharides (vegetable or animal) such as cellulose and derivatives, carrageenans and derivatives, alginates, dextran, acacia and preferably chitosan or any of its derivatives, both natural and synthetic, more preferably the salts of chitosan and even more preferably the acetate of chitosan, and proteins both derived from plants and animals such as corn proteins (zein), those derived from gluten, such as gluten or its gliadin and glutenin fractions and more preferably gelatin, casein and soy proteins and derivatives thereof, as well as natural or synthetic polypeptides preferably of the elastin type obtained by chemical or genetic modification of microorganisms or plants, lipids such as beeswax, carnauba wax, candelilla wax, shellac and fatty acids and monoglycerides and / or mixtures of all the previous.

In the case of chitosan the degree of deacetylation will preferably be greater than 80% and more preferably greater than 87%. The penetration of the precursors will be accelerated by the use of temperature, a homogenizer of turbulent regime, ultrasound, pressure or mixing of the above.

In a subsequent or alternative step to the dissolution of the fines pre-treated with the previously proposed precursors and modifiers, optionally low molecular weight substances that have an active or bioactive character will be added so that they are either intercalated or released in a controlled manner. giving rise to nanocomposites with active or bioactive capacity. The active substances will be ethanol, or ethylene, or of the essential oil type (preferably thymol, carvacrol, linalol and mixtures), or antimicrobial peptides of reduced size (preferably bacteriocins) natural or obtained by genetic modification (preferably nisins, enterocins, lacticins and lysozyme. ), or natural or synthetic antioxidants (preferably polyphenols, preferably flavonoids, rosemary or other plant extract and vitamins, preferably ascorbic acid or vitamin C), or drugs, or enzymes or bioavailable calcium compounds, or prebiotics (indigestible fiber), or organic and inorganic metal salts (preferably silver, copper, nickel or cobalt). These elements are expected to remain fixed to later be released from the nanocomposite to the product in a controlled way (matrix control) and exert their active or bioactive role,

ES 2 331 284 A1 and / or that can be released from the matrix and that the nanoparticles control the kinetics (nanoadditive control). The contents to be added are generally less than 80% by volume of the solution, preferably less than 12% and more preferably less than 8%. The penetration of these substances will be accelerated and without limiting sense through the use of temperature, a homogenizer with a turbulent regime, ultrasound, pressure or a mixture of the above.

8) Add the result of the previous stages in solid or liquid state to a plastic or ceramic matrix. Alternatively, they can also be included on the matrix containing the nanoadditives and additionally organic and inorganic antimicrobial metal salts (preferably silver, copper, nickel or cobalt) and / or any other type of active and bioactive substances without limiting sense of the mentioned above with the aim of reinforcing or complementing the active or bioactive effect of the nanocomposite. In the case of plastic matrices, both the nanoadditives and the aforementioned complementary compounds can be added during their processing using any manufacturing method related to the plastics processing industry such as extrusion, application and curing processes typically used to manufacture and forming thermosets and elastomers, injection, blowing, compression molding, resin transfer molding, calendering, thermal shock, internal ultrasonic mixing, co-extrusion, co-injection and mixing of these.

According to a preferred embodiment, the plastic matrix can be made of any thermoplastic, thermoset or elastomer or derived from biomass and biodegradable materials such as proteins, polysaccharides, lipids and biopolyesters or mixtures of all these and can contain all kinds of additives that improve the barrier properties to electromagnetic radiation and resistance to fire and / or other nanoadditives different from those described in this application and that are typically added to plastics. to improve its processing or its properties. Alternatively, an evaporative precipitation of the resulting set of nanoadditives and modifiers and, optionally, of the plastic matrix in solution can be carried out, using drying methodologies such as heating and / or centrifugation and / or gravimetric processes in solution or turbo-dryers and / or atomization; by cooling or by adding a precipitating agent to form either a powder of the additive or a masterbatch or what is the same, a concentrate of the nanoadditive in a plastic matrix.

In the case of the ceramic matrix, organic and / or inorganic metal salts with antimicrobial properties can be added together with other active or bioactive substances in any of the stages of the manufacture or processing of ceramic materials, although they will preferably be added during the preparation of powders before atomization.

Additive concentrates in polymeric matrix can be treated in the following ways:

a) is ground to give a particulate product by milling.

b) It is processed using any plastic processing methodology to obtain solid-state pellets.

c) is processed by any manufacturing process related to the plastics processing industry such as extrusion, injection, blowing, compression molding, resin transfer molding, calendering, thermal shock, internal mixing, ultrasound, co-extrusion, co-injection and mixing of these.

d) it is used as an additive on any plastic matrix (including the aforementioned biopolymers and biomedical materials) in a conventional plastics processing route such as those mentioned above.

Finally, when the nanocomposite material is reinforced with nanoadditives that contain metals such as silver or organic and / or inorganic salts of silver, copper, cobalt, nickel or other metals with antimicrobial power, it can be applied, whether it has been done before or if no, a physical or chemical treatment to change the oxidation state, totally or partially, of the metallic center intercalated in the plastic or ceramic matrix either before, during or after forming. These treatments include but are not limited to: annealing at high temperatures (2501200 ° C), UV radiation, infrared radiation, microwave radiation, chemical reduction by ethanol and / or NaBH₄ and / or other chemical reducing agents. At the end of any of these treatments, the degree of oxidation of the metal center (silver, copper, nickel, zinc, cobalt, or other metal used) will have been modified, giving the material advantageous antimicrobial properties.

Finally, a third essential aspect of the present invention refers to the use of the nanocomposite materials obtained to reinforce antimicrobial activity in multisectoral applications in which it is required to limit microbial proliferation through the use of plastic materials and ceramic compounds, particularly in applications of packaging and packaging in general of food and food components (in the case of polymeric materials), in biomedical applications, medical-surgical and pharmaceutical, or in antifouling applications, in construction applications for glazes and tiles, in applications for personal hygiene products and in contact applications in busy places such as supermarkets, trolleys, stands, linear, escalators or airports, in textile applications, as a barrier to gases, vapors, solvents and organic products, such as aromas and components of aromas, oils, fats and hydrocarbons, and mixed products of organic and inorganic character, for applications that require biodegradable or compostable character, for active containers that require an organic character

ES 2 331 284 A1 thymicrobial, antioxidant or of another type that requires the controlled release of substances of low molecular weight, preferably volatile, for applications that require antimicrobial capacity and for the use of biopolymers either without the need for the use of plasticizers or requiring lower amounts of these.

Also these nanocomposite materials will serve as materials with barrier properties to electromagnetic radiation and resistance to fire.

All the characteristics and advantages set forth, as well as others specific to the invention, may be better understood with the following examples. On the other hand, the examples shown below are not limiting but illustrative so that the present invention can be better understood.

Brief description of the figures

The invention is described below with reference to the attached figures, in which:

Figure 1 corresponds to the X-ray diffractograms (WAXS) obtained from a sample of montmorillonite type clay modified with hexadecyltrimethylammonium bromide (organic antimicrobial, blowing agent and compatibilizer) and silver nitrate (temperature resistant antimicrobial), using ethanol as a reducing agent by the method described in Example 1, and a sample of the same type of unmodified clay. This graph shows how the antimicrobial system intercalates in the clay and moves the natural peak of the clay to lower angles.

Figure 2 is an image obtained by transmission electron microscope (TEM) in which the main morphologies that can be observed in the nano-fillers obtained according to the present invention are presented. The image corresponds to an aggregate of montmorillonite-type clay sheets modified with hexadecyltrimethylammonium bromide and silver nitrate, using ethanol as a reducing agent, by the method described in Example 1. You can see the silver nanoparticles formed on the surface.

Figure 3 corresponds to the X-ray diffractograms (WAXS) obtained from a kaolinitic type clay sample (pretreated with DMSO) modified with hexadecyltrimethylammonium bromide (organic antimicrobial, blowing agent and compatibilizer) and with silver nitrate (resistant antimicrobial at temperature), using UV radiation as a reducing agent by the method described in Example 2, and a sample of the same type of unmodified clay (pretreated with DMSO). In this graph it is observed how the antimicrobial system intercalates in the clay and as a result leads to the disappearance of the peak of the natural clay pretreated with DMSO.

Figure 4 is an image obtained by transmission electron microscope (TEM) in which the main and typical morphologies that can be observed in the nano-fillers obtained according to the present invention are presented. The image corresponds to an aggregate of kaolinite-type clay sheets (pretreated with DMSO) modified with hexadecyltrimethylammonium bromide and silver nitrate, using UV radiation as a reducing agent, by the method described in Example 2.

Figure 5 is a transmission electron microscope (TEM) image of an aggregate of montmorillonite-type clay sheets interspersed with silver nitrate, using ethanol as a reducing agent, by the method described in Example 3.

Figure 6 is a transmission electron microscope (TEM) image of an aggregate of kaolinite-type clay sheets (pretreated with DSMO) interspersed with silver nitrate, using UV radiation as a reducing agent, by the method described in Example 4.

Figure 7 corresponds to an image obtained by transmission electron microscope (TEM) of a film obtained by casting a polylactic acid nanocomposite with 10% kaolinite-type clay (pre-treated with DSMO) interspersed with silver nitrate, by the method described in Example 5.

Figure 8 shows the improvement in permeability to water vapor obtained in a polylactic acid nanocomposite film with 10% kaolinite type clay (pretreated with DMSO) interspersed with silver nitrate with respect to a pure polylactic acid film (Example 5).

Examples

Example 1

Synthesis and intercalation of metallic silver nanoparticles in montmorillonite-type clays modified with 33% by mass of hexadecyltrimethylammonium bromide, using ethanol as reducing agent. Initially, the already modified clay was dispersed with 33% hexadecyltrimethylammonium bromide in ethanol, at ambient conditions, at a ratio of 1 g of clay per 100 g of solvent, and 0.05 g of AgNO were added to the dispersion.₃. The dispersion was refluxed at 70 ° C for 6 hours; The dispersion was subsequently allowed to settle, the excess solvent was removed and the clay was dried in a convection oven for 1 h at 70 ° C. The clay obtained was characterized using diffraction

ES 2 331 284 A1 X-ray (see Figure 1) and transmission electron microscopy (see Figure 2). The diffractograms of Figure 1 demonstrate that the modifying agents (silver particles and hexadecyltrimethylammonium bromide) have intercalated between the sheets, according to the displacement of the basal peak at lower angles (from 6.38 to 5.26). Through TEM images, it was determined that in this case the silver nanoparticles reached between 3 and 23 nm, the average size being 16 nm; and that said nanoparticles are presumably located in the interlaminar spaces of the clay, on the surface and edges.

In another study, the antimicrobial capacity of this clay was determined with 5% silver nitrate against Salmonella spp. A food-borne pathogenic microorganism was used, such as Salmonella spp. CECT 554, which was obtained from the Spanish Collection of Type Cultures (Valencia, Spain). The study conditions were set in the use of the bacterium in the medium exponential phase and with an initial concentration of the microorganism of approximately 10⁵ CFU / mL. The experimental part was carried out using an adaptation of the macrodilution method established for the determination of the bactericidal activity of antimicrobial agents approved in 1999 by the National Committee for Clinical Laboratory Standards. According to this method, 100 mg of the clay having a final concentration of 5% silver and 33% hexadecyltrimethylammonium bromide was introduced into a sterile tube containing 10 mL of Mueller Hinton Broth (MHB) culture broth. Subsequently, and after 5 h, the tube was inoculated with 0.1 mL of a culture of Salmonella spp. under the conditions described above. At the same time, two tubes containing a sample without silver were inoculated (one with clay of the same type without any modification and the other with clay of the same type modified with 33% hexadecyltrimethylammonium bromide), and another tube without sample that would serve as a control. Once the samples were inoculated, all tubes were incubated at 37 ° C for 24 hours. Then 0.1 mL of each sample was seeded on Tryptone Soy Agar (TSA) plates. After 24 hours of incubation at 37 ° C, the viable cells were counted on the plate. Except for the control without sample and the control of clay without modification, a significant reduction in the number of viable was obtained after incubation at 37 ° C for 24 h (see Table 2). The modified clay control with 33% hexadecyltrimethylammonium bromide shows a reduction of three orders of magnitude in the number of viable ones after the incubation period, demonstrating that this modifying agent has some antibacterial activity. The clay sample modified with 5% silver nitrate and 33% hexadecyltrimethylammonium bromide reduced the number of viable ones by more than 99.9%, demonstrating the bactericidal capacity of this clay.

TABLE 2

<td>Sample</td><td>Initial CFU / mL</td><td>Final CFU / mL</td>

<td>Control without sample</td><td> 7,0* 10⁵</td><td> 3,8 * 10⁹</td>

<td>Montmorillonite-type clay control, no modification</td><td> 7,0* 10⁵</td><td> 2,0* 10⁹</td>

<td>Control of montmorillonite-type clay modified with 33% hexadecyltrimethylammonium bromide</td><td> 7,0* 10⁵</td><td> 1,4 * 10²</td>

<td>Montmorillonite type clay modified with 33% hexadecyltrimethylammonium bromide and 5% AgNO₃</td><td> 7,0* 10⁵</td><td> &lt; 1</td>

Example 2

Synthesis and intercalation of metallic silver nanoparticles in kaolinite-type clays pretreated with dimethylsulfoxide (DMSO) and modified with 33% by mass of hexadecyltrimethylammonium bromide, using UV radiation as reducing agent. Initially, the kaolinite clay was pretreated with dimethylsulfoxide to increase the interlaminar space. For this, 60 g of clay were dispersed in 300 ml of dimethylsulfoxide, and kept under magnetic stirring and at 65 ° C for 24 h. Subsequently, the clay was filtered by suction, washed with methanol and dried in a convection oven at 80 ° C for 6 hours.

Once dry, the clay pretreated with dimethylsulfoxide is dispersed in water, at a ratio of 1 g of clay per 100 g of solvent, and subsequently 0.05 g of AgNO were added.₃ and 0.33 g of hexadecyltrimethylammonium bromide. The dispersion was maintained under vigorous and constant magnetic stirring under a source of UV radiation of 30 W and 235 nm wavelength. The exposure time to UV radiation was 24 h, then the solid was filtered by suction and dried in a convection oven at 70 ° C for 1 h. The clay obtained was characterized using diffraction of

ES 2 331 284 A1 X-ray (see Figure 3) and transmission electron microscopy (see Figure 4). The disappearance of the basal peak signal of kaolinite pretreated with dimethylsulfoxide and unmodified with hexadecyltrimethylammonium bromide in the diffractograms of Figure 3 (2θ = 8.06), indicates that after the simultaneous intercalation of hexadecyltrimethylammonium bromide and silver particles clay sheets find steric impediments to be able to hold together. Through TEM images, it was determined that in this case the silver nanoparticles reached between 3 and 24 nm, the average size being 10 nm; and that said nanoparticles are located on the surface, edges, and presumably, in the interlaminar spaces of the clay. In another study, the antimicrobial capacity of this clay was determined with 5% silver nitrate against Salmonella spp. A food-borne pathogenic microorganism was used, such as Salmonella spp. CECT 554, which was obtained from the Spanish Collection of Type Cultures (Valencia, Spain). The study conditions were set in the use of the bacterium in the medium exponential phase and with an initial concentration of the microorganism of approximately 10⁵ CFU / mL. The experimental part was carried out using an adaptation of the macrodilution method established for the determination of the bactericidal activity of antimicrobial agents approved in 1999 by the National Committee for Clinical Laboratory Standards. According to this method, 100 mg of the clay having a final concentration of 5% silver nitrate and 33% of hexadecyltrimethylammonium bromide were introduced into a sterile tube containing 10 mL of Mueller Hinton Broth (MHB) culture broth. . Subsequently, and after 5 h, the tube was inoculated with 0.1 mL of a culture of Salmonella spp. under the conditions described above. At the same time, two tubes containing sample without silver were inoculated (one with clay of the same type without any modification but pretreated with DSMO and the other with clay of the same type pretreated with DMSO and modified with 33% hexadecyltrimethylammonium bromide), and another tube without sample. that would serve as a control. Once the samples were inoculated, all tubes were incubated at 37 ° C for 24 hours. Then 0.1 mL of each sample was seeded on Tryptone Soy Agar (TSA) plates. After 24 hours of incubation at 37 ° C, the viable cells were counted on the plate. Except for the control without sample and the control of clay pretreated with dimethylsulfoxide but without modification, a significant reduction in the number of viable (&gt; 99.9%) was obtained, both in the control of clay pretreated with DMSO and modified with hexadecyltrimethylammonium bromide. as in the clay sample pretreated with DMSO and modified with hexadecyltrimethylammonium bromide and silver nitrate after incubation at 37 ° C for 24 h (see Table 3). These results show that both hexadecyltrimethylammonium bromide and silver nanoparticles intercalated in kaolinite have strong antimicrobial effects.

TABLE 3

<td>Sample</td><td>Initial CFU / mL</td><td>Final CFU / mL</td>

<td>Control without sample</td><td> 7,0* 10^b</td><td> 3,8* 10⁹</td>

<td>Control of kaolinite-type clay pretreated with dimethylsulfoxide without modification with hexadecyltrimethylammonium bromide</td><td> 7,0* 10⁵</td><td> 3,9* 10⁹</td>

<td>Control of kaolinite type clay pretreated with dimethylsulfoxide and modified with 33% hexadecyltrimethylammonium bromide.</td><td> 7,0* 10⁵</td><td> &lt; 1</td>

<td>Kaolinite type clay pretreated with dimethylsulfoxide, modified with 33% hexadecyltrimethylammonium bromide and 5% AgNO₃</td><td> 7,0* 10⁵</td><td> &lt; 1</td>

Example 3

Synthesis and intercalation of metallic silver nanoparticles in unmodified montmorillonite-type clays, using ethanol as a reducing agent. Initially, the clay was dispersed in ethanol, at ambient conditions, at a ratio of 1 g of clay per 100 g of solvent, and 0.1 g of AgNO were added.₃ to the dispersion. This was refluxed at 70 ° C for 6 hours; the dispersion was subsequently allowed to settle, the excess solvent was removed and the clay was dried in a convection oven for 1 h at 70 ° C. The clay obtained was characterized using X-ray diffraction (see Figure 5). The diffractograms in Figure 5 indicate that there is no shift in the signal from the basal peak (6.38; 2θ) after the incorporation of silver nanoparticles into the clay.

In another study, the antimicrobial capacity of this clay was determined with 5% silver nitrate against Salmonella spp. A food-borne pathogenic microorganism was used, such as Salmonella spp. CECT

ES 2 331 284 A1

554, which was obtained from the Spanish Collection of Type Crops (Valencia, Spain). The study conditions were set in the use of the bacterium in the medium exponential phase and with an initial concentration of the microorganism of approximately 10⁵ CFU / mL. The experimental part was carried out using an adaptation of the macrodilution method established for the determination of the bactericidal activity of antimicrobial agents approved in 1999 by the National Committee for Clinical Laboratory Standards. According to this method, 100 mg of the clay having a final concentration of silver nitrate of 5% was introduced into a sterile tube containing 10 mL of Mueller Hinton Broth (MHB) culture broth. Subsequently, and after 5 h, the tube was inoculated with 0.1 mL of a culture of Salmonella spp. under the conditions described above. At the same time, a tube containing sample without silver and another without sample was inoculated, both would serve as controls. Once the samples were inoculated, all tubes were incubated at 37 ° C for 24 hours. Then 0.1 mL of each sample was seeded on Tryptone Soy Agar (TSA) plates. After 24 hours of incubation at 37 ° C, the viable cells were counted on the plate. Except for both controls, a significant reduction in the number of viable (&gt; 99.9%) was obtained after incubation at 37 ° C for 24 h (see Table 4).

TABLE 4

<td>Sample</td><td>Initial CFU / mL</td><td>Final CFU / mL</td>

<td>Control without sample</td><td> 7,0* 10^b</td><td> 3,8* 10⁹</td>

<td>Control of clay of the same montmorillonite type without modification</td><td> 7,0* 10⁵</td><td> 2,0* 10⁹</td>

<td>Montmorillonite-type clay modified with 5% AgNO₃</td><td> 7,0* 10⁵</td><td> &lt; 1</td>

Example 4

Synthesis and intercalation of metallic silver nanoparticles in kaolinite-type clays pretreated with dimethylsulfoxide, using UV radiation as a reducing agent. Initially, the kaolinite clay was pretreated with dimethylsulfoxide to increase the interlaminar space. For this, 60 g of clay were dispersed in 300 ml of dimethylsulfoxide, and kept under magnetic stirring and at 65 ° C for 24 h. Subsequently, the clay was filtered by suction, washed with methanol and dried in a convection oven at 80 ° C for 6 hours.

Once dry, the clay pretreated with dimethylsulfoxide is dispersed in water at ambient conditions, at a ratio of 1 g of clay per 100 g of solvent, and then 0.05 g of AgNO were added.₃. The dispersion was maintained under vigorous and constant magnetic stirring under a source of UV radiation of 30 W and 235 nm wavelength. The exposure time to UV radiation was 24 h, after which the solid was filtered by suction and dried in a convection oven at 70 ° C for 1 h. The TEM image of Figure 6 shows an average size of reduced silver particles of 15 nm, and that these are found on the surfaces and edges of the clay sheets. In another study, the antimicrobial capacity of this clay pretreated with DMSO and modified with 5% silver nitrate against Salmonella spp. A food-borne pathogenic microorganism was used, such as Salmonella spp. CECT 554, which was obtained from the Spanish Collection of Type Cultures (Valencia, Spain). The study conditions were set in the use of the bacterium in the medium exponential phase and with an initial concentration of the microorganism of approximately 10⁵ CFU / mL. The experimental part was carried out using an adaptation of the macrodilution method established for the determination of the bactericidal activity of antimicrobial agents approved in 1999 by the National Committee for Clinical Laboratory Standards. According to this method, 100 mg of the DMSO pretreated clay having a final silver concentration of 5% was introduced into a sterile tube containing 10 mL of Mueller Hinton Broth (MHB) culture broth. Subsequently, and after 5 h, the tube was inoculated with 0.1 mL of a culture of Salmonella spp. in. the conditions described above. At the same time, a tube containing a clay sample of the same type pretreated with dimethylsulfoxide but without silver and another without sample was inoculated, both would serve as a control. Once the samples were inoculated, all tubes were incubated at 37 ° C for 24 hours. Then 0.1 mL of each sample was seeded on Tryptone Soy Agar (TSA) plates. After 24 hours of incubation at 37 ° C, the viable cells were counted on the plate. Except for the two controls, a significant reduction in the number of viable (&gt; 99.9%) was obtained (see Table 5).

TABLE 5

<td>Sample</td><td>Initial CFU / mL</td><td>Final CFU / mL</td>

<td>Control without sample</td><td> 7,0* 10⁵</td><td> 3,8 * 10⁹</td>

<td>Kaolinite-type clay control</td><td> 7,0* 10⁵</td><td> 3,9* 10⁹</td>

ES 2 331 284 A1

<td>pretreated with dimethylsulfoxide, unmodified with hexadecyltrimethylammonium bromide</td><td></td><td></td>

<td>Kaolinite type clay pretreated with dimethylsulfoxide and modified with 5% AgNO₃.</td><td> 7,0* 10⁵</td><td> &lt; 1</td>

Example 5

Preparation of polylactic acid films with 10% kaolinite type clay pretreated with dimethylsulfoxide and intercalated with silver nanoparticles. Initially, a dispersion in chloroform at 10% by weight (with respect to the dry weight of polymer) of clay pretreated with dimethylsulfoxide and to which silver nanoparticles had been intercalated under the method of reduction with UV radiation was prepared. Subsequently, the polymer (polylactic acid) was added at 5% by weight in the clay dispersion in chloroform. A polylactic acid / silver nanoclay nanocomposite film was obtained by evaporation of the solvent at ambient conditions, a process called "casting". These nanocomposites were characterized by studying their morphology by transmission electron microscopy (TEM, see Figure 7), as well as their water vapor barrier and antimicrobial properties. Additionally, the water permeability (see Figure 8) of this film of polylactic acid and 10% by weight of clay with antimicrobial properties was studied, using the ASTM E96 standard, at 25 ° C and 75% relative humidity. The addition of antimicrobial clay to the polymeric matrix causes a reduction in permeability of 26.8%, which is why the composite material presents a better barrier to water than pure polylactic acid.

To evaluate the antimicrobial capacity of the PLA films, 600 mg of film were weighed, both from the control without clay and from the sample with antimicrobial clay, and were introduced into 10 mL of sterile culture medium. They were stored at 4 ° C for four weeks, prior to their inoculation with Salmonella spp. Considering that the films contained 10% clay, and in turn that clay contained 5% silver nitrate, the final concentration of silver nitrate that has been used is 300 ppm, being the minimum bactericidal concentration (in this case, reduce the population to zero) of Salmonella around 100 ppm. The films contain an amount of silver 3 times higher than the bactericidal dose when used in suspension. After four weeks of storage and continuous release, the controls show an increase in the number of viable ones of three orders of magnitude, while in the sample of PLA film with 10% clay interspersed with silver, the viable ones are reduced by three orders of magnitude. magnitude (see Table 6).

TABLE 6

<td>Sample</td><td>Initial CFU / mL</td><td>Final CFU / mL</td>

<td>Control without sample</td><td> 2,0* 10⁵</td><td> 4,7* 10⁸</td>

<td>PLA film control (without clay)</td><td> 2,0* 10^b</td><td> 6,6* 10⁸</td>

<td>PLA film with 10% kaolinite pretreated with dimethylsulfoxide and interspersed with silver nanoparticles.</td><td> 2,0* 10⁵</td><td> 3,5* 10²</td>

Example 6

Preparation of chitosan films with 10% kaolinite type clay pretreated with dimethylsulfoxide and intercalated with silver nanoparticles. Initially, a 0.9% solution of chitosan in 1% acetic acid is prepared at 70 ° C. This solution was filtered and a dispersion in water at 10% by weight (with respect to the dry weight of polymer) of kaolinite type clay pretreated with dimethylsulfoxide and intercalated with silver nanoparticles was added under vigorous stirring. It is poured into Petri dishes and the solvent is allowed to evaporate at ambient conditions. A chitosan nanocomposite / silver nanoclay film was obtained by the process called "casting". To evaluate the antimicrobial capacity of the chitosan films, different portions of films were weighed and stored at 4 ° C for 12 hours before inoculation with Salmonella spp. The weights used were: 25, 50 and 75 mg of film, which were placed in 10 mL tubes with sterile culture medium. The chitosan films contained 10% clay, which in turn contained 5% silver, so the final concentrations of silver nitrate used are as follows: 25 mg of chitosan film contained 0.125 mg of silver nitrate ; 50 mg of film contained 0.25 mg of silver; and 75 mg of film contained 0.375 mg of silver. The results of table 7 show that the chitosan control films (without clay) have a certain antimicrobial activity, since as the weight of the film increases, the number of viable bacteria decreases, with 75 mg of film being sufficient quantity to totally inhibit bacterial growth in the

ES 2 331 284 A1 medium to the given conditions. Samples with 10% clay have a greater capacity to inhibit growth, with 25 mg of film the number of viable is reduced by three orders of magnitude and from 50 mg of film total inhibition of growth is obtained.

TABLE 7

<td>Sample</td><td>Initial CFU / mL</td><td>Final CFU / mL</td>

<td>Control without sample</td><td> 2,0* 10^b</td><td> 4,7* 10⁸</td>

<td>Chitosan control without clay (25 mg)</td><td> 2,0* 10⁵</td><td> 3,5 *10⁶</td>

<td>Chitosan control without clay (50 mg)</td><td> 2,0*10^b</td><td> 2,5 *10³</td>

<td>Chitosan control without clay (75 mg)</td><td> 2,0* 10^b</td><td>NG</td>

<td>Chitosan film with 10% clay pretreated with dimethylsulfoxide and interspersed with silver nanoparticles (sample 25 mg)</td><td> 2,0* 10⁵</td><td> 1,9*10²</td>

<td>Chitosan film with 10% clay pretreated with dimethylsulfoxide and interspersed with silver nanoparticles (sample 50 mg)</td><td> 2,0* 10⁵</td><td>NG</td>

<td>Chitosan film with 10% clay pretreated with dimethylsulfoxide and interspersed with silver nanoparticles (sample 75 mg)</td><td> 2,0* 10⁵</td><td>NG</td>

Example 7

Evaluation of the antimicrobial capacity of PVOH and EVOH films with 10% kaolinite type clay pretreated with dimethylsulfoxide and intercalated with silver nanoparticles. A 5% PVOH and EVOH solutions were uniformly incorporated 10% (based on polymer weight) of kaolinite type clay pretreated with dimethylsulfoxide and interspersed with silver nanoparticles. Subsequently, the respective films were obtained by evaporation of the solvent (casting process) and stored in a desiccator at 0% relative humidity and at room temperature.

For the evaluation of the antimicrobial capacity, 100 mg of each of the films obtained were introduced into 10 mL of sterile culture broth and stored at 4 ° C for 72 hours before inoculation with Salmonella spp. In parallel, control tubes without sample and control tubes of PVOH and EVOH films without clays were prepared. A set of samples and controls was additionally prepared under the same conditions and processed immediately, in order to study the release of the antimicrobial agent from the matrices with respect to time. The results in table 8 show that the control films without clays allow the multiplication of the number of viable ones up to two orders of magnitude, regardless of the storage time. EVOH film samples with 10% clay interspersed with silver nitrate reduced the number of viable 100 times at the time of inoculation and then 100 times more after 72 hours of incubation. On the other hand, samples of PVOH films with 10% silver clay showed a reduction of four orders of magnitude in the number of viable at the time of inoculation of the sample and total inhibition after 72 hours of incubation. These results show the bactericidal activity of clays interspersed with silver incorporated in EVOH and PVOH matrices, with the most pronounced antimicrobial effect in the latter polymer.

ES 2 331 284 A1

TABLE 8

<td>Sample</td><td>Initial CFU / mL</td><td>CFU / mL to Oh</td><td>CFU / mL at 72h</td>

<td>Control without sample</td><td> 1,6*10⁶</td><td> 3,9*10⁸</td><td> 4,5*10⁸</td>

<td>PVOH control</td><td> 1,6*10⁶</td><td> 1,3*10⁸</td><td> 2,7*10⁸</td>

<td>EVOH control</td><td> 1,6*10⁶</td><td> 3,6*10⁸</td><td> 1,9*10⁸</td>

<td>PVOH + 10% kaolinite pretreated with dimethylsulfoxide and intercalated with silver nanoparticles</td><td> 1,6*10⁶</td><td> 7,3*10²</td><td>NG</td>

<td>EVOH + 10% kaolinite pretreated with dimethylsulfoxide and intercalated with silver nanoparticles</td><td> 1,6*10⁶</td><td> 3,4*10⁴</td><td> 2,5*10²</td>