EP4680030A1

EP4680030A1 - Functional clays with controlled release of natural additives

Info

Publication number: EP4680030A1
Application number: EP24709789.2A
Authority: EP
Inventors: Cástor SALGADO SONEIRA; José Francisco FERNÁNDEZ LOZANO; Julián JIMÉNEZ REINOSA; Daniel ALONSO DOMINGUEZ
Original assignee: Consejo Superior de Investigaciones Cientificas CSIC
Current assignee: Consejo Superior de Investigaciones Cientificas CSIC
Priority date: 2023-03-13
Filing date: 2024-03-12
Publication date: 2026-01-21
Also published as: WO2024189037A1; CN121218876A

Abstract

The invention relates to compositions comprising one or more essential oils (EOs). Further disclosed are processes for generating said compositions and incorporating the same in a substrate, a material incorporating said compositions and uses thereof.

Description

FUNCTIONAL CLAYS WITH CONTROLLED RELEASE OF NATURAL ADDITIVES

DESCRIPTION

TECHNICAL FIELD OF THE INVENTION

The invention relates to compositions comprising essential oils (EOs) deposited onto a nanoclay. The compositions of the invention are particularly useful in agriculture and food industry.

BACKGROUND OF THE INVENTION

The development of active composite materials is a promising strategy in the packaging industry for improving the physical properties of the package, such as mechanical strength, thermal stability or barrier properties.

Some of the most widely used materials in this sector are polymers such as polyethylene (PE), polypropylene (PP), polystyrene (PS), polyvinyl (PVC) and polyethylene terephthalate (PET). The main functional applications that are demanded in the plastics sector intended for agriculture and food industry are the repellent capacity against pests, the photostability against UV radiation and the effective incorporation of additives to the packaging that allow the conservation of the product without altering its organoleptic properties.

Antimicrobial essential oils provide a great opportunity for the development of these additives, since they are generally non-toxic and because they have important properties for food preservation. Some essential oils are antimicrobial and antioxidant and can be used to produce active antimicrobial or high-performance antioxidant packaging.

However, an important drawback in the use of essential oils is their high volatilization rate (the speed with which essential oils evaporate when they come into contact with air) that affect durability. Another important limitation in the use of essential oils is the need to use a reduced dose to avoid annoying side effects, but their inability to dilute in water strongly restricts their possible applications. One of the trends to solve these limitations is the incorporation of essential oils in polymer matrices for packaging. However, if pure oil is added during the manufacture of conventional packaging, it will be volatilized and degraded during the thermal process, generating significant mass loss and compromising the antimicrobial function. The main causes of decomposition are the high shear rates and high temperatures applied during processing in the manufacture of packaging. The polymers typically used for active packaging such as PP or LDPE require processing temperatures > 150°C to develop materials with desired properties or shape. In this scenario, the use of natural additives (such as essential oils) mixed with the polymer pellets would provide active properties to the final material. Nevertheless, the evaporation of volatile compounds during thermoplastic processing would cause the formation of bubbles in the final product, worsening its physical properties.

Some studies have shown that these compounds can be protected via encapsulation using different encapsulating compounds, such as liposomes, solid-lipid nanoparticles, nano-emulsions, cyclodextrins, and nanostructured lipid nano-carriers [Zanetti, et al.; Trends in Food Science & Technology, 2018, 81 , 51-60.].

EP 2 301 368 A 1 discloses antimicrobial compositions for preventing microbial spoilage of food which comprise (i) an organic acid such as tartaric acid or fumaric acid and (ii) an essential oil such as cinnamon or oregano oil.

Document WO 2006/000032 A1 discloses antimicrobial packaging material comprising 0.05 - 1.5 wt.% of an essential oil such as thymol or carvacrol in LDPE.

Polymer based food packaging materials which comprise silicate clays onto which essential oils or their components have been absorbed have also been reported.

De Oliveira et al., disclose compositions comprising plant essential oils (e.g. oregano oil, thyme oil), absorbed onto the silicate clay mineral montmorillonite and their incorporation into either synthetic or natural polymers [Colloids & Surfaces B: Biointerfaces, 2021 , 209, 1 12186], Shemesh et al., describe antimicrobial active films based on low-density polyethylene (LDPE), organo-modified montmorillonite clays (MMT) and carvacrol essential oil [Polym. Adv. Technol., 2014, 26, 110-116], lamareerat et al., relates to a reinforced cassava starch based edible film incorporated with cinnamon essential oil and sodium bentonite nanoclay as food packaging material [J. Food Sci. & Technol., 2018, 55, 1953-1959], Document US 2015/257381 A1 discloses antimicrobial films based on the organically modified clay cloisite and thymol. Document EP 3 395 170 A1 discloses degradable packaging film for fruit and vegetables which comprises a polyolefin matrix incorporating an essential oil (especially cinnamon or oregano oil) which has been encapsulated in a cyclodextrin or silica clay. Komadel et al. teaches the use of mineral acids to activate clay materials including bentonite and sepiolite, by replacing divalent calcium and leaching out ferric, ferrous and aluminium ions thus increasing the specific surface area and porosity of the clay [Appl. Clay Sci., 2016, 131 , 84-99],

Giannakas et al., presented a method for the preparation of sodium exchanged montmorillonite (NaMt) and commercial organically modified montmorillonite (OrgMt) hybrids with essential oils (EO) such as oregano oil, thyme oil and basil oil for controlled release applications. The authors found that the adsorption of EO in the NaMt and OrgMt clays led to release of EO molecules at temperatures above 220 °C by strong bonding between the surface hydroxyl groups of the clay and the EO, [Giannakas; A. et al.; Appl. Clay Sci., 2017, 146, 362-370], The presence of organic amino chains in OrgMt by using dimethyl dialkyl ammonium compound (to compatibilize the surface of hydrophilic nanoclays with the hydrophobic polymer matrices) reduces the thermal stability of the hybrids due to a weak interaction of the EO with the clay surface. Thus, this method does not solve the problem of EO incorporation into a polymeric matrix. These materials also fail to solve solvent immiscibility issues of the EO and prolonged stability in contact with air.

U.S. patent application publication no. US 2018112068 proposes the use of halloysite nanotubes (HNTs), which are natural aluminosilicate clay minerals, as nanoscale carriers for carvacrol. The entrapment of carvacrol within HNTs allowed the incorporation of this highly volatile essential oil into different polymer matrices. Similarly, US 2016007591 A1 refers to polymeric packaging materials combining halloysite nanotubes (HNTs) having ethylene removal and barrier properties and HNTs loaded with essential oils as antibacterial agents.

However, although these approaches slow out-diffusion of essential oils (EOs) to some extent, they are not able to prevent the EOs from being released by chemical contact into a liquid medium when embedded in a polymer matrix. This problem can make it difficult to produce suitable packaging for specific foods, as the active compounds in EOs can migrate into the food products and cause organoleptic modification of food or toxicity. On the other hand, the use of EO absorbed in clays and regardless of the absorption method shows limitations in long-term stability when exposed to air and requires nonaqueous solvents for safe dosage due to water/oil immiscibility problems. Therefore, there is a need in the art to provide new compositions that allow to improve the physico-chemical properties, such as thermal stability, of essential oils, as well as to achieve a safe and controlled release.

FIGURES

Figure 1 is a scheme illustrating the encapsulation of essential oils (EOs) in a nanoclay according to the invention and the incorporation thereof into a polymeric matrix.

Figure 2: A) FTIR spectra of sepiolite and SEP-OCDA-CI. B) 13C CP-MAS spectra of sepiolite, SEP-OCDA and SEP-OCDA-CI. C) SEM micrographs of sepiolite (1) and SEP- OCDA-CI (2) and TEM micrographs of sepiolite (3) and SEP-OCDA-CI (4). C) TGA weight loss curves of sepiolite and SEP-OCDA-CI. D) DTG curves of sepiolite and SEP- OCDA-CI.

Figure 3: A) FTIR spectra of the bentonite and BNT-FUM-CI. B) SEM micrograph of BNT- FUM-CI sample. C) TGA weight loss curves of bentonite, BNT-FUM and BNT-FUM-CI. D) DTG curves of bentonite, BNT-CI, BNT-FUM and BNT-FUM-CI.

Figure 4: A) FTIR spectra of LDPE-BNT-FUM-CI-1%, LDPE and BNT-FUM-CI films. B) SEM micrograph of LDPE-BNT-FUM-CI-1 %.

BRIEF DESCRIPTION OF THE INVENTION

After extensive research, the present inventors have developed functional clays which allow the effective encapsulation of volatile essential oils (EOs) and increased thermal resistance properties. Advantageously, encapsulated EOs supported in nanoclays showed increased thermal resistance properties and time-controlled release. This improvement made possible its use as antimicrobial product with lasting effectivity or allows their incorporation into a thermoplastic packaging, as LDPE matrix, without any evaporation phenomena affecting the integrity of the extruded material.

Thus, in a first aspect, the present invention refers to a composition comprising:

(i) a plurality of nanoclay particles,

(ii) optionally an organic surfactant,

(iii) at least one solid organic acid, and

(iv) at least one essential oil; wherein the at least one essential oil and the at least one organic acid are deposited on the surface of the nanoclay particles.

In a second aspect, the present invention relates to a composite material comprising the composition of the invention incorporated in a substrate.

In third aspect, the present invention relates to a process for the preparation of a composition of the present invention comprising the steps of:

(a) providing a suspension of nanoclay particles;

(b) optionally adding an organic surfactant to the suspension obtained in step (a) and mixing to obtain an organo-modified nanoclay suspension; and

(c) adding at least one solid organic acid and at least one essential oil to the suspension obtained in step (a) or in step (b) and mixing to deposit the at least one essential oil and the at least one organic acid on the nanoclay; said process optionally further comprising:

(d) filtering the nanoclay composition obtained from step (c), followed by drying the recovered nanoclay composition; and/or

(e) thermally treating the composition obtained in step (d) by calcination in air atmosphere at a temperature of 80 to 250 °C.

In another aspect, the present invention refers to a composition obtainable by the process of the third aspect.

In a further aspect, the present invention relates to the use of a composition as defined above as a bioprotectant. This use can be for crop protection in agriculture; for the protection and preservation of plant seeds; for the preservation of food products, in particular fruits and vegetables or for the preservation of fodder.

These aspects and preferred embodiments are additionally described further down in the description and defined in the claims.

DETAILED DESCRIPTION OF THE INVENTION

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. As used herein, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise.

As defined above, in a first aspect, the present invention relates to a composition comprising:

(i) a plurality of nanoclay particles,

(ii) optionally an organic surfactant,

(iii) at least one solid organic acid, and

Nanoclay

In the context of the present invention, the term "nanoclay" refers to a wide family of natural silicates being arranged in a fibrillary or layered stacking, which features one or more dimensions, such as length, width, thickness, in the nanometer size.

The basic building blocks of clay minerals are tetrahedral silicates and octahedral aluminum-oxygen-hydroxide sheets. The arrangement of tetrahedral and octahedral sheets gives rise to various classes of clay minerals such as 1 :1 and 2:1. The 1 :1 clay consists of one tetrahedral and one octahedral sheet (e.g., kaolinite and serpentine) whereas the 2:1 clay consists of an octahedral sheet sandwiched between two tetrahedral sheets (e.g., smectite, illite, chlorite and vermiculite). Typical morphology of clay minerals corresponds to a laminar or sheet-like structure. However, different morphologies are possible, such as the fibrous, or rather expressed the needlelike, sepiolite and palygorskite silicates as well as the nanotubular halloysite.

In a particular embodiment, the nanoclay of the invention is a smectite type clay, which is a layered 2:1 phylosilicate with an expandable structure. Non-limiting examples of smectite-type clays are montmorillonite, nontronite, beidellite, volkonskoite, hectorite, bentonite, saponite, sauconite, magadite, kenyaite and vermiculite. In a preferred embodiment, the nanoclay is bentonite.

In another particular embodiment, the clay is a palygorskite-sepiolite type clay, /. e., a fibrous 2:1 phyllosilicate. Non-limiting examples of this group of clays are palygorskite, sepiolite. In a preferred embodiment, the nanoclay is sepiolite. In another particular embodiment, the clay is a kaolinite type clay, i. e., a layered 1 :1 phyllosilicate. Non-limiting examples of this group of clays are kaolinite and serpentinite. In a preferred embodiment, the nanoclay is kaolin.

In a preferred embodiment, the nanoclay is selected from the group consisting of bentonite, sepiolite and mixtures thereof.

In an embodiment, the nanoclay particles have a mean thickness of 1 to 100 nm. The mean thickness can be determined using Scanning Electron Microscopy (SEM). The mean thickness can be calculated from the individual values of at least 100 randomly chosen nanoclay particles.

In a particular embodiment, the mean thickness of the nanoclay layered particles is about 1-60 nm and the longest length typically ranges from 100 nm to 600 nm as determined using Scanning Electron Microscopy (SEM), resulting in aspect ratios ranging between 100 to 600. It should be noted that these mean sizes refer to a mean calculated from the size of the specific dimension being measured of the different particles found in the nanoclay of the invention. The mean sizes can be calculated from the individual values of at least 100 randomly chosen nanoclay particles.

In another particular embodiment, the mean thickness of the nanoclay fiber particles is ranged between 10 to 40 nm and the longest length typically ranges from 200 nm to 1500 nm as determined using Scanning Electron Microscopy (SEM), resulting in aspect ratios ranging between 5 to 150 nm.

In a particular embodiment, the content of the nanoclay in the composition is between 60 to 95% by weight based on the total weight of the composition. Preferably, the content of the nanoclay in the composition is 70 to 90% by weight based on the total weight of the composition.

In addition, due to their structure and physicochemical properties, clay minerals can have different surface charge and exchangeable ions, and are able to interact and intercalate different inorganic and organic substances. These substances can be adsorbed on the surface, edges, or interlayer spaces of clay particles.

In a particular embodiment, the surface of the clay particles is activated by a surface activator or alkali compound. In the context of the present invention, the term “alkali compound” refers to a base which contains a hydroxide or a salt of an alkaline metal used to increase the pH value of the clay in presence of water to a value of pH>9 in order to facilitate the de-agglomeration of the nanoclay particles and to exchange clay structural alkaline-earth cations for alkaline cations.

In a preferred embodiment, the alkali compound is selected from the group consisting of hydroxide or carbonate of sodium, potassium or lithium and mixtures thereof. In a preferred embodiment, the alkali compound is sodium carbonate.

Surfactant

In a particular embodiment, the clay is modified with a surfactant. In the context of the present invention, the term “surfactant” refers to an inorganic or organic molecule used to modify the clay surface by replacing the cations present in the clay through ion exchange. The surfactant may be a cationic, an anionic or a non-ionic surfactant. Nonlimiting examples of inorganic molecules as surfactants are the polyphosphoric acids and their salts such as sodium hexametaphosphate, tetrasodium pyrophosphates, low molecular weight sodium polyacrylate, phosphonic and phosphinic derivatives such as diphosphonic acids and their salts, diphosphonic acids and their salts, as well as phosphonic acid salts. Non-limiting examples of organic surfactants are phospholipid, ammonium or phosphonium surfactants.

In a particular embodiment, the organic surfactant is selected from quaternary ammonium salts that are cationic compounds containing alkyl groups in a chain length of C8-C18, polyamide compounds, polyglycols, silicone oil, polydimethylsiloxanes, organo-metallic compounds, amines and polyamines and any combination thereof. In a preferred embodiment, the organic surfactant is selected from the group consisting of octadecylamine (OCDA), didodecyldimethylammonium bromide (DDAB), sodium dodecyl sulfate (SDS), polypropylene glycol ethoxylated and propoxylated (Ultraric PE 105), silicon-based surfactant (Niax L-595), bis-(2- hydroxyethyl)methyltallowalkylammonium chloride (HMAC), polyoxyethylene octyl phenyl ether (Triton X-100), acid phosphate ester of ethoxylated nonylphenol (Beycostat A B09), lauric arginate (LAE), poly(ethylene glycol) monooleate (PEGMONO), triblock copolymers of poly(ethylene oxide) and polypropylene oxide) (Pluronic), oleic acid (OA), polysiloxane-polyether copolymer, (AK8805), stearic acid (SA), palmitic acid (PA), sorbitan monostearate (Span 60), benzimidazolium-N,N’-hexadecane-2-hydroxy-ethyl bromide (BHHB), poly(ethylene glycol) (PEG), dodecylbenzene sulfonic acid (DBSA), sodium cholate (SC), or mixtures thereof. In a more preferred embodiment, the organic surfactant is octadecylamine (OCDA).

Essential Oil (EO)

The term “essential oil” means natural, volatile oils, which are responsible for many of the fragrances produced by plants. Essential oils (EOs) are mainly composed of lipophilic and highly volatile secondary plant metabolites, primarily monoterpenes and sesquiterpenes; however, other compounds like allyl and isoallyl phenols, along with alkaloids and coumarins, are also common. Essential oils can be pure single compounds or mixtures of compounds. Typically used essential oils, their chemistry and plant families are known in the art.

Plant essential oils may be routinely prepared by the skilled person by employing known methods. Exemplary methods for deriving an essential oil include, but are not limited to, distillation such as steam distillation, solvent extraction, absolute oil extraction, resin tapping, wax embedding or cold pressing. The term "essential oil" also encompasses synthetic variants or mimics of such compounds. The term "essential oil" is also meant to include derivatives thereof, including racemic mixtures, enantiomers, diastereomers, hydrates, salts or solvates, thereof. When the essential oil is a mixture of compounds, the present invention encompasses each of the constituent compounds of the essential oil.

Non-limiting examples of essential oils include lavender (Lavandula angustifolia), peppermint (Mentha piperita), tea tree (Melaleuca alterni folia), clove (Syzygium aromaticum), sage (Salvia officinalis), cinnamon (Cinnamomum zeylanicum), eucalyptus (Eucalyptus globulus), lemongrass (Cymbopogon flexuosos), and origanum vulgar.

In a particular embodiment, the essential oil is selected from clove, cinnamon, mint, red thyme, oregano, rosemary, citronella, geraniol, strawberry oil or a combination thereof.

In a particular embodiment, the at least one essential oil is deposited on the nanoclay in the form of drops of nano-size dimensions, particularly, the dimensions are equal to or less than 100 nm, as determined using TEM. In a preferred embodiment, the essential oil nanodrops have a mean size equal to or less than 80 nm, preferably equal to or less than 60 nm as determined using TEM. In a particular embodiment, the essential oil nanodrops have a mean size between 5 and 80 nm, and even more preferably between 10 and 60 nm. The individual nanodrops deposited on the nanoclay of the invention can present sizes lower or higher than these mean values, provided that the mean size of the nanodrops deposited on the nanoclay of the invention is within the above specified mean values. Preferably, the size of 95%, 90%, 80% or 70% of the deposited nanodrops falls within these mean values.

In a particular embodiment, the essential oil is present in an amount of between 0.1 and 25% by weight based on the total weight of the composition. In a more particular embodiment, the essential oil is present in an amount of between 2 and 20% by weight based on the total weight of the composition. In another more particular embodiment, the essential oil is present in an amount of between 4 and 16% by weight based on the total weight of the composition. The content of essential oil in the composition may be determined by thermogravimetric analysis (TGA). In a particular embodiment, the content of essential oil in the nanoclay composition is measured by TGA taking into account mass losses at 220-700 °C (heating up to 900°C with heating rate of 10°C/min in air atmosphere) by comparison with pure nanoclay thermographs).

Organic acid

The compositions of the present invention comprise at least one solid organic acid. The term solid in the context of organic acids of the present invention is meant to refer to an acid which is in a solid state at room temperature (i.e. about 25° C). Non-limiting examples of solid organic acids include sorbic acid, lactic acid, ascorbic acid, oxalic acid, tartaric acid, citric acid, adipic acid, succinic acid, maleic acid or fumaric acid. In a particular embodiment, the organic acid is selected from the list of acids approved as food additives and registered as E-additives for the EFSA.

In a particular embodiment, the at least one solid organic acid may be selected from the group consisting of: aliphatic, cycloaliphatic and aromatic carboxylic acids and derivatives thereof. In a particular embodiment, the at least one solid organic acid is a Ci-Ce linear or branched aliphatic carboxylic acid. In another particular embodiment, the organic acid is a dicarboxylic, tricarboxylic or tetracarboxylic acid. In another particular embodiment, the organic acid is selected from the group consisting of sorbic acid, benzoic acid, dihydroacetic acid, lactic acid, ascorbic acid, erythrobic acid, oxalic acid, tartaric acid, citric acid, adipic acid, succinic acid, nicotinic acid, tetracetric ethylene diamine acid, thiodipropionic acid, phytic acid, alginic acid, glutamic acid, guanylic acid, inosinic acid, cyclamic acid, cholic acid, maleic acid, fumaric acid and mixtures thereof. In a more particular embodiment, the solid acid is fumaric acid, citric acid, tartatic acid or mixtures thereof. In a preferred embodiment, the solid acid is fumaric acid. In another preferred embodiment, the solid acid is citric acid. In a further preferred embodiment, the solid acid is tartaric acid.

In a particular embodiment, the organic acid has a melting point of at least 50 °C, at least 75 °C, at least 100 °C or at least 150 °C. The melting point is the temperature where the solid-liquid phase change occurs. Measurement of a solid compound's melting point is a standard practice in the organic chemistry laboratory, and organic acids melting points can be found in reference books, such as The Engineering ToolBox (2017). Melting points of Hydrocarbons, Alcohols and Acids. [online] Available at: https://www.engineeringtoolbox.com/melting-temperature-hydrocarbons-alkane-alkene- benzene-aromatic-alcohol-acid-naphthalene-d_1965.html [Accessed 11 march 2024) or in the Material Safety Data Sheet of the organic acids.

In a particular embodiment, the at least one organic acid has a solubility in water at 20 °C of less than 1 g/L, less than 5 g/L, less than 10 g/L, less than 50 g/L, less than 100 g/L, less than 500 g/L or less than 1000 g/L.

In a particular embodiment, the at least one organic acid has a solubility in water at 20 °C of at least 1 g/L, at least 5 g/L, at least 10 g/L, at least 50 g/L, at least 100 g/L, at least 500 g/L or at least 1000 g/L.

In a particular embodiment, the organic acid is a combination of at least one organic acid with a solubility in water at 20 °C of at least 500 g/L, and at least one organic acid with a solubility in water of less than 50 g/L.

Advantageously, the authors of the present invention have found that depending on the solubility of the organic acids, the release mechanism of the essential oils can be modulated in time

In a particular embodiment, the at least one organic acid is present in an amount of between 1-8% with respect to the total weight of the composition. In a more particular embodiment, the organic acid is present in an amount of between 1-5 % with respect to the total weight of the composition. The content of organic acid is determined by TGA. In a particular embodiment, the content of organic acid in the nanoclay composition is measured by TGA taking into account mass losses at 350°C and 560°C (heating up to 900°C with heating rate of 10°C/min in air atmosphere) by comparison with pure nanoclay thermographs).

In a particular embodiment, the organic acid is adhered to the essential oil, preferably the essential oil nanodrops, deposited on the nanoclay. In a preferred embodiment, the organic acid encapsulates the essential oil nanodrops deposited on the nanoclay surface, as determined using TEM.

The present inventors have now surprisingly and advantageously found, the release of an essential oil from the composition of the invention is substantially delayed when said essential oil is deposited together with an organic acid. In addition, the essential oil thermal stability is increased by encapsulation in the compositions of the invention. Without wishing to be bound by theory, the authors believe this greater efficiency in the encapsulation and sustained release of the essential oil is caused by the positive interaction between the polar groups of the acid and the essential oil components as well as the physical characteristics of the acids (solid at room temperature), which interact with the essential oil deposited on the clay.

The term "sustained release" is used in a conventional sense relating to a gradual release of a compound during a period of time and preferably, although not necessarily, with relatively constant compound release levels over a long period of time.

Alternatively or additionally, the compositions of the present invention can be characterized by their essential oil release rate when the release of essential oil is measured according to UV-vis spectroscopy applying the Lambert-Beer law by using the molar absorptivity coefficient of the specific component in the essential oil in methanol, under accelerate aging conditions on exposure to air at 80 °C. More specifically, the release rate may be measured under the accelerated aging conditions according to Comparative Example 1 below.

In an embodiment, for the same aforementioned experimental conditions, the essential oil comprised in the composition of the present invention is released in an amount of less than 20%, by weight, preferably less than 15% by weight, more preferably less than 10% by weight, still more preferably less than 5% by weight, even more preferably less than 1 % by weight relative to the total essential oil contained in the composition, within 10 days of exposure to air at 80°C. In an embodiment, for the same aforementioned experimental conditions, the essential oil comprised in the composition of the present invention is released in an amount of less than 20%, by weight, preferably less than 15% by weight, more preferably less than 10% by weight, still more preferably less than 5% by weight, even more preferably less than 1 % by weight relative to the total essential oil contained in the composition, within 15 days of exposure to air at 80°C.

In a preferred embodiment, the composition of the invention comprises a bentonite nanoclay in a 77-94% by weight, fumaric acid in a 1-3% by weight, and 5-20% by weight essential oil, all weight percentages based on the total weight of the composition. In a more preferred embodiment, the essential oil is selected from clove oil or oregano oil.

In a preferred embodiment, the composition of the invention comprises a bentonite nanoclay in a 77-94% by weight, citric acid in a 1-3% by weight, and 5-20% by weight essential oil, all weight percentages based on the total weight of the composition.

In a preferred embodiment, the composition of the invention comprises a bentonite nanoclay in a 77-94% by weight, tartaric acid in a 1-3% by weight, and 5-20% by weight essential oil, all weight percentages based on the total weight of the composition.

In a preferred embodiment, the composition of the invention comprises a sepiolite nanoclay in a 80-97% by weight, octadecylamine (OCDA) as organic surfactant in a 1- 5% by weight; fumaric acid in a 1-5% by weight, and 1-10% by weight essential oil, all weight percentages based on the total weight of the composition. In a more preferred embodiment, the essential oil is selected from clove oil or oregano oil.

In a preferred embodiment, the composition of the invention comprises a sepiolite nanoclay in a 80-97% by weight, octadecylamine (OCDA) as organic surfactant in a 1- 5% by weight; citric acid in a 1-5% by weight, and 1-10% by weight essential oil, all weight percentages based on the total weight of the composition.

In a preferred embodiment, the composition of the invention comprises a sepiolite nanoclay in a 80-97% by weight, octadecylamine (OCDA) as organic surfactant in a 1- 5% by weight; tartaric acid in a 1-5% by weight, and 1-10% by weight essential oil, all weight percentages based on the total weight of the composition. Composite material

In a second aspect, the present invention relates to a composite material comprising the composition as defined in the first aspect incorporated to a substrate.

As used herein, the term “substrate” refers to such any suitable support material such as organic polymer, inorganic polymer or cellulose based materials. In a preferred embodiment, the substrate is a polymer suitable for forming plastic products by thermoforming. In a more preferred embodiment, the substrate is one of acrylonitrile butadiene styrene (ABS), ethylene-vinyl acetate (EVA), polyethylene, polypropylene, polycarbonate, PVC, acrylic polymers (such as PMMA), polyethylene terephthalate (PET), polyesters, polyurethanes, polystyrene and biopolymers such as polylactic acid (PLA), polycaprolactone (PCL), polysaccharides, polyvinyl alcohol (PVA), celluloids, thermoplastic starch, polyterpenes, polyhydroxyalkanoates, polybutylene succinate (PBS), as well as their mixtures and combinations.

In an even more preferred embodiment, the substrate is selected from polyethylene (PE), low-density polyethylene (LDPE), high-density polyethylene (HDPE), polylactic acid (PLA), polybutylene adipate terephthalate (PBAT) and polypropylene (PP) polymers. In a most preferred embodiment, the substrate is low-density polyethylene (LDPE).

In a particular embodiment, the nanoclay composition is incorporated in at least a portion of the substrate. In a preferred embodiment, the nanoclay composition is homogeneously distributed in the substrate.

In a particular embodiment, the content of the nanoclay composition in the composite material is between 0.01 and 25% by weight based on the total weight of the composite material. In a preferred embodiment, the content of the nanoclay composition in the composite material is 0.2%, 0.5%, 1 %, 2% or 5% by weight based on the total weight of the composite material.

In a particular embodiment, the composite material can further comprise additives, such as a tackifier, a filler, an antioxidant, a thickener, a colouring agent or a photostabilizing agent (e. g., HALS o hindered amine light stabilizers).

In a particular embodiment, the composite material of the invention is suitable for forming plastic products by extrusion, preferably, it is suitable for forming films by extrusion. The present inventors have surprisingly found that encapsulation of essential oils along with a solid organic acid in the nanoclay compositions of the invention allows their incorporation into a thermoplastic packaging, such as a LDPE matrix without any evaporation phenomena affecting the integrity of the extruded material. Advantageously, the inventors have also found that encapsulation of essential oils with a solid organic acid in the nanoclay compositions of the invention favors the essential oil’s vapor controlled release and inhibits the liquid or solid migration. Therefore, it results in an advantage over the state of the art because it does not present migration both in contact with liquids and in contact with solids and thus allows its use in contact with food, for example, without altering its organoleptic properties.

The present invention further relates to a process for preparing a composite material comprising mixing a nanoclay composition with a substrate material and meltcompounding the mixture. Preferably, the substrate and the nanoclay composition are mixed in a mass ratio of 99:1 to 75:25. In a preferred embodiment, the mixture is compounded by hot-melt extrusion, preferably at a temperature of 100 to 250 °C.

Process

A third aspect of the present invention is directed to a process for preparing the composition of the invention, wherein said process comprises the following steps:

(a) providing a suspension of nanoclay particles;

(e) thermally treating the composition obtained in step (d) by calcination in air atmosphere at a temperature of 80 to 250 °C. In a particular embodiment, the nanoclay particles of step (a) are suspended in water or in an aqueous solution. In a particular embodiment, the suspension may comprise one or more additives such as deflocculants, pH adjusting agents, surface activators, antimicrobial agent. In a particular embodiment, the concentration of the nanoclay in the suspension is from 1 to 70% w/v, more preferably from 5 to 50% w/v. In a particular embodiment, the suspension comprises 3-5% of sodium carbonate as a surface activator of the nanoclay.

In another particular embodiment, step (a) comprises suspending a clay raw material in water and subjecting said raw clay material to a delamination treatment to maximize dispersion of the clay particles. For the purposes of the present invention, the term “delamination” is also meant to refer to “defibrillation” in the context of a fibrous clay. The skilled person in the art will readily understand if a specific clay undergoes delamination (e. g., bentonite) or defibrillation (e. g., sepiolite) based on its morphology.

Delamination (or defibrillation) of the clay can be achieved in a variety of ways. The delamination can be a chemical, physical or mechanical delamination or a combination thereof. Preferably, the delamination is a mechanical delamination. Mechanical delamination comprises for instance subjecting the clay raw material to agitation, mixing (e. g., hand mixer, cowles mixer, extruder, high shear mixer), sonication, milling (e.g. ball milling) or grinding (e.g. ring milling, hammer milling).

In a preferred embodiment, the delamination is performed by mechanical mixing at 600 to 1800 rpm. Although it will depend on the amount of clay to be delaminated, the delamination is preferably carried out in an interval from 5 to 180 min, and more preferably from 10 to 30 min.

In a particular embodiment, the nanoclay of step (a), before or after delamination, is a layered nanoclay as described further above with a mean thickness between 1-60 nm and longest length between 100-600 nm.

In a particular embodiment, the nanoclay of step (a), before or after delamination, is a fibrillar nanoclay as described further above with a mean thickness between 10 to 40 nm and a longest length between 200 nm and 1500 nm.

In a particular embodiment, the method comprises a step (b) of adding an organic surfactant to the suspension obtained in step a) and mixing to obtain an organo-modified nanoclay suspension. The surfactant used in step (b) can be any suitable surfactant known in the art. Preferably, the surfactant used is an organic surfactant as described above. In a particular embodiment, the surfactant is selected from the group consisting of quaternary ammonium salts. In a most preferred embodiment, the surfactant used is octadecylamine (OCDA).

The surfactant may be added directly within a few seconds to the suspension prepared in step (a) or can be added slowly to the aqueous suspension prepared in step (a), with constant stirring of the suspension during the entire time the addition takes place. In one embodiment of the present invention, the surfactant is added to the suspension prepared in step (a) of the method at a concentration range between 0.1 and 20% by weight, and more preferably between 5 and 10% by weight based on the total weight of the nanoclay particles in the suspension. In a particular embodiment, the organic surfactant is dissolved in a solvent prior to being added to the suspension in step (a). In a more particular embodiment, the organic surfactant is added as an aqueous solution.

The method of the present invention comprises a step (c) of adding at least one solid organic acid and at least one essential oil to the suspension obtained in step (a) or in step (b) and mixing to deposit the at least one essential oil and the at least one organic acid on the nanoclay. The essential oil and the organic acid can be added directly and within a few seconds to the suspension obtained in step (a) or (b) or can be added slowly with constant stirring of the suspension during the entire time the addition takes place. In a particular embodiment, the at least one essential oil and the solid organic acid are added simultaneously. In another particular embodiment, the essential oil and the organic acid are added in sequence. In a preferred embodiment, step (c) comprises adding, firstly, the organic acid and, secondly, the essential to the nanoclay suspension of step (a) or step (b).

The at least one essential oil added in step (c) may be added as a solution of the essential oil diluted in a solvent. Any suitable solvent for the dilution of a specific essential oil can be used. In a particular embodiment, the solvent is an organic solvent, preferably selected from methanol, ethanol, isopropanol. In a most preferred embodiment, the organic solvent is ethanol.

In one embodiment of the present invention, the at least one essential oil is added to the suspension prepared in step (a) or (b) of the method at a concentration ranging from between 5-50% by weight, and more preferably between 10-20% by weight based on the total weight of the nanoclay particles in the suspension.

In a particular embodiment, the at least one organic acid in step (c) is added in solid form. In another particular embodiment, the organic acid in step (c) is added as an aqueous solution, preferably in a concentration of 25-125% w/v. In a particular embodiment, the at least one organic acid is added to the suspension prepared in step (a) or (b) of the method at a concentration ranging from between 0.2 and 20% by weight, and more preferably between 1 and 10% by weight based on the total weight of the nanoclay particles in the suspension.

In a preferred embodiment of step (c) of the method of the invention, the at least one essential oil and the nanoclay are in a mass ratio in the suspension between 0.005:1 to 5:1 , preferably in a mass ratio between 0.01 :1 and 1 :1 more preferably in a mass ratio of 0.25:1.

In a particular embodiment, step (c) is performed at a temperature between room temperature and 100 °C, preferably at a temperature between 40 and 80 °C.

In a particular embodiment, the method of the present invention further comprises a step

(d) of filtering the nanoclay composition obtained from step (c), optionally subsequent washing with water, preferably distilled water, followed by drying the recovered nanoclay composition, preferably in an air atmosphere and at a temperature between 40 and 100°C. In a particular embodiment, the washing is performed under continuous stirring, preferably during an interval from 10 to 50 hours. In a particular embodiment, the drying is preferably carried out in an interval from 10 to 100 hours, and more preferably from 24 to 48 hours.

(e) of thermally treating the composition obtained in step (d) by means of a calcination process in an air atmosphere, and at controlled temperatures. The calcination process is preferably carried out at temperatures between 80 and 250°C, and more preferably between 100 and 160°C. Although it will depend on the amount of nanoclay composition to be calcined, this calcination is preferably carried out in an interval from 0.5 to 12 hours, and more preferably from 1 to 8 hours. Optionally, the process comprises a further step of micronizing the composition obtained in step (d) or (e). In a preferred embodiment, the composition obtained in step (d) or (e) is ground and sieved through a 100 micrometers sieve.

In a preferred embodiment, the process comprises: a) providing a suspension of nanoclay particles, preferably sepiolite, by suspending a raw clay material in water and delaminating said raw clay material; c) adding at least one solid organic acid, preferably fumaric acid, and at least one essential oil to the suspension obtained in step (a) and mixing to deposit the at least one essential oil and the at least one organic acid on the nanoclay; d) filtering the nanoclay composition obtained from step (c) and drying the recovered nanoclay composition; and/or e) thermally treating the composition obtained in step (d) by calcination in air atmosphere at a temperature of 80 to 250 °C.

In another preferred embodiment, the process comprises: a) providing a suspension of nanoclay particles, preferably bentonite, by suspending a raw clay material in water and delaminating said clay material;

(b) adding an organic surfactant, preferably octadecylamine, in water under stirring to the nanoclay suspension of step (a) and mixing to obtain an organo- modified nanoclay suspension; c) adding at least one solid organic acid, preferably fumaric acid, and at least one essential oil to the suspension obtained in step (b) to deposit the at least one essential oil and the at least one organic acid on the nanoclay; d) filtering the nanoclay composition obtained from step (c), followed by drying the recovered nanoclay composition; and/or e) thermally treating the composition obtained in step (d) by calcination in air atmosphere at a temperature of 80 to 250 °C.

In a more preferred embodiment, the above processes further comprise micronizing the composition obtained in step (d). In a different aspect, the invention refers to a composition obtainable by a process as defined above in any of its embodiments, in particular in the first embodiment of the process of the present invention.

Preferences for each component of the composition are those described for the composition as defined in the first aspect of the invention.

Uses

In another aspect, the present invention relates to the use of a composition of the invention as a bioprotectant.

In the context of the present invention, the term bioprotectant refers to a substance or composition used to manage pest problems by means of an antimicrobial and/or a pestrepellent activity.

Accordingly, in a particular embodiment, the compositions of the invention are used as a pest repellent agent. As used herein, the term pest repellent is used to refer to a nontoxic substance which has the effect of deterring pests from attacking or even approaching said substance. Pest repellents may function by providing a bad taste (e.g. a bitter taste) or smell, or both, to a pest. Pest repellents may also mimic odours related to predators of said pests. Particular pests against which the compositions of the invention may be used include rodents such as mice, rats, squirrels, and insects, preferably insects attacking crops including, but not limited to, insects from Tribolium spp. such as Tribolium castaneum (red fluor beetle), Tribolium audax (american black flour beetle), Tribolium confusum (confused flour beetle); and insects from Sitophilus spp., such as Sitophilus granaries (wheat weevil), Sitophilus linearis (tamarind weevil), Sitophilus zeamais (Maize weevil), Sitophilus oryzae (rice weevil), or disease-carrying insects including, but not limited to, flies, sand flies, mosquitoes, ticks, and fleas.

In another particular embodiment, the compositions of the invention are used as an antimicrobial agent, i. e., having activity against pathogenic microorganisms. In a more particular embodiment, the antimicrobial activity is antifungal, antiviral and/or antibacterial activity. In a particular embodiment, the compositions of the invention are used as an antimicrobial agent against gram positive bacteria and gram negative bacteria. In a particular embodiment, the composition of the invention is used as and antimicrobial agent against gram positive bacteria of the genus Bacillus, Clostridium, Corynebacterium, Enterococcus, Listeria and Staphylococcus. In a particular embodiment, the composition of the invention is used as an antimicrobial agent against gram negative bacteria selected from the group consisting of Bartonella henselae, Bordetella pertussis, Borrelia burgdorferi, Brucella abortus, Campylobacter jejuni, Escherichia coli, Erwinia amylovora, Francisella tularensis, Klebsiella pneumonia, Haemophilus influenza, Helicobacter pylori, Legionella pneumophila, Leptospira interrogans, and Pseudomonas aeruginosa, Rickettsia rickettsia, Salmonella spp., Treponema pallidum, Vibrio cholera, Yersinia pestis and Xanthomonas citri. In a preferred embodiment, the bacteria are Escherichia coli and Listeria innocua.

The use of the compositions of the invention can advantageously be in agriculture for crop protection. Another use of the compositions of the invention can also advantageously be in the preservation of food products, in particular of fruits and vegetables, or for the preservation of fodder. The use in the preservation of food products may advantageously further provide a fragrance to the product. In a preferred embodiment, the compositions of the invention may be used in a packaging material, preferably for the preservation of food products. Accordingly, the present invention further refers to the use of a composite material according to the invention in a packaging material, preferably for the preservation of food products.

The following examples are merely illustrative of certain embodiments of the invention and cannot be considered as restricting it in any way.

EXAMPLES

Materials and methods

The following materials have been used: Sepiolite and bentonite clays provided by SEPIOLSA; Essential oils of cinnamon, clove, arvensis mint, red thyme, oregano, strawberry aroma and citronella supplied by the Arocival company; Sodium triphosphate (TPF), octadecylamine (OCDA), fumaric acid, and absolute ethanol, methanol, acetone, and hexane HPLC solvents; supplied by Sigma Aldrich; LDPE provided by Encapsulae S.L; Poly (2,6-diphenyl-p-phenylene oxide) (TENAX) supplied by Sigma Aldrich.

The compositions, prepared by any of the Examples described below, were characterized by the following techniques: FTIR: Infrared spectra were recorded using a Perkin Elmer Spectrum 100 FTIR spectrophotometer, with Universal ATR. The data were processed with Spectrum 10 software, from Perkin Elmer.

NMR: Solid-state Cross Polarization Magnetic Angle Spinning C-13 and Si-29 Nuclear Magnetic Resonance (13C/29Si CP/MAS NMR) spectra were obtained using a Bruker AV 400 WB spectrometer.

SEM/TEM: Scanning electron microscopy (SEM) measurements were carried out using a Hitachi S-4700 cold cathode field emission scanning electron microscope (FE-SEM) equipped with Noran EDS and WDS microanalysis systems. The sample preparation was done as follows: 0.01 wt% clay aqueous suspensions were prepared, a drop was deposited on a glass sample holder and the sample was dried in an oven at 60 °C. The samples were plated using an EMScope SC500, depositing a gold layer of about 10 pm by physical vapor deposition. Transmission electron microscopy (TEM) images were obtained in a JEOL model Transmission Electron Microscope JEM 2100. The samples were deposited in 0.01wt% clay aqueous suspension on a sample-holder grid.

DTA/TGA: Thermogravimetric and differential thermal analysis were performed on a TGA-DCS-DTA Q600 equipment from TA Instruments. Tests were performed with approximately 15-20 mg of clay sample under nitrogen gas or 2-3 mg film sample under air and at temperature ramp of about 10 °C/min from 25 to 900 °C. Data were processed with the TA Universal analysis software. TGA-Mass experiments were carried out on a TA Instruments Q500 thermobalance equipped with an EGA oven coupled to a ThermoStar Omnistar GSD 301 O/301T mass spectrometer. Pt sample holders and N2 as purge gas with a flow of 90 mL/min were used at temperature ramp of 10°C/min from room temperature to 600°C.

Example 1 : Octadecylamine (OCDA)-organo-sepiolite modified with clove oil (SEP- OCDA-CI)

In two 5 L beakers, 423 g of raw sepiolite and 8.4 g of sodium carbonate were suspended in 3.3 L of distillated water for each beaker. Both suspensions were mixed with a hand blender for 10 minutes and let stand for 24 hours. Afterwards, the suspensions were stirred with a Cowles mixer at 1200 rpm for 20 minutes and filtered with a 100 pm sieve. The slurries were magnetically filtered and transferred to a 15 L pot equipped with a hot plate, mechanic mixer and an ultra-turrax homogenizer and the suspension was heated up to 60 °C. Then, 23 g of OCDA were suspended in 1 L of distilled water at 70 °C and transferred to the pot under mechanical mixing. Afterwards, 25 g of fumaric acid and 250 g of clove essential oil diluted in 100 g of ethanol were added to the mixture. The slurry was mixed for 2 hours at 60 °C and vacuum filtered with a Buchner funnel to obtain a cake that was dispersed in 5 L of hot distillated water with a hand blender. The mixture was then stirred for 24 h at room temperature, filtered and dried at 80 °C for 48 hours. Finally, the modified clay was grounded, sieved (100 pm) and thermally treated at 120 °C for 3 hours to obtain 537 g of modified sepiolite (SEP-OCDA-CI). Organo-modified sepiolite without essential oil (SEP-OCDA) was similarly synthesized for comparison with the SEP-OCDA-CI sample in the characterization process.

The effect of incorporating clove oil being encapsulated by sepiolite clay is shown in Figure 2. The FTIR spectra (Figure 2A) show a displacement of the Si-O-Si band from 1004 cm^-1 in the sepiolite to 1014 cm^-1 in the modified clay, due to the interaction of the eugenol with the groups Si-OH from sepiolite; and a displacement of the eugenol (-C=C- aromatic) signal from 1609 to 1616 cm-1 in the SEP-OCDA-CI sample, suggesting a positive interaction between eugenol and the polar groups in sepiolite and OCDA.

The SEP-OCDA-CI composition and the corresponding starting materials (SEP-OCDA and clove oil) were analyzed by solid state NMR (Figure 2B). In particular, the 13C-MAS spectrum of the SEP-OCDA-CI shows the methylene groups of the OCDA signals and the appearance of a signal at 30.3 ppm due to oil interaction clove with the methylene groups of the OCDA giving rise to two types of methylene signals, confirming that the nano-deposition of clove oil on the surface of sepiolite is favored by the interaction with the hydrophilic groups of the OCDA.

Sepiolite has a characteristic fibrillary structure and present a good separation of the rods as shown by SEM and TEM micrographs (Figure 2C, 1 and 3) micrographs. Meanwhile the formation of agglomerates is clearly observed in the essential oil modified clay (Figure 2C, 2 and 4) in which the deposition of the organic nanodrops performs an appreciable surface roughness.

The TGA curves show three distinct zones (Figure 2D). From room temperature to 70 °C there is a loss of adsorbed water on the clay surface. Between 70 and 220 °C losses of organic matter bound by weak interactions at the clay surface (including any excess EO not adsorbed to the clay). Above 220 °C the volatilization of the essential oil components adsorbed on the clay takes place (as well as decomposition of the OCDA, loss of bound water and dehydroxylation of the clay around 700 °C). The content of clove oil incorporated in the clay composition was calculated to be a 4%wt from the difference in mass loss in the region of 220-700 °C with respect to the clay without essential oil (SEP- OCDA).

Example 2: OCDA-organosepiolite nanoclay modified with different essential oils

Following the procedure of Example 1 , sepiolite clay was modified with different essential oils (EOs): oregano, citronella, strawberry, rosemary, red thyme, cinnamon and peppermint and the content of essential oil incorporated in the nanoclay studied by thermogravimetry as explained above in Example 1. The results obtained are summarized in Table 1. The columns on the right show the thermal properties of EOs: Degradation initiation temperature (T95%) and temperature of maximum degradation (Tmax).

Table 1 . Weight losses of encapsulated EOs within sepiolite and thermal properties of pure EOs.

In general, modification of sepiolite with the majority of essential oils did not imply changes in the experimental process, with the exception of rosemary and oregano oils. The high volatility of these oils meant that the temperature of the mixture did not reach 70 °C during the modification process. It can be observed that the mass loss between 70 and 220 °C, where desorption of essential oil weakly bound to the clay occurs, is less than 2% for all EOs. Such low desorption values are very advantageous for the subsequent incorporation of the modified clay into a polymeric matrix; since high amounts of volatile elements present during processing would result in a greater tendency to the formation of bubbles in the polymeric film decreasing its final properties.

Example 3. Bentonite nanoclay modified with clove oil (BNT-FUM-CI)

In two 5 L beakers, 411 g of raw bentonite and 8.4 g of sodium carbonate were suspended in 2.9 L of distilled water for each beaker. The both suspensions were mixed with a hand blender for 10 minutes and let stand for 24 hours. Afterwards, the suspensions were stirred with a Cowles mixer at 1200 rpm for 20 minutes and filtered with a 100 pm sieve. The slurries were magnetically filtered and transferred to a 15 L pot equipped with a mechanic mixer and an ultra-turrax homogenizer. Then, 25 g of fumaric acid (FUM) and 250 g of clove essential oil diluted in 100 g of ethanol were added to the mixture. The slurry was mixed for 2 hours and vacuum filtered with a Buchner funnel to obtain a cake that was dried at 80 °C for 48 hours. Finally, the modified clay was grounded, sieved (100 pm) and thermally treated at 120 °C for 3 hours to obtain 537 g of modified bentonite (BNT-FUM-CI). Organo-modified bentonite without clove oil (BNT- FUM) was similarly synthesized for comparison with the BNT-FUM-CI sample in the characterization process.

Firstly, the FTIR spectra shown in Figure 3A shows the disappearance of the bentonite structural water band (3621 cm^-1) in the BNT-FUM-CI composition and the appearance of bands at 2931 cm^-1 , and at 1513 cm^-1 belonging to the group -CH3 and to the aromatic group -C=C- of eugenol. As can be seen from the SEM micrographs in Figure 3B, the modification of the clay and the incorporation of clove oil in BNT-FUM-CI alters the morphology of the bentonite, which shows a more rounded surface in the stack edges and a higher degree of packing This effect is similar to that observed for the sepiolite composition of Example 1 .

The TGA thermograms in Figure 3C show that the encapsulated amount of clove oil is approximately a 15-17 wt%. The presence of fumaric acid favors the clove oil preservation on the bentonite surface. DTG curves of BNT-FUM-CI showing Tmax values corresponding to fumaric acid at 447 °C and 481 °C are presented in Figure 3D.

Example 4: Antimicrobial activity tests

Following the procedure of Example 3, a bentonite nanoclay product including Oregano (thymus capitatus) essential oil in combination with fumaric acid (BNT-FUM-Ore) was obtained. The total amount of Ore in the product was calculated to be 6%wt as measured by thermogravimetric losses at 220-700 °C temperatures.

The effectiveness of the BNT-FUM-Ore product as antimicrobial product was studied by the determination of the MIC methodology (Minimum Inhibitory Concentration). The MIC is defined as the lowest concentration of an antimicrobial agent that inhibits the growth of a microorganism. a. Escherichia coli

For this methodology, Escherichia coli was used as pathogen microorganism in a concentration of 10² cfu/mL in an enriching medium of Tryptone Soy Broth (TSB), 3% wt. The tubes were incubated for 24 hours at 35°C with shaking every half hour. After the incubation time, dilutions were prepared and plated. The tubes with BNT-FUM-Ore and E. coli were seeded in TBX (Tryptone Bile X-Glucuronide Agar) and incubated for 24 hours at 44°C. Different concentrations of BNT-FUM-Ore in distilled water where studied, and the data obtained are summarized in Table 2.

Table 2. BNT-FUM-Ore antimicrobial activity results against E. coli.

The results in Table 2 show that E. coli growth is fully inhibited with BNT-FUM-Ore diluted in water at 3000 ppm (0.5 % wt). The MIC is 3000 ppm. This turns out to be very convenient as pure Oregano essential oil is immiscible with water and its use as antimicrobial agent requires dilution with ethanol. In addition to the flammability of ethanol, its use in contact with plants could cause burn damage to the flowers and plant leaves. Moreover, the durability of diluted essential oil in ethanol is limited in time due the high volatility of such components.

Following the procedure of the Example 3, a bentonite nanoclay product including Thyme oil or Thymol (2-isopropyl-5-methylphenol) essential oil in combination with fumaric acid (BNT-FUM-Thy) was obtained. The total amount of Thymol in the product was calculated to be 6% wt. Following the previous procedure, the MIC for E. coli of BNT-FUM-Thy is 1000 ppm. That fact is convenient because it allows using a reduced dosage of the composition of the present invention. b. Listeria innocua

In another assay, Listeria innocua was used as pathogen microorganism in a concentration of 10² cfu/mL in an enriching medium of Tryptone Soy Broth (TSB), 3% wt. The tubes were incubated for 24 hours at 37°C with shaking every half hour. After the incubation time, dilutions were prepared and plated. The tubes with BNT-FUM-Ore and L. innocua were seeded in MLAB (Microinstant Listeria Agar Base) and incubated for 24 hours at 374°C. Table 3 summarizes the obtained data:

Table 3. BNT-FUM-Ore antimicrobial activity results against L. innocua.

The results in Table 3 show that L. innocua growth is fully inhibited with BNT-FUM-Ore diluted in water at 1000 ppm (0.1 wt.%). The MIC is 1000 ppm. This turns out to be very convenient as the composition of this invention is antimicrobial against both gram positive bacteria as L. innocua and gram negative bacteria as E. coli. Thus the composition signaled BNT-FUM-Ore has a broad antimicrobial effect.

Example 5: Preparation of LDPE/nanoclay composite films

Modified clays LDPE-SEP-OCDA-CI and LDPE-BNT-FUM-CI were embedded in a LDPE matrix. Firstly, the polymer pellets were mixed with the clay (ratio LDPE/clay

90/10) in an acoustic type mixer (Resodyn LabRAMII H) and a masterbatch was prepared by extrusion at 170 °C. Finally, films with a thickness of 90 pm were blown to obtain composites with 3 concentrations of each clay: 0.5 wt%, 1 wt% and 2 wt%.

The thickness of the films was measured with a Mitutoyo Absolute meter, with a resolution of 0.001 mm. 5 measurements were taken in different regions of the film establishing an average value with standard deviation.

LDPE-clay-EO films were further characterized by FTIR as shown in Figure 4A, showing spectra for LDPE, BNT-FUM-CI and LDPE-BNT-FUM-CI. A blue shift of the vibrational mode belonging to the Si-O-Si bond (1013 cm-1) of the nanoclay when embedded in the polymeric matrix can be observed. The clay is well dispersed and is oriented in the direction of the material blowing as can be observed in the SEM micrograph of LDPE- BNT-FUM-CI-1 % in Figure 4B. The presence of encapsulated clove oil into the LDPE films was analyzed by UV-vis spectroscopy. The absorbance at 280 nm in the films was measured and the concentration of encapsulated clove oil into the LDPE matrix was calculated. The thickness of the film was taken into account, as well as the molar absorptivity coefficient of the clove oil in solution. The obtained results were compared with the theoretical amount of clove oil, calculated by the mass losses of the modified clays obtained by TGA. These data are shown in the Table 4.

Table 4. Characterization of the LDPE/clay films.

The amount of clove into the films measured by UV-vis spectroscopy is lower than that calculated theoretically based on the results of thermal degradation of the clays (TGA).

This difference is the percentage of clove oil that has been evaporated during the composite processing. The greater is the amount of clove oil embedded into the polymer matrix, the greater is the amount released during polymer processing. Since the amount of clove oil incorporated in bentonite is higher (16 wt%) than in sepiolite (4 wt%), a higher release rate is achieved in the former case.

Example 6. Pest repellence

By using the BEN-FUM-Ore composition of the Example 3 and the procedure of Example 5, LDPE plastic films with different contents BEN-FUM-Ore contents have been prepared in order to evaluate the barrier effect of the film against a specific pest.

To carry out the tests, three concentrations (1 wt%, 2 wt% and 5 wt%) of BEN-FUM-Ore in LDPE were used, with two different textures, in non-microperforated plastic and in microperforated plastic. As comparative film, pure LDPE film was produced in similar conditions without the incorporation of the composition from the present invention. The film thickness was in all the cases 60 pm, measured with a Mitutoyo Absolute meter as explained in Example 5.

For each of the concentrations and for each type of plastic (microperforated and non- microperforated), a total of 10 repetitions were carried out with their corresponding 10 LDPE films as controls. A flour sack and 20 T. castaneum adult individuals were placed in a glass container, and daily checks were performed for a week. During the period of maintenance of the T. castaneum colonies, the temperature of the climate chamber oscillated between 26°C and 28°C with a relative humidity located in a range between 65%RH and 80%RH, and a photoperiod of 12:12 hours. During each test, the individuals found inside and outside the flour sack were counted to see if the treatments had a repellent effect.

These tests measure the spatial repellence that the insects experience against chemical substances, which results in the insects avoiding contact with the product and thereby in a protection of the food. This repellence drives the insect away or can also induce a response linked to feeding.

Subsequently, using the SPSS statistical analysis program, an ANOVA was carried out on the results obtained from the tests. This allowed to verify the significant differences in the movement of these insects depending on the concentration of the tested materials used and therefore if they are effective as repellents and to what extent The results of a Tukey HSD (Honestly-significant-difference) test comparing the means of the t levels of a factor after having rejected the null hypothesis of equality of means using the ANOVA technique are summarized in Table 5.

Table 5. Values of the homogeneous subsets within the bag (Assumption: greater mobility. Subset for alpha=0.05). In all cases the repetition number is N=10. Sigma for subset 1 was 0.584 and for subset 2 was 1 .000.

Taking as a basis the assumption of comparison with the mean of controls, it can be concluded that the incorporation of the composition of the present invention in the plastic is effective in reducing individuals inside the bags of flour. There are no significant differences between the different amounts of BEN-FUM-Ore 1 wt%, 2 wt %, 2 wt% microperforated, 5 wt% and 5 wt% microperforated, but between them with the control and 1 wt% microperforated.

The product of the present invention thus shows repellency against pests when it is used as a food container.

Example 7. Migration tests

In order to evaluate migration of species from the LDPE-nanoclay films different tests were performed. The Commission Regulation (Ell) No 10/2011 of 14 January 2011 on plastic materials and articles intended to come into contact with food indicates that the overall limit has been 10 mg/dm². Forthat reason, an overall migration assay was carried out on films processed according to Example 5.

Food simulant migration studies were carried out for the LDPE films with a 2 wt% content of BEN-FUM-CI and SEP-OCDA-CL nanoclay compositions. Two Overall migration (OM) tests were performed with food simulants type A (FW/EtOH 10% v/v) and D1 (FW/EtOH 50% v/v). Simulant A is applied to packaging containing hydrophilic foods capable of extracting hydrophilic substances, such as sugars and derivatives, dried fruit in paste form, fresh vegetables, fresh and canned fish, meats or cheeses. Simulant D1 is assigned for foods that have a lipophilic character and are able to extract lipophilic substances. Food simulant D1 shall be used for alcoholic foods with an alcohol content of above 20% and for oil in water emulsions.

Both test consisted of weighing 1 dm² of each film and immersing it completely in the simulant mixture for 10 days at 40 °C in a closed container. Subsequently, the materials were dried in an oven at 40 °C for 24 h and weighed, calculating the difference in mass of each film before and after the test. Regulation (Ell) No. 10/2011 i establishes that in order to be used as food packaging, the overall migration limit is 10 mg/dm² of film. Once the assay has been completed, the washing waters from the type A migration study were analysed (ICP-OES - Termo Jarrel Ash IRIS ADVANTAGE) to determine if there are migrations of inorganic species from the films. The concentrations of some metals were analysed in its oxidized state: Al³⁺, Mg²⁺, Na⁺ and Si⁴⁺.

Table 6 shows the mass losses obtained by the films in the overall (OM) migration tests. The OM tests indicate that these losses are less than 10 mg/dm² of film, therefore the films comply with the migration standard.

Table 6. Overall migration (OM) results for the LDPE films:

The washing waters obtained from the OM with the simulant A assay were analyzed (ICP-OES) to determine if there are migrations of inorganic species from the samples. Films reinforced with sepiolite nanoclay and bentonite nanoclay but without the modification with essential oils were also analysed. The results are shown in Table 7.

Table 7. ICP-OES analysis of main cations expressed in the form of oxides (AI2O3, MgO, Na2O and SiO2) migrated from composite films to the washing waters for the OM assay with a type A simulant. For comparative purposes the corresponding analysis of the used pure water was performed.

The analysis reveals low amounts of inorganic specimens released to the aqueous media. The maximum amount of Al³⁺ (0.07 mg/L) was considerably lower than the 1 mg/L that is allowed by the Regulation No 10/2011. These results can be altered by the presence of other analytes, such as salts dissolved in the distilled water or impurities released from the LDPE matrix. Comparative Example 1 : Air exposure durability experiments

For comparative purposes, clove oil encapsulation experiments were performed in the same experimental conditions as in Example 3 using bentonite clay without any acid treatment or bentonite clays activated with different commercial acids.

The encapsulation efficiency of the differently treated clays was studied under accelerated aging conditions (under air exposure at 80 °C for 15 days).

Approximately, 2 g of each clay were exposed to the air in an oven at 80 °C. Samples were collected at 1 , 5, 10 and 15 days. The release of clove oil was studied by UV-vis spectroscopy using a Perkin Elmer Lambda950 instrument. The main component in clove oil is eugenol (84%) which has a UV absorption band at 280 nm, due to the conjugation of the aromatic ring. The spectra were recorded in absorbance mode between 250 nm and 400 nm with a resolution of 1 nm. The samples were extracted by triplicate with MeOH (HPLC) in sealed flasks by employing a standardized protocol: 1) Magnetic stirring (5 min). 2) Ultrasonication bath (5 min). 3) Magnetic stirring (5 min). Then, the supernatant is filtered and measured with the UV-spectrometer in absorbance mode to calculate the concentration of the EO in the clay. The amount of eugenol in the extracted samples was calculated by interpolating the absorbance at 280 nm in a calibration line obtained from different concentrations of clove oil in MeOH.

Tables 8 and 9 below show the amount of clove oil encapsulated in the compositions (wt%) and the percentage of clove oil lost, respectively, during the accelerated aging tests.

Table 8. Clove oil encapsulation efficiency during the accelerated aging test in bentonite clay (BNT-CI) using different acids.

Table 9. Clove oil losses during the accelerated aging test of BNT-CI with different acids. n.d.l. No detectable loss Pure clove essential oil (CO) fully evaporated at 80°C after 20 hours. At 4 hours, >70% of the CO was evaporated. As consequence, the encapsulation in the nanoclay results in a higher thermal stability. The clays treated with different acids released between 14- 20% of CO after 15 days of aging, with the exception of solid organic acid in which fumaric acid release only 7%, and citric acid or tartaric acid which showed no detectable losses. The incorporation of solid organic acid showed a more sustained retention of clove oil essential oil over time as compared to other non-solid organic and inorganic acids.

Comparative Example 2: Air exposure durability experiments with different organic acids

Clove oil encapsulation experiments were performed in similar experimental conditions as in Example 3 using bentonite clay without any acid treatment or bentonite clays activated with different organic acids (Sigma-Aldrich was used as supplier). The organic acids selected were characterized by a different amount of carboxylic unit in its formulae. Organic acid with 1 , 2 and 3 carboxylic acid units were selected. The different organic acids were selected according with their melting point, that it’s the temperature in which the organic acid experience a transformation from the solid state to a liquid state. In this sense, the lactic acid is the only organic acid that it is liquid at room temperature. The clays were dried at 80 °C for 1 hour instead 24 hours. This shorter drying may result in higher losses due to the presence of excess essential oil weakly interacting with the clay through physical forces (London dispersion force). The adsorbed CO refers to the essential oil that interacts with the polar groups of the clay (as described in the example 1 of the invention) and it is encapsulated by the organic acid.

The encapsulation efficiency of the differently treated clays was studied under accelerated aging conditions (under air exposure at 80 °C for 15 days). The samples were collected and analyzed as described in the Comparative Example 1.

Table 10. Clove oil losses after 15 days of accelerated aging (at 80°C in air atmosphere) test of BNT-CI samples with different organic acids. Characteristic data of organic acids are summarized as the melting point (Mp), the molecular weight, (Mw), the carboxylic units contained in its formulae (COOH units), and their solubility in water (S).

Clay without acid treatment show the highest wt % of CO loss after 15 days of accelerated weathering (only 20% of the CO remains in the composition). The samples that used organic acids that were in a solid state during the accelerated aging test showed an increase in the proportion of retained essential oil compared to the samples without acid or with a non-solid organic acid (liquid). In particular, the increase in essential oil comprised up to 100% more essential oil for samples with essential oil melting point > greater than 152°C.

The authors have not found a relationship between the number of carboxylic acid units of the organic acids and the greater capacity to retain the essential oil. Likewise, no dependence has been observed on this retention capacity and the molecular weight of organic acids. The direct relationship observed by the inventors correlates the melting point of organic acids with a greater retention capacity of the encapsulated essential oil.

This greater retention of the essential oil encapsulated in organic acids that are in a solid state at the temperature of the aging test represents an advantage for the product of the invention since it allows a longer period of effectiveness. An additional advantage of essential oil samples encapsulated in clays with organic acids in a solid state is that depending on the solubility of the organic acids, the release mechanism of the essential oils can be modulated in time.

This latter advantage lies in that the antimicrobial response of the materials of the present invention is activated in the aqueous media where microorganisms proliferate. In this sense, organic acids with high solubility can be available that will therefore quickly release the essential oil into the aqueous medium, organic acids with low solubility that will slowly release into the aqueous medium, and combinations that allow for products with a time adjusted to the required application. A controlled release rate thus allows the useful life of these products to be prolonged under optimal conditions of functional response.

Claims

1. Composition comprising:

(i) a plurality of nanoclay particles,

(ii) optionally an organic surfactant,

(iii) at least one solid organic acid, and

2. A composition obtainable by a process comprising the steps of:

(a) providing a suspension of nanoclay particles;

3. Composition according to claim 1 or 2, wherein the nanoclay is selected from the group consisting of bentonite, sepiolite or mixtures thereof.

4. Composition according to any one of claims 1 to 3, wherein the nanoclay particles have a mean thickness of 1 to 100 nm.

5. Composition according to any of the previous claims, wherein the organic acid is selected from an acid having a solubility in water of less than 50 g/L at 20 °C, an acid having a solubility in water of at least 500 g/L at 20 °C or a combination thereof.

6. Composition according to any of the previous claims, wherein the organic acid is a dicarboxylic, tricarboxylic or tetracarboxylic acid.

7. Composition according to any of the previous claims, wherein the organic acid is selected from the group consisting of sorbic acid, benzoic acid, dihydroacetic acid, lactic acid, ascorbic acid, erythrobic acid, oxalic acid, tartaric acid, citric acid, adipic acid, succinic acid, nicotinic acid, tetracetric ethylene diamine acid, thiodipropionic acid, phytic acid, alginic acid, glutamic acid, guanylic acid, inosinic acid, cyclamic acid, cholic acid, maleic acid, fumaric acid and mixtures thereof.

8. Composition according to any of the previous claims, wherein the solid organic acid is fumaric acid, citric acid, tartaric acid, or mixtures thereof.

9. Composition according to any of the previous claims, wherein the solid organic acid is present in an amount between 1-5 wt% with respect to the total weight of the composition.

10. Composition according to any of the previous claims, wherein the essential oil is selected from the group consisting of clove oil, oregano oil, citronella oil, strawberry oil, rosemary oil, red thyme oil, cinnamon oil, peppermint oil, or mixtures thereof.

11. Composition according to any of the previous claims, wherein the essential oil is present in an amount of 0.1 to 25 wt% with respect to the total weight of the composition.

12. Composition according to any of the previous claims, wherein the essential oil is in the form of nanodrops with mean sizes between 10 and 60 nm.

13. Composition according to claim 12, wherein the organic acid is adhered to the essential oil nanodrops deposited on the nanoclay.

14. Composite material comprising a composition as defined in any of claims 1 to 13 incorporated into a plastic substrate.

15. Composite material according to claim 14, wherein the plastic substrate comprises a thermoplastic polymer selected from acrylonitrile butadiene styrene (ABS), ethylenevinyl acetate (EVA), polyethylene, polypropylene, polycarbonate, PVC, acrylic polymers (such as PMMA), polyethylene terephthalate (PET), polyesters, polyurethanes, polystyrene and biopolymers such as polylactic acid (PLA), polycaprolactone (PCL), polysaccharides, polyvinyl alcohol (PVA), celluloids, thermoplastic starch, polyterpenes, polyhydroxyalkanoates, polybutylene succinate (PBS), or a combination thereof.

16. Process for the preparation of a composition according to any one of claims 1 to 13 comprising the steps of

(a) providing a suspension of nanoclay particles;

17. Use of a composition according to any one of claims 1 to 13 as a bioprotectant, preferably as an antimicrobial and/or a pest-repellent agent.

18. Use of the composition according to any one of claims 1 to 13 or the material according to any one of claims 14 or 15 in a packaging material, preferably for the preservation of food products.