EP4337009A1 - Aqueous nanodispersions and nanoemulsions for water treatment - Google Patents

Abstract

Disclosed is a nanoemulsion with a continuous aqueous phase and a dispersed lipid phase, wherein the dispersed lipid phase contains mineral particles consisting of clusters of phyllosilicate sheets swollen with water and exfoliated, at least one essential oil and an amphiphilic dispersing agent.

Description

Aqueous nano-dispersions and nano-emulsions for water treatment

Field of invention

The present invention relates to aqueous nano-dispersions and nanoemulsions intended to decontaminate water or to prevent its contamination by pathogenic agents, in particular water used for aquaculture, or the drinking water of terrestrial animals, which can according to the dosage be used as a preventive treatment for animal infections.

State of the art

[0002] Worldwide, the demand for fish is increasing due to the combination of several factors: demographic growth, urbanization and multiplication of wealth. Research on fish supply and demand suggests that aquaculture production will need to double by 2030 to meet growing global demand and needs. Currently, fish accounts for nearly 20 percent of animal foods consumed worldwide. This makes it an interesting target, as described later in this document, knowing that the principles also apply to terrestrial animal farms and domestic animals, each with their specificity.

[0003] According to current estimates, yields from marine capture fishing should increase only very slightly. Only aquaculture will therefore be able to meet the demand.

[0004] Aquaculture represents 50% of the fish consumed in the world. The sector is developing like all intensive farming with the use of antibiotics to deal with the diseases that affect farms. This overexposure to antibiotics contributes to the development of antibiotic resistance and the spread of pathogens. It has become a major concern in this sector, under pressure from consumers.

[0005] We can cite the major families of recurring pathogens of livestock, encountered in farms: photobacterium (for example under species damselae), vibrionacea (for example alginolyticus, photobacterium, or vibrio), pseudomonas (for example anguilliseptica ). Faced with these pathogens, fish farmers most often resort to the use of antibiotics. But, at the end of the treatment, the flora generally shows an increased resistance to the antibiotic used, many resistance genes are carried by plasmids which can be transmitted from one bacterial species to another. The risk of selecting resistant pathogenic strains therefore encourages limiting the use of antibiotics (Pham 2017). Researchers are therefore faced with the need to find other alternatives and active ingredients to fight against pathogens and multi-resistance.

[0007] The treatment of choice for microbial diseases remains antibiotic therapy. Abuses in its use have unfortunately led to the appearance of resistant strains and frequent treatment failures. There is also a risk of transmission of resistance plasmids to other bacteria, including those pathogenic for humans, and of accumulation of residues in fish flesh and in fish farm effluents. Severe legislation has considerably restricted the use of antibiotics in the aquatic environment, posing major problems for fish farmers whose antibacterial arsenal is increasingly limited.

[0008] Among the alternatives used to replace antibiotics, there are essential oils. The use of cinnamon oil, mainly containing cinnamaldehyde, has already been incorporated into the diet of tilapia infected with streptococcus iniae and has reduced the very high fish mortality rate (Rattanachaikunsopon & Phumkhachom, 2010). The effects of essential oils on broiler chickens have also been described (Brenes and Roura, Animal Feed Science and Technology, 158, 2010, 1-14).

[0009] The treatments are usually carried out in balneotherapy in aquaculture farms. But as these are volatile and hydrophobic molecules, soaking fish in a tank containing essential oils remains complex, difficult to implement and not recommended on a large scale in farms using cages in the open sea, or connected to a watercourse.

[0010]There is therefore a growing need for alternative systems making it possible to effectively protect farms, particularly aquaculture farms, from these pathogens in a targeted manner, replacing the systematic use of antibiotic drugs, and particularly in a preventive approach, which does not does not induce antibiotic resistance, it is an issue coupled with the constraints of implementing the oils that must be administered efficiently and simply to target species (aquaculture, and distribution in drinking water for terrestrial species).

Brief description of the invention

The subject of the invention is an aqueous nano-dispersion comprising mineral particles, an amphiphilic dispersing agent and active molecules, such that the particles are clusters of exfoliated phyllosilicate sheets of size D90 in number less than 1000 nanometers, and such that the active molecules include essential oils.

[0012] The clusters of very small phyllosilicate sheets serve as a support for the molecules of essential oils which are physically bound to the surface of the phyllosilicate sheets with an amphiphilic surfactant, this has the advantage of stabilizing these molecules and to concentrate them on their surface. The small size of the clusters of sheets thus makes it possible to widely disperse the active molecules, which when they are hydrophobic and of low density tend to group together to form large drops, or even lipid films on the surface of the water. These active molecules can be essential oils or combinations of essential oils.

Advantageously, the size D50 in number of the particles is less than 750 nm, preferably less than 200 nm and very preferably less than 100 nm. In some embodiments, the size D50 in number of the particles ranges from 10 to 800 nm, preferably from 10 to 750 nm, preferably from 20 to 500 nm, preferably from 20 to 400 nm, preferably from 20 to 300 nm, preferably 20 to 200 nm, preferably 20 to 100 nm, preferably 20 to 90 nm, preferably 25 to 70 nm, preferably 25 to 50 nm. [0015] The reduction in size of the clusters of phyllosilicate sheets has the advantage of promoting the dispersion of the active molecules while having a high local concentration of active molecule, and thus of enhancing their effectiveness. Using these nanometric size phyllosilicate sheets, we increase the contact surface with the essential oils, which remain labile, because they can be desorbed, which promotes their effectiveness. Moreover, they are thus dispersed in the volume of water on their phyllosilicate support, and can interact with pathogens. [0016] This aqueous nano-dispersion is very simple to implement in a basin of aquaculture water or in the drinking water of terrestrial animals. The small size of the clusters of sheets facilitates their suspension in water with slow sedimentation.

The invention also relates to a nano-emulsion with a continuous aqueous phase and a dispersed lipid phase, in which the dispersed lipid phase contains lipids, mineral particles consisting of clusters of phyllosilicate sheets swollen with water and exfoliated, at least one essential oil and one amphiphilic dispersing agent, and in which the size D50 in number of the drops of the dispersed lipid phase is less than 800 nm and preferably less than 300 nm.

In some embodiments, the size D50 in number of the drops of the dispersed lipid phase ranges from 10 to 800 nm, preferably from 20 to 700 nm, preferably from 50 to 500 nm, preferably from 75 to 400 nm. nm, preferably 50 to 300 nm, preferably 100 to 300 nm.

[0019] The water of the lipid phase is physisorbed between the layers of phyllosilicates to swell them (interlayer space), which makes it possible to achieve smaller particle sizes by exfoliation, and the dispersing agent, with a part hydrophilic and a hydrophobic part, is adsorbed on the surface of the clusters of sheets by its hydrophilic part. This makes the sheets partially hydrophobic, promotes exfoliation of the phyllosilicates and allows good dispersion of the phyllosilicate sheets in the lipids.

[0020] In other words, the lipid phase does not include any water other than the water adsorbed in the phyllosilicate sheets.

The amphiphilic dispersing agent of the aqueous nano-dispersion and of the nanoemulsion can be chosen from the group of ethyl lauroyl arginate (LAE), cationic surfactants based on arginine with 16 carbons and more, phospholipids, e-polylysine and combinations thereof.

[0022] Advantageously, the dispersing agent is a phosphoglyceride.

Very advantageously, the dispersing agent is a phosphatidyl choline and preferentially lecithin. [0024] These dispersing agents will bind to the silanolate functions of the phyllosilicates via the cationic part of their structure.

Preferably, the phyllosilicates of the aqueous nano-dispersion and of the nanoemulsion mainly have sodium cations in the interfoliar position. The interfoliar sodium cations have the advantage of offering less resistance to the increase in the interfoliar distance of the so-called sodium phyllosilicate sheets and thus of promoting their swelling by water molecules as well as their exfoliation. Sodium phyllosilicates thus have advantages on the size of the dispersion to obtain clusters of smaller sheets, this favors fine dispersions of essential oil and thus a greater surface-to-volume ratio of the phyllosilicate clusters which support the molecules is obtained. essential oil molecules, and this increases the encounter statistics between the active molecules and the pathogens.

[0027] Very preferably, the phyllosilicates are smectites which are mainly sheets of montmorillonites. Advantageously, the amphiphilic dispersing agent content of the aqueous nanodispersion is between 0.2 and 2.0% by weight relative to the weight of the mineral particles.

Advantageously, the dispersing agent content of the dispersed lipid phase of the nano-emulsion is advantageously between 10% and 400% and preferably between 20% and 300% by weight relative to the weight of particles of the lipid phase.

[0030] When the content of dispersing agent such as lecithin is insufficient, the hydrophobic character of the clay particles is insufficient, which does not stabilize them in the lipid phase. high, the hydrophilic nature of the particles is lost and the molecules of essential oils find themselves in competition with the dispersing agent on the surface of the particles, which does not promote their availability on the surface of the phyllosilicate particles.

[0032] For exfoliated phyllosilicate particles with a specific surface of the order of 300 m2/g, the preferred range of dispersing agent content corresponds to a rate of coverage of the silanol functions by the dispersing agent between 20 and 60%, ie between 30 and 130% of the mass of clay used to provide a dispersing agent such as soya lecithin.

Advantageously, the water content of the dispersed lipid phase of the nanoemulsion is between 10% and 300% and preferably between 20% and 200% by weight relative to the weight of particles of the lipid phase.

[0034] Below 10% water by weight relative to the weight of the particles, the phyllosilicate sheets are not sufficiently pre-swollen and it is not possible to obtain a sufficient reduction in the particle sizes during their exfoliation. .

[0035] Beyond 300%, the amount of water no longer allows the molecules of the dispersing agent to physically bind to the surface of the sheets and the sheets are not sufficiently hydrophobic to serve as a support for the molecules. active like essential oils.

[0036] Aqueous nano-dispersions and nanometric emulsions can be used as an agent for decontaminating aquaculture water and drinking water for terrestrial animals, or as a means of preventive treatment of pathologies in animals through its daily hydration, or in the preventive treatment of animals against the development of contamination agents in their organism. They can thus be used to protect these waters from the proliferation of pathogens. In certain indirect applications, these systems can constitute a palatant to encourage the water intake of animals.

[0037] Nanometric emulsions have the advantage of being able to remain in homogeneous suspension under the force of thermal agitation, which makes it possible to store them over long periods of time while remaining very easy to homogenize during their use. According to another mode of use, the aqueous nano-dispersions and the nanometric emulsions can complete a food, a food supplement or a premix.

According to yet another mode of use, aqueous dispersions and nanometric emulsions can be used as a natural preservative, based on essential oils for preparations whose water activity parameter is greater than 0.45 . The water activity does not represent the water content (or humidity) but rather the availability of this water. The higher the water activity, the greater the amount of free water and the greater the ability for microorganisms to grow.

[0041] The nanometric emulsions obtained by this approach are very stable particles mechanically, as well as over time, and can be easily introduced into an extruder to be coextruded at the heart of granulated foods. They will thus be ideal vehicles for the incorporation of essential oils into foods in the form of a premix, which facilitates handling (because it reduces the risk of inhalation or evaporation of essential oils). The premixes are thus also found in a galenic form which promotes their metabolization with drop sizes of the order of a hundred nanometers.

Their presence in a food, a food supplement or a premix has the advantage of favoring the preservation over time of these foods, supplements or premix, the bacteriostatic properties of essential oils in the form of particles dispersed at the nanometric scale constitute a considerable advantage in the preservation of food, which limits the use of heat treatments, which degrade the nutritional quality in particular the micro-nutrients, or preservatives which can be controversial despite their massive use.

The invention also relates to a method for preparing a nano-emulsion, comprising the following steps: dissolving the dispersing agent in the aqueous phase of the lipid phase and obtaining a homogeneous solution; add the phyllosilicates and mix the dough obtained with a kneader; add the lipids of the lipid phase; shearing the composition obtained; add the aqueous phase; provide shear energy to obtain a micrometric emulsion; and resume the micrometric emulsion with a high pressure homogenizer or a microfluidizer to obtain a nanometric emulsion.

The objective of the invention is a mode of use of essential oils which makes it possible to limit their volatility while having a high efficiency with quantities used under control. This implementation of essential oils in a stably nanoscale dispersed form promotes the encounter statistics with pathogens. The storage and incorporation of essential oils in animal nutrition practices will be greatly facilitated by the stability and homogeneity of the dispersions.

Description of Figures

The invention is described below with the aid of Figures 1 to 10, given solely by way of illustration:

The figure. 1 shows a structural diagram of a bentonite;

The figure. 2 presents the formula of lecithin;

The figure. 3 schematically presents the evolution of the lecithin content as a function of the specific surface of the clay and for several coverage rates; The figure. 4 schematically presents the evolution of the size of the drops of the dispersed phase according to the size and the concentration of the mineral particles;

The figure. 5 presents the size of the mineral particles for two concentrations of clay;

The figure. 6 shows the measurement of the size of different clays dispersed in water;

The figure. 7 shows the size of the lipid drops in an emulsion;

The figure. 8 shows a diagram of the emulsion obtained;

The figure. 9 schematically presents the evolution of the size of the drops of the dispersed phase according to the size and the concentration of the mineral particles; and

The figure. 10 shows a diagram for measuring the contact angle between a drop of pure water and the clay surface.

Detailed description of the invention

An emulsion is of the “oil in water” type, when (i) the dispersing phase is an aqueous phase and (ii) the dispersed phase is an organic phase (hydrophobic, lipidic or oily). Such an emulsion is also commonly referred to as “direct emulsion” or by the abbreviation “O/W”. In the context of the present invention, the term “labile” molecule is understood to mean a molecule bound to a substrate by physical, ionic interactions, or Van der Waals forces, non-covalent, which gives it a capacity to grip or reversible organization. In the context of the present invention, the composition comprises exfoliated phyllosilicates, that is to say phyllosilicates whose layers have been separated by exfoliation.

The term "exfoliated" means phyllosilicates having undergone exfoliation, that is to say a more or less complete separation of its individual sheets. The exfoliation process usually includes three phases:

- (1) swelling of the phyllosilicate sheets with water,

- (2) The adsorption of a hydrophobic molecule on the surface of the phyllosilicate particles, to make it compatible with lipids, for example lecithin, and - (3) The supply of shear energy to separate the phyllosilicate particles in the dispersing phase.

In the context of the invention, the term “clay” or “mineral particles” is used interchangeably to designate and describe phyllosilicates.

In what follows, the D50 in number of a sample of particles represents the size of the particles for which 50% of the particles of the sample have a particle size smaller than this value, by analogy a D90 in number represents the size particles for which 90% of the particles of the sample have a particle size below this value.

[0052] The term “nanometric emulsion” or “nano-emulsion” means an emulsion in which the drops of the dispersed phase have a size D50 in number of less than 800 nanometers (nm), preferably less than 500 nanometers (nm), of particularly less than 300 nanometers (nm) and a D90 number less than 1000 nm.

The term "nano-dispersion" or "nanometric dispersion" means a dispersion in which the colloidal particles of phyllosilicates are dispersed with a size D50 in number of less than 1000 nanometers (nm). The water activity (symbol for activity of water) represents the water vapor pressure p of a wet product divided by the saturation vapor pressure po at the same temperature:

[0055] aw=p/p ₀ [0056] This parameter reflects the interactions of the water with the matrix of the wet product.

[0057] Water activity is one of the main parameters influencing the preservation of food or pharmaceutical products. Microorganisms need “free” water (free for biochemical reactions) to grow. Water activity does not represent the water content (or humidity) but rather the availability of this water. The higher the water activity, the greater the amount of free water (1 being the maximum) and the more microorganisms will grow.

[0058] In embodiments, the water activity is 0 to 1, preferably 0.45 to 1, preferably 0.6 to 1, preferably greater than 0.45. The present invention thus relates to an aqueous nano-dispersion and a nanoemulsion comprising particles and active liposomal molecules. The particles consist of clusters of phyllosilicate sheets of D50 size in number less than 1,000 nanometers (nm). The active molecules can be essential oils (EO) or combinations of essential oils. Lipids

The lipids of the lipid, or hydrophobic, or oily, or organic phase of the nanometric emulsions, are chosen according to the applications envisaged from vegetable oils, mineral oils, synthetic oils, hydrophobic organic solvents and liquid polymers. hydrophobic. The lipid phase of the nano-emulsion includes bacteriostatic agents, such as essential oils. It may also contain unsaturated fatty acids, antioxidants and vitamins.

In the examples, sunflower oil is used. Essential oils

[0062] For centuries, man has used plants as a means of prevention and or cure against diseases. More precisely the extracts of its plants, were used as remedies in many civilizations across the globe. [0063] The Afnor NF T 75 - 006 (2000) standard defines essential oils as being "products obtained either from natural plant materials by steam distillation, or by mechanical processes from epicarp of citrus or by dry distillation. The essential oil thus obtained is separated from the aqueous phase by physical processes. An essential oil is an odorous and volatile, non-greasy substance extracted from a plant in liquid form. It comes from a secretion produced by certain plants and contained in specialized structures (hairs, pockets and secretory ducts). Depending on the essential oil, the entire aromatic plant, or more specifically some of its organs (root, bark, leaf, flower, fruit, seed, etc.) will be selected to extract the aromatic compounds. An essential oil has a complex molecular composition which gives it unique virtues. It does not contain proteins, fats or carbohydrates, contains no minerals or vitamins: it therefore has no nutritional value.

[0065] At room temperature, the essential oils are liquid, apart from a few special cases: the essential oils of myrrh and sandalwood are rather viscous and those of rose and camphor can be crystallized. At low temperature, some essential oils crystallize: those of anise, field mint or saturated thyme when the bottles are stored in the refrigerator.

[0066] Essential oils are volatile, this volatility explains their fragrant character as well as their method of obtaining by steam distillation. They are very soluble in fatty oils (which constitute a very good support when one wishes to dilute them), lipids (hence the principle of enfleurage formerly used to extract essential oils by putting them in contact with a fat animal such as lanolin), ether, most organic solvents as well as in alcohol (of high titer). Character liposo fad: essential oils do not dissolve in water, it is therefore imperative to use a surfactant to allow their implementation suspended in water. They have a high refractive index and often have a rotatory power. Most of them are colored.

Given the great complexity of the chemotypic composition of essential oils, despite possible synergies, some authors prefer to study the effect of an isolated compound in order to then be able to compare it to the overall activity of the oil.

The activity of EO components depends on their chemical structure. Oxygenated terpenes (e.g. carvacrol, thujone) exhibit higher antimicrobial activity than non-oxygenated terpenes (e.g. a- and b-pinene, limonene). In addition, the lipophilic character of their hydrocarbon skeleton as well as the hydrophilic character of their functional groups are decisive with respect to their antimicrobial activity. Thus, the order of antimicrobial activity of these compounds is as follows: phenols > aldehydes > ketones > alcohols > ethers > hydrocarbons.

Among essential oil constituents, phenolic compounds have been shown to possess significant antimicrobial activity. It seems that the presence of the hydroxyl group is related to the inactivation of microbial enzymes. Most likely, this group interacts with the cell membrane, causing leakage of cell components, modification of fatty acids and phospholipids, as well as impaired energy metabolism and influence on the synthesis of genetic material. The phyllosilicates

[0070] Phyllosilicates are clay minerals of the group of silicates built by stacking tetrahedral layers ("T") where the tetrahedra share three peaks out of four ("basal" oxygens), the fourth peak ("apical" oxygen ) being connected to an octahedral (“O”) layer occupied by different cations (Al, Mg, Le, Ti, Li, etc.). Figure 1 shows an example of a phyllosilicate structure. These stacked structures form organized sheets (as described in detail below) whose surface charge is negative over a wide pH range (4<pH<9), which will be stabilized by cationic counterions. These counter-ions will be monovalent, or divalent, which gives the clay an ability to be swollen in water more or less strongly, by inserting water molecules between the layers. [0071] Smectites are a group of clay minerals, and therefore silicates, more precisely phyllosilicates.

Their typical composition is where A represents a cation interlayer (alkali or alkaline-earth element), D an octahedral cation, T a tetrahedral cation, O oxygen and Z a monovalent anion (generally OH-).

They crystallize in the monoclinic system.

These are phyllosilicates of TOT (or 2:1) structure, that is to say made up of sheets comprising two tetrahedral layers head to tail, bonded together by octahedral cations. The sheets are bound together by the interfoliar cations.

A distinction is made between dioctahedral (beïdellite, montmorillonite, nontronite, etc.) and trioctahedral (hectorite, saponite, etc.) smectites.

Montmorillonite is a 2/1 type clay, also called TOT (for tetrahedron/octahedron/tetrahedron). This means that a montmorillonite sheet is made up of three layers: an octahedral Al(OH)sO layer: 7 atoms for 6 vertices + aluminum in the center. The OH- and oxygen being shared between the different octahedra that make up the layer. and two tetrahedral layers which cover on each side the octahedral layer at its base; S1O4: 5 atoms for 4 vertices + silicon in the middle. The oxygens being shared between the different tetrahedrons that make up the layer.

The imperfections in the crystal are compensated by interfoliar cations, generally monovalent or divalent, which ensure the electrical neutrality of the mineral.

All of the phyllosilicates can be used, but the smectites and particularly the montmorillonites have the advantage, due to their lamellar structure with a spacing between the layers greater than the other phyllosilicates, of being able to be swollen by small molecules with hydrophobic properties which will improve the exfoliation of the clay platelets and thus facilitate their dispersion in the lipid composition. The other phyllosilicates, but also the micas and the talcs can also be exfoliated in this way, but the energy which would be necessary to disperse the lamellar layers in the lipid phase would be much higher. In the examples, bentonite is used as phyllosilicate. Bentonites are clays mainly composed of montmorillonite, whose interlayer cations are usually either calcium, or sodium, potassium, their combination or other metal ions. Bentonite is negatively charged on the surface (on the length) and positively on the sides (width) which allows it to interact with other charged molecules.

[0081] Clays are hydrophilic and smectites, including bentonite, have a swelling capacity. This particularity makes it possible to adsorb water-soluble molecules in the interfoliar space of clays via an aqueous phase. The water is said to be physisorbed on the surface of the clay sheets via the silanol groups.

In the description, the term “clay” will also be used to refer to phyllosilicates.

Dispersing or Surface Agent The dispersing or surface agent usually used is a molecule with a hydrophobic part and a hydrophilic part. The adhesion of this dispersing agent by physical interaction to the mineral particles makes it possible to make the clay sheets hydrophobic and to obtain a good dispersion of these clay sheets in a lipid phase. It is advantageous to use a dispersing agent having a cationic polar head and a hydrophobic chain, soluble in the lipid phase, such as phospholipids having cationic polar functions such as for serine, ethanolamine or even choline, we thus obtain phosphatidylserine, phosphatidylethanolamine, or even phosphatidylcholine, better known as “lecithin”. It is a lipid of the class of phosphoglycerides. Arginine grafted on a long alkyl chain (Cl 6 and more) can also play this role of dispersing agent. You can also use ethyl lauroyl arginate (LAE). E-polylysine can also be used as a surfactant.

[0085] All these dispersing agents are labile because they bind to the surface of the clusters of phyllosilicate sheets by non-covalent interactions, which gives them a capacity for adhesion or reversible organization depending on the physicochemical conditions (pH , ionic strength, temperature, etc.). Phosphatidylcholine has (FIG. 2): a hydrophilic pole: choline (1) and the phosphate group (2); a hydrophobic tail: fatty acid residues (here, palmitic (5) and oleic (4) acid residues); and glycerol (3) connects these two hydrophilic and hydrophobic poles.

The phosphate group is negatively charged, while choline is positively charged. Phosphatidylcholine is therefore zwitterionic.

It is both hydrophilic and lipophilic, and its hydrophilic-lipophilic balance (HLB) can vary between 2 and 9.5 depending on the fatty acid residues of the hydrophobic tail.

Process for obtaining an emulsion

To stabilize the emulsions, one approach consists in using so-called “emulsifying” or “emulsifying” compounds.

These emulsifying compounds are most often emulsifying surfactants (also called “surfactants”) which, thanks to their amphiphilic structure, are placed at the oil/water interface and stabilize the dispersed droplets.

However, emulsifying compounds of this type do not always offer the desired stability over time, with a permanent balance of surfactants between the interface to be stabilized and the micelles in solution. In addition, synthetic surfactants often have disadvantages from an ecological or food point of view, since they disrupt biological systems through a strong interaction with cell membranes.

These emulsifying/emulsifying compounds can also consist of solid particles, which make it possible to obtain so-called “Pickering emulsions”.

[0093] Pickering emulsions are emulsions which are stabilized by particles in colloidal suspension in the aqueous phase which become anchored at the oil/water interface, interpreted as a wetting effect at the interface of the two phases, with a strong stability.

[0094] Unlike surfactants which are continuously adsorbed and desorbed under the effect of thermal agitation, the particles in suspension colloidal particles strongly adsorb at the interfaces and the desorption energy of the particles becomes high enough to make the phenomenon irreversible.

The phyllosilicate particles thus have an emulsifying role to stabilize the emulsions according to an object of the invention. These emulsions are thus Pickering emulsions.

The objective of this step is to obtain a stable emulsion with a dispersed phase in the form of drops in a continuous phase. The lipid phase comprises the dispersion of phyllosilicates in oil previously described. The aqueous phase is composed of water which can be supplemented with a monovalent salt, with a concentration between 0 and 100 mM in water, advantageously with NaCl at a concentration of less than 50 mM and very advantageously at 25 mM. This ionic strength was chosen to limit the electrostatic repulsions due to the surface charges of the clay particles. To disperse the two immiscible phases, we will provide a shear energy applied in batch, at room temperature, with for example a rotor / stator device with an air gap of 150 micrometers with a mobile of 30 mm, at 4500 rpm for 4 min .

To obtain an emulsion according to one of the objects of the invention, a lipid phase is always used in which clay particles are dispersed and stabilized with a dispersing or surface agent in the presence of water as previously describe.

The direct or reverse character of the emulsion obtained is mainly a function of the relative viscosities of the continuous phase and of the dispersed phase, of the proportion of dispersed phase relative to the continuous phase at the start of the emulsification, knowing that the dispersed phase can then be added drop by drop to increase the proportion, it is thus possible to produce emulsions with more than 65% by weight of dispersed phase.

The size of the drops of the dispersed phase obtained is a function of the size of the clay particles and of the concentration of these clay particles. Figure 9 schematically presents the changes observed. [00100] For a given clay particle size, the diameter of the drops decreases with the concentration; the more particles are added, the more interfaces they can stabilize and therefore drops of the dispersed phase result in smaller diameters. However, the size of the clay particles will dictate a minimum droplet size; you can't make drops smaller than the stabilizing particles.

[00101] It should be noted that the stabilization of emulsion drops by clay particles induces a stiffening of the interface with drops which lose their sphericity. In addition, the size limit of drops observed on the plateau with a high concentration of clay depends on the shear energy provided to fragment the drops.

[00102] Emulsions stabilized by phyllosilicates organized on the surface of the droplets, make it possible to have lipid droplets stabilized against coalescence by a physical barrier of dominant negative charge on the surface for a wide range of pH ranging from pH 4 and pH 10 This negative charge is provided by the surface silanolate bonds of the clay platelets. These negatively charged silanolates can interact with molecules (L-arginine, L-Lysine) or cationic polymers (chitosan, hyaluronic acid, polylysine, etc...), which makes it possible to change the surface interactions of the droplets and functionalize them or change their attractiveness for different supports. It is also possible to functionalize them by covalent bonding by condensation of silanes prepared to provide specific functions. A variety of silanes which can be condensed by one or more silanes are possible. Mention may be made, by way of example, of mono, di or tri ethoxy aminopropylsilanes. Many molecules can be used to then covalently couple the amine function provided by the silanes. Simple chemistry can be used with coupling agents like isothiocyanates, N-hydroxysuccimide ester (NHS-ester), isocyanates, acyl azides, sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, aryl halides, imidoesters, carbodiimides , anhydrides, and fluorophenyl esters

[00103] The surfaces of the particles can thus be functionalized to interact with surface antigens on bacteria, or bacterial biofilms. Aqueous nano-dispersion of phyllosilicate particles

[00104] The preparation of an aqueous nano-dispersion of phyllosilicate particles comprises the following steps:

[00105] (1) Prepare an aqueous solution of phyllosilicates: homogenize the solution with a rotor stator and leave to stand; [00106] (2) Add to the aqueous solution of phyllosilicates a given quantity of the dispersing agent, for example e-polylysine; homogenize with a vortex;

[00107] (3) Add to the previous solution a given quantity of essential oil;

[00108] (4) Homogenize the completed solution in an ultrasound bath to obtain the aqueous nano-dispersion of phyllosilicates. [00109] In embodiments, the phyllosilicate content in the aqueous nanodispersion ranges from 0.005 to 20% by weight.

[00110] In embodiments, the content of phyllosilicates in the aqueous nanodispersion ranges from 0.5 to 20% by weight, preferably from 1 to 20% by weight. [00111] In embodiments, the content of phyllosilicates in the aqueous nanodispersion ranges from 0.005 to 5% by weight, preferably from 0.005 to 2% by weight.

[00112] In embodiments, the content of phyllosilicates in the aqueous nano-dispersion ranges from 0.5 and 35% by weight and preferably from 0.5 to 15% by weight, relative to the weight of the lipid composition. .

Method for preparing a nano-emulsion

The invention also relates to nanometric emulsions. These emulsions consist of a lipid phase and a continuous aqueous phase. [00114] The objective is to obtain a nanometric emulsion according to one of the objects of the invention.

[00115] To combine two systems which originally have no affinity, one apolar (lipids - essential oils) and the other polar (aqueous phase - phyllosilicates), we will: (1) Dissolve the dispersing agent in water for swelling the clay and obtaining a homogeneous mixture;

(2) Add the clay and mix the composition obtained with a kneader to swell the phyllosilicate sheets with water, and adsorb the dispersing agent on the surface of the phyllosilicate particles, to make them compatible with the lipid phase;

(3) Add lipids from the lipid phase;

(4) Bring shear energy to the resulting composition to disperse/exfoliate the clay sheets in the lipid phase;

(5) Add water from the external aqueous phase with, for example, an oily phase/aqueous phase ratio of 40/60;

(6) Bring shear energy to the composition obtained to disperse the lipid phase in the aqueous phase and obtain a micrometric emulsion; and

(7) Take this emulsion and shear it with even more energy by treatment in a high pressure homogenizer or a microfluidizer and obtain a nanometric emulsion. The purpose of steps (1) to (4) is to prepare the lipid phase of the nanoemulsion according to one of the subjects of the invention.

This lipid phase comprises lipids, for example sunflower oil to which at least one essential oil is added as an antibacterial agent, phyllosilicates, swelling water of the phyllosilicates and an amphiphilic dispersing agent.

[00118] Step (1) is obtained by adding water in sufficient quantity to solubilize the dispersing agent and impregnate the clay sheets.

[00119] Clays are known for their water absorbing properties, and they can swell depending on their chemical and structural composition between 2 times their mass in water, up to 20 times their mass in water. The clays thus swollen form a gel whose more or less swollen and more or less exfoliated sheets incorporate the entire volume of water. It is not necessary to saturate all of the water absorption capacity of the clays to obtain a satisfactory dispersion of the clay in the lipid phase, which is why we limit the water supply to 300 % by weight relative to clay. Conversely, clays need to be at least impregnated with water to promote their dispersion. Exfoliable clays are usually stored at a humidity level of 10%. It is essential not to drop below the 5% threshold to avoid the collapse of the phyllosilicate sheets, leading to a structure that loses its ability to exfoliate.

[00120] An insufficient water content of the swelling water of the lipid phase does not allow good exfoliation of the phyllosilicates.

[00121] When the water content is too high, the clay sheets are easier to exfoliate but they are impregnated with water and they have less capacity to adsorb the molecules of the amphiphilic dispersing agent. They keep their hydrophilic character.

Preferably, the water content is between 10 and 400% and very preferably between 20% and 200% relative to the weight of phyllosilicates or clays of the lipid phase. In step (2) a dispersing agent is used, such as lecithin, as dispersing agent/exfo binder. This will be adsorbed on the surface of the clay sheets by ionic interaction between the ammonium functions and the silanol groups of the clays. The lecithin or Ge-polylysine is pre-dissolved in the water of step (1) to facilitate its incorporation. The amount of lecithin or e-polylysine can vary from 5% to 100% clay surface coverage. This coverage rate is calculated according to the total outer surface of clay after exfoliation, and the number of anionic charges at the surface of the sheets (usually there are about five silanol functions per square nanometer, 5 /nm ² ) accessible by lecithin or e-polylysine molecules (the most swollen layers (spread apart by water)). It therefore depends on the specific surface of the clay accessible by the dispersing agent.

[00124] It is therefore necessary to work on different levels of dispersing agent, depending on the clays used, which will have quantities of silanols sufficiently accessible to the lecithin molecules. It is necessary to aim for optimal coverage rates between 20% and 60% of the surface silanols of the particles depending on the particle size and the desired hydrophobation. Thus different types of particles can be prepared to stabilize direct emulsions. [00125] Figure 3 schematically shows the evolution of the necessary content of lecithin by weight relative to the content by weight of clay as a function of the specific surface of the exfoliated clays for several coverage rates of the silanols of the clay sheets by lecithin.

This figure shows that the optimal lecithin content to obtain good exfoliation followed by stable direct emulsification is between 13% and 129% by mass relative to the mass of the clay for a coverage rate of 20% and respectively a specific clay surface of 100 m ² /g and 1000 m ² /g. For a coverage rate of 60%, the optimal lecithin content is between 39% and 387% by mass relative to the mass of the clay and respectively a specific clay surface of 100 m ² /g and 1000 m ² / g.

[00127] When the content of dispersing agent such as lecithin is insufficient, the hydrophobic character of the clay particles is insufficient.

When the amount of dispersing agent such as lecithin makes it possible to cover the surface silanols of the particles, the dispersion of the clays in the lipid phase is promoted. At coverage rates close to 100%, the hydrophilic character of clays is lost, they become very stable in a lipid environment, which makes them less likely to migrate to the water-oil interface to stabilize the emulsion. [00129] There is a competition between the dispersing agent such as lecithin and the water molecules for physisorption on the surface of the clays, so an excess of dispersing agent will be unfavorable to the quantity of adsorbable water. and consequently to the quality of the exfoliation of the clay particles into particles of nanometric size. [00130] For the bentonite used, with a specific surface of the order of 300 m ² /g, there must therefore be an optimum lecithin content of the order of 30 to 130% by weight relative to the mass of the clay with a coverage rate of between 20 and 60% of the surface silanols of the clay.

[00131] A measurement of the contact angle between a drop of pure water and the surface of the clays at the end of step (2), that is to say before adding the lipids (3) and the shear energy (4), makes it possible to check that the quantity of dispersant or surface agent is satisfactory

In step (3), the lipids of the lipid phase of the nano-emulsion are added to the paste obtained in step (2). The resulting lipid phase is such that the level of phyllosilicates, that is to say the weight of phyllosilicates, is greater than 0.2% by weight relative to the weight of the lipid phase and preferably between 0.2% and 20% by weight. These levels of phyllosilicates make it possible to easily obtain direct emulsions in step (6).

[00133] Step (4) can be obtained by shearing applied in batch by means for example of a Silverson (rotor-stator shearing), an additional treatment by ultrasound or using a high pressure homogenizer is also possible to reduce particle size.

In step (5) the water from the aqueous phase is added. Advantageously, this aqueous phase can be supplemented with a monovalent salt, with a concentration between 0 and 100 mM in water, advantageously with NaCl at a concentration of less than 50 mM and very advantageously 25 mM.

[00135] In step (6) the supply of shear energy can be achieved with a rotor/stator with an air gap of 150 micrometers and a mobile of 30 mm, at a speed of 2000 to 5000 rpm for 3 to 30 min, preferably 4000 rpm for 5 min, even more preferably at 4500 rpm for 4 min.

[00136] In order to reduce the average diameters of the drops, in step (7) preformed emulsions were passed through a high pressure (HP) homogenizer (GEA Niro Soavi Panda Plus 2000, Italy) up to 5 times at 1000 bar at room temperature. The HP homogenizer was equipped with a high pressure valve (HP valve) and a low pressure valve (LP valve). In this study, the pressure at the LP valve was adjusted to 100 bar and the HP valve was used to arrive at a treatment pressure of 1000 bar which corresponds to the sum of the pressures at the two valves.

By shearing of a liquid composition is meant the application of shearing forces in an air gap positioned between a rotor and a stator. This air gap can be between 0.1 mm and 2 mm depending on the equipment. This shear force in the air gap is expressed in the form of a shear gradient, which will be all the stronger as the speed of rotation of the rotor is high, as the diameter of the rotor is large, and as the air gap is weak. In commercial equipment, the speed of the rotor can vary between a few rpm up to 12000 rpm. To translate the parameters into universal values that can be used on any type of equipment, the speed at the end of the rotor is determined, which must be of the order of 2.5 m/s, for an air gap of 150 μm on the M5 equipment from Silverson.

[00138] It is important to apply the right speed, to provide the right level of energy which makes it possible to fracture the emulsion droplets to the right size, while the treatment time makes it possible to obtain good treatment homogeneity. volume to be emulsified. [00139] A speed variation (shear gradient) makes it possible to modulate the size of the emulsions, which will vary between 1 μm at 10,000 rpm, and 70 μm at 1,000 rpm. The processing time is determined for this equipment for maximum emulsion volumes of 3 L. Methods

Method 1: Particle size measurements of an aqueous or lipid dispersion (DLS)

The size of the mineral particles obtained in an aqueous or lipid medium is measured by dynamic light scattering (Dynamic Light Scattering or DLS). The experiments were performed with a Malvem Nano ZS instrument. All measurements were performed at a temperature of 20°C with a detection angle of 173°. The hydrodynamic diameter was obtained from the analysis of the correlation function using the Malvem DTS software, and by approximating a spherical shape of the particles or clusters of phyllosilicate sheets by taking into account the largest dimensions of the particles. The viscosity of sunflower oil is 66 cSt.

The sample tested is brought by dilution to a concentration of 0.1% by weight of particles relative to the weight of the medium (water or oil). 1 min before the measurement, the sample tested is stirred with a vortex.

[00142] Figures 5 and 6a to 6d presented give the evolution of the number of particles as a function of their size in semi-logarithmic coordinates.

[00143] Figure 6 shows the particle sizes of four different phyllosilicates after dispersion and exfoliation in water as previously described.

[00144] The concentrations of phyllosilicates are 5% by weight relative to the entire composition during exfoliation and 0.1% when measuring the particle sizes.

[00145] Fig. 6(a) shows the size curve obtained for a bentonite from Wyoming. The D50 is: 255 nm.

[00146] Fig. 6(b) presents the size curve obtained for a superfine green clay of French origin, montmorillonite - illite. The D50 is: 295 nm.

[00147] Fig. 6(c) shows the size curve obtained for a Lafaure bentonite. The D50 is: 295 nm.

[00148] Fig. 6(d) shows the size curve obtained for a Smectagri bentonite. The D50 is: 712 nm.

[00149] For these four figures we have the abscissa axis which represents the diameter of the particles, while the ordinate axis represents the percentage of the population by number of particles at the diameter considered.

[00150] In the four tests, the size D90 of the particles is well below 1 micrometer.

Method 2: Measurement of contact angles

[00151] A measurement of the contact angle between a drop of pure water and the surface of the clays at the end of step (2), that is to say before adding the lipids (3) and the energy of shear (4), makes it possible to check that the quantity of dispersing or surface agent is satisfactory. For the clay layers to be able to fulfill their role of mineral emulsifying particles, it is necessary for the contact angle to be between 35 and 45 degrees and preferably between 37 and 42 degrees. Above and below the values indicated, the stability of the emulsions is not sufficient. A contact angle less than 30 degrees indicates that the surface of the clays is too hydrophilic to stabilize the emulsions. An angle greater than 50 degrees indicates that the surface is too hydrophobic to stabilize the emulsions.

[00152] The clays are deposited in a thin layer using a spatula on a flat solid support. In order to limit the effects due to the irregularity (roughness) of the surface, drops of pure water of only 2 pL are deposited on the clays. The images obtained during the deposits also make it possible to consider that the wetting obeys the Wenzel model. As the roughnesses of the surfaces obtained with the different clays can be considered as relatively similar, the contact angles measured are considered to be representative of the wettability of the clays, even if the values are slightly lower than the angles which would be obtained on the same surfaces at smooth state.

[00153] When the equilibrium state is reached, the deposited drop is observed using a high magnification digital camera and the equation of the envelope of the drop is obtained by nonlinear regression assuming that the envelope of the drop follows the shape of an ellipse. The contact angle is obtained by measuring the slope of the tangent to the envelope of the drop at the point of intersection with the straight line parallel to the plane of the clay layer (see figure 17).

[00154] Each liquid is deposited at 2 different places in the clay layer, and the contact angle of each drop is measured 3 times. The absolute error on each angle measurement can be estimated at +/- 2 degrees.

[00155] The measured contact angle is 37 to 39 degrees. Thus, the clay particles obtained according to the method of the invention will lead to the production of stable nano-emulsions. EXAMPLES

Example 1: In vitro test of the activity of essential oils bound to particles of phyllosilicates in an aqueous dispersion

[00156] Several tests have been carried out to show the interest of phyllosilicate particles as a support for essential oils and thus to improve their specific effectiveness on the one hand and to illustrate the obtaining of lipid drops of appropriate size on the other hand.

[00157] The choice of microorganisms was based on three bacterial species causing frequent pathologies in fish, more specifically sea bream (sparus aurata).

[00158] These are species that cause diseases inducing massive mortality in most cultures of different species of fish. They are predominantly gram negative. The microorganisms were provided to us by the Pasteur Institute, in the form of bacterial lyophilisate, with the following references: [00159] Vibrio alginolyticus CIP 103336T

[00160] Photobacterium damselae ssp. Piscicida CIP 104404T

[00161] Pseudomonas anguilliseptica CIP 106711T

[00162] In addition to the strains studied, two control strains were used:

[00163] Escherichia coli, provided by the microbiology laboratory at Polytech Clermont Ferrand.

[00164] Staphylococcus aureus (Ref.: cip 53-156), supplied by LMGE, Clermont Auvergne University.

The test of the effectiveness of a composition according to the invention, of an essential oil, bound to bentonite particles was carried out by the aromatogram method.

[00166] It is a method inspired by the antibiogram which makes it possible to determine the growth-inhibiting activity of essential oils by measuring the diameter of inhibition around a central well into which the dispersion containing the oil will be introduced. essential. A suspension of the germ under consideration is prepared in sterile distilled water and adjusted to, for example, 10 ⁸ bacteria/ml. Each suspension (100 mΐ) is plated on a Petri dish 90 mm in diameter comprising a nutrient medium for the germs (agar). A well 6 mm in diameter is created in the agar into which a composition according to the invention is introduced.

[00167] The growth inhibitory activity is determined by the inhibition diameters around the wells after 24 to 48 h.

[00168] The aqueous nano-dispersion according to one of the objects of the invention consists of:

Sterile distilled water: 10 g

Bentonite: 0.5 g - e-Polylysine: 1 ml of solution (at 0.36 g/l)

Essential oil: 50mg.

[00169] The order of addition is respected according to the above composition, and each ingredient is vigorously stirred before adding the next ingredient. Thus the bentonite is swollen and exfoliated and then physically binds the essential oil molecules to the surface of the particles via the dispersing agent. This protocol is the one previously described. The order of magnitude of the D50 size in number of bentonite particles is 300 nm.

[00170] This constitutes a stock solution of supported essential oil dispersion. For the tests, 200 mΐ of this dispersion of essential oil at 4.55 mg/ml are introduced into the well of the Petri dish, which represents 0.9 lmg of essential oil.

A control is also carried out with antibiotics: Chloromphenicol for V. alginolyticus and P. anguilliseptica in quantities of 30 pg and 8 pg respectively in the well of the Petri dish, and Ciprofloxacin with a quantity of 5 pg for P damselae spp. piscicida [00172] The following table shows the mean inhibition diameters obtained for the essential oils which have demonstrated significant inhibitory activity.

[00173] The 7 essential oils (EO), described in the table above, gave a significant and common growth inhibition signature for the three species studied. With regard to P. anguilliseptica, we observe more sensitivity towards the majority of essential oils. The seven most inhibiting essential oils are: Chinese cinnamon, compact oregano, lemongrass, palmarosa, thyme with thymol and mountain savory.

[00174] These results show all the advantages of using an aqueous nano-dispersion according to one of the objects of the invention comprising particles of phyllosilicate carriers of essential oils. These results make it possible to determine the optimal conditions for the preparation and the obtaining of the nano-emulsions according to the invention. Example 2: Exfoliation of phyllosilicates in oil

To measure the size of the particles in the lipid phase of a nanoemulsion, the protocol for preparing a nano-emulsion was followed, stopping at the end of step (4) of adding high shear energy. Then samples were taken to qualify the size of the phyllosilicate particles dispersed in the lipid phase.

[00177] FIG. 5 presents the result of measurements of the size of bentonite particles dispersed in the lipid phase for two concentrations of bentonite: 1% and 10%. At a concentration of 1%, the size distribution has a maximum around 1 micrometer. At a concentration of 10%, the maximum clearly shifted towards the small sizes. The maximum number of particles has a size of the order of 25 nanometers. The increase in concentration for a given shear energy leads to a reduction in particle size (by friction between the particles) which reflects an improvement in dispersion. Example 3: Preparation of a nanometric emulsion

[00178] Direct emulsions were prepared using a lipid phase/aqueous phase ratio of 40/60. The emulsification was carried out by supplying shear energy with a rotor/stator device with an air gap of 150 micrometers and a spindle of 30 mm, at 4500 rpm for 4 minutes. The emulsions thus obtained remain micrometric, because the energy provided is insufficient. To reduce the size, the emulsions thus obtained are then sheared with greater and more efficient energy, with a high pressure homogenizer or a microfluidizer.

The size of the drops thus obtained is measured as for the particles by dynamic light scattering (Dynamic Light Scattering or DLS). Figure 7 shows the average diameter of the dispersed phase lipid drops. The D50 is 300 nanometers.

[00180] As the size of the drops is directly linked to that of the particles, the size of these particles can be estimated to be of the order of 50 nanometers.

FIG. 8 schematically presents the emulsion 20 obtained. We see the lipid particles 28 dispersed in an aqueous phase 26. The lipid particles or drops, which comprise a composition as previously described, are stabilized by the dispersed phyllosilicate mineral particles. These particles of very small size which retain a certain hydrophilic character will preferentially arrange themselves at the water/oil interface, this is what is represented at reference 24.

This lipid product is obtained from a direct oil/water emulsion obtained by dispersion in water of a lipid composition as previously described. The emulsion can be concentrated by separation of the aqueous phase, this separation can be carried out by any means, in particular by ultrafiltration.

[00185] The concentrated nanometric emulsions thus obtained make it possible to work on demanding applications on the quantity of water provided by the emulsion, such as dry food preparations, dry food coatings. Concentrated solutions also make it possible to reduce volumes and concentrate active ingredients, which facilitates product logistics, and makes it possible to work on applications requiring larger quantities and concentrations of active ingredients.

[00186] Nanometric emulsions are an excellent means of maximizing the encounter statistics between the pathogens and the active principles, because by diluting the latter in a lipid matrix and by supporting them on the phyllosilicate sheets at the interface, then by reducing the size of objects at the nanometric scale, we multiply by several orders of magnitude the number of particles that can interact with pathogens. In fact, emulsions of 20 microns (size of drops usually obtained with rotor-stator shearing), the drops carry around a volume of 510 mht', whereas a drop of 300 nm (size obtained after high pressure homogenization) encapsulates a volume of approximately 0.11 μm ³ . We deduce that for the same volume of dispersed lipid phase we have about 4640 times more particles in suspension. It is this large number of drops which promotes the encounter statistics between the pathogens and the active ingredients contained in the nanometric dispersions and makes them more effective (at the same concentration of active ingredient, i.e. 5 to 50 mg HE/ml of lipid phase) pathogen treatments in demanding environments. For example, we can cite the decontamination of zooplankton production basins such as Rotifers, Copepods or Artemia, which produce significant quantities of pathogens, in particular Vibrio. Today Artemia farms are washed with fresh water, then with hydrogen peroxide, which is a very aggressive treatment for this zooplankton.

Claims

1. Nano-emulsion with a continuous aqueous phase and a dispersed lipid phase, characterized in that the dispersed lipid phase contains mineral particles consisting of clusters of phyllosilicate sheets swollen with water and exfoliated, at least one essential oil and an amphiphilic dispersing agent, and in that the size D50 in number of the drops of the dispersed lipid phase is less than 800 nm and preferably less than 300 nm.

2. Nano-emulsion according to any one of the preceding claims, in which the amphiphilic dispersing agent is chosen from the group of ethyl lauroyl arginate (LAE), cationic surfactants based on arginine with 16 carbons and more , phospholipids, e-polylysine and combinations thereof.

3. Nano-emulsion according to claim 2, wherein said dispersing agent is a phosphoglyceride.

4. Nano-emulsion according to Claim 3, in which the said dispersing agent is a phosphatidyl choline and preferentially lecithin.

5. Nano-emulsion according to any one of the preceding claims, in which the sheets of phyllosilicates are sheets with interlayer sodium cations and very preferentially mainly sheets of montmorillonites.

6. Nano-emulsion according to any one of the preceding claims, in which the content of dispersing agent is between 10% and 400% and preferably between 20% and 300% by weight relative to the weight of particles.

7. Nano-emulsion according to any one of the preceding claims, in which the dispersed lipid phase comprises a water content of between 10% and 300% and preferably between 20% and 200% by weight relative to the weight of particles.

8. Nano-emulsion according to any one of the preceding claims, in which the dispersed lipid phase comprises a level of phyllosilicates greater than 0.5% by weight relative to the weight of said lipid phase and preferably between 2% and 20 %.

9. Use of a nano-emulsion according to any one of the preceding claims as an agent for decontaminating aquaculture water and drinking water for terrestrial animals.

10. Nano-emulsion as defined in any one of claims 1 to 8, for its use in the preventive treatment of animals against the development of contamination agents in their organism.

11. Use of a nano-emulsion according to any one of claims 1 to 8, to supplement a food, a food supplement or a premix.

12. Use of a nano-emulsion according to any one of claims 1 to 8, as a natural preservative based on essential oils, for preparations whose water activity parameter is greater than 0.45.

13. Process for preparing a nano-emulsion according to any one of claims 1 to 8, comprising the following steps:

- dissolve the dispersing agent in the aqueous phase of the lipid phase and obtain a homogeneous solution;

- add the phyllosilicates and mix the dough obtained with a kneader;

- add the lipids of the lipid phase;

- shearing the composition obtained;

- add the aqueous phase; - provide shear energy to obtain a micrometric emulsion; and

- resume the micrometric emulsion with a high pressure homogenizer or a microfluidizer to obtain a nanometric emulsion.