AQUEOUS NANO-DISPERSIONS AND NANO-EMULSIONS for water treatment

Abstract

Lipid composition comprising particles and lipophilic active molecules, such that the particles consist of clusters of D90-sized phyllosilicate sheets with a number less than 1000 nanometers in size, such that the active molecules comprise essential oils, and such that the active molecules are physically bound to the surface of the particles with an amphiphilic dispersing agent. Figure 8

Description

Title of the invention: AQUEOUS NANO-DISPERSIONS AND NANO-EMULSIONS for water treatment Field of invention

[0001] The present invention relates to aqueous nano-dispersions and nano-emulsions intended to decontaminate water or to prevent its contamination by pathogenic agents, in particular water used for aquaculture, or drinking water for terrestrial animals, which can, depending on the dosage, be used as a preventive treatment for animal infections. State of the art

[0002] Globally, demand for fish is increasing due to a combination of population growth, urbanization, and increasing 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 the world's animal-based foods. This makes it an attractive target, as described later in this document, although the principles also apply to terrestrial animal farming and domestic animals, each with its own specific characteristics.

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

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

[0005] We can cite large families of recurring pathogens in livestock farming, encountered in farms: photobacterium (for example subspecies damselae), vi-brionacea (for example alginolyticus, photobacterium, or vibrio), pseudomonas (for example anguilliseptica).

[0006] 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 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 combat pathogens and multi-resistance.

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

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

[0009] Usually, treatments are carried out in balneotherapy in aquaculture farms. But since these are volatile and hydrophobic molecules, soaking fish in a basin containing EOs 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 to effectively protect farms, particularly aquaculture, from these pathogens in a targeted manner, as a substitute for the systematic use of antibiotic drugs, and particularly in a preventive approach, which does not induce antibiotic resistance. This is a challenge coupled with the constraints of implementing the oils which must be administered effectively and simply to the target species (aquaculture farming, and distribution in drinking water for terrestrial species). Brief description of the invention

[0011] 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 1,000 nanometers, and such that the active molecules comprise essential oils.

[0012] The clusters of very small phyllosilicate sheets serve as support for the essential oil molecules that are physically bound to the surface of the phyllosilicate sheets with an amphiphilic surfactant, this has the advantage of stabilizing these molecules and concentrating them on their surface. The small size of the sheet clusters thus allows the active molecules to be widely dispersed, 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.

[0013] 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.

[0014] The reduction in the size of the phyllosilicate sheet clusters has the advantage of promoting the dispersion of the active molecules while having a high local concentration of active molecule, and thus of reinforcing their effectiveness. Using these nanometric-sized phyllosilicate sheets, the contact surface with the essential oils is increased, which remain labile, because they can desorb, which promotes their effectiveness. Furthermore, they are thus dispersed in the volume of water on their phyllosilicate support, and can interact with pathogens.

[0015] This aqueous nano-dispersion is very simple to implement in an aquaculture water basin or in terrestrial animal drinking water. The small size of the leaflet clusters facilitates their suspension in the water with slow sedimentation.

[0016] 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 by water and exfoliated, at least one essential oil and an amphiphilic dispersing agent, and in which the number-average size D50 of the drops of the dispersed lipid phase is less than 800 nm and preferably less than 300 nm.

[0017] The water of the lipid phase is physisorbed between the phyllosilicate sheets to swell them (interfoliar space), which makes it possible to achieve smaller particle sizes by exfoliation, and the dispersing agent, with a hydrophilic part and a hydrophobic part, is adsorbed on the surface of the sheet clusters by its hydrophilic part. This makes the sheets partially hydrophobic and allows good dispersion of the phyllosilicate sheets in the lipids.

[0018] The amphiphilic dispersing agent of the aqueous nano-dispersion and the nanoemulsion can be selected from the group of ethyl lauroyl arginate (LAE), cationic surfactants based on 16-carbon and higher arginine, phospholipids, e-polylysine and combinations thereof.

[0019] Advantageously, the dispersing agent is a phosphoglyceride.

[0020] Very advantageously, the dispersing agent is a phosphatidyl choline and preferably lecithin.

[0021] These dispersing agents will bind to the silanolate functions of the phyllosilicates via the cationic part of their structure.

[0022] Preferably, the phyllosilicates of the aqueous nano-dispersion and of the nanoemulsion have predominantly sodium cations in interfoliar positions.

[0023] Interfoliar sodium cations have the advantage of offering less resistance to increasing the interfoliar distance of the so-called sodium phyllosilicate sheets and thus promoting their swelling by water molecules as well as their exfoliation. Sodium phyllosilicates thus have advantages in terms of dispersion size to obtain smaller sheet clusters, this promotes fine dispersions of essential oil and thus a greater surface area to volume ratio of the phyllosilicate clusters which support the active molecules of essential oil is obtained, and this increases the statistics of encounter between the active molecules and the pathogens.

[0024] Very preferably, the phyllosilicates are smectites which are mainly montmorillonite sheets.

[0025] Advantageously, the content of amphiphilic dispersing agent in the aqueous nano-dispersion is between 0.2 and 2.0% by weight relative to the weight of the mineral particles.

[0026] Advantageously, the content of dispersing agent in 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.

[0027] 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.

[0028] At excessively high dispersing agent contents, the hydrophilic character of the particles is lost and the essential oil molecules 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.

[0029] For exfoliated phyllosilicate particles with a specific surface area of ​​the order of 300 m2 / g, the preferred range of dispersing agent content corresponds to a coverage rate of the silanol functions by the dispersing agent of between 20 and 60%, or between 30 and 130% of the mass of clay used to provide dispersing agent such as soy lecithin.

[0030] 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.

[0031] 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 particle sizes during their exfoliation.

[0032] Beyond 300%, the quantity 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 active molecules such as essential oils.

[0033] Aqueous nano-dispersions and nanometric emulsions can be used as a decontamination agent for aquaculture waters and drinking waters for terrestrial animals, or as a means of preventive treatment of pathologies in animals through their daily hydration, or in the preventive treatment of animals against the development of contaminating agents in their organism. They can thus be used to protect these waters from the proliferation of pathogenic agents. In certain indirect applications, these systems can constitute an appetizer to encourage the animals to take water.

[0034] Nanometric emulsions have the advantage of being able to remain in homogeneous suspension under the force of thermal agitation, which allows them to be stored for long periods while remaining very easy to homogenize during their use.

[0035] According to another mode of use, the aqueous nano-dispersions and the nanometric emulsions can supplement a food, a food supplement or a premix.

[0036] According to yet another mode of use, the aqueous dispersions and nanometric emulsions can be used as a natural preservative agent, based on essential oils for preparations having a high water activity.

[0037] Water activity does not represent 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 capacity for microorganisms to develop.

[0038] The nanometric emulsions obtained by this approach are particles that are very stable mechanically, as well as over time, and can be easily introduced into an extruder to be co-extruded into the heart of granulated foods. They will thus be ideal vehicles for the incorporation of essential oils into foods in the form of premix, which facilitates handling (because it reduces the risk of inhalation, or evaporation of the essential oils). The premixes are thus also found in a galenic form that promotes their metabolization with drop sizes of the order of a hundred nanometers.

[0039] Their presence in a food, a food supplement or a premix has the advantage of promoting the preservation over time of these foods, supplements or premixes, 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 micro-nutrients, or of agents of conservation which can be controversial despite their massive use.

[0040] The invention also relates to a process for preparing a nano-emulsion, comprising the following steps:

[0041] - dissolving the dispersing agent in the aqueous phase of the lipid phase and obtain a homogeneous solution;

[0042] - add the phyllosilicates and mix the resulting dough with a kneader;

[0043] - add the lipids of the lipid phase;

[0044] - shear the composition obtained;

[0045] - add the aqueous phase;

[0046] - providing shear energy to obtain a micrometric emulsion; and

[0047] - resume the micrometric emulsion with a high pressure homogenizer or a microfluidizer to obtain a nanometric emulsion.

[0048] The objective of the invention is a method of using essential oils which makes it possible to limit their volatility while having great effectiveness with controlled quantities used. This implementation of essential oils in a form dispersed at the nanometric scale in a stable manner promotes the statistics of encounters 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

[0049] The invention is described below with the aid of figures 1 to 8, given solely for illustration purposes:

[0050] [Fig-1] shows a structural diagram of a bentonite;

[0051] [Fig.2] shows the formula of lecithin;

[0052] [Fig.3] schematically shows the evolution of the lecithin content as a function of the specific surface area of ​​the clay and for several coverage rates;

[0053] [Fig.4] schematically shows the evolution of the size of the drops of the phase dispersed according to the size and concentration of the mineral particles;

[0054] [Fig.5] shows the size of mineral particles for two concentrations of clay;

[0055] [Fig. 6] shows the measurement of the size of different clays dispersed in water;

[0056] [Fig.7] shows the size of lipid drops in an emulsion;

[0057] [Fig.8] shows a diagram of the emulsion obtained. Detailed description of the invention

[0058] 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 a “direct emulsion” or by the acronym “O / W”.

[0059] 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 non-covalent Van der Waals forces, which gives it a capacity for reversible attachment or organization.

[0060] In the following, 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 less than this value, by analogy a D90 in number represents the size of the particles for which 90% of the particles of the sample have a particle size less than this value.

[0061] The term “nanometric emulsion” or “nano-emulsion” means an emulsion in which the drops of the dispersed phase have a size D50 in number less than 500 nanometers (nm), and a D90 in number less than 1000 nm.

[0062] The term “nano-dispersion” or “nanometric dispersion” means a dispersion in which the colloidal particles of phyllosilicates are dispersed with a D50 size in number less than 1000 nanometers (nm).

[0063] The present invention thus relates to an aqueous nano-dispersion and a nanoemulsion comprising liposoluble particles and active molecules. The particles consist of clusters of phyllosilicate sheets of size D50 in number less than 1,000 nanometers (nm). The active molecules can be essential oils (EO) or combinations of essential oils. Lipids

[0064] The lipids of the lipidic, or hydrophobic, or oily, or organic phase of the nanometric emulsions are chosen according to the intended applications from vegetable oils, mineral oils, synthetic oils, hydrophobic organic solvents and hydrophobic liquid polymers. The lipidic phase of the nanoemulsion comprises bacteriostatic agents, such as essential oils. It may also contain unsaturated fatty acids, antioxidants and vitamins.

[0065] In the examples, sunflower oil is used. Essential oils

[0066] For centuries, man has used plants as a means of preventing and / or curing diseases. More specifically, extracts from these plants have been used as remedies by many civilizations across the globe.

[0067] 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 the epicarp of citrus fruits, or by dry distillation. The essential oil thus obtained is separated from the aqueous phase by physical processes."

[0068] An essential oil is a 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 certain 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 contains no proteins, lipids, or carbohydrates, does not contain minerals or vitamins: it therefore has no nutritional value.

[0069] At room temperature, essential oils are liquid, except for a few special cases: myrrh and sandalwood essential oils are rather viscous and rose and camphor essential oils can be crystallized. At low temperatures, some essential oils crystallize: anise, field mint or saturated thyme essential oils when the bottles are stored in the refrigerator.

[0070] Essential oils are volatile, this volatility explains their odorous 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 EO by putting them in contact with an animal fat such as lanolin), ether, most organic solvents as well as in alcohol (of high titer). Liposoluble in nature: essential oils do not dissolve in water, it is therefore essential to use a surfactant to allow them to be suspended in water. They have a high refractive index and often have a rotatory power. Most of them are colored.

[0071] 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.

[0072] The activity of the components of EOs depends on their chemical structure. Oxygenated terpenes (e.g., carvacrol, thujone) exhibit higher antimicrobial activity than non-oxygenated terpenes (e.g., α- and β-pinene, limonene). In addition, the lipophilic nature of their hydrocarbon skeleton and the hydrophilic nature of their functional groups are decisive for their antimicrobial activity. Thus, the order of antimicrobial activity of these compounds is as follows: phenols > aldehydes > ketones > alcohols > ethers > hydrocarbons.

[0073] Among the essential oil constituents, phenolic compounds have been shown to possess significant antimicrobial activity. It appears that the presence of the hydroxyl group is linked to the inactivation of microbial enzymes. Most likely, this group interacts with the cell membrane, causing leakage of cellular components, a change in fatty acids and phospholipids, as well as an alteration of energy metabolism and an influence on the synthesis of genetic material. Phyllosilicates

[0074] Phyllosilicates are clay minerals of the silicate group built by stacking tetrahedral ("T") layers where the tetrahedra share three out of four vertices (the "basal" oxygens), the fourth vertex (the "apical" oxygen) being connected to an octahedral ("O") layer occupied by different cations (Al, Mg, Fe, Ti, Li, etc.). [Fig.l] 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), qui seront stabilisés par des contres-ions cationiques. Ces contres-ions seront monovalents, ou divalents, ce qui confère à l’argile une capacité à être gonflée dans l’eau plus ou moins fortement, par insertion de molécules d’eau entre les feuillets.

[0075] Smectites are a group of clay minerals, and therefore silicates, more precisely phyllosilicates.

[0076] Their typical composition is A03D2 3T4OioZ2n H2O, where A represents an interlayer cation (alkali or alkaline earth element), D an octahedral cation, T a tetrahedral cation, O oxygen and Z a monovalent anion (generally OH-).

[0077] They crystallize in the monoclinic system.

[0078] These are phyllosilicates with a TOT (or 2:1) structure, i.e. made up of sheets comprising two head-to-tail tetrahedral layers, linked together by octahedral cations. The sheets are linked together by interlayer cations.

[0079] A distinction is made between dioctahedral smectites (beidellite, montmorillonite, nontronite, etc.) and trioctahedral smectites (hectorite, saponite, etc.).

[0080] Montmorillonite is a 2 / 1 type clay, also called TOT (for tetrahedron / octahedron / tetrahedron). This means that a montmorillonite sheet is formed of three layers:

[0081] - an octahedral layer A1(OH )5O: 7 atoms for 6 vertices + aluminum at center. The OH and oxygen are shared between the different octahedra that make up the layer.

[0082] - and two tetrahedral layers which cover on each side the octahedral layer at its base; SiO4: 5 atoms for 4 vertices + silicon in the middle. The oxygens are shared between the different tetrahedra that make up the layer.

[0083] Imperfections in the crystal are compensated by monovalent or divalent interlayer cations, which ensure the electrical neutrality of the mineral.

[0084] All phyllosilicates can be used, but smectites and particularly montmorillonites have the advantage, due to their lamellar structure with a greater spacing between the sheets than other phyllosilicates, to be 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. Other phyllosilicates, but also micas and talcs can also be exfoliated in this way, but the energy that would be necessary to disperse the lamellar sheets in the lipid phase would be much higher.

[0085] In the examples, bentonite is used as phyllosilicate. Bentonites are clays mainly composed of montmorillonite, the interlayer cations of which are usually either calcium, sodium, potassium, their combination or other metal ions.

[0086] Bentonite is negatively charged on the surface (along the length) and positively charged on the sides (width) which allows it to interact with other charged molecules.

[0087] Clays are hydrophilic and smectites, including bentonite, have a swelling capacity. This characteristic makes it possible to adsorb water-soluble molecules in the interlayer space of clays via an aqueous phase. Water is said to be physisorbed on the surface of the clay sheets via the silanol groups.

[0088] In the description, the term “clay” will also be used to refer to phyllosilicates. Dispersion or surfactant

[0089] A molecule with a hydrophobic part and a hydrophilic part is usually used as a dispersing or surfacing agent. The adhesion of this dispersing agent by physical interaction with the mineral particles makes it possible to make the clay sheets hydrophobic and to obtain good dispersion of these clay sheets in a lipid phase.

[0090] 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 choline, thus obtaining phosphatidylserine, phosphatidylethanolamine, or phosphatidylcholine, better known under the name of "lecithin". It is a lipid of the phosphoglyceride class. Arginine grafted onto a long alkyl chain (Cl6 and more) can also play this role of dispersing agent. Ethyl lauroyl arginate (LAE) can also be used. E-polylysine can also be used as a surfactant.

[0091] All these dispersing agents are labile because they bind to the surface of the phyllosilicate sheet clusters by non-covalent interactions, which gives them a reversible attachment or organization capacity depending on the physicochemical conditions (pH, ionic strength, temperature, etc.).

[0092] Phosphatidylcholine has ([Fig.2]):

[0093] - a hydrophilic pole: choline (1) and the phosphate group (2);

[0094] - a hydrophobic tail: fatty acid residues (here, the acid residues palmitic (5) and oleic (4)); and

[0095] - glycerol (3) links these two hydrophilic and hydrophobic poles.

[0096] The phosphate group is negatively charged, while choline is positively charged. Phosphatidylcholine is therefore zwitterionic.

[0097] 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.

[0098] Measurements of the particle size of an aqueous or lipid dispersion (DLS)

[0099] The size of mineral particles obtained in an aqueous or lipid medium is measured by dynamic light scattering (DLS). The experiments were carried out with a Malvem NanoZS 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 Malvern 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.

[0100] The tested sample 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 minute before the measurement, the tested sample is stirred with a vortex.

[0101] The figures presented give the evolution of the number of particles as a function of their size in semi-logarithmic coordinates.

[0102] Aqueous nano-dispersion of phyllosilicate particles

[0103] (1) Prepare an aqueous solution of phyllosilicates: homogenize the solution with a rotor stator and let it rest;

[0104] (2) Add to the aqueous solution of phyllosilicates a given quantity of the agent of dispersion, e.g. e-polylysine; homogenize by vortexing;

[0105] (3) Add to the previous solution a given quantity of essential oil;

[0106] (4) Homogenize the completed solution in an ultrasonic bath to obtain the aqueous nano-dispersion of phyllosilicates.

[0107] [Fig. 6] shows the particle sizes of four different phyllosilicates after dispersion and exfoliation in water as previously described.

[0108] The concentrations of phyllosilicates are 5% by weight relative to the whole composition during exfoliation and 0.1% when measuring the particle sizes.

[0109] [Fig.6(a)] shows the size curve obtained for a Wyoming bentonite. The D50 is: 255 nm.

[0110] [Fig.6(b)] shows the size curve obtained for a superfine green clay of origin France, montmorillonite - illite. The D50 is: 295 nm.

[0111] [Fig.6(c)] shows the size curve obtained for a Lafaure bentonite. The D50 is: 295 nm.

[0112] [Fig.6(d)] shows the size curve obtained for a Smectagri bentonite. The D50 is: 712 nm.

[0113] 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 in number of particles at the diameter considered.

[0114] In all four tests, the D90 particle size is well below 1 micrometer. Preparation of a nano-emulsion

[0115] The objective is to obtain a nanometric emulsion according to one of the objects of the invention.

[0116] To combine two systems which originally have no affinity, one apolar (lipids - essential oils) and the other polar (aqueous phase - phyllosilicates), we will:

[0117] (1) Dissolve the dispersing agent and obtain a homogeneous mixture;

[0118] (2) Add the clay and mix with a kneader the composition obtained for pre-swelling the phyllosilicate sheets by swelling in water, and adsorbing the dispersing agent on the surface of the phyllosilicate particles, to make it compatible with the lipid phase;

[0119] (3) Add the lipids of the lipid phase;

[0120] (4) Providing shear energy to the resulting composition to disperse / exfoliate the clay sheets in the lipid phase;

[0121] (5) Add the water from the external aqueous phase with e.g. a phase ratio oily / aqueous phase of 40 / 60;

[0122] (6) Providing shear energy to the resulting composition to disperse the lipid phase into the aqueous phase and obtain a micrometric emulsion; and

[0123] (7) Take this emulsion and shear it with even more energy by treatment in a high-pressure homogenizer or microfluidizer and obtain a nanometric emulsion.

[0124] Step (1) is obtained by adding water in sufficient quantity to solubilize the dispersing agent and impregnate the clay sheets.

[0125] 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 layers integrate the entire volume of water. It is not necessary to saturate the entire water absorption capacity of the clays to obtain a satisfactory dispersion of the clay in the lipid phase, This is why we limit the water intake to 300% by weight relative to the clay. Conversely, it is necessary for the clays to be minimally impregnated with water to promote their dispersion. Exfoliable clays are usually stored at a humidity level of 10%. It is essential not to go below the 5% threshold to avoid the collapse of the phyllosilicate sheets, leading to a structure that loses its exfoliation capacity.

[0126] An insufficient water content of the lipid phase does not allow the solubilization of the hydrophilic molecules of interest.

[0127] When there is too high a water content, the clay sheets are easier to exfoliate but they are impregnated with water and they have less capacity to adsorb the molecules of the dispersing agent (usually hydrophobic). They retain their hydrophilic character.

[0128] In step (2) a dispersing agent is used, such as lecithin as a dispersing / exfoliating agent. 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 e-polylysine is pre-dissolved in the water from step (1) to facilitate its incorporation. The quantity of lecithin or e-polylysine can vary between a coverage rate of the clay surface of 5% and 100%. This coverage rate is calculated based on the total external clay surface after exfoliation, and the number of anionic charges on the surface of the sheets (usually there are about five silanol functions per nanometer squared, 5 / nm2) accessible by the lecithin or e-polylysine molecules (the most swollen sheets (separated by water)). It therefore depends on the specific surface area of ​​the clay accessible by the dispersing agent.

[0129] It is therefore necessary to work on different rates 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.

[0130] [Fig.3] schematically presents the evolution of the necessary lecithin content by weight relative to the clay content by weight as a function of the specific surface area of ​​the exfoliated clays for several rates of coverage of the silanols of the clay sheets by lecithin.

[0131] 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 area of ​​100 m2 / g and 1000 m2 / g. For a coverage rate of 60% the optimal lecithin content is between 39% and 387% in mass relative to the mass of the clay and respectively a specific surface area of ​​clay of 100 m2 / g and 1000 m2 / g.

[0132] When the content of dispersing agent such as lecithin is insufficient, the hydrophobic character of the clay particles is insufficient.

[0133] When the amount of dispersing agent such as lecithin allows the surface silanols of the particles to be covered, the dispersion of the clays in the lipid phase is promoted. At coverage rates close to 100%, the hydrophilic character of the 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.

[0134] 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 nano-sized particles.

[0135] For the bentonite used, with a specific surface area of ​​the order of 300 m2 / g, we must therefore have an optimal 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.

[0136] Step (4) can be obtained by shearing applied in batch using, for example, a Silverson (rotor-stator shear), additional treatment by ultrasound or using a high-pressure homogenizer is also possible to reduce the particle size.

[0137] 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 Na Cl at a concentration of less than 50 mM and very advantageously 25 mM.

[0138] In step (6) the supply of shear energy can be achieved with a rotor / stator with an air gap of 150 micrometers and a 30 mm mobile, at 4,500 rpm for 4 minutes.

[0139] In order to reduce the average droplet diameters, 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 processing pressure of 1000 bar which corresponds to the sum of the pressures at both valves. Tests

[0140] Several tests have been carried out to show the interest of phyllosilicate particles as a support for essential oils and thus improve their specific effectiveness. on the one hand and illustrate the obtaining of lipid drops of appropriate size on the other hand.

[0141] In vitro test of the activity of essential oils bound to phyllosilicate particles in an aqueous dispersion

[0142] The choice of microorganisms was based on three bacterial species causing frequent pathologies in fish, more specifically sea bream (sparus aurata).

[0143] These are species that cause diseases inducing massive mortality in most cultures of different species of fish. They are mainly gram negative. The microorganisms were provided to us by the Pasteur Institute, in the form of bacterial lyophilisate, with the following references:

[0144] Vibrio alginolyticus CIP 103336T

[0145] Photobacterium damselae ssp. Piscicida CIP 104404T

[0146] Pseudomonas anguilliseptica CIP 1067 HT

[0147] In addition to the strains studied, two control strains were used:

[0148] Escherichia coli, provided by the microbiology laboratory at Polytech Clermont Ferrand.

[0149] Staphylococcus aureus (Ref.: cip 53-156), provided by LMGE, University of Clermont Auvergne.

[0150] The test of the effectiveness of a composition according to the invention, of an essential oil, linked to bentonite particles was carried out by the aromatogram method.

[0151] This is a method inspired by the antibiogram which makes it possible to determine the growth-inhibiting activity of essential oils by measuring the inhibition diameter around a central well into which the dispersion containing the essential oil will be introduced. A suspension of the germ in question is prepared in sterile distilled water and adjusted to, for example, 108 bacteria / ml. Each suspension (100 μl) is spread on a Petri dish 90 mm in diameter containing 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.

[0152] Growth inhibitory activity is determined by the inhibition diameters around the wells after 24 to 48 hours.

[0153] The aqueous nano-dispersion according to one of the objects of the invention consists of:

[0154] Sterile distilled water: 10 g

[0155] Bentonite: 0.5 g

[0156] e-Polylysine: 1 ml of solution (at 0.36 g / l)

[0157] Essential oil: 50 mg.

[0158] The order of addition is respected according to the composition above, and each ingredient is vigorously stirred before the addition of the next ingredient. Thus the bentonite is pre-swollen and exfoliated 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.

[0159] This constitutes a mother solution of supported essential oil dispersion. For the tests, 200 μl of this essential oil dispersion at 4.55 mg / ml are introduced into the well of the Petri dish, which represents 0.9 μg of essential oil.

[0160] A control with antibiotics is also carried out: Chloromphenicol for V. algi-nolyticus and P. anguilliseptica at 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

[0161] The following table shows the average inhibition diameters obtained for the essential oils which demonstrated notable inhibitory activity.

[0162] [Tables 1] P. damselae V. algi-nolyticus P. Anguilliseptica E.coli S. aureus Ajowan (Carum Opticum) 28 32 31 25 30 Chinese cinnamon (cinnamomum cassia L.) 30 30 32 28 28.5 Lemongrass (Cymbopogon flexuosus) 30 28 32 24 22.5 Compact oregano (Origanum compactum L.) 31 28 32 28 25 Palmarosa (Cymbopogon martinii (Roxb.)Will. Watson var. motia) 28 30 32 28 25 Palmarosa (Cymbopogon martinii) 29 30 33 26 23.5 Mountain savory (Satureja montana L.) 27 29 32 28 25 Thymol Thyme (Thymus zygis L.) 28 25 30 26 25.5 Reference ATB (Chloramphenicol) 30 30 30 30 Reference ATB (Ciprofloxacin) 30

[0163] Among the 38 essential oils tested, at this concentration of essential oil (EO), only 7 EOs gave a significant growth inhibition signature common to the three species studied. Regarding P. anguilliseptica, more sensitivity was observed towards the majority of EOs. The seven most inhibited EOs The varieties are: Chinese Cinnamon, Compact Oregano, Lemongrass, Palmarosa, Thymol Thyme and Mountain Savory.

[0164] These results show the full interest of using an aqueous nano-dispersion according to one of the objects of the invention comprising phyllosilicate particles supporting essential oils. Exfoliation of phyllosilicates in oil

[0165] To measure the particle size in the lipid phase of a nanoemulsion, the previous protocol was followed, stopping at step (4) of supplying high shear energy. Then samples were taken to qualify the size of the phyllosilicate particles dispersed in the lipid phase.

[0166] [Fig.5] shows 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 has 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 decrease in particle size (by friction between the particles) which reflects an improvement in dispersion. Preparation of a nanometric emulsion

[0167] The invention also relates to nanometric emulsions. These emulsions are composed of a dispersed lipid phase - the previous lipid composition - and a continuous aqueous phase.

[0168] Direct emulsions were prepared using a lipid phase / aqueous phase ratio of 40 / 60. Emulsification was carried out by supplying shear energy with a rotor / stator device with a 150 micrometer air gap and a 30 mm rotor, at 4,500 rpm for 4 minutes. The emulsions thus obtained remain micrometric, because the energy supplied 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.

[0169] The size of the drops thus obtained is measured as for the particles by dynamic light scattering (DLS). [Fig.7] shows the average diameter of the lipid drops of the dispersed phase. The D50 is 300 nanometers.

[0170] Since the size of the drops is directly related to that of the particles, the size of these particles can be estimated to be of the order of 50 nanometers.

[0171] [Fig.8] schematically presents the emulsion 20 obtained.

[0172] The lipid particles 28 are seen dispersed in an aqueous phase 26.

[0173] The lipid particles or drops, which have a composition as previously described, are stabilized by the dispersed phyllosilicate mineral particles. These very small particles, which retain a certain hydrophilic character, will preferentially be arranged at the water / oil interface, this is what is represented in reference 24.

[0174] This lipid product is obtained from a direct oil / water emulsion obtained by dispersing in water 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.

[0175] The concentrated nanometric emulsions thus obtained make it possible to work on applications requiring the quantity of water provided by the emulsion, such as dry food preparations and dry food coatings. The concentrated solutions also make it possible to reduce volumes and concentrate the active ingredients, which facilitates product logistics and makes it possible to work on applications requiring larger quantities and concentrations of active ingredients.

[0176] Nanometric emulsions are an excellent means of maximizing the statistics of encounters between pathogens and active ingredients, because by diluting the latter in a lipid matrix and supporting them on the phyllosilicate sheets at the interface, then reducing the size of the objects to the nanometric scale, the number of particles that can interact with the pathogens is multiplied by several orders of magnitude. Indeed, in emulsions of 20 microns (drop size usually obtained with rotor-stator shear), the drops carry a volume of approximately 510 pm3, while a drop of 300 nm (size obtained after high-pressure homogenization) encapsulates a volume of approximately 0.11 pm3. We deduce that for the same volume of dispersed lipid phase we have around 4640 times more particles in suspension.It is this large number of drops that promotes the statistical encounter between pathogens and the active ingredients contained in nanometric dispersions and makes more effective (at the same concentration of active ingredient, i.e. 5 to 50 mg HE / ml of lipid phase) treatments against pathogens in demanding environments. For example, we can cite the decontamination of zooplankton production tanks such as Rotifers, Copepods or Artemia, which produce significant quantities of pathogens, particularly Vibrio. Today, Artemia farms are washed with fresh water, then with hydrogen peroxide, which is a very aggressive treatment for this zooplankton.

Claims

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 exfoliated phyllosilicate sheets, at least one essential oil and an amphiphilic dispersing agent, and in that the number-wise size D50 of the drops of the dispersed lipid phase is less than 800 nm and preferably less than 300 nm.

2. The nanoemulsion of claim 1, wherein the amphiphilic dispersing agent is selected from the group of ethyl lauroyl arginate (LAE), cationic surfactants based on 16-carbon and higher arginine, phospholipids, e-polylysine and combinations thereof.

3. A nanoemulsion according to claim 2, wherein said dispersing agent is a phosphoglyceride.

4. Nano-emulsion according to claim 3, wherein said dispersing agent is a phosphatidyl choline and preferably lecithin.

5. Nano-emulsion according to any one of the preceding claims, in which the phyllosilicate sheets are sheets with interfoliar sodium cations and very preferably predominantly montmorillonite sheets.

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

9. Nano-emulsion as defined in any one of claims 1 to 7, for its use in the preventive treatment of animals against the development of contaminating agents in their organism.

10. Use of a nanoemulsion according to any one of the claims- indications 1 to 7, to complete a food, a food supplement or a premix.

11. Use of a nano-emulsion according to any one of claims 1 to 7, as a natural preservative, based on essential oils for preparations having a water activity allowing microorganisms to develop.

12. A method for preparing a nano-emulsion according to any one of claims 1 to 7, comprising the following steps: - dissolving the dispersing agent in the aqueous phase of the lipid phase and obtaining a homogeneous solution; - adding the phyllosilicates and mixing the resulting dough with a kneader; - adding the lipids of the lipid phase; - shearing the resulting composition; - adding the aqueous phase; - providing shear energy to obtain a micrometric emulsion; and - reworking the micrometric emulsion with a high-pressure homogenizer or a microfluidizer to obtain a nanometric emulsion.

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