EP3743377A1

EP3743377A1 - Encapsulation of active substances and/or micro-organisms in a lamellar material

Info

Publication number: EP3743377A1
Application number: EP19709998.9A
Authority: EP
Inventors: Sylvain K. PLUCHON; Jean-Claude Yvin; Jocelyne Brendle; Lionel Limousy; Patrick DUTOURNIE; Anne MAILLARD; Marion BRUNEAU; Simona BENNICI
Original assignee: Centre National de la Recherche Scientifique CNRS; Universite de Haute Alsace; Agro Innovation International SAS
Current assignee: Centre National de la Recherche Scientifique CNRS; Universite de Haute Alsace; Agro Innovation International SAS
Priority date: 2018-01-23
Filing date: 2019-01-22
Publication date: 2020-12-02
Also published as: WO2019145636A1; US20210032178A1; ZA202004541B; FR3077067A1; BR112020014279A2; CA3088222A1; US11649196B2; FR3077067B1

Abstract

The present invention relates to a process for encapsulating a compound selected from the group consisting of at least one active substance, at least one micro-organism and their mixtures in a hybrid organic-inorganic material having a lamellar 2:1 structure, this material having the following general formula: Na_x[(Mg₃)(Al_x(RSi)_4-x)O₈+x(OH)₂] (I), the process comprising the following steps: (a) sol-gel synthesis of the hybrid organic-inorganic material having a lamellar 2:1 structure in the presence of the compound; (b) recovery of the compound encapsulated in the material of general formula (I). The invention further relates to the compound encapsulated in a hybrid organic-inorganic material having a 2:1 lamellar structure of general formula (I), a composition comprising this compound and its use in plant fertilisation, nutrition, growth stimulation and/or prophylaxis and/or the improvement of physical, chemical and/or biological properties of the soil or a plant cultivation substrate.

Description

ENCAPSULATION OF ACTIVE SUBSTANCES AND / OR

MICROORGANISMS IN LAMELLAR MATERIAL

The present invention relates to the encapsulation in lamellar materials of active substances and / or microorganisms for the growth or treatment of plants.

The encapsulation of active substances in clays such as synthetic clays is already known (US 2009/0233107). However, such encapsulation takes place after the synthesis of the clay, which therefore requires the use of a two-step process.

In addition, there is no document mentioning the encapsulation of microorganisms within a clay, whether natural or synthetic.

However, the inventors have surprisingly discovered that it is possible to encapsulate such microorganisms and / or active substances in talc or saponite-type synthetic clay during the synthesis of these clays. that is to say in one step, while maintaining the activity of the active substances and microorganisms.

The present invention therefore relates to a method of encapsulation of a compound selected from the group consisting of at least one active substance, at least one microorganism and their mixtures in an organic-inorganic hybrid material of lamellar structure of type 2: 1, said material having the following general formula I:

Na _x [(Mg ₃ ) (Al _x (RSi) _4- x) 0 ₈₊ x (OH) ₂ ) (I)

in which

x is a number such that 0 <x <1, 2 and R represents a C 1 -C ₃₀ alkyl group, an aryl group, a C ₁ -C ₃ alkyl aryl group or an O-C 1 -C ₃₀ alkyl group; the alkyl group may be substituted by a group selected from a phenyl, vinyl, aminopropyl or mercaptopropyl group,

the method comprising:

a) sol-gel synthesis of the organic-inorganic hybrid material of 2: 1 lamellar structure in the presence of the compound;

b) recovering the compound encapsulated in the material of general formula I.

The present invention thus relates to a method of encapsulation in an organic-inorganic hybrid material of lamellar structure of type 2: 1, said material having the following general formula I:

Na _x [(Mg ₃ ) (Al _x (RSi) _4- x) 0 ₈₊ x (OH) ₂ ) (I)

in which

x is a number such that 0 <x <1, 2 and

R is alkyl _{Cl-C30 /} preferably n-hexadecyl group or a methyl group, especially a n-hexadecyl; an aryl group, advantageously a phenyl group; a (C ₁ -C ₃ ) alkyl aryl group, advantageously an ethylphenyl group; or a C ₁ -C ₃ O-alkyl group, advantageously an ethoxy group; the alkyl group may be substituted with a group selected from phenyl, vinyl, aminopropyl or mercaptopropyl.

For the purposes of the present invention, the term "C 1 -C ₃₀ alkyl group" means any linear or branched saturated alkyl group having from 1 to 30 carbon atoms, such as the methyl, ethyl, n-propyl, isopropyl or n-butyl group. isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, sec-pentyl, sec-isopentyl, neopentyl, n-hexyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl , n-heptyl, n-octyl, n-nonyl, n- decyl, n-undecyl, n-dodecyl, n-pentadecyl, n-hexadecyl, n-icosyl and n-triacontyl. Advantageously it is methyl, ethyl, n-propyl, isopropyl or n-hexadecyl, even more preferably methyl, ethyl, or n-hexadecyl, more particularly the ethyl or n-hexadecyl group.

By the term "aryl group" is meant in the sense of the present invention one or more aromatic rings having 5 to 20 carbon atoms, which can be contiguous or fused. In particular, the aryl groups can be monocyclic, bicyclic or polycyclic groups. Preferably it is phenyl, bi-phenyl, naphthyl, anthracenyl, phenanthrenyl, tetracenyl, chrysenyl, triphenylenyl, pyrenyl, benzofluorenyl, benzopyrenyl. Advantageously, it is a phenyl group.

The organic-inorganic hybrid material of lamellar structure is of type 2: 1. It is therefore silicates formed by stacks of layers that are constituted by an octahedral layer surrounded on both sides by two tetrahedral layers. The Si and Al atoms (if present) are found in the tetrahedral layer and the Mg atom in the octahedral layer. The atom of Na, if it is present, is in the interfoliar space. The material can therefore be either talc (when x = 0) or saponite (x¹0) type.

Advantageously, it is of talc type that is to say that x = 0.

The source of silicon necessary for the synthesis of the material of formula I is advantageously an organoalkoxysilane or a mixture of organoalkoxysilanes of the following general formula II: RSi (OR ₃ (II) in which

R is as defined above and

R 'is a methoxy or ethoxy group. Advantageously, the silicon source is chosen from the group consisting of:

phenyltrimethoxysilane (PhenylTMS) of formula (a) below:

Phenyl-Si (OCH ₃ ) ₃ (a);

tetraethylorthosilicate or tetraethylsilane (TEOS) of the following formula (b): Si (OC ₂ H ₅ ) ₄ (b);

hexadecyltrimethoxysilane (CI ₆ TMS) of formula (c) below:

CH ₃ (CH ₂ ) ₄ CH ₂ -Si (OCH ₃ ) ₃ (c)

the methyltriethoxysilane (MTES) of formula (d) below:

CH ₃ -Si (OCH ₃ ) ₃ (c)

the phenethyltrimethoxysilane of formula (e) below:

Phenyl- (CH ₂ ) ₂ Si (OCH ₃ ) ₃ (e)

triethoxyphenylsilane of formula (f) below:

Phenyl-Si (OC ₂ H ₅ ) ₃ (f);

and their mixtures.

In particular it is selected from the group consisting of methyltriethoxysilane, phenyltrimethoxysilane, tetraethylorthosilicate, hexadecyltrimethoxysilane, triethoxyphenylsilane and their mixtures, more particularly in the group consisting of phenyltrimethoxysilane, tetraethylorthosilicate, triethoxyphenylsilane and hexadecyltrimethoxysilane, and mixtures thereof. more advantageously in the group consisting of phenyltrimethoxysilane, tetraethylorthosilicate, hexadecyltrimethoxysilane and mixtures thereof. In the case of mixtures, it may be in particular a mixture between tetraethylorthosilicate and phenyltrimethoxysilane or between tetraethylorthosilicate and hexadecyltrimethoxysilane or between triethoxyphenylsilane and hexadecyltrimethoxysilane, advantageously it is a mixture between tetraethylorthosilicate and hexadecyltrimethoxysilane or between triethoxyphenylsilane and hexadecyltrimethoxysilane, still more advantageously between tetraethylorthosilicate and hexadecyltrimethoxysilane.

These mixtures can be made in any proportion.

The compound will therefore be encapsulated within the material of formula I and in particular if its size allows it (of the order of Angstrom) within the interfoliar space of the material (this is the case of tryptophan and folic acid). The compound according to the invention is chosen from the group consisting of at least one active substance, at least one microorganism and their mixtures.

For the purposes of the present invention, the term "active substance" is intended to mean any organic substance that is biologically active, that is to say any organic substance capable of reacting with living organisms, in particular plants, and therefore having a specific role in metabolism. for example, by acting directly on the plant, or by acting on the soil or the cultivation substrate, or by acting on pests or on the contrary on beneficial organisms. This type of substance can thus allow fertilization, nutrition, growth stimulation and / or plant prophylaxis and / or the improvement of the physical, chemical and / or biological properties of the soil or the substrate for growing plants. The active substance may be a molecule, but also a plant extract, an algae extract, a humic extract or any other type of extract or co-product. It can be of natural or synthetic origin, such as a hormone, a protein, an oligosaccharide, a lipid such as an essential oil, an enzyme, an amino acid such as tryptophan, a vitamin such as folic acid or the active substance of a drug or plant protection product such as a pesticide, fungicide, herbicide, nematicide. In an advantageous embodiment, the active substance is chosen from the group consisting of an amino acid, in particular tryptophan, a vitamin, in particular folic acid, an essential oil and mixtures thereof, more advantageously in the group consisting of an amino acid, in particular tryptophan, an essential oil and their mixtures advantageously it is tryptophan.

In a particular embodiment, the compound according to the invention is therefore an active substance. It can also be a mixture of 2, 3 or 4 active substances, in particular of 2 or 3 active substances.

In an advantageous embodiment, a single active substance is encapsulated, such as, for example, tryptophan or folic acid.

In the case where the encapsulated compound is an active substance, the source of silicon necessary for the synthesis of the material of formula I is advantageously phenyltrimethoxysilane or a mixture of organoalkoxysilanes of general formula II, advantageously a mixture of phenyltrimethoxysilane (a) and tetraethylorthosilicate (b). Advantageously, the mixture comprises less than 80 mol% of tetraethylorthosilicate and more than 20 mol% of phenyltrimethoxysilane, more preferably less than 60 mol% of tetraethylorthosilicate and more than 40 mol% of phenyltrimethoxysilane, still more advantageously less than 50 mol%. mol% of tetraethylorthosilicate and more than 50 mol% of phenyltrimethoxysilane, relative to the total number of mol of the mixture.

In an advantageous embodiment, the mixture comprises between 20 and 60 mol% of tetraethylorthosilicate and between 40 and 80 mol% of phenyltrimethoxysilane, in particular between 20 and 40 mol% of tetraethylorthosilicate and between 60 and 80 mol% of phenyltrimethoxysilane, more particularly 20 mol% of tetraethylorthosilicate and 80 mol% of phenyltrimethoxysilane, relative to the total number of mol of the mixture.

In the case where the encapsulated compound is an active substance, step a) comprises the following successive steps:

al) Adding a source of magnesium, advantageously magnesium nitrate hexahydrate, of the active substance, of the silicon source, in the case where x¹0, of the aluminum source, advantageously aluminum acetylacetonate, and a possible solvent;

a2) adjusting the pH to between 8 and 14, advantageously between 9 and 12, in particular 10, for example using an aqueous NaOH solution; a3) stirring the mixture, advantageously for a period of between 1 and 24 hours, in particular between 12 and 24 hours, more particularly at least 2 hours, still more particularly at least 12 hours, so as to obtain a gel;

a4) recovering the solid phase of the gel obtained in step a3);

a5) drying of the solid phase of the gel obtained in step a4).

The solvent of step a1) may be a polar solvent such as water, alcohol (in particular ethanol or glycerol), propylene glycol, methyl-5- (dimethylamino) -2-methyl- 5-oxopentanoate (such as RHODISOLV® Polarclean marketed by Solvay), propylene carbonate or mixtures thereof, advantageously water, alcohol (in particular ethanol or glycerol), methyl-5- (dimethylamino) -2-methyl-5-oxopentanoate (such as RHODISOLV® Polarclean marketed by Solvay) or their mixtures, more particularly water, alcohol (in particular ethanol or glycerol) or their mixtures. It may be a biobased and / or biodegradable solvent such as glycerol, propylene glycol, methyl-5- (dimethylamino) -2-methyl-5-oxopentanoate (such as RHODISOLV® Polarclean marketed by Solvay) , propylene carbonate or their mixtures, in particular glycerol, methyl-5- (dimethylamino) -2-methyl-5-oxopentanoate (such as RHODISOLV® Polarclean sold by Solvay) or mixtures thereof. It can also be a mixture of alcohol and oil.

In the case where the organic substance comprises an oil or is in liquid form, the solvent of step a1) may not be present.

On the other hand, in the case where the active substance is a solid, the solvent of step a1) must be present.

Step a3) can be carried out at a temperature between room temperature and the boiling temperature of the solvent, preferably at room temperature.

Step a4) may be carried out by any method well known to those skilled in the art, such as by filtration or centrifugation, in particular by centrifugation.

Step a5) can be carried out in an oven, for example at a temperature of 40 ° C, or by air drying or lyophilization.

An intermediate step a4) bis can be added between steps a4) and a5) which consists of washing the solid phase of the gel obtained in step a4) with the solvent of step a1).

In another particular embodiment, the compound according to the invention is therefore at least one microorganism. Advantageously, the microorganism is chosen from the group consisting of a bacterium such as Bacillus subtilis (for example accessible under the number CIP 52.62 from the Pasteur Institute), a microalga such as spirulina (for example cyanobacterium Arthrospira platensis sold under the name SPIRULINA NATURAL by the company EARTHRISE®), a fungus such as Piriformospora indica (for example accessible under the number DSM 11827 at the Max-Planck-Institut für terrestrische Mikrobiologie), and their mixtures, advantageously the microorganism is in vegetative form, in encysted form or in spore form, in particular in vegetative form, more particularly it is a bacterium such as Bacillus subtilis, still more particularly in vegetative form, or a fungus such as Piriformospora indica, more particularly a bacterium such as Bacillus subtilis.

The compound according to the invention may therefore consist of a single type of microorganism such as Bacillus subtilis bacteria. It may also consist of a mixture of 2, 3 or 4 different types of microorganisms, in particular of 2 or 3 types of microorganisms. different. In an advantageous embodiment, it consists of a single type of microorganism.

In the case where the encapsulated compound is a microorganism, the source of silicon necessary for the synthesis of the material of formula I is advantageously chosen from the group consisting of methyltriethoxysilane, phenyltrimethoxysilane, triethoxyphenylsilane and hexadecyltrimethoxysilane, and their mixtures, more advantageously the phenyltrimethoxysilane, triethoxyphenylsilane, hexadecyltrimethoxysilane and mixtures thereof, more advantageously it is hexadecyltrimethoxysilane or a mixture of triethoxyphenylsilane and hexadecyltrimethoxysilane, in particular it is hexadecyltrimethoxysilane.

In the case where the encapsulated compound is a microorganism, step a) comprises the following successive steps:

al) adding a source of magnesium, advantageously magnesium nitrate hexahydrate, the microorganism, a solvent, a source silicon and in the case where x¹0, the aluminum source, preferably aluminum acetylacetonate;

a2) adjusting the pH to between 8 and 14, advantageously between 9 and 12, in particular 10, for example using an aqueous NaOH solution; a3) stirring the mixture, advantageously for a period of between 1 and 24 hours, in particular between 12 and 24 hours, more particularly at least 2 hours, even more particularly at least 12 hours, advantageously 24 hours, so as to obtain a gel .

The method according to the invention may further comprise the following successive steps after step a3) of:

a4) recovering the solid phase of the gel obtained in step a3);

a5) drying of the solid phase of the gel obtained in step a4), advantageously by freeze-drying.

The solvent of step a1) may be a polar solvent such as water, alcohol (in particular ethanol or glycerol), propylene glycol, methyl-5- (dimethylamino) -2-methyl- 5-oxopentanoate (such as RHODISOLV® Polarclean marketed by Solvay), propylene carbonate or mixtures thereof, advantageously water, alcohol (in particular ethanol or glycerol), propylene glycol, methyl-5 - (dimethylamino) -2-methyl-5-oxopentanoate (such as RHODISOLV® Polarclean marketed by Solvay) or their mixtures, more particularly water, alcohol (in particular ethanol or glycerol), methyl- 5- (dimethylamino) -2-methyl-5-oxopentanoate (such as RHODISOLV® Polarclean sold by Solvay) or mixtures thereof, more particularly water, alcohol (in particular ethanol or glycerol) or their mixtures. It may be a biobased and / or biodegradable solvent such as glycerol, propylene glycol, methyl-5- (dimethylamino) -2-methyl-5-oxopentanoate (such as RHODISOLV® Polarclean marketed by Solvay) propylene carbonate or mixtures thereof, in particular glycerol, propylene glycol, methyl-5- (dimethylamino) -2-methyl-5-oxopentanoate (such as RHODISOLV® Polarclean marketed by Solvay) or their mixtures, more particularly glycerol, methyl-5- (dimethylamino) -2-methyl-5 oxopentanoate (such as RHODISOLV® Polarclean marketed by Solvay) or their mixtures. It can also be a mixture of alcohol and oil.

In an advantageous embodiment and in particular when the microorganism is in vegetative form, the microorganism of step a1) is in the form of a preculture of said microorganism, advantageously having a microorganism content of between 10 ³ and 10 ^10. ufc / ml, in particular between 10 ⁶ and 10 ⁹ cfu / ml, advantageously 10 ⁸ cfu / ml.

In this case, the method according to the invention may also comprise a preliminary step prior to step a) of preparing the microorganism preculture. This step can be carried out by methods that are well known to those skilled in the art. It includes in particular the seeding of the microorganism in a nutrient medium and the incubation for a period sufficient to obtain the desired microorganism concentration.

Step a3) can be carried out at room temperature.

Another step a4) ter can also be added between steps a4) or a4) bis and a5) which consists in the freezing of the solid phase of the gel obtained in step a4) or step a4) bis, if the latter is implemented.

Step a5) can be carried out in an oven, for example at a temperature of 40 ° C, or by air drying or lyophilization, Advantageously it is carried out by lyophilization.

The process according to the invention can be implemented in a bio-reactor.

The present invention further relates to a compound encapsulated in an organic-inorganic hybrid material of lamellar structure type 2: 1, said material having the general formula I as described above, the encapsulated compound being selected from the group consisting of at least one active substance, at least one microorganism and mixtures thereof.

The material, the active substance and the microorganism are as described above.

Advantageously, the encapsulated compound is obtainable, in particular obtained, by the process according to the invention as described above. The encapsulation rate is advantageously at least 20 mg of compound / g of material, more preferably at least 30 mg of compound / g of material, more advantageously at least 40 mg of compound / g of material, in particular at least 50 mg compound / g material, more particularly at least 60 mg compound / g material, more particularly at least 65 mg compound / g material.

The encapsulated microorganism can be revived by methods well known to those skilled in the art, such as for example by seeding on solid nutrient medium (petri dish) or liquid (bioreactor).

The encapsulated compound can be stored between 0 and 12 months at a temperature between 4 ° C and room temperature. When the compound is a microorganism or comprises a microorganism, it must be stored at a temperature of 4 ° C.

The present invention furthermore relates to a composition, in particular a phytopharmaceutical composition, advantageously intended for fertilization, nutrition, growth stimulation and / or plant prophylaxis and / or the improvement of the physical, chemical and / or biological properties of the plant. soil or plant culture substrate, comprising the compound encapsulated in a hybrid organic-inorganic material of lamellar structure type 2: 1 according to the invention.

This composition comprises any excipient suitable for administration to a plant or soil or plant culture substrate, for example by foliar application, root, field or above ground. It is in particular a phytopharmaceutically acceptable excipient. For the purposes of the present invention, the term "phytopharmaceutically acceptable", which is acceptable for use on plants or soil, that is to say non-polluting for the environment and non-toxic to humans ( users).

It may further comprise other active compounds having a synergistic or complementary action on the plant or the soil or plant culture substrate, such as, for example, nutrients advantageously chosen from the group consisting of nitrogen, phosphorus, potassium, calcium, magnesium, silicon, trace elements and their mixtures, organic raw materials and / or mineral raw materials, pesticides, fungicides, herbicides, nematicides, hormones, humic substances, seaweed extracts , amino acids, plant extracts, salicylic acid and the precursors or analogs of salicylic acid, nitric oxide and precursors or analogues of nitric oxide, cyclic nucleotides and mixtures thereof.

This composition can therefore be in the form of a fertilizer or a biostimulant.

It can be in solid form, in particular in the form of powder, granules or microgranules, in liquid form or in gel form.

Thus, it may for example be in solid form of the powder type, granulated, microgranulated in fertilizers or culture media for nutrition or stimulation of plants for use in full or localized.

It can also be in the form of a liquid or gel of fertilizers or biostimulants for use in foliar or root application.

It can also be in the form of a water-soluble fertilizer for use in fertigation field or above ground.

It can also be in a solid or liquid form of amendment for the improvement of the physical, chemical or biological properties of the soil or the growing medium.

It can be a phytosanitary type composition or a biocontrol composition, PNPP (natural preparations of low concern), SDN (stimulation of natural defenses), SDP (stimulation of plant defenses), in the case of plant prophylaxis .

The compositions according to the invention can therefore be used:

by direct application to a soil, over the entire soil surface or, preferably, in a localized manner in the root region of the plants to be treated; or

by application at the level of the leaves and / or the plants to be treated, by any suitable means of distribution, such as for example by spraying in the case of a liquid formulation. These compositions may further be introduced into the water irrigation system and / or into fertilizer formulations.

In general, the amount of composition to be used depends on the nature of the plant to be treated, the nature of the encapsulated compound and the intended mode of administration.

Those skilled in the art will know how to adapt the quantities to be used according to the mode of application chosen. In particular, relatively smaller amounts are used when the composition is applied in the root region while relatively larger amounts are used when the composition is applied over the entire soil surface. These compositions can be used in a single application or in sequential application.

The present invention finally relates to the use of the compound according to the present invention or of the composition according to the present invention for the fertilization, the nutrition, the stimulation of the growth and / or the prophylaxis of the plants and / or the improvement of the physical properties , chemical and / or biological soil or plant growth substrate, preferably cultivated plants or agronomic interest.

In particular this use can be implemented by foliar application, root, full field or above ground.

Bacillus subtilis bacterium stimulates plant growth and protects against biotic (pathogenic) and abiotic (lack of water) stresses. It makes it possible to solubilize in the water phosphate forms insoluble in the water of the phosphorus.

The fungus Piriformospora indica improves the ability of plants to tolerate environmental stress, stimulates plant growth and promotes the absorption of nutrients. Tryptophan is a precursor to the production of auxin, a hormone that regulates root development in particular.

The essential oil especially thyme is a stimulator of natural defenses.

Spirulina is a producer of chemical compounds with a biological activated on plants.

Folic acid inhibits primary root development and increased development and secondary root maturation (horizontal stretching) by redistribution of auxin (growth hormone) in primary roots.

The present invention will be better understood on reading the description of the drawings and examples which follow, which are given by way of non-limiting indication.

FIG. 1 represents the follow-up in time (in days) of the population (in cfu / ml) of Bacillus subtils CIP 52.62 encapsulated (compound according to the invention) and not encapsulated in the presence of phosphorus under the conditions of example 3 .

FIG. 2 represents the evolution of the pH of the culture medium as a function of time (in days) in the presence of Bacillus subtilis Cl. 52.62 encapsulated (compound according to the invention) and not encapsulated and in the presence of phosphorus under the conditions of Example 3.

FIG. 3 represents the concentration difference between the solubilized phosphorus and the phosphorus immobilized by the bacterial flora as a function of time (in days) in the presence of encapsulated Bacillus subtilis CIP 52.62 (compound according to the invention) and not encapsulated under the conditions of Example 3 Comparative Example 1: Effect of Different Biodegradable Solvents and Ethanol on the Chemical Synthesis of Lamellar-Inorganic Oral-Inorganic Compounds

In a 1000 ml beaker, 19.44 g of magnesium nitrate hexahydrate (MgNu ₃ , 6H ₂ O) (99%, Sigma) and 200 ml of biodegradable solvent (glycerol (Quaron> 99.5%) or propylene glycol (VWR)) were added. or methyl-5- (dimethylamino) -2-methyl-5-oxopentanoate (RHODISOLV® Polarclean sold by Solvay) or propylene carbonate (Quaron> 99.7%)) or ethanol with stirring at 55 ° C. at 220 rpm per minute for 15 minutes. Addition, with stirring for 15 minutes, of 4.58 g of triethoxyphenylsilane (97%, Sigma) and 15.42 g of hexadecyltrimethoxysilane (CI ₆ TMS) (> 85%, Sigma). Addition of 100mL of NaOH (97%, Sigma) IM and stirring 24h. After 15 days of storage at room temperature and relative humidity, in the dark, each synthesis is controlled to validate the good formation of the compounds and certain physicochemical properties (pH, viscosity, appearance, volume, stability) (Table 1). A product is considered stable if there is no apparent change in appearance and viscosity and there is no phase separation or settling.

Table 1: Physico-chemical properties of lamellar materials

In view of the results, the use of biodegradable solvents or ethanol during the syntheses does not therefore negatively affect the formation of the compounds. The substitution of ethanol is possible using glycerol, propylene glycol, propylene carbonate or methyl-5- (dimethylamino) -2-methyl-5-oxopentanoate (RHODISOLV® Polarclean marketed by Solvay).

Example 1 Encapsulation of an Active Molecule

Example 1.1: encapsulation of tryptophan

a) 100% compound Phenyl TRYPTO

1.944 g of magnesium nitrate hexahydrate (99%, Sigma Aldrich) are added to 20 ml of absolute ethanol (99.9%, Carlo Erba), the mixture is stirred until complete dissolution. 200 mg of L-tryptophan (> 98%, Sigma Aldrich) are introduced with stirring and then 2 g of phenyltrimethoxysilane (PhenylTMS) (98%, ABCR) are added. The whole is left stirring and the pH of the solution is brought to a value of 10 by addition of 15 ml of an aqueous solution of sodium hydroxide (> 97%, Sigma Aldrich) IM concentration. After 24h stirring at room temperature, the solid is separated from the solution by centrifugation (speed of 10,000 rpm for 10 min). The solid is washed three times with ethanol before being dried in an oven at 40 ° C for 48 hours. The compound obtained is then milled in an agate mortar before being characterized. 1.35 g of compound known as 100% phenyl TRYPTO is recovered.

The X-ray diffractogram of the sample shows several diffraction peaks in the angular domains 2-10 ° 2 theta, 15-25 ° 2 theta, 30-40 ° 2 theta and 55-652 theta. These peaks respectively correspond to reflections on the lattice planes (001), (020; 110), (130; 220) and (060: 330), characteristic of the presence of a lamellar phase. The value of the do6o periodicity is 0.156 nm, a characteristic value for a phase Lamellar organic-inorganic hybrid type talc type structure of the formula Mg ₃ (RSi) 40 ₈ (0H) 2 wherein R is phenyl. The periodicity of the invention is of the order of 1.32 nm.

The amount of tryptophan in the compound (encapsulation rate) was determined by UV spectrophotometry at a wavelength of 280 nm. The compound contains 63.4 mg of tryptophan per g of material. The thermogravimetric analysis carried out under air between 30 and 800 ° C at a rate of rise in temperature of 5 ° C / min shows that the most significant loss of mass (decomposition of the products) begins only from 300 ° C, which confirms that tryptophan has been encapsulated within the material (Table 2).

Table 2: Result of thermogravimetric analyzes carried out under nitrogen and under air

Comparison between the ¹³ C solid NMR spectrum of tryptophan alone and the 100% Phenyl TRYPTO compound indicates the presence of a broad resonance at about 111 ppm, attributed to the presence of tryptophan. The mobility of the latter is greatly reduced, which shows that tryptophan is present in the interfoliar space. So we get well tryptophan encapsulated in a hybrid material organic-inorganic lamellar structure type 2: 1 of the formula Mg ₃ (RSi) 40 ₈ (0H) 2.

b) compound 80Ph-20TEOS TRYPTO

2 g of magnesium nitrate hexahydrate (99%, Sigma Aldrich) are added to 20 ml of absolute ethanol (99.9%, Carlo Erba), the mixture is stirred until complete dissolution. 200 mg of L-tryptophan (> 98%, Sigma Aldrich) are added with stirring then a mixture consisting of 1.646 g of phenyltrimethoxysilane (PhenylTMS) (98%, ABCR) and 0.432 g tetraethylsilane (TEOS) (98%, ABCR) is added (mass mixture of 79.2% of PhenylTMS and 20.8% of TEOS which represents 80% of PhenylTMS and 20% of TEOS in mol). The whole is left stirring and the pH of the solution is brought to a value of 10 by addition of 15 ml of an aqueous solution of sodium hydroxide (> 97%, Sigma Aldrich) IM concentration. After 24h stirring at room temperature, the solid is separated from the solution by centrifugation (speed of 10,000 rpm for 10 min). The solid is washed three times with ethanol before being dried in an oven at 40 ° C for 48 hours. The compound obtained is then milled in an agate mortar before being characterized. 1.43 g of compound known as 80Ph-20TEOS TRYPTO is recovered.

The X-ray diffractogram of the sample shows several diffraction peaks in the angular domains 2-10 ° 2 theta, 15-25 ° 2 theta, 30-40 ° 2 theta and 55-652 theta. These peaks respectively correspond to reflections on the lattice planes (001), (020; 110), (130; 220) and (060: 330), characteristic of the presence of a lamellar phase. The value of the periodicity d ₀ 06 is 0.156 nm, a characteristic value for an organic-inorganic hybrid lamellar phase of talc type structure of formula Mg ₃ (RSi) 408 (OH) ₂ in which R represents a mixture of phenyl group and O-ethyl group. The periodicity is equal to 1.4 nm.

The amount of tryptophan in the compound (encapsulation rate) was determined by UV spectrophotometry at a wavelength of 280 nm, the compound comprises 68.7 mg of tryptophan per g of material.

The thermogravimetric analysis carried out under air between 30 and 800 ° C at a rate of rise in temperature of 5 ° C / min shows that the most significant loss of mass (decomposition of the products) begins only from 300 ° C, which confirms that tryptophan has been encapsulated within the material (Table 3).

Table 3: Result of thermogravimetric analyzes carried out under nitrogen and under air

Comparison between the ¹³ C solid NMR spectrum of tryptophan alone and the 80Ph-20TEOS TRYPTO compound indicates the presence of a wide resonance at about 111 ppm, attributed to the presence of tryptophan. The mobility of the latter is greatly reduced, a sign that tryptophan is present in the interfoliar space. Tryptophan encapsulated in an organic-inorganic hybrid material of 2: 1 type lamellar structure of the formula Mg ₃ (RSi) ₄ O ₈ (OH) 2 is thus obtained.

c) MTES compounds TRYPTO, CI ₆ TMS TRYPTO, TEOS TRYPTO, 20PH-80TEOS TRYPTO, 40Ph-60TEOS TRYPTO and 60Ph-40TEOS TRYPTO

By a method identical to that used to prepare the compound 100% Phenyl TRYPTO other compounds according to the invention were prepared by replacing phenyltrimethoxysilane as a silicon source with methyltriethoxysilane (MTES) or hexadecyltrimethoxysilane (CI ₆ TMS) or tetraethylsilane (TEOS). The compounds obtained were named respectively MTES TRYPTO, CI ₆ TMS TRYPTO and TEOS TRYPTO.

By a method identical to that used to prepare the compound 80Ph-20TEOS TRYPTO other compounds according to the invention were prepared by replacing the mixture 20% TEOS and 80% PhenylTMS as source of silicon with a mixture 80% TEOS and 20% PhenylTMS in mol, a mixture 60% TEOS and 40% PhenylTMS in mol and a mixture 40% TEOS and 60% PhenylTMS in mol. The resulting compounds were named respectively 20Ph-80TEOS TRYPTO, 40Ph-60TEOS TRYPTO and 60Ph-40TEOS TRYPTO.

The quantity of compounds recovered and the encapsulation levels determined by UV spectrophotometry at a wavelength of 280 nm are collated in Table 4 below.

Table 4

The X-ray diffractogram of the samples of each of the compounds exhibited several diffraction peaks in the angular domains 2-10 ° 2 theta, 15-25 ° 2 theta, 30-40 ° 2 theta and 55-652 theta. These peaks respectively correspond to reflections on the lattice planes (001), (020; 110), (130; 220) and (060: 330), characteristic of the presence of a lamellar phase. The value of the periodicity d ₀ 06 is 0.156 nm, a characteristic value for an organic-inorganic hybrid lamellar phase of talc type structure of formula Mg ₃ (RSi) ₄ 0 ₈ (OH) ₂ . The periodicity is equal to 1.4 nm.

The comparison between the ¹³ C solid NMR spectrum of tryptophan alone and the compounds according to the invention indicates the presence of a broad resonance at about 111 ppm, attributed to the presence of tryptophan. The mobility of the latter is greatly reduced, a sign that tryptophan is present in the interfoliar space.

Thus, for each compound according to the invention, tryptophan encapsulated in an organic-inorganic hybrid material of lamellar structure of type 2: 1 having the formula Mg 3 (RSi) ₄ O ₈ (OH) ₂ is obtained.

In the case where the silicon source is a mixture between TEOS and PhenylTMS, there is a linear correlation between the amount of encapsulated tryptophan and the% of TEOS used, with the exception of the case where the TEOS content is 0. % (100% Phenyl TRYPTO compound).

Example 1.2: Encapsulation of an essential oil

2 g of magnesium nitrate hexahydrate (99%, Sigma Aldrich) are added to 20 ml of a mixture composed of absolute ethanol (99.9%, Carlo Erba) and oil (Greenfix 3000) in equal proportions at 0%, 25%, 75% or 100% by volume of oil. The mixture is stirred until complete dissolution. 1.646 g of phenyltrimethoxysilane (98%, ABCR) and 0.432 g of tetraethylsilane (98%, ABCR) are then added (mass mixture of 79.2% of PhenylTMS and 20.8% of TEOS which represents 80% of PhenylTMS and % of TEOS in mol). The whole is left stirring and the pH of the solution is brought to a value of 10 by addition of 15 ml of an aqueous solution of sodium hydroxide (> 97%, Sigma Aldrich) IM concentration. After 24h stirring at room temperature, the solid is separated from the solution by centrifugation (speed of 10,000 rpm for 10 min). The solids are washed three times with ethanol before being dried in an oven at 40 ° C. for 48 hours. The compounds obtained are then milled in an agate mortar before being characterized and are designated SHE5 (25% oil), SHE15 (75% oil), SH20 (100% oil) and 80%. 20% T (0% oil). The quantities obtained for the different samples are respectively 2.24 g, 2.62 g, 0.12 g and 1.43 g.

Comparison between X-ray diffractograms of samples SHE5 (25% oil), SHE15 (75% oil), SH20 (100% oil) and 80% P-20% T (0% oil ) shows the presence of diffraction peaks in the angular domains 2-10 ° 2 theta, 15-25 ° 2 theta, 30-40 ° 2 theta and 55- 65 ° 2 theta. These peaks respectively correspond to reflections on the lattice planes (001), (020; 110), (130; 220) and (060: 330), characteristic of the presence of a lamellar phase. The value of the periodicity d ₀₆ o is 0.156 nm, a characteristic value for a lamellar phase of organic-inorganic hybrid type of talc type structure. The periodicities dooi are of the order of 1.4 nm. It should be noted that the intensity of the diffraction peaks decreases with the oil content in the mixture. The thermogravimetric analysis carried out under air between 30 and 800 ° C at a rate of rise in temperature of 5 ° C / min shows that the most significant loss of mass (decomposition of the products) begins only from 300 ° C, which confirms that the oil has been encapsulated within the material (Table 5).

Table 5: Result of thermogravimetric analyzes carried out under nitrogen and under air

Example 1.3 Encapsulation of Folic Acid

a) with ethanol as solvent

1.60 g of magnesium nitrate hexahydrate (99%, Sigma Aldrich) are added to 20 ml of absolute ethanol (99.9%, Carlo Erba), the mixture is stirred until complete dissolution. 200 mg of folic acid (> 97%, Sigma Aldrich) are introduced with stirring and then 2 g of phenyltrimethoxysilane (PhenylTMS) (98%, Sigma Aldrich) are added. The whole is left stirring and the pH of the solution is brought to a value of 10 by addition of 10 ml of an aqueous solution of sodium hydroxide (> 97%, Sigma Aldrich) of IM concentration. After 24h stirring at room temperature, the solid is separated from the solution by centrifugation (speed of 10,000 rpm for 10 min). The solid is dried in an oven at 60 ° C for 24 hours. The compound obtained (2.1 g) is then milled in an agate mortar before being characterized and is noted Eth-Ph 200AF in situ.

The X-ray diffractogram of the sample shows several diffraction peaks in the angular domains 2-10 ° 2 theta, 15-25 ° 2 theta, 30-40 ° 2 theta and 55-65 ° 2 theta. These peaks respectively correspond to reflections on the lattice planes (001), (020; 110), (130; 220) and (060; 330), characteristic of the presence of a lamellar phase. The value of the periodicity d ₀ 6o is 0.156 nm, a characteristic value for a lamellar phase of the organic-inorganic hybrid type of talc-type structure of formula Mg ₃ (RSi) 40 ₈ (OH) 2 in which R represents a phenyl group . The periodicity d ₀ oi is of the order of 1.14 nm. The amount of folic acid in the compound (encapsulation rate) was determined by UV spectrophotometry at a wavelength of 280 nm, the compound comprises 108.3 mg of folic acid per g of material. The thermogravimetric analysis carried out under air between 30 and 800 ° C at a rate of rise in temperature of 5 ° C / min shows that the most significant loss of mass (decomposition of the products) begins only from 300 ° C, which confirms that the folic acid has been encapsulated within the material (Table 6).

Table 6: Result of thermogravimetric analyzes carried out under nitrogen and under air

Comparison between the ¹³ C solid-state NMR spectrum of folic acid alone and the reference compound Eth-Ph 200AF in situ indicates the presence of broad resonances at about 46, 97, 112, 150 and 166 ppm, attributed to the presence of folic acid. The mobility of the latter is greatly reduced, a sign that folic acid is present in the interfoliar space. Folic acid encapsulated in a hybrid organic-inorganic material of lamellar structure of type 2: 1 of formula Mg ₃ (RSi) 40 ₈ (OH) _{2 is} thus obtained.

b) with glycerol as solvent

1.60 g of magnesium nitrate hexahydrate (99%, Sigma Aldrich) are added to 20 ml of glycerol (87%, Fluka), the mixture is maintained under agitation until complete dissolution. 200 mg of folic acid (> 97%, Sigma Aldrich) are introduced with stirring and then 2 g of phenyltrimethoxysilane (PhenylTMS) (98%, Sigma Aldrich) are added. The whole is left stirring and the pH of the solution is brought to a value of 10 by addition of 10 ml of an aqueous solution of sodium hydroxide (> 97%, Sigma Aldrich) of IM concentration. After 24h stirring at room temperature, the solid is separated from the solution by centrifugation (speed of 10,000 rpm for 10 min). The solid is washed four times with deionized water before being dried in an oven at 60 ° C for 24 hours. The compound obtained (2.2 g) is then milled in an agate mortar before being characterized and is noted Gly-Ph 200AF in situ.

The X-ray diffractogram of the sample shows several diffraction peaks in the angular domains 2-10 ° 2 theta, 15-25 ° 2 theta, 30-40 ° 2 theta and 55-65 ° 2 theta. These peaks respectively correspond to reflections on the lattice planes (001), (020; 110), (130; 220) and (060; 330), characteristic of the presence of a lamellar phase. The value of the periodicity do6o is 0.155 nm, a characteristic value for a lamellar phase organic-inorganic hybrid type talc type structure of the formula Mg ₃ (RSi) 40 ₈ (0H) 2 wherein R is phenyl. The periodicity d ₀ oi is of the order of 1.24 nm.

The amount of folic acid in the compound (encapsulation rate) was determined by UV spectrophotometry at a wavelength of 280 nm, the compound comprises 56.66 mg of folic acid per g of material. The thermogravimetric analysis carried out under air between 30 and 800 ° C at a rate of rise in temperature of 5 ° C / min shows that the most significant loss of mass (decomposition of the products) begins only from 300 ° C, which confirms that the folic acid has been encapsulated within the material (Table 7). Table 7: Result of thermogravimetric analyzes carried out under air

Comparison between the ¹³ C solid NMR spectrum of folic acid alone and the reference compound Gly-Ph 200AF in situ indicates the presence of a broad resonance at about 46, 97, 112, 150 and 166 ppm, attributed to the presence of folic acid. The mobility of the latter is greatly reduced, a sign that folic acid is present in the interfoliar space. We thus obtain well-encapsulated folic acid in an organic-inorganic hybrid material of lamellar structure type 2: 1 of the formula Mg ₃ (RSi) ₄ 0 ₈ (OH) _2.

Example 2 Encapsulation of a Microoraanism

Example 2.1: Encapsulation of Bacillus subtilis

a) 100% Phenyl BS compound

A preculture of Bacillus subtilis (accessible under the number CIP 52.62 of the Pasteur Institute) is prepared from cryotubes containing 400 μl of a suspension of Bacillus subtilis maintained at -20 ° C. in 1.6 ml of glycerol. The nutrient medium in which the contents of the cryotube are incorporated is an LB broth (lysogeny broth). It is composed of 10 g of tryptone (pancreatic hydrolyzate casein type peptone), 5 g of yeast extract and 10 g of NaCl for 1 liter of demineralized water. The yeast extract is obtained from yeast autolysates. This is suspended yeast biomass driven to autolysis by passage at 50 ° C for several hours, the liquid phase of which is recovered. This medium is prepared directly in the Erlenmeyer flasks and then autoclaved for 20 minutes at 121 ° C. The seeding is then carried out with the flame to avoid any contamination. An enumeration in the middle solid on petri dish (90 mm diameter) is performed after 18 hours of incubation at 37 ° C to determine the initial concentration of bacteria that is added during the synthesis.

The synthesis is carried out in a bioreactor. The first step is to clean the bioreactor with absolute ethanol. The preculture is introduced into the bioreactor (at 10% of the final volume, ie 200 ml of LB medium containing the bacteria at a level of 10 ³ cfu / ml). In a 2L bottle, 97g of magnesium nitrate (99%, Sigma Aldrich) are dissolved in one liter of absolute ethanol (99.9%, Carlo Erba). 100 g of phenyltrimethoxysilane (PhenylTMS) (98%, ABCR) are then added to this solution and the whole is poured into the bioreactor. Part of the sodium hydroxide is rapidly introduced manually, to pH = about 9.5, then the remainder is gradually added via the pump until a pH = 10 (volume of aqueous sodium hydroxide solution: 750 ml) is obtained. . After stirring for 24 hours, the gel is centrifuged for 10 minutes at a speed of 9500 rpm, washed three times with deionized water and then the pellet is frozen before being lyophilized. The sample thus prepared is called 100% Phenyl BS and contains the same amount of bacteria as at the beginning.

Characterization of the sample by X-ray diffraction indicates the formation of an organic-inorganic type lamellar phase of talc type structure of formula Mg ₃ (RSi) ₄ 0 ₈ (OH) ₂ in which R represents a phenyl group (presence of the characteristic reflections of the lattice planes (001), (020, 110), (130.220) and (060)) with a periodicity d ₀₀₁ equal to 1.3 nm.

Few isolated bacteria are observable on scanning electron microscopy. These are trapped in the agglomerates.

b) CieTMS BS compound A preculture of Bacillus subtilis (accessible under the number CIP 52.62 of the Pasteur Institute) is prepared from cryotubes containing 400 μl of a suspension of Bacillus subtilis maintained at -20 ° C. in 1.6 ml of glycerol. The nutrient medium in which the contents of the cryotube are incorporated is an LB broth (lysogeny broth) as described above. This medium is prepared directly in the Erlenmeyer flasks and then autoclaved for 20 minutes at 121 ° C. The seeding is then carried out with the flame to avoid any contamination. An enumeration in a solid medium on petri dishes (90 mm in diameter) is carried out after 18 hours of incubation at 37 ° C. in order to determine the initial concentration of bacteria which is added during the synthesis (10 ³ cfu / ml).

The synthesis is carried out in a bioreactor. The first step is to clean the bioreactor with absolute ethanol. The preculture is introduced into the bioreactor (at 10% of the final volume, ie 200 ml of LB medium containing the bacteria). In a 2L bottle, 55.65 g of magnesium nitrate hexahydrate (99%, Sigma Aldrich) are dissolved in one liter of absolute ethanol (99.9%, Carlo Erba). 100 g of hexadecyltrimethoxysilane ( _C16 TMS) (> 85%, Sigma) are then added to this solution and the whole is poured into the bioreactor. Part of the sodium hydroxide is rapidly introduced manually, to pH = about 9.5, then the remainder is gradually added via the pump until a pH = 10 (volume of 1M sodium hydroxide solution: 450 ml) is reached. Once this pH is reached, the pump is stopped. After stirring for 24 hours, the gel is centrifuged for 10 minutes at a speed of 9500 rpm, washed three times with deionized water and then the pellet is frozen before being lyophilized. The sample thus prepared is for reference C ₁₀ TMS BS. The amount of bacteria present in the sample is 10 ² cfu / ml.

Characterization of the sample by X-ray diffraction indicates the formation of a lamellar phase of organic-inorganic type of talc type structure of formula Mg ₃ (RSi) ₄ 0 ₈ (OH) 2 in which R represents a CH ₃ (CH 2) 4 CH ₂ ((presence of characteristic reflections of the lattice planes (001), (020, 110), (130.220 ) and (060) with a periodicity d _00i equal to 1.5 nm.

Few bacteria are observable on scanning electron microscopy snapshots these are trapped in the agglomerates,

c) CieTMS -triethoxyphenylsilane compound

cl) Effect of different biodegradable solvents in substitution of ethanol on the viability of Bacillus subtilis after synthesis

In a 500 ml beaker, add 9.72 g of magnesium nitrate hexahydrate (MgN0 ₃ , 6H ₂ O) (99%, Sigma Aldrich) and 100 ml of biodegradable solvent (Glycerol (Quaron> 99.5%) or propylene glycol (VWR) or methyl-5- (dimethylamino) -2-methyl-5-oxopentanoate (RHODISOLV® Polarclean sold by Solvay) with stirring at 55 ° C. at 220 rpm for 15 minutes. 7.71 g of triethoxyphenylsilane (97%, Sigma Aldrich) and 2.29 g of hexadecyltrimethoxysilane (> 85%, Sigma). Addition of 20 ml of culture medium of Bacillus subtilis (accessible under the number CIP 52.62). Pasteur Institute) at 6.50 × 10 ⁷ cfu / ml (the culture medium was prepared in a 3L bioreactor by seeding Bacillus subtilis in 20 ml of BHI medium (OXOID) and incubating for 18-24 h at 30 ° C. 200 rpm) 70 ml of 1M NaOH (> 97%, Sigma Aldrich) (pH 10) are added and the mixture is stirred for 24 hours. A petri dish (90 mm diameter) is made at 37 ° C in the dark with 60% relative humidity to determine the concentration of microorganisms at To (No), To + 24h (N _24h ) and To + 14 days (N _i4j ) after synthesis. The results are shown in Table 8 below. Table 8: concentration of microorganisms at To (No), To + 24h (N _24h ) and To + 14 days (NMJ) after synthesis according to the solvent used for the synthesis

The viability of the microorganisms is maintained at least 14 days after synthesis with glycerol, propylene glycol or Polarclean as solvent. This result confirms that biodegradable solvents can be used for the encapsulation of Bacillus subtilis.

c2) Effect of various biodegradable solvents in substitution of ethanol on the viability of Bacillus subtilis after synthesis and storage at 4 ° C. and 22 ° C. for 15 days

In a 2000 ml beaker, add 97.2 g of magnesium nitrate hexahydrate (MgN0 ₃ , 6H ₂ O) (99%, Sigma Aldrich) and 1000 ml of biodegradable solvent (glycerol (Quaron> 99.5%) or methyl 5- (dimethylamino) -2-methyl-5-oxopentanoate (RHODISOLV® Polarclean)) with stirring at 55 ° C at 220 rpm for 15 minutes. 77.1 g of triethoxyphenylsilane (97%, Sigma Aldrich) and 22.9 g of hexadecyltrimethoxysilane (> 85%, Sigma) were added with stirring for 15 minutes. Addition of 200 ml BaciHus subtilis culture medium (accessible under the CIP 52.62 number from the Pasteur Institute) at 6.50 × 10 ⁷ cfu / ml (the culture medium was prepared in a 3L bioreactor by seeding BaciHus subtilized in 20 mL of BHI medium (OXOID) and incubated 18-24 h at 30 ° C, 200 rpm). Add 700 mL of 1M NaOH (97%, Sigma Aldrich) (pH 10) and stirring 24h. 2 500 mL samples of the mixture are made. In the dark, a sample is kept in an oven at + 22 ° C ± 2 ° C with a relative humidity of 30%, the other is placed at +4 ± 2 ° C with a relative humidity of 65% for 15 days. An enumeration in solid medium on petri dish (90 mm in diameter) is carried out at 37 ° C to determine the concentration of bacteria at To (N ₀ ), To + 24h (N _24h ) and To + 15j (Nis _j ) . The results are shown in Table 9 below.

Table 9: concentration of microorganisms at To (N ₀ ), To + 24h (N _24h ) and To + 15 days (Nisj) after synthesis according to the solvent used for the synthesis and the storage temperature.

The viability of Baciiius subtiiis is maintained at + 4 ° C and + 22 ° C at least 15 days after synthesis with glycerol or Polarclean as solvent. This result confirms that biodegradable solvents can be used for the encapsulation of Bacillus subtilis.

c3) Effect of encapsulation (synthesis by biodegradable solvents in substitution of ethanol) on the viability of Baciiius subtiiis after heat treatment

The synthesis is identical to that of Example 2.1c2 with glycerol (Quaron> 99.5%) or methyl-5- (dimethylamino) -2-methyl-5-oxopentanoate (RHODISOLV® Polarclean) as solvent. 2 samples of 500mL of the mixture are made. In the dark, 45 samples of 9mL are taken. 3 batches of 15 samples are made and respectively placed in an oven (30% relative humidity) at + 40 ° C, + 60 ° C and + 80 ° C ± 2 ° C. In addition to the darkness, 3 batches of 15 samples of non-encapsulated microorganisms are also constituted and respectively placed in an oven (20% relative humidity) at + 60 ° C. and + 80 ° C. ± 2 ° C. The climbs at the setpoint temperatures are calibrated on a a 30-minute period in which the tubes are held for 2min, 5min and 10 minutes at core temperature before removal of the tubes for enumeration. 3 tubes are thus collected for each modality of microorganisms and temperature. A solid medium count on petri dish (90 mm in diameter) is carried out at 37 ° C in order to determine the concentration of bacteria at To (Nomin), To + 2min (N2min), To + 5min (N _5mi n) and To + 10 min (Niomin) · The results are shown in Table 10 below.

Table 10: concentration of microorganisms to To (Name _/ nX To + 2min (N 2min) _/ To + 5min (Nsmin) and To + 10 min (N _10mi n) after heat treatment depending on the solvent used for your synthesis and ia temperature of the heat treatment.

For the glycerol and Polarclean syntheses, the treatments reveal a quasi thermal insensitivity of Bacillus subtiis encapsulated compared to non-encapsulated Bacillus subtilis (at 10 min, respectively 2.00 × 10 ⁶ cfu / ml and 4.80 × 10 ⁶ cfu / ml) whereas the concentration of Bacillus subtiiis unencapsulated strongly decreases (4, 24xio ⁴ cfu / ml at 2min). When reading the results, the thermal protection provided by the encapsulation is confirmed. Example 2.2 Encapsulation of Piriformospora Indica

a) compound CI ₆ TMS PI

The fungus Piriformospora indica (P. indica) (accessible under the number DSM 11827 from Max-Planck-Institut für Terrestrische Mikrobiologie) is initially placed in an incubator (box 140 mm in diameter) for 72 hours at 28 ± 1 ° C, with YCG agar to obtain a fresh culture. The mycelium is collected using a sterile spatula and introduced into an Erlenmeyer flask containing 0.5% tween 80 solution and beads. After stirring for 2 minutes, an enumeration in a solid medium is carried out in order to determine the starting concentration (N0, in cfu / ml).

5.56 g of magnesium nitrate hexahydrate (99%, Sigma Aldrich) are dispersed in 100 ml of absolute ethanol (99.9%, Carlo Erba). The mixture is stirred until complete dissolution. 10 g of hexadecyltrimethoxysilane (C ₁₆ TMS) (> 85%, Sigma) are then added. The whole is left stirring and then the pH of the solution is brought to a value of 10 by addition of an aqueous solution of sodium hydroxide. sodium (> 97%, Sigma Aldrich) of IM concentration. The formulation is then seeded at 10% with a preculture of P. Indica. After 24h stirring at room temperature, the solid is separated from the solution by centrifugation (speed of 9000 rpm for 10 min), washed three times with distilled water before being frozen and lyophilized for 48 hours (sample of reference CI ₆ TMS PI). For comparison, a sample is prepared under the same conditions in the absence of P. Indica.

X-ray diffraction analysis indicates that in both cases a lamellar phase is obtained with a periodicity of 1.53 nm for the sample CI ₆ TMS PI and at 1.60 nm for the compound without P. Indica. . The SEM images of the CI ₆ TMS PI sample show the presence of shapes very similar to the hyphae observed with the only Hyphes mushroom (about 2 μm wide) covered with particles of material. The fungus is thus encapsulated in its vegetative form (hyphae + conidiophores) in the material of formula I according to the invention,

b) compound CI ₆ TMS -triethoxyphenylsilane

(bl) Effect of alvcerol as a substitute for ethanol on the viability of

Piriformospora indica after synthesis

In a 500 ml beaker, 9.72 g of magnesium nitrate hexahydrate (MgNC, 6H ₂ O) (99%, Sigma Aldrich) and 100 ml of biodegradable solvent (Glycerol (Quaron> 99.5%)) were added with stirring. at 55 ° C at 220 rpm for 15 minutes. Add with stirring for 15 minutes of 7.71 g of triethoxyphenylsilane (97%, Sigma Aldrich) and 2.29 g of hexadecyltrimethoxysilane (> 85%, Sigma). Addition of 20 ml of culture medium of Piriformospora indica (accessible under the number DSM 11827 from Max-Planck-Institut für Terrestrische Mikrobiologie) at 3.00x10 ⁷ cfu / ml. Add 70 mL of 1M NaOH (> 97%, Sigma Aldrich) and stir 24 hours. An enumeration in solid medium on petri dish (90 mm diameter) is carried out at 37 ° C in the dark with 60% relative humidity in order to determine the concentration of microorganisms at To (N ₀ ), To + 24h ( N ₂ 4 _h ) and To + 14 days (NHJ) after synthesis. The results are shown in Table 11 below.

Table 11: concentration of microorganisms at To (N ₀ ), To + 24h (N ₂ 4h) and To + 14 days (Ni) after synthesis

The viability of the microorganisms is maintained at least 14 days after synthesis with glycerol as solvent. This result confirms that Biodegradable solvents can be used for the encapsulation of Piriformospora indica.

b2) Effect of alvcerol in substitution of ethanol on viability of Piriformospora indica after synthesis and storage at 4 ° C and 22 ° C for 30 days

In a 2000 ml beaker, 97.2 g of magnesium nitrate hexahydrate (MgNO 3, 6H 2 O) (99%, Sigma Aldrich) and 1000 ml of biodegradable solvent (glycerol (Quaron> 99.5%)) were added with stirring at 55 ° C. ° C at 220 rpm for 15 minutes. 77.1 g of triethoxyphenylsilane (97%, Sigma Aldrich) and 22.9 g of hexadecyltrimethoxysilane (> 85%, Sigma) were added with stirring for 15 minutes. Addition of 200 ml culture medium of Piriformospora indica (accessible under the number DSM 11827 from Max-Planck-Institut für Terrestrische Mikrobiologie) at 3.00x10 ⁷ cfu / ml. Addition of 700 mL of 1M NaOH (97%, Sigma Aldrich) and stirring 24h. 2 500 mL samples of the mixture are made. In the dark, one sample is kept in an oven at + 22 ° C ± 2 ° C with a relative humidity of 30%, the other is placed at +4 ± 2 ° C with a relative humidity of 65% for 30 days . A solid medium count on petri dish (90 mm in diameter) is carried out at 37 ° C to determine the concentration of mushrooms at To (N ₀ ), To + 24h (N24 _h ), To + 15j (Ni¾) and To + 30j (N _30j ). The results are shown in Table 12 below.

Table 12: concentration of microorganisms at To (No), To + 24h (N ₂ 4h), To + ₁₅ days (N ₁₅ J) and To + 30j (N30j) after synthesis as a function of the storage temperature.

The viability of Piriformospora indica is maintained at + 4 ° C. and + 22 ° C. at least 15 days after synthesis with glycerol as solvent. This result confirms that glycerol can be used for the encapsulation of Piriformospora indica.

b3) Effect of encapsulation (synthesis by alvcerol in substitution of ethanol) on viability of Piriformospora indica after heat treatment

The synthesis is identical to that of Example 2.2b2. 2 samples of 500mL of the mixture are made. In the dark, 45 samples of 9mL are taken. 3 batches of 15 samples are made and respectively placed in an oven (30% relative humidity) at + 40 ° C, + 60 ° C and + 80 ° C ± 2 ° C. In addition to darkness, 3 batches of 15 samples of non-encapsulated microorganisms are also constituted and respectively placed in an oven (20% relative humidity) at + 40 ° C, + 60 ° C and + 80 ° C ± 2 ° C . The setpoint temperature increases are calibrated over a period of 30 minutes whereby the tubes are held for 2min, 5min and 10 minutes at the core setpoint before removal of the count tubes. 3 tubes are thus collected for each modality of microorganisms and temperature. An enumeration in solid medium on petri dish (90 mm diameter) is carried out at 37 ° C to determine the concentration of fungi at To (Nomin), To + 2min (N _2m in), To + 5min (N _5m in ) and To + 10 min (Ni _0mi n). The results are shown in Table 13 below.

Table 13: microorganism concentration at To (Nomin), To + ₂ min (N ₂ min) To + ₅ min (N 5 mm) and To-h ₁₀ min (N ₁₀ min) after heat treatment as a function of the temperature of the heat treatment.

For the glycerol syntheses, the treatments reveal the lower thermal sensitivity of piriformospora indica encapsulated compared to non-encapsulated Piriformospora indica (at 60 ° C./2 min, 4.40 × 10 ⁴ cfu / ml against 7.10 × 10 ³ cfu / ml; C / 5min 4.55xl0 ⁴ cfu / ml against 9.06xl0 ³ cfu / ml, at 60 ° C / 10min l, 95xl0 ⁴ cfu / ml against 1.31xl0 ² cfu / ml, at 80 ° C / 2min, 1, 50x1o ¹ cfu / ml against 0.00x100 cfu / ml). When reading the results, the thermal protection provided by the encapsulation is confirmed.

Example 2.3; encapsulation of spirulina

a) compound C-16 algae

2 g of magnesium nitrate hexahydrate (99%, Sigma Aldrich) are dispersed in 20 ml of absolute ethanol (99.9%, Carlo Erba). The mixture is stirred until complete dissolution. Spirulina Ig (cyanobacterium Arthrospira platensis sold under the name SPIRULINA NATURAL by the company EARTHRISE®) is introduced into the medium and then 1.11 g of hexadecyltrimethoxysilane (CI ₆ TMS) (> 85%, Sigma) are then added to each mixture. The whole is left stirring and the pH of the solution is brought to a value of 9 by adding 9 ml of an aqueous solution of sodium hydroxide (> 97%, Sigma Aldrich) IM concentration. After 24 hours stirring at room temperature, the solid is separated from the solution by centrifugation (speed of 10,000 rpm for 10 min), washed three times with ethanol and dried in an oven at 40 ° C during 48h. The compound obtained is then ground in an agate mortar before being characterized and called C-16 algae. For comparison, a sample was also prepared in the absence of spirulina (standard Talc reference sample).

X-ray diffraction analysis indicates that in both cases a lamellar phase of talc type structure is obtained with a periodicity of 1.6 nm in both cases (presence of diffraction peaks in the angular domains 2-10). 2 theta, 15-25 ° 2 theta, 30-40 ° 2 theta and 55-65 ° 2 theta corresponding respectively to reflections on the (001), (020; 110), (130; 220) and ( 060: 330)). The presence of spirulina does not inhibit the formation of the material. The presence of Spirulina does not induce increase of the periodicity of ₀ oi · Spirulina is therefore encapsulated in its vegetative form (twisted structure) in the material according to the invention.

Example 3 Evaluation of the Solubilization Capability of an Insoluble Phosphate in Water Using a Compound According to the Invention

a) Preparation of the encapsulated bacterium

10 g of magnesium nitrate hexahydrate (99%, Sigma Aldrich) are added to 20 ml of absolute ethanol (99.9%, Carlo Erba). The mixture is stirred until complete dissolution. 10 g of phenyltrimethoxysilane (98%, ABCR) are then added with stirring. With stirring, the mixture is seeded at 10% v / v of a preculture of Bacillus subtilis CIP 52.62 concentration 10 ⁸ cfu / ml. The whole is left stirring then the pH of the solution is brought to a value of 10 by addition of an aqueous solution of sodium hydroxide (> 97%, Sigma Aldrich) of IM concentration. After 24h stirring at room temperature, a bacterial count is performed on the whole seeded defining the bacterial concentration to 1.7 x 10 ⁵ cfu / ml. b) Solubilization test:

In 200mL Erlenmeyer flasks, a liquid culture medium (glucose, 10g / L, MgCl ₂ .6H ₂ O, 5g / L, MgSO ₄ .7H ₂ O, 0.25g / L, KCl, 0.2g / L; (NH ₄ ) ₂ S0 _{4.0 (} g / L) pH 7.0, supplemented with an insoluble phosphorus source in an aqueous medium (NH ₄ MgPO _{4 •} 6H ₂ O 8.9g / Erlenmeyer) is prepared.

Each Erlenmeyer flask is then inoculated with 1 ml of a preculture of Bacillus subtHis CIP 52.62 (Institut Pasteur) at a concentration of 6.5 × 10 ⁴ cfu / ml (free) or 382.3 ml of a preculture of Bacillus subtilis CIP. 52.62 at the concentration of 1.7 × 10 ⁵ cfu / ml (compound according to the invention 100% Phenyl BS: encapsulated bacterium prepared according to Example 3a). The Erlenmeyer flasks are kept in an oven with stirring. A colony count is made every 2 days as well as a measurement of the pH and concentration of solubilized phosphorus and phosphorus immobilized by the bacterial flora (measurement of total phosphorus by ICP and measurement of inorganic phosphate in solution by HPIC). The results are shown in FIGS. 1 to 3. The developmental comparison of Bacillus subtilis indicates a better solubilization capacity of the water insoluble phosphorus (FIG. 1). The comparison of development of Bacillus subtilis indicates a drop in the pH of the solution to be correlated with solubilization of bacterial origin of phosphorus (FIG. 2). The comparison of the phosphorus concentration indicates a greater bacterial solubilization than its immobilization by the bacterial flora (FIG. 3). There is therefore a better growth of the encapsulated bacteria according to the invention and a better solubilization of phosphorus when such bacteria are used.

Claims

A method of encapsulating a compound selected from the group consisting of at least one active substance, at least one microorganism and mixtures thereof in an organic-inorganic hybrid material of lamellar structure of type 2: 1, said material having the formula following general I:

Na _x [(Mg ₃ ) (Al _x (RSi) _4- x) 0 ₈₊ x (OH) ₂ ) (I)

in which

x is a number such that 0 <x <1, 2 and

R represents an alkyl group C1-C30, an aryl group, an (alkyl-C ₃₎ aryl or O-alkyl C1-C30, the alkyl group may be substituted with a group selected from a group phenyl, vinyl, aminopropyl or mercaptopropyl.

the method comprising:

b) recovering the compound encapsulated in the material of general formula I.

2. Method according to claim 1, characterized in that x = 0;

3. Method according to any one of claims 1 or 2, characterized in that the compound is an active substance, preferably selected from the group consisting of an amino acid, in particular tryptophan, an essential oil and mixtures thereof, so advantageous, it is tryptophan.

4. Method according to any one of claims 1 or 2, characterized in that the compound is a microorganism, preferably selected from the group consisting of a bacterium such as Bacillus subtiüs, a microalga such as spirulina, a mushroom such as the Piriformospora indica, and mixtures thereof, advantageously, the microorganism is in vegetative form, more particularly it is a bacterium.

5. Method according to any one of claims 1 to 4, characterized in that the silicon source necessary for the synthesis of the material of formula I of step a) is an organoalkoxysilane or a mixture of organoalkoxysilanes of general formula II following: RSi (OR ') ₃ (II) in which

R is as defined in claim 1 and

R 'is a methoxy or ethoxy group.

6. Method according to claim 5, characterized in that the silicon source is selected from the group consisting of phenyltrimethoxysilane of formula (a) below: Phenyl-Si (OCH3) 3 (a), the tetraethylorthosilicate of formula (b) following: Si (OC ₂ H ₅ ) 4 (b), hexadecyltrimethoxysilane of formula (c) below: CH ₃ (CH 2) i CH 2 -Si (OCH ₃ ) 3 (c) and mixtures thereof.

7. Method according to claim 6, characterized in that the encapsulated compound is an active substance and in that the silicon source is a mixture of organoalkoxysilanes of general formula II, advantageously a mixture between phenyltrimethoxysilane (a) and tetraethylorthosilicate (b) advantageously the mixture comprises 20 mol% of tetraethylorthosilicate and 80 mol% of phenyltrimethoxysilane relative to the total number of mol of the mixture.

8. The method of claim 6, characterized in that the encapsulated compound is a microorganism and in that the silicon source is selected from the group consisting of phenyltrimethoxysilane (a) and hexadecyltrimethoxysilane (c).

9. Process according to claim 1, wherein the encapsulated compound is an active substance and in that step a) comprises the following successive steps:

al) Adding a source of magnesium, advantageously magnesium nitrate hexahydrate, of the active substance, of the silicon source, in the case where x¹0, of the aluminum source, advantageously aluminum acetylacetonate and a possible solvent;

a2) adjusting the pH to between 8 and 14, advantageously 10;

a3) stirring the mixture, advantageously for a period of between 1 and 24 hours, in particular at least 12 hours, so as to obtain a gel;

a4) recovering the solid phase of the gel obtained in step a3);

a5) drying of the solid phase of the gel obtained in step a4).

10. Process according to any one of claims 1, 2, 4 to 6 and 8, characterized in that the encapsulated compound is a microorganism and in that step a) comprises the following successive steps:

al) adding a magnesium source, advantageously magnesium nitrate hexahydrate, the microorganism, a solvent, a silicon source and in the case where x¹0, from the aluminum source, advantageously acetylacetonate aluminum;

a2) adjusting the pH to between 8 and 14, advantageously 10; a3) stirring the mixture, advantageously for a period of between 1 and 24 hours, in particular at least 12 hours, so as to obtain a gel.

11. The method of claim 10, characterized in that it comprises the following successive steps after step a3) of:

a4) recovering the solid phase of the gel obtained in step a3);

a5) drying of the solid phase of the gel obtained in step a4).

12. Method according to any one of claims 10 or 11, characterized in that the microorganism is in the form of a preculture of said microorganism, preferably having a microorganism content of between 10 ³ and 10 ¹⁰ cfu / ml, and in that it comprises a preliminary step prior to step a) of preparing the microorganism preculture.

13. Method according to any one of claims 11 or 12, characterized in that the drying step a5) consists of lyophilization.

14. Compound encapsulated in an organic-inorganic hybrid material of lamellar structure of type 2: 1, said material having the following general formula I:

Na _x [(Mg ₃ ) (Al _x (RSi) _4- x) 0 ₈₊ x (OH) ₂ ) (I)

in which

x is a number such that 0 <x <1, 2 and

R represents a C1-C30 alkyl group, an aryl group, a C1-C3 alkyl aryl group or a C1-C30 O-alkyl group, the alkyl group which may be substituted with a group selected from phenyl, vinyl, aminopropyl or mercaptopropyl.

the encapsulated compound being selected from the group consisting of at least one active substance, at least one microorganism and mixtures thereof.

15. Compound encapsulated in an organic-inorganic hybrid material of lamellar structure type 2: 1 according to claim 14, characterized in that it is obtainable by the method according to any one of claims 1 to 13.

16. Composition, advantageously intended for fertilization, nutrition, growth stimulation and / or plant prophylaxis and / or improvement of the physical, chemical and / or biological properties of the soil or the substrate for growing plants, comprising the compound encapsulated in an organic-inorganic hybrid material of lamellar structure type 2: 1 according to any one of claims 14 or 15 and a suitable excipient.

17. Composition according to claim 16, characterized in that it is in solid form, in particular in the form of powder, granules or microgranules, in liquid form or in gel form.

18. Composition according to any one of claims 16 or 17, characterized in that it further comprises nutrients advantageously selected from the group consisting of nitrogen, phosphorus, potassium, calcium, magnesium, silicon , trace elements and mixtures thereof, organic raw materials and / or mineral raw materials.

19. Use of the compound according to any one of claims 14 or 15 or the composition according to any one of claims 16 to 18 for the fertilization, nutrition, growth stimulation and / or prophylaxis of plants and / or improving the physical, chemical and / or biological properties of the soil or plant growing substrate.

20. Use according to claim 19 by foliar application, root, full field or above ground.