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

Abstract

The present invention relates to a method for 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 2:1 lamellar structure, said material having the following general formula I:
Na.sub.x[(Mg.sub.3)(Al.sub.x(RSi).sub.4−x)O.sub.8+x(OH).sub.2]  (I)
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) recovery of the compound encapsulated in the material of general formula I. It further relates to the compound encapsulated in an organic-inorganic hybrid material of 2:1 lamellar structure of general formula I, a composition comprising same and its use for fertilizing, feeding, stimulating growth and/or prophylaxis of plants and/or improvement of the physical, chemical and/or biological properties of the soil or of the culture substrate of plants.

Claims

1. 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 2:1 lamellar structure, said organic-inorganic hybrid material having the following general formula I:
Na.sub.x[(Mg.sub.3)(Al.sub.x(RSi).sub.4−x)O.sub.8+x(OH).sub.2]  (I) in which x is a number such that 0≤x<1.2 and R represents a C.sub.1-C.sub.30 alkyl group, an aryl group, a (C.sub.1-C.sub.30 alkyl)aryl group or a C.sub.1-C.sub.30 O-alkyl group, and the alkyl group may be substituted with a group selected from a phenyl, vinyl, 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; and b) recovery of the compound encapsulated in the material of general formula I, wherein the source of silicon necessary for synthesis of the material of formula I of step a) is an organoalkoxysilane or a mixture of organoalkoxysilanes of the following general formula II:
RSi(OR′).sub.3  (II) in which R′ is a methoxy or ethoxy group.

2. The method as claimed in claim 1, wherein the encapsulated compound is a microorganism and step a) comprises the following successive steps: a1) adding a source of magnesium, the microorganism, a solvent, a source of silicon and in the case when x≠0, the source of aluminum; a2) adjusting the pH to between 8 and 14; a3) stirring the mixture so as to obtain a gel.

3. The method as claimed in claim 2, wherein it comprises the following successive steps after step a3): a4) recovering the solid phase of the gel obtained in step a3); a5) drying the solid phase of the gel obtained in step a4).

4. The method as claimed in claim 3, wherein the drying step a5) consists of lyophilization.

5. The method as claimed in claim 2, wherein the microorganism is in the form of a preculture of said microorganism and said method comprises a preliminary step before step a) of preparing the preculture of microorganism.

6. The method as claimed in claim 1, wherein the source of silicon is selected from the group consisting of phenyltrimethoxysilane of the following formula (a): phenyl-Si(OCH.sub.3).sub.3 (a), tetraethylorthosilicate of the following formula (b): Si(OC.sub.2H.sub.5).sub.4 (b), hexadecyltrimethoxysilane of the following formula (c): CH.sub.3(CH.sub.2).sub.14CH.sub.2—Si(OCH.sub.3).sub.3 (c) and mixtures thereof.

7. The method as claimed in claim 6, wherein the encapsulated compound is an active substance and the source of silicon is a mixture of phenyltrimethoxysilane (a) and tetraethylorthosilicate (b).

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

9. The method as claimed in claim 1, wherein x=0.

10. The method as claimed in claim 1, wherein the compound is an active substance selected from the group consisting of an amino acid, an essential oil, a vitamin and mixtures thereof.

11. The method as claimed in claim 1, wherein the compound is a microorganism selected from the group consisting of a bacterium, a microalga, a fungus, and mixtures thereof.

12. The method as claimed in claim 1, wherein the encapsulated compound is an active substance and step a) comprises the following successive steps: a1) adding a source of magnesium, the active substance, the source of silicon, in the case when x≠0, the source of aluminum, and an optional solvent; a2) adjusting the pH to between 8 and 14; a3) stirring the mixture, so as to obtain a gel; a4) recovering the solid phase of the gel obtained in step a3); a5) drying the solid phase of the gel obtained in step a4).

13. The method as claimed in claim 1 wherein the source of silicon is selected from the group consisting of phenyltrimethoxysilane (a), methyltriethoxysilane (c) and a mixture of phenyltrimethoxysilane (a) with tetraethylorthosilicate (b).

14. A compound encapsulated in an organic-inorganic hybrid material of 2:1 lamellar structure, said organic-inorganic hybrid material having the following general formula I:
Na.sub.x[(Mg.sub.3)(Al.sub.x(RSi).sub.4−x)O.sub.8+x(OH).sub.2]  (I) in which x is a number such that 0≤x<1.2 and R represents a C.sub.1-C.sub.30 alkyl group, an aryl group, a (C.sub.1-C.sub.30 alkyl)aryl group or a C.sub.1-C.sub.30 O-alkyl group, and the alkyl group may be substituted with a group selected from a phenyl, vinyl, or mercaptopropyl group; the encapsulated compound being selected from the group consisting of at least one active substance, at least one microorganism and mixtures thereof.

15. A composition comprising the compound encapsulated in an organic-inorganic hybrid material of 2:1 lamellar structure as claimed in claim 14 and an excipient.

16. The composition as claimed in claim 15, which is in solid form, in liquid form or in the form of gel.

17. The composition as claimed in claim 15, which further comprises nutrients, organic raw materials and/or mineral raw materials.

18. The compound encapsulated in an organic-inorganic hybrid material of 2:1 lamellar structure as claimed in claim 14, which is obtained by a method comprising: a) sol-gel synthesis of the organic-inorganic hybrid material of 2:1 lamellar structure in the presence of the compound; and b) recovery of the compound encapsulated in the material of general formula I, wherein the source of silicon necessary for synthesis of the material of formula I of step a) is an organoalkoxysilane or a mixture of organoalkoxysilanes of the following general formula II:
RSi(OR′).sub.3  (II) in which R′ is a methoxy or ethoxy group.

19. A method for fertilizing, feeding, stimulating growth and/or prophylaxis of plants and/or improving the physical, chemical and/or biological properties of the soil or of the culture substrate of plants comprising administration of an effective amount of a compound as claimed in claim 14, or of a composition comprising the compound encapsulated in an organic-inorganic hybrid material of 2:1 lamellar structure as claimed in claim 14 and a suitable excipient, to a plant in need thereof or to the soil or culture substrate of a plant in need thereof.

20. The method as claimed in claim 19, wherein the administration is an application to the leaves, to the roots, in the open or outside the soil of the plant in need thereof.

Description

(1) The present invention will be better understood on reading the description of the drawings and the following examples, which are given as a guide and are nonlimiting.

(2) FIG. 1 shows monitoring over time (in days) of the population (in CFU/ml) of Bacillus subtilis CIP 52.62, encapsulated (compound according to the invention) and unencapsulated, in the presence of phosphorus in the conditions of example 3.

(3) FIG. 2 shows the variation of culture medium pH as a function of time (in days) in the presence of Bacillus subtilis CIP 52.62, encapsulated (compound according to the invention) and unencapsulated, and in the presence of phosphorus in the conditions of example 3.

(4) FIG. 3 shows the difference in concentration between solubilized phosphorus and phosphorus immobilized by the bacterial flora as a function of time (in days) in the presence of Bacillus subtilis CIP 52.62, encapsulated (compound according to the invention) and unencapsulated, in the conditions of example 3.

COMPARATIVE EXAMPLE 1

Effect of Various Biodegradable Solvents and of Ethanol on the Chemical Synthesis of Organic-Inorganic Compounds of the Lamellar Type

(5) In a 1000 mL beaker, addition of 19.44 g of magnesium nitrate hexahydrate (MgNO.sub.3, 6H.sub.2O) (99%, Sigma) and 200 mL of biodegradable solvent (Glycerol (Quaron >99.5%) or propylene glycol (VWR) or methyl-5-(dimethylamino)-2-methyl-5-oxopentanoate (RHODISOLV® Polarclean marketed by Solvay) or propylene carbonate (Quaron >99.7%)) or ethanol with stirring at 55° C. at 220 rpm for 15 minutes. Addition, with stirring for 15 minutes, of 4.58 g of triethoxyphenylsilane (97%, Sigma) and 15.42 g of hexadecyltrimethoxysilane (C.sub.16TMS) (>85%, Sigma). Addition of 100 mL of 1M NaOH (97%, Sigma) and stirring for 24 h. After 15 days of storage at ambient temperature and relative humidity, in the dark, each synthesis is monitored to validate proper formation of the compounds and certain physicochemical properties (pH, viscosity, appearance, volume, stability) (Table 1). A product is considered to be stable if there is no apparent change in appearance and viscosity and if there is no phase separation or decanting.

(6) TABLE-US-00001 TABLE 1 physicochemical properties of the lamellar materials Solvents Propylene Propylene Ethanol Glycerol glycol carbonate Polarclean Final volume 316 357 392 733 442 (mL) pH 9.84 9.75 11.1 8.45 10.1 Viscosity (cP) 4.68 15.9 18.9 4.41 10.5 Appearance milky milky Milky milky milky Stability at Stable Stable Stable Stable Stable 4° C. at 15 d Stability at Stable Stable Stable Stable Stable room temper- ature at 15 d

(7) It can be seen from the results that the use of biodegradable solvents or of ethanol during the syntheses therefore does not have an adverse effect on formation of the compounds. Ethanol can be replaced with 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

(8) a) Compound 100% Phenyl TRYPTO

(9) 1.944 g of magnesium nitrate hexahydrate (99%, Sigma Aldrich) is added to 20 mL of absolute ethanol (99.9%, Carlo Erba), and the mixture is stirred until completely dissolved. 200 mg of L-tryptophan (>98%, Sigma Aldrich) is introduced with stirring and then 2 g of phenyltrimethoxysilane (PhenyITMS) (98%, ABCR) is added. The whole is stirred and then the solution pH is adjusted to a value of 10 by adding 15 mL of an aqueous solution of sodium hydroxide (>97%, Sigma Aldrich) with a concentration of 1M. After stirring at room temperature for 24 h, the solid is separated from the solution by centrifugation (speed of 10000 rpm for 10 min). The solid is washed three times with ethanol before being dried in a stove at 40° C. for 48 h. The compound obtained is then ground in an agate mortar before being characterized. 1.35 g of compound designated 100% phenyl TRYPTO is recovered.

(10) The X-ray diffraction pattern of the sample has several diffraction peaks in the angle domains 2-10°2 theta, 15-25°2 theta, 30-40°2 theta and 55-652 theta. These peaks correspond respectively to reflections on the (001), (020; 110), (130; 220) and (060:330) lattice planes, characteristic of the presence of a lamellar phase. The value of the periodicity d.sub.060 is 0.156 nm, a typical value for a lamellar phase of the hybrid organic-inorganic type with a structure of the talc type of formula Mg.sub.3(RSi).sub.4O.sub.8(OH).sub.2 in which R represents a phenyl group. The periodicity d.sub.001 is of the order of 1.32 nm.

(11) The amount of tryptophan in the compound (rate of encapsulation) was determined by UV spectrophotometry at a wavelength of 280 nm. The compound comprises 63.4 mg of tryptophan per g of material. Analysis by thermogravimetry, carried out under air between 30 and 800° C. at a rate of temperature rise of 5° C./min, shows that the greatest weight loss (decomposition of the products) only begins starting from 300° C., which confirms that the tryptophan has been properly encapsulated in the material (Table 2).

(12) TABLE-US-00002 TABLE 2 result of thermogravimetric analyses carried out under nitrogen and under air Sample Peaks (° C.) Range used (° C.) Weight loss (%) Under N.sub.2 100% Phenyl 80  30-200 6.81 TRYPTO 358-480 200-800 43.83 Under air 70  30-200 6.84 364-450-604 200-800 45.24

(13) Comparison between the NMR spectrum of the solid of the .sup.13C of tryptophan alone and of the compound 100% Phenyl TRYPTO 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 the tryptophan is present in the interlayer space. Therefore we have indeed obtained tryptophan encapsulated in an organic-inorganic hybrid material of 2:1 lamellar structure of formula Mg.sub.3(RSi).sub.4O.sub.8(OH).sub.2.

(14) b) Compound 80Ph-20TEOS TRYPTO

(15) 2 g of magnesium nitrate hexahydrate (99%, Sigma Aldrich) is added to 20 mL of absolute ethanol (99.9%, Carlo Erba), and the mixture is stirred until completely dissolved. 200 mg of L-tryptophan (>98%, Sigma Aldrich) is added with stirring and then a mixture consisting of 1.646 g of phenyltrimethoxysilane (PhenyITMS) (98%, ABCR) and 0.432 g tetraethylsilane (TEOS) (98%, ABCR) is added (mixture, by weight, of 79.2% of PhenyITMS and 20.8% of TEOS, which represents 80% of PhenyITMS and 20% of TEOS in mol). The whole is stirred and then the solution pH is adjusted to a value of 10 by adding 15 mL of an aqueous solution of sodium hydroxide (>97%, Sigma Aldrich) with a concentration of 1M. After stirring at room temperature for 24 h, the solid is separated from the solution by centrifugation (speed of 10000 rpm for 10 min). The solid is washed three times with ethanol before being dried in a stove at 40° C. for 48 h. The compound obtained is then ground in an agate mortar before being characterized. 1.43 g of compound designated 80Ph-20TEOS TRYPTO is recovered.

(16) The X-ray diffraction pattern of the sample has several diffraction peaks in the angle domains 2-10°2 theta, 15-25°2 theta, 30-40°2 theta and 55-65°2 theta. These peaks correspond respectively to reflections on the (001), (020; 110), (130; 220) and (060:330) lattice planes, characteristic of the presence of a lamellar phase. The value of the periodicity d.sub.006 is 0.156 nm, a typical value for a lamellar phase of the hybrid organic-inorganic type with a structure of the talc type of formula Mg.sub.3(RSi).sub.4O.sub.8(OH).sub.2 in which R represents a mixture of phenyl group and O-ethyl group. The periodicity d.sub.001 is equal to 1.4 nm.

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

(18) Analysis by thermogravimetry, carried out under air between 30 and 800° C. at a rate of temperature rise of 5° C./min, shows that the greatest weight loss (decomposition of the products) only begins starting from 300° C., which confirms that the tryptophan has been properly encapsulated in the material (Table 3).

(19) TABLE-US-00003 TABLE 3 result of the thermogravimetric analyses carried out under nitrogen and under air Sample Peaks(° C.) Range used (° C.) Weight loss (%) Under N.sub.2 80Ph—20TEOS 80  30-200 8.47 TRYPTO 358-480 200-800 39.32 Under air 69  30-200 8.30 364-450 200-800 41.30

(20) Comparison between the NMR spectrum of the solid of the .sup.13C of the tryptophan alone and of the compound 80Ph-20TEOS TRYPTO 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 the tryptophan is present in the interlayer space.

(21) Therefore tryptophan encapsulated in an organic-inorganic hybrid material of 2:1 lamellar structure of formula Mg.sub.3(RSi).sub.4O.sub.8(OH).sub.2 is indeed obtained.

(22) c) Compounds MTES TRYPTO, C.sub.16TMS TRYPTO, TEOS TRYPTO, 20Ph-80TEOS TRYPTO, 40Ph-60TEOS TRYPTO and 60Ph-40TEOS TRYPTO

(23) Using a method identical to that used for preparing the compound 100% Phenyl TRYPTO, other compounds according to the invention were prepared by replacing phenyltrimethoxysilane as the source of silicon with methyltriethoxysilane (MTES) or hexadecyltrimethoxysilane (C.sub.16TMS) or tetraethylsilane (TEOS). The compounds obtained were named MTES TRYPTO, C.sub.16TMS TRYPTO and TEOS TRYPTO, respectively.

(24) Using a method identical to that used for preparing the compound 80Ph-20TEOS TRYPTO, other compounds according to the invention were prepared by replacing the mixture 20% TEOS and 80% PhenyITMS as the source of silicon with a mixture 80% TEOS and 20% PhenyITMS in mol, a mixture 60% TEOS and 40% PhenyITMS in mol and a mixture 40% TEOS and 60% PhenyITMS in mol. The compounds obtained were named 20Ph-80TEOS TRYPTO, 40Ph-60TEOS TRYPTO and 60Ph-40TEOS TRYPTO, respectively.

(25) The amount of compounds recovered and the rates of encapsulation determined by UV spectrophotometry at a wavelength of 280 nm are presented in Table 4 below.

(26) TABLE-US-00004 TABLE 4 rate of mass encapsulation (mg source of recovered tryptophan/g of Compound silicon (g) material) MTES TRYPTO MTES 1.123 45.9 C.sub.16TMS TRYPTO C.sub.16TMS 1.173 24.7 TEOS TRYPTO TEOS 1.074 23.4 20Ph—80TEOS 20Ph—80TEOS 1.36 37.6 TRYPTO 40Ph—60TEOS 40Ph—60TEOS 1.49 48.9 TRYPTO 60Ph—40TEOS 60Ph—40TEOS 1.36 58 TRYPTO

(27) The X-ray diffraction pattern of the samples of each of the compounds has several diffraction peaks in the angle domains 2-10°2 theta, 15-25°2 theta, 30-40°2 theta and 55-65°2 theta. These peaks correspond respectively to reflections on the (001), (020; 110), (130; 220) and (060:330) lattice planes, characteristic of the presence of a lamellar phase. The value of the periodicity d.sub.006 is 0.156 nm, a typical value for a lamellar phase of the hybrid organic-inorganic type with a structure of the talc type of formula Mg.sub.3(RSi).sub.4O.sub.8(OH).sub.2. The periodicity d.sub.001 is equal to 1.4 nm.

(28) Comparison between the NMR spectrum of the solid of the .sup.13C of the tryptophan alone and of 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 the tryptophan is present in the interlayer space.

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

(30) In the case when the source of silicon is a mixture of TEOS and PhenyITMS, we observe a linear correlation between the amount of tryptophan encapsulated and the percentage of TEOS used, except for the case when the content of TEOS is 0% (compound 100% Phenyl TRYPTO).

Example 1.2: Encapsulation of an Essential Oil

(31) 2 g of magnesium nitrate hexahydrate (99%, Sigma Aldrich) is added to 20 mL of a mixture made up of absolute ethanol (99.9%, Carlo Erba) and oil (Greenfix 3000) in proportions equal to 0, 25, 75 or 100 vol % of oil. The mixture is stirred until completely dissolved. 1.646 g of phenyltrimethoxysilane (98%, ABCR) and 0.432 g of tetraethylsilane (98%, ABCR) are then added (mixture, by weight, of 79.2% of PhenyITMS and 20.8% of TEOS, which represents 80% of PhenyITMS and 20% of TEOS in mol). The whole is stirred and then the solution pH is adjusted to a value of 10 by adding 15 mL of an aqueous solution of sodium hydroxide (>97%, Sigma Aldrich) with a concentration of 1M. After stirring at room temperature for 24 h, the solid is separated from the solution by centrifugation (speed of 10000 rpm for 10 min). The solids are washed three times with ethanol before being dried in a stove at 40° C. for 48 h. The compounds obtained are then ground in an agate mortar before being characterized and are called SHE5 (25% of oil), SHE15 (75% of oil), SH20 (100% of oil) and 80% P-20% T (0% of oil). The amounts obtained for the different samples are 2.24 g, 2.62 g, 0.12 g and 1.43 g, respectively.

(32) Comparison between the X-ray diffraction patterns of the samples SHE5 (25% of oil), SHE15 (75% of oil), SH20 (100% of oil) and 80% P-20% T (0% of oil) shows the presence of diffraction peaks in the angle domains 2-10°2 theta, 15-25°2 theta, 30-40°2 theta and 55-65°2 theta. These peaks correspond respectively to reflections on the (001), (020; 110), (130; 220) and (060:330) lattice planes, characteristic of the presence of a lamellar phase. The value of the periodicity d.sub.060 is 0.156 nm, a typical value for a lamellar phase of the hybrid organic-inorganic type of structure of the talc type. The periodicities d.sub.001 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.

(33) Analysis by thermogravimetry, carried out under air between 30 and 800° C. at a rate of temperature rise of 5° C./min, shows that the greatest weight loss (decomposition of the products) only begins starting from 300° C., which confirms that the oil has been properly encapsulated in the material (Table 5).

(34) TABLE-US-00005 TABLE 5 result of the thermogravimetric analyses carried out under nitrogen and under air Sample Peaks (° C.) Range used (° C.) Weight loss (%) Under N.sub.2 SHE5 67  30-200 12.95 361  200-800 37.86 Under air 76  30-200 12.69 339  200-800 45.24 Under N.sub.2 SHE15 74  30-200 14.02 345-521 200-800 38.46 Under air 75  30-200 13.89 339-460-598-761 200-800 55.60 Under N.sub.2 SHE20 46  30-200 8.76 349-597 200-800 36.83 Under air 50  30-200 8.58 353-599 200-800 55.94

Example 1.3: Encapsulation of Folic Acid

(35) a) With Ethanol as Solvent

(36) 1.60 g of magnesium nitrate hexahydrate (99%, Sigma Aldrich) is added to 20 mL of absolute ethanol (99.9%, Carlo Erba), and the mixture is stirred until completely dissolved. 200 mg of folic acid (>97%, Sigma Aldrich) is introduced with stirring and then 2 g of phenyltrimethoxysilane (PhenyITMS) (98%, Sigma Aldrich) is added. The whole is stirred and then the solution pH is adjusted to a value of 10 by adding 10 mL of an aqueous solution of sodium hydroxide (>97%, Sigma Aldrich) with a concentration of 1M. After stirring at room temperature for 24 h, the solid is separated from the solution by centrifugation (speed of 10000 rpm for 10 min). The solid is dried in a stove at 60° C. for 24 h. The compound obtained (2.1 g) is then ground in an agate mortar before being characterized and is designated Eth-PH 200AF in situ.

(37) The X-ray diffraction pattern of the sample has several diffraction peaks in the angle domains 2-10°2 theta, 15-25°2 theta, 30-40°2 theta and 55-65°2 theta. These peaks correspond respectively 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.sub.060 is 0.156 nm, a typical value for a lamellar phase of the hybrid organic-inorganic type with a structure of the talc type of formula Mg.sub.3(RSi).sub.4O.sub.8(OH).sub.2 in which R represents a phenyl group. The periodicity door is of the order of 1.14 nm.

(38) The amount of folic acid in the compound (rate of encapsulation) was determined by UV spectrophotometry at a wavelength of 280 nm; the compound comprises 108.3 mg of folic acid per g of material. Analysis by thermogravimetry, carried out under air between 30 and 800° C. at a rate of temperature rise of 5° C./min, shows that the greatest weight loss (decomposition of the products) only begins starting from 300° C., which confirms that the folic acid has been properly encapsulated in the material (Table 6).

(39) TABLE-US-00006 TABLE 6 result of the thermogravimetric analyses carried out under nitrogen and under air Peaks (° C.) Range used (° C.) Weight loss (%) Under N.sub.2 80  30-200 4.8 375-400-505-620 200-800 45.5 Under air 70  30-200 5.2 385-545-620 200-800 56.4

(40) Comparison between the NMR spectrum of the solid of the .sup.13C of the folic acid alone and of 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 interlayer space. Therefore folic acid encapsulated in an organic-inorganic hybrid material of 2:1 lamellar structure of formula Mg.sub.3(RSi).sub.4O.sub.8(OH).sub.2 is indeed obtained.

(41) b) With Glycerol as Solvent

(42) 1.60 g of magnesium nitrate hexahydrate (99%, Sigma Aldrich) is added to 20 mL of glycerol (87%, Fluka), and the mixture is stirred until completely dissolved. 200 mg of folic acid (>97%, Sigma Aldrich) is introduced with stirring and then 2 g of phenyltrimethoxysilane (PhenyITMS) (98%, Sigma Aldrich) is added. The whole is stirred and then the solution pH is adjusted to a value of 10 by adding 10 mL of an aqueous solution of sodium hydroxide (>97%, Sigma Aldrich) with a concentration of 1M. After stirring at room temperature for 24 h, the solid is separated from the solution by centrifugation (speed of 10000 rpm for 10 min). The solid is washed four times with demineralized water before being dried in a stove at 60° C. for 24 h. The compound obtained (2.2 g) is then ground in an agate mortar before being characterized and is designated Gly-PH 200AF in situ.

(43) The X-ray diffraction pattern of the sample has several diffraction peaks in the angle domains 2-10°2 theta, 15-25°2 theta, 30-40°2 theta and 55-65° 2 theta. These peaks correspond respectively 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.sub.060 is 0.155 nm, a typical value for a lamellar phase of the hybrid organic-inorganic type with a structure of the talc type of formula Mg.sub.3(RSi).sub.4O.sub.8(OH).sub.2 in which R represents a phenyl group. The periodicity d.sub.001 is of the order of 1.24 nm.

(44) The amount of folic acid in the compound (rate of encapsulation) was determined by UV spectrophotometry at a wavelength of 280 nm; the compound comprises 56.66 mg of folic acid per g of material. Analysis by thermogravimetry, carried out under air between 30 and 800° C. at a rate of temperature rise of 5° C./min, shows that the greatest weight loss (decomposition of the products) only begins starting from 300° C., which confirms that the folic acid has been properly encapsulated in the material (Table 7).

(45) TABLE-US-00007 TABLE 7 result of the thermogravimetric analyses carried out under air Peaks (° C.) Range used (° C.) Weight loss (%) 70  30-200 7.3 380-620 200-800 46.2

(46) Comparison between the NMR spectrum of the solid of the .sup.13C of the folic acid alone and of 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 interlayer space. Therefore folic acid encapsulated in an organic-inorganic hybrid material of 2:1 lamellar structure of formula Mg.sub.3(RSi).sub.4O.sub.8(OH).sub.2 is indeed obtained.

EXAMPLE 2: ENCAPSULATION OF A MICROORGANISM

Example 2.1: Encapsulation of Bacillus subtilis

(47) a) Compound 100% Phenyl BS

(48) A preculture of Bacillus subtilis (accessible under number CIP 52.62 from 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 made up of 10 g of tryptone (peptone of the casein pancreatic hydrolyzate type), 5 g of yeast extract and 10 g of NaCl to one liter of demineralized water. The yeast extract is obtained from yeast autolyzates. It is biomass of yeasts in suspension induced to autolysis by passage at 50° C. for several hours, from which the liquid phase is recovered. This medium is prepared directly in conical flasks and then is autoclaved for 20 minutes at 121° C. Seeding is then effected by flame to prevent any contamination. A solid medium count on a Petri dish (90 mm diameter) is carried out after 18 h of incubation at 37° C. to determine the initial concentration of bacterium that is added during the synthesis.

(49) Synthesis is carried out in a bioreactor. The first step consists of cleaning the bioreactor with absolute ethanol. The preculture is fed into the bioreactor (to 10% of the final volume, i.e. 200 mL of LB medium containing the bacteria at a content of 10.sup.3 CFU/ml). In a 2 L bottle, 97 g of magnesium nitrate (99%, Sigma Aldrich) is dissolved in a liter of absolute ethanol (99.9%, Carlo Erba). 100 g of phenyltrimethoxysilane (PhenyITMS) (98%, ABCR) is then added to this solution and then the whole is poured into the bioreactor. A portion of the soda is quickly introduced manually, up to about pH=9.5, and then the remainder is added gradually by pump until pH=10 is reached (volume of 1M aqueous solution of soda: 750 mL). After stirring for 24 h, the gel is centrifuged for 10 minutes at a speed of 9500 rpm, washed three times with demineralized 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 bacterium as at the start.

(50) Characterization of the sample by X-ray diffraction indicates formation of a lamellar phase of the organic-inorganic type with a structure of the talc type of formula Mg.sub.3(RSi).sub.4O.sub.8(OH).sub.2 in which R represents a phenyl group (presence of the reflections characteristic of the lattice planes (001), (020,110), (130,220) and (060)) with a periodicity d.sub.001 equal to 1.3 nm. Few isolated bacteria are observable on the photographs from scanning electron microscopy. The latter are embedded in the agglomerates.

(51) b) Compound C.sub.16TMS BS

(52) A preculture of Bacillus subtilis (accessible under number CIP 52.62 from 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 conical flasks and then is autoclaved for 20 minutes at 121° C. Seeding is then effected by flame to prevent any contamination. A solid medium count on a Petri dish (90 mm diameter) is carried out after 18 h of incubation at 37° C. to determine the initial concentration of bacterium that is added during the synthesis (10.sup.3 CFU/ml).

(53) Synthesis is carried out in a bioreactor. The first step consists of cleaning the bioreactor with absolute ethanol. The preculture is fed into the bioreactor (to 10% of the final volume, i.e. 200 mL of LB medium containing the bacteria). In a 2 L bottle, 55.65 g of magnesium nitrate hexahydrate (99%, Sigma Aldrich) is dissolved in a liter of absolute ethanol (99.9%, Carlo Erba). 100 g of hexadecyltrimethoxysilane (C.sub.16TMS) (>85%, Sigma) is then added to this solution and then the whole is poured into the bioreactor. A portion of the soda is quickly introduced manually, up to about pH=9.5, and then the remainder is added gradually by pump until pH=10 is reached (volume of 1M soda solution: 450 mL). Once this pH is reached, the pump is stopped. After stirring for 24 h, the gel is centrifuged for 10 minutes at a speed of 9500 rpm, washed three times with demineralized water and then the pellet is frozen before being lyophilized. The sample thus prepared has the reference C.sub.16TMS BS. The amount of bacterium present in the sample is 10.sup.2 CFU/ml.

(54) Characterization of the sample by X-ray diffraction indicates formation of a lamellar phase of the organic-inorganic type with a structure of the talc type of formula Mg.sub.3(RSi).sub.4O.sub.8(OH).sub.2 in which R represents a group CH.sub.3(CH.sub.2).sub.14CH.sub.2 ((presence of the reflections characteristic of the lattice planes (001), (020,110), (130,220) and (060)) with a periodicity d.sub.001 equal to 1.5 nm.

(55) Few bacteria are observable on the photographs from scanning electron microscopy; the latter are embedded in the agglomerates.

(56) c) Compound C.sub.16TMS—Triethoxyphenylsilane

(57) c1) Effect of Different Biodegradable Solvents in Ethanol Substitution on the Viability of Bacillus subtilis after Synthesis

(58) In a 500 mL beaker, addition of 9.72 g of magnesium nitrate hexahydrate (MgNO.sub.3, 6H.sub.2O) (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 marketed by Solvay) with stirring at 55° C. at 220 rpm for 15 minutes. Addition, 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 Bacillus subtilis (accessible under number CIP 52.62 from the Pasteur Institute) at 6.50×10.sup.7 CFU/ml (the culture medium was prepared in a 3 L bioreactor by seeding Bacillus subtilis in 20 mL of BHI medium (OXOID) and then incubation for 18-24 h at 30° C., 200 rpm). Addition of 70 mL of 1M NaOH (>97%, Sigma Aldrich) (pH 10) and stirring for 24 h. A solid medium count on a Petri dish (90 mm diameter) is carried out at 37° C. in the dark with 60% relative humidity to determine the concentration of microorganisms at To (N.sub.0), To+24 h (N.sub.24 h) and To+14 days (N.sub.14d) after synthesis. The results are presented in Table 8 below.

(59) TABLE-US-00008 TABLE 8 concentration of microorganisms at To (N.sub.0), To + 24 h (N.sub.24 h) and To + 14 days (N.sub.14 d) alter synthesis as a function of the solvent used for synthesis Bacillus subtilis (CFU/ml) N.sub.0 N.sub.24 h N.sub.14 d Glycerol 1.4 × 10.sup.6 1.4 × 10.sup.6 1.2 × 10.sup.6 Propylene glycol 3.1 × 10.sup.6 1.4 × 10.sup.6 1.5 × 10.sup.6 Polarclean 2.9 × 10.sup.6 1.2 × 10.sup.6 7.2 × 10.sup.5

(60) The microorganisms remain viable for at least 14 days after synthesis with glycerol, propylene glycol or Polarclean as solvent. This result confirms that biodegradable solvents may be used for encapsulation of Bacillus subtilis.

(61) c2) Effect of Different Biodegradable Solvents in Ethanol Substitution on the Viability of Bacillus subtilis after Synthesis and Storage at 4° C. and 22° C. for 15 Days

(62) In a 2000 mL beaker, addition of 97.2 g of magnesium nitrate hexahydrate (MgNO.sub.3, 6H.sub.2O) (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 rm for 15 minutes. Addition, with stirring for 15 minutes, of 77.1 g of triethoxyphenylsilane (97%, Sigma Aldrich) and 22.9 g of hexadecyltrimethoxysilane (>85%, Sigma). Addition of 200 mL of culture medium of Bacillus subtilis (accessible under number CIP 52.62 from the Pasteur Institute) at 6.50×10.sup.7 CFU/ml (the culture medium was prepared in a 3 L bioreactor by seeding Bacillus subtilis in 20 mL of BHI medium (OXOID) and then incubation for 18-24 h at 30° C., 200 rpm). Addition of 700 mL of 1M NaOH (97%, Sigma Aldrich) (pH 10) and stirring for 24 h. Two 500-mL samples of the mixture are taken. In darkness, one sample is kept in a stove at +22° C.±2° C. with a relative humidity of 30%, and the other is kept at +4±2° C. with a relative humidity of 65% for 15 days. A solid medium count on a Petri dish (90 mm diameter) is carried out at 37° C. to determine the concentration of bacteria at To (N.sub.0), To+24 h (N.sub.24 h) and To+15d (N.sub.15d). The results are presented in Table 9 below.

(63) TABLE-US-00009 TABLE 9 concentration of microorganisms at To (N.sub.0), To + 24 h (N.sub.24 h) and To + 15 days (N.sub.15 d) after synthesis as a function of the solvent used for synthesis and the storage temperature. Storage Bacillus subtilis (CFU/ml) temperature N.sub.0 N.sub.24 h N.sub.15 d Glycerol 4° C. 6.5 × 10.sup.7 2.1 × 10.sup.7 2.9 × 10.sup.7 22° C. 6.5 × 10.sup.7 2.1 × 10.sup.7 2.5 × 10.sup.7 Polarclean 4° C. 1.0 × 10.sup.7 6.1 × 10.sup.6 5.6 × 10.sup.6 22° C. 6.5 × 10.sup.7 1.0 × 10.sup.7 6.2 × 10.sup.6

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

(65) c3) Effect of Encapsulation (Synthesis by Biodegradable Solvents as Ethanol Replacement) on the Viability of Bacillus subtilis after Thermal Treatment

(66) Synthesis is identical to that in example 2.1c2 with glycerol (Quaron >99.5%) or methyl-5-(dimethylamino)-2-methyl-5-oxopentanoate (RHODISOLV® Polarclean) as solvent. Two 500 mL samples of the mixture are taken. In darkness, 45 samples of 9 mL are taken. 3 batches of 15 samples are made up and are put in a stove (30% relative humidity) at +40° C., +60° C. and +80° C.±2° C., respectively. In parallel, in darkness, 3 batches of 15 samples of unencapsulated microorganisms are also made up and are put in a stove (20% relative humidity) at +60° C. and +80° C.±2° C., respectively. The rise to the set temperatures is calibrated over a time of 30 minutes, at the end of which the tubes are kept at the set core temperature for 2 min, 5 min and 10 minutes before taking out the tubes for counting. 3 tubes are thus taken for each variant of microorganisms and temperature. A solid medium count on a Petri dish (90 mm diameter) is carried out at 37° C. to determine the concentration of bacteria at To (N.sub.0 min), To+2 min (N.sub.2 min), To+5 min (N.sub.5 min) and To+10 min (N.sub.10 min). The results are presented in Table 10 below.

(67) TABLE-US-00010 TABLE 10 concentration of microorganisms at To (N.sub.0 min), To + 2 min (N.sub.2 min), To + 5 min (N.sub.5 min) and To + 10 min (N.sub.10 min) after thermal treatment as a function of the solvent used for synthesis and the temperature of the thermal treatment. Bacillus subtilis (CFU/ml) N.sub.0 min N.sub.2 min N.sub.5 min N.sub.10 min Glycerol Encapsulated 40° C. 2.90 × 10.sup.7 2.20 × 10.sup.7 2.10 × 10.sup.7 2.40 × 10.sup.7 60° C. 6.50 × 10.sup.7 6.50 × 10.sup.7 6.50 × 10.sup.7 6.50 × 10.sup.7 80° C. 6.50 × 10.sup.7 2.00 × 10.sup.7 2.00 × 10.sup.7 2.00 × 10.sup.7 Polarclean Encapsulated 40° C. 6.50 × 10.sup.7 5.40 × 10.sup.7 7.60 × 10.sup.7 6.20 × 10.sup.7 60° C. 6.50 × 10.sup.7 1.80 × 10.sup.7 1.80 × 10.sup.7 6.20 × 10.sup.6 80° C. 6.50 × 10.sup.7 4.80 × 10.sup.6 4.80 × 10.sup.6 4.80 × 10.sup.6 No synthesis Unencapsulated 60° C. 6.50 × 10.sup.7 1.30 × 10.sup.7 1.30 × 10.sup.7 2.00 × 10.sup.7 80° C. 6.50 × 10.sup.7 4.24 × 10.sup.4 1.73 × 10.sup.3 7.53 × 10.sup.3

(68) For the syntheses with glycerol and with Polarclean, the treatments reveal encapsulated Bacillus subtilis to be almost insensitive to heat compared to unencapsulated Bacillus subtilis (at 10 min, respectively 2.00×10.sup.6 CFU/mL and 4.80×10.sup.6 CFU/ml) whereas the concentration of unencapsulated Bacillus subtilis decreases considerably (4.24×10.sup.4 CFU/ml at 2 min). The results confirm that encapsulation provides thermal protection.

Example 2.2: Encapsulation of Piriformospora indica

(69) a) Compound C.sub.16TMS PI

(70) The fungus Piriformospora indica (P. Indica) (accessible under number DSM 11827 from the Max-Planck-Institut für terrestrische Mikrobiologie) is first put in an incubator (140-mm diameter dish) for 72 h at 28±1° C., with YCG agar in order to obtain a fresh culture. The mycelium is collected using a sterile spatula and then introduced into a conical flask containing a 0.5% solution of Tween 80 as well as beads. After stirring for 2 minutes, a solid medium count is performed to determine the starting concentration (N0, in CFU/mL).

(71) 5.56 g of magnesium nitrate hexahydrate (99%, Sigma Aldrich) is dispersed in 100 mL of absolute ethanol (99.9%, Carlo Erba). The mixture is stirred until completely dissolved. 10 g of hexadecyltrimethoxysilane (C.sub.16TMS) (>85%, Sigma) is then added. The whole is stirred and then the solution pH is adjusted to a value of 10 by adding aqueous solution of sodium hydroxide (>97%, Sigma Aldrich) with a concentration of 1M. The formulation is then seeded at 10% with a preculture of P. indica. After stirring at room temperature for 24 h, the solid is separated from the solution by centrifugation (speed of 9000 rpm for 10 min), washed with distilled water three times before being frozen and lyophilized for 48 h (reference sample C.sub.16TMS PI). For comparison, a sample is prepared in the same conditions in the absence of P. indica.

(72) X-ray diffraction analysis indicates that in both cases a lamellar phase is obtained with a periodicity d.sub.001 equal to 1.53 nm for the sample C16TMS PI and 1.60 nm for the compound without P. indica.

(73) The SEM photographs of the sample C.sub.16TMS PI show the presence of forms very similar to the hyphae observed with the fungus alone Hyphae (about 2 μm wide) covered with particles of material. The fungus is therefore encapsulated in its vegetative form (hyphae+conidiophores) in the material of formula I according to the invention.

(74) b) Compound C.sub.16TMS—Triethoxyphenylsilane

(75) b1) Effect of Glycerol as Replacement for Ethanol on the Viability of Piriformospora indica after Synthesis

(76) In a 500 mL beaker, addition of 9.72 g of magnesium nitrate hexahydrate (MgNO.sub.3, 6H.sub.2O) (99%, Sigma Aldrich) and 100 mL of biodegradable solvent (Glycerol (Quaron >99.5%)) with stirring at 55° C. at 220 rpm for 15 minutes. Addition, 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 number DSM 11827 from the Max-Planck-Institut für terrestrische Mikrobiologie) at 3.00×10.sup.7 CFU/ml. Addition of 70 mL of 1M NaOH (>97%, Sigma Aldrich) and stirring for 24 h. A solid medium count on a Petri dish (90 mm diameter) is carried out at 37° C. in the dark with 60% relative humidity to determine the concentration of microorganisms at To (N.sub.0), To+24 h (N.sub.24 h) and To+14 days (N.sub.14d) after synthesis. The results are presented in Table 11 below.

(77) TABLE-US-00011 TABLE 11 concentration of microorganisms at To (N.sub.0), To + 24 h (N.sub.24 h) and To + 14 days (N.sub.14 d) after synthesis Piriformospora indica (CFU/ml) N.sub.0 N.sub.24 h N.sub.14 d Glycerol 3.3 × 10.sup.6 2.2 × 10.sup.6 1.8 × 10.sup.6

(78) The microorganisms remain viable for at least 14 days after synthesis with glycerol as solvent. This result confirms that biodegradable solvents may be used for encapsulation of Piriformospora indica.

(79) b2) Effect of Glycerol as Replacement for Ethanol on the Viability of Piriformospora indica after Synthesis and Storage at 4° C. and 22° C. for 30 Days

(80) In a 2000 mL beaker, addition of 97.2 g of magnesium nitrate hexahydrate (MgNO.sub.3, 6H.sub.2O) (99%, Sigma Aldrich) and 1000 mL of biodegradable solvent (Glycerol (Quaron >99.5%)) with stirring at 55° C. at 220 rpm for 15 minutes. Addition, with stirring for 15 minutes, of 77.1 g of triethoxyphenylsilane (97%, Sigma Aldrich) and 22.9 g of hexadecyltrimethoxysilane (>85%, Sigma). Addition of 200 mL of culture medium of Piriformospora indica (accessible under number DSM 11827 from the Max-Planck-Institut für terrestrische Mikrobiologie) at 3.00×10.sup.7 CFU/ml. Addition of 700 mL of 1M NaOH (97%, Sigma Aldrich) and stirred for 24 h. 2 samples of 500 mL of the mixture are taken. In darkness, one sample is kept in a stove at +22° C.±2° C. with a relative humidity of 30%, and the other is kept at +4±2° C. with a relative humidity of 65% for 30 days. A solid medium count on a Petri dish (90 mm diameter) is carried out at 37° C. in order to determine the concentration of fungi at To (N.sub.0), To+24 h (N.sub.24 h), To+15d (N.sub.15d) and To+30d (N.sub.30d). The results are presented in Table 12 below.

(81) TABLE-US-00012 TABLE 12 concentration of microorganisms at To (N.sub.0), To + 24 h (N.sub.24 h), To + 15 days (N.sub.15 d) and To + 30 d (N.sub.30 d) after synthesis as a function of storage temperature Storage Piriformospora indica (CFU/ml) temperature N.sub.0 N.sub.24 h N.sub.15 d N.sub.30 d Glycerol 4° C. 3.75 × 10.sup.6 5.30 × 10.sup.6 4.60 × 10.sup.6 1.14 × 10.sup.6 22° C. 3.75 × 10.sup.6 5.30 × 10.sup.6 5.60 × 10.sup.5 4.85 × 10.sup.4

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

(83) b3) Effect of Encapsulation (Synthesis by Glycerol as a Replacement for Ethanol) on the Viability of Piriformospora indica after Thermal Treatment

(84) Synthesis is identical to that in example 2.2b2. Two 500 mL samples of the mixture are taken. In darkness, 45 samples of 9 mL are taken. 3 batches of 15 samples are made up and are put in a stove (30% relative humidity) at +40° C., +60° C. and +80° C.±2° C., respectively. In parallel, in darkness, 3 batches of 15 samples of unencapsulated microorganisms are also made up and are put in a stove (20% relative humidity) at +40° C., +60° C. and +80° C.±2° C., respectively. The rise to the set temperatures is calibrated over a time of 30 minutes, at the end of which the tubes are kept for 2 min, 5 min and 10 minutes at the set core temperature before taking out the tubes for counting. 3 tubes are thus taken for each variant of microorganisms and temperature. A solid medium count on a Petri dish (90 mm diameter) is carried out at 37° C. in order to determine the concentration of fungi at To (N.sub.0 min), To+2 min (N.sub.2 min), To+5 min (N.sub.5 min) and To+10 min (N.sub.10 min). The results are presented in Table 13 below.

(85) TABLE-US-00013 TABLE 13 concentration of microorganisms at To (N.sub.0 min), To + 2 min (N.sub.2 min) To + 5 min (N.sub.5 min) and To + 10 min (N.sub.10 min) after thermal treatment as a function of the temperature of the thermal treatment. Piriformospora indica (CFU/ml) N.sub.0 min N.sub.2 min N.sub.5 min N.sub.10 min Glycerol Encapsulated 40° C. 3.00 × 10.sup.7 1.10 × 10.sup.6 1.10 × 10.sup.6 1.00 × 10.sup.6 60° C. 3.00 × 10.sup.7 4.40 × 10.sup.4 4.55 × 10.sup.4 1.95 × 10.sup.4 80° C. 3.00 × 10.sup.7 1.50 × 10.sup.1 0.00 × 10.sup.0 0.00 × 10.sup.0 No synthesis Unencapsulated 40° C. 5.14 × 10.sup.6 1.69 × 10.sup.6 1.53 × 10.sup.6 1.32 × 10.sup.6 60° C. 5.14 × 10.sup.6 7.10 × 10.sup.3 9.06 × 10.sup.3 1.31 × 10.sup.2 80° C. 5.14 × 10.sup.6 0.00 × 10.sup.0 0.00 × 10.sup.0 0.00 × 10.sup.0

(86) For the syntheses with glycerol, the treatments reveal lower thermal sensitivity of encapsulated Piriformospora indica compared to unencapsulated Piriformospora indica (at 60° C./2 min, 4.40×10.sup.4 CFU/mL versus 7.10×10.sup.3 CFU/ml; at 60° C./5 min 4.55×10.sup.4 CFU/mL versus 9.06×10.sup.3 CFU/ml; at 60° C./10 min 1.95×10.sup.4 CFU/mL versus 1.31×10.sup.2 CFU/ml; at 80° C./2 min, 1.50×10.sup.1 CFU/mL versus 0.00×10° CFU/ml). The results confirm that encapsulation provides thermal protection.

Example 2.3: Encapsulation of Spirulina

(87) a) Compound C-16 Alga

(88) 2 g of magnesium nitrate hexahydrate (99%, Sigma Aldrich) is dispersed in 20 mL of absolute ethanol (99.9%, Carlo Erba). The mixture is stirred until completely dissolved. 1 g of Spirulina (cyanobacterium Arthrospira platensis marketed under the name SPIRULINA NATURAL by the company EARTHRISE®) is introduced into the medium and then 1.111 g of hexadecyltrimethoxysilane (C.sub.16TMS) (>85%, Sigma) is added to each mixture. The whole is stirred and then the solution pH is adjusted to a value of 9 by adding 9 mL of an aqueous solution of sodium hydroxide (>97%, Sigma Aldrich) with a concentration of 1M. After stirring at room temperature for 24 h, the solid is separated from the solution by centrifugation (speed of 10000 rpm for 10 min), washed three times with ethanol before being dried in a stove at 40° C. for 48 h. The compound obtained is then ground in an agate mortar before being characterized and is designated C-16 alga. For comparison, a sample was also prepared in the absence of Spirulina (reference sample Talc conventional).

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

EXAMPLE 3: EVALUATION OF THE CAPACITY FOR SOLUBILIZATION OF A Water-Insoluble Phosphate Using a Compound According to the Invention

(90) a) Preparation of the Encapsulated Bacterium:

(91) 10 g of magnesium nitrate hexahydrate (99%, Sigma Aldrich) is added to 20 mL of absolute ethanol (99.9%, Carlo Erba). The mixture is stirred until completely dissolved. 10 g of phenyltrimethoxysilane (98%, ABCR) is then added with stirring. While stirring, the mixture is seeded at 10% v/v of a preculture of Bacillus subtilis CIP 52.62 of concentration 10.sup.8 CFU/mL. The whole is stirred and then the solution pH is adjusted to a value of 10 by adding aqueous solution of sodium hydroxide (>97%, Sigma Aldrich) with a concentration of 1M. After stirring at room temperature for 24 h, a bacterial count is performed on the seeded ensemble defining the bacterial concentration at 1.7×10.sup.5 CFU/ml.

(92) b) Solubilization Test:

(93) In 200 mL conical flasks, a liquid culture medium (glucose, 10 g/L; MgCl.sub.2.6H.sub.2O, 5 g/L; MgSO.sub.4.7H.sub.2O, 0.25 g/L; KCl, 0.2 g/L; (NH.sub.4).sub.2SO.sub.4, 0.1 g/L) of pH 7.0, supplemented with a source of phosphorus insoluble in an aqueous medium (NH.sub.4MgPO.sub.4.Math.6H.sub.2O, 8.9 g/conical flask) is prepared. Each conical flask is then seeded with 1 mL of a preculture of Bacillus subtilis CIP 52.62 (Pasteur Institute) at a concentration of 6.5×10.sup.4 CFU/mL (free) or with 382.3 μL of a preculture of Bacillus subtilis CIP 52.62 at a concentration of 1.7×10.sup.5 CFU/ml (compound according to the invention 100% Phenyl BS: encapsulated bacterium prepared according to example 3a). The conical flasks are kept in a stove with stirring. A colony count is carried out every 2 days as well as measurement of the pH and of the concentration of solubilized phosphorus and of phosphorus immobilized by the bacterial flora (measurement of total phosphorus by ICP and measurement of inorganic phosphate in solution by HPIC). The results are presented in FIGS. 1 to 3. Comparison of the growth of Bacillus subtilis indicates better capacity for solubilization of water-insoluble phosphorus (FIG. 1). Comparison of the growth of Bacillus subtilis indicates a lowering of the solution pH correlated with solubilization of bacterial origin of the phosphorus (FIG. 2). Comparison of the phosphorus concentration indicates bacterial solubilization greater than its immobilization by the bacterial flora (FIG. 3). We therefore find better growth of the bacteria encapsulated according to the invention and better solubilization of phosphorus when such bacteria are used.