FR2785292A1 - CAPSULES WITH POROUS MINERAL SHELL IN WHICH ARE IMMOBILIZED IN A LIQUID BIOLOGICAL MEDIUM, ONE OR MORE BIOLOGICAL MATERIALS, THEIR PREPARATION PROCESS AND USES - Google Patents

Abstract

The invention concerns a mineral capsule consisting of a mineral cortex and a liquid core wherein is immobilised at least an active biological material. The invention also concerns a method for preparing said capsules and their uses.

Description

 The present invention relates to new capsules with porous mineral bark in which are immobed! one or more biological active materials in a liquid medium.

 In general, the techniques for immobilizing active materials aim to limit and more preferably to prevent their free migration into a surrounding medium.

There are two general methods of immobilization:
The first method consists in binding the active material to be immobilized to a support type surface either by adsorption (ionic interactions, Van der Waals bonds), or by covalent bond or via an intermediate compound.

 The second method, on the other hand, aims to physically retain the active material inside a solid or porous matrix such as a stabilized gel.

 This second approach is widely used for the immobilization of biological active materials such as cells or enzymes for example. Most commonly, a physical encapsulation of the biological active materials considered is carried out inside a polymer matrix.

The encapsulation material must in fact meet several requirements:
-11 must contribute to ensuring the stability of the structure and the activity of the immobilized active material;
-11 must have sufficient porosity to control the diffusion of the immobilized active material and, where appropriate, its exchanges with the surrounding medium and
-11 must allow, where appropriate, easy reuse of the immobilized active material.

 The materials most frequently used to carry out this type of immobilization are hydrocolloid gels. By way of illustration of these gels, mention may in particular be made of natural gels such as alginates and carrageenans. However, other polymeric networks of a synthetic nature have also been developed, such as that based on polyacrylamide.

 This type of encapsulation is generally obtained by adding the active material to be immobilized in the form of a suspension in an aqueous solution of a precursor of the encapsulation material. The precursor solution is then transformed there into droplets generally by dispersion. Finally, these droplets are stabilized in the form of beads in which the active material is trapped, either by polymerization or any other type of crosslinking.

 It has also been proposed to encapsulate enzymes in a porous structure of a mineral nature. The encapsulation technique chosen is then related to the sol-gel technique. According to this mode of encapsulation, the hydrolysis and polycondensation of a metal alkoxide are initiated in water or in a hydroalcoholic medium, the enzyme is dispersed there and the resulting composition is then gelled and then dried.

 Compared to organic matrices 6 based on a natural gel of alginate or carrageenan type, these inorganic matrices are particularly advantageous. Their mechanical resistance is considerably increased. They generally have a hydrophilic character as well as better stability to solvents and to pH. What is more, they are stable in concentrated saline medium unlike organic matrices based on alginate for example.

 However, just like organic matrix encapsulation systems, these inorganic matrices are unsuitable for the packaging of biological active materials that need to be stored in a liquid medium. Furthermore, the low porosity of these systems, due to their matrix nature, constitutes an obstacle to the growth of biological active materials and to the maintenance of their activity.

 The object of the present invention is precisely to propose a new encapsulation mode, which is particularly advantageous insofar as it precisely allows biological active materials to be preserved in a liquid environment conducive to the conservation of their structure and activity and, where appropriate, their development. internal while effectively protecting them against any degradation likely to be caused to them by the surrounding environment.

 The present invention therefore has for first object a mineral capsule consisting of a mineral shell and a liquid core in which is immobilized at least one biological active material.

 For the purposes of the present invention, it is intended to cover, under the name biological active material, any molecule, cell or organism having an industrial interest due to its biological activity.

 By way of illustration of these biological active materials, mention may in particular be made of cellular organisms such as microorganisms of the bacteria, yeast, fungus and algae type, cells of animal or vegetable origin, enzymes or proteins of the antibody type for example.

 It is more preferably cells or living organisms.

 As examples of biologically active molecules, mention may more particularly be made of enzymes such as hydrolases, nucleases, oxidases, proteases, isomerases and the like.

 It may in particular be oxidoreductases such as alcohol dehydrogenases, oxygenases and glucose dehydrogenases, transferases such as D-glutamyl transferase, lyases such as fumarase and aspartase, hydrolases such as lipases, nitrilehydratases, lactases and acylases as well as isomerases. It can also be proteins or protein complexes of the cytochrome C, hemoglobin, myoglobin, transferrin or superoxide dismutase type or of antibodies.

 As an example of bacteria, mention may more particularly be made of lactic acid bacteria and environmental bacteria.

 In contrast to the conventional encapsulation systems mentioned above, the biological active material is in the case of the present invention not adsorbed in a form dispersed within a matrix but concentrated in a liquid medium which is isolated from the surrounding medium with mineral bark.

 For the purposes of the present invention, the term “liquid medium” means a medium capable of ensuring the conservation of the activity and the structure and / or the survival and, where appropriate, the internal development of a biological active material, this medium being under a fluid form of the liquid type. As such, it differs from alginate or carrageenan type media which are similar to media in gel form.

 It can in particular be the natural biological medium of the immobilized biological active material. Generally this liquid biological medium is or is derived from an aqueous medium.

 Of course, this liquid medium can be buffered and / or supplemented with trace elements, sugars, salts and any other nutritive agent capable of being necessary for the conservation of the activity and the structure and / or the survival and if necessary internal development of the immobilized biological active material.

 These active materials, depending on their water-soluble nature, are either dissolved or dispersed in the liquid medium.

 The mineral bark obtained according to the method of the invention has the double advantage of effectively protecting the liquid medium and the active material (s) it contains, and if necessary allowing it to be exchanged with the medium. surrounding capsules. This is in particular achieved by adjusting the degree of porosity of the mineral capsules obtained according to the invention.

 According to a first variant of the invention, the mineral capsules obtained can be non-porous. This specificity is more particularly advantageous when it is essentially desired to ensure effective protection of the liquid medium, incorporating at least one biological active material, with respect to its surrounding medium. In contrast to the microcapsules obtained by interfacial polycondensation and which consist of an organic shell, the capsules obtained according to the invention are much more resistant mechanically, thermally and chemically due to the mineral character of their shell. In this particular case, the aqueous medium and, where appropriate, the active material which it contains are generally released by fractionation of the capsule or by induced degradation of the latter.

 According to a second variant of the invention, the capsules obtained can be porous and this porosity is controllable. This is of significant interest when it is desired, in addition to protecting the liquid medium and the active material which it contains, to allow its exchanges with the surrounding medium of said capsules.

 In fact, this adjustment of the porosity as well as that of the size of the capsules is accomplished through mainly the choice of the constituent mineral material. I bark mineral or more precisely the choice of the precursor of this material. This aspect of the invention is discussed in more detail below.

 The present invention is therefore particularly advantageous insofar as it provides a reliable packaging method, compatible with the internal development of the immobilized active ingredients and adjustable according to the nature and the amount of immobilized active ingredient (s). ).

 The preservation within the capsule of a liquid medium which can be the natural biological medium of the immobilized active material is a guarantee of prolonged functioning of said active material, functioning which can for example result in the production of metabolites or be enzymatic.

 The mineral nature of the bark of the claimed capsules constitutes an effective protective barrier to the biological active material vis-à-vis the surrounding environment while allowing, where appropriate, its exchanges with it.

 The size of the capsules being controllable, it can be adjusted as a function of the constraints linked to the size of the active ingredients to be encapsulated or also to the number of these biological active ingredients.

 Finally, this capsule is prepared in the context of the present invention under operating conditions which are sufficiently gentle to not affect the integrity of said active materials. The active ingredient to be immobilized is in fact not exposed during its conditioning to temperature and, where appropriate, pH values likely to harm it.

 Regarding the composition of the mineral bark. it consists of at least one oxide and / or hydroxide of aluminum, silicon, zirconium and / or a transition metal.

 By transition metal is meant more particularly the metals of the fourth period ranging from scandium to zinc insofar as these are of course compatible in terms of safety with the intended application. It is more particularly an oxide or hydroxide of titanium, manganese, iron, cobalt, nickel or copper.

 Of course, this mineral shell can comprise oxides and / or hydroxides of different natures.

 Particularly suitable for the present invention are the oxides and / or hydroxides of silicon, aluminum, titanium and zirconium.

 According to a preferred embodiment of the invention, the capsules comprise a mineral shell based on at least one silicon oxide.

 In general, the size of the capsules according to the invention can be between 1 and several tens of micrometers. The particle size of the mineral material constituting the bark of these capsules can vary for its part between 1 and 200 nm.

 As regards more particularly the thickness of the mineral shell, it can vary between 1 and 200 nm.

 These capsules can also be characterized by the amount of liquid medium that the mineral shell retains by introducing the retention parameter of the capsule. This corresponds to the ratio of the mass of the liquid medium contained in the capsule to that of the mineral bark.

 When the bark is too porous, the retention of the capsule is low. One can thus vary the retention of the capsules by fixing the size of the particles of the material constituting the mineral shell as well as the thickness of the latter.

A subject of the present invention is also a process useful for preparing minor capsules in accordance with the present invention, said process comprising
1) the emulsification of a liquid medium containing at least one biological active material within a second phase immiscible with said liquid medium so as to disperse it in the form of droplets,
2) bringing into contact, within the inverse emulsion thus obtained, at least one hydrolyzable and polycondensable compound of zirconium, silicon, aluminum and / or a transition metal under conditions of temperature and pH suitable for formation of a precipitate consisting of the corresponding oxide or hydroxide and
3) the recovery of the mineral capsules thus formed and if necessary their purification, said process being characterized in that the formation of the mineral precipitate in the second step is carried out in the presence of an amphiphilic surfactant system, present at the level of the emulsion and capable of concentrating the deposit of mineral particles of said precipitate formed at the interface of the droplets and of the second phase and of effectively blocking their diffusion within said droplets.

 The amphiphilic surfactant system proposed according to the invention has the advantage of blocking the phenomenon of natural diffusion of mineral particles towards the center of the droplets.

 For the purposes of the invention, the term “amphiphilic surfactant system” is intended to denote either a single compound at the level of which two regions coexist with very different solubilities and sufficiently distant from each other to behave independently, or an association of '' at least two compounds having very different solubilities such as a first compound with hydrophilic character and a second compound with hydrophobic character. Generally these two regions or compounds respectively comprise at least one hydrophilic group and one or more long chains of hydrophobic nature.

 Consequently, the surfactant system used according to the invention can be represented by a single compound and which will then be introduced before carrying out the second stage, that is to say the hydrolysis and polycondensation stage, or further resulting from an in situ interaction of at least two compounds such as for example an organosoluble surfactant initially present in the second generally organosoluble phase and a water-soluble compound present in the generally aqueous liquid medium. One can also envisage a coupling or a complexation between a first organosoluble agent and a second organosoluble agent of ionic nature such as a quaternary ammonium. The two compounds meet at the interface of the droplets formed during the emulsification. Due to their interaction, they help to stabilize the system by decreasing the interfacial tension at the interface of the droplets and probably act as a steric or electrostatic barrier.

 In the claimed process, the embodiment using at least two distinct compounds is more particularly preferred capable of interacting to lead to a surfactant system capable of effectively opposing the diffusion of mineral particles in the aqueous droplets and of stabilizing said emulsion.

 The surfactant system proposed according to the invention preferably comprises at least one surfactant with an HLB value of less than 7.

 The term HLB designates the ratio of the hydrophilicity of the polar groups of the surfactant molecule to the hydrophobicity of the lipophilic part of this same molecule.

 In this particular case, the two compounds are preferably respectively present in the liquid medium, generally aqueous and the second phase generally organosoluble and interact with each other during the emulsification of the liquid medium in said second phase.

 This option has the advantage of giving the corresponding emulsion satisfactory stability as soon as it is formed. Furthermore, it is possible, if necessary, by appropriately selecting the agents constituting the amphiphilic surfactant system, to adjust the pH to a value compatible with the active material.

 As regards the emulsification, it can be carried out by applying a mechanical energy of intense agitation to the two initial phases, and / or a sonication. The size of the droplets obtained at the end of the emulsification step can be between approximately 0.1 and ten μm.

 The compound present in the liquid, generally aqueous medium preferably has a viscosifying action.

 More particularly, this compound can be a compound chosen from sugars and their derivatives. As such, the oses (or monosaccharides), the osides and the highly depolymerized polyholosides are suitable. Compounds are understood to have a molar mass by weight which is more particularly less than 20,000 g / mole.

 Among the dares, there may be mentioned aldoses such as glucose, mannose, galactose and ketoses such as fructose.

 Osides are compounds which result from the condensation, with elimination of water, of ose molecules with non-carbohydrate molecules. Among the osides, the holosides which are formed by the combination of exclusively carbohydrate units are preferred, and more particularly the oligoholosides (or oligosaccharides) which contain only a limited number of these units, that is to say a general number. less than or equal to 10. As examples of oligoholosides, mention may be made of sucrose, lactose, cellobiose, maltose.

 The highly depolymerized polyholosides (or polysaccharides) suitable are described for example in the work of P. ARNAUD entitled "Course of organic chemistry", GUTHIER-VILLARS editors, 1987. More particularly, polyholosides are used whose molecular mass in weight is more particularly less than 20,000 g / mole.

 By way of nonlimiting example of highly depolymerized polyholosides, mention may be made of dextran, starch, xanthan gum and galactomannans such as guar or locust bean. These polysaccharides preferably have a melting weight greater than 100 C and a solubility in water of between 10 and 500 gA.

 Also suitable for the invention are gum arabic, gelatin and their fatty derivatives such as the fatty acid sucroesters, the carbohydrate alcohols of sorbitol, mannitol, ether carbohydrates such as methyl-, ethyl-, carboxymethyl-, hydroxyethyl- and hydroxypropyl ethers of cellulose and glycerols, pentaerythrol, propylene glycol, ethylene glycol, non-viscous diols and / or polyvinyl alcohols.

 It is preferably a hydrocolloid. Mention may in particular be made, for example of this type of compound, of alginates, polysaccharides of the natural gum type such as carrageenans, xanthan and guar and very particularly cellulose derivatives.

 Preferably, it is a cellulose derivative and more preferably hydroxyethylcellulose.

 The generally organosoluble surfactant (s) present in the second phase can be chosen from fatty alcohols, triglycerides, fatty acids, sorbitan esters, fatty amines, these compounds being or not in a polyalkoxylated form, liposoluble lecithins, polyalkylene dipolyhydroxystearates, quaternary ammonium salts, monogtycerides, polyglycerol esters, polyglycerol polyricinoleate and lactic esters.

 Fatty alcohols generally contain from 6 to 22 carbon atoms. The triglycerides can be triglycerides of plant or animal origin (such as lard, tallow, peanut oil, butter oil, cottonseed oil, linseed oil, olive oil, fish oil, coconut oil, coconut oil).

 Fatty acids are fatty acid esters (such as for example oleic acid, stearic acid).

 Sorbitan esters are cyclized fatty acid sorbitol esters comprising from 10 to 20 carbon atoms such as auric acid, stearic acid or oleic acid.

 According to a preferred embodiment of the invention, this surfactant is a sorbitan ester as defined above and more preferably sorbitan sesquioleate.

 As emerges from the preceding description, the compound present in the generally aqueous liquid medium must interact with the surfactant present in the second generally hydrophobic phase to lead to a surfactant system capable of constituting an effective diffusion barrier with respect to the particles. mineral precipitate. Consequently, their respective choices must be made taking this imperative into account.

 Of course, the nature of the active material to be encapsulated, as well as the composition of the mineral bark of the capsules prepared according to the invention are also determining factors in the choice of the surfactant system and the assessment of the respective amounts of the two corresponding compounds. These adjustments in fact fall within the competence of a person skilled in the art.

dans laquelle :
? 2 représente un radical alkyle ou alcényle comprenant 7 à 22 atomes de carbone, Ri représente un atome d'hydrogène ou un radical alkyle comprenant 1 à 6 atomes de carbone, A représente un groupement (CO) ou (OCH2CH2), n vaut 0 ou 1, x vaut 2 ou 3, y vaut 0 à 4, Q représente un radical -R3-COOM avec R2 représentant un radical alkyle comprenant 1 à 6 atomes de carbone, M représente un atome d'hydrogène, un métal alcalin, un métal alcalino-terreux ou encore un groupement ammonium quaternaire dans lequel les radicaux liés à I'atome d'azote, identiques ou différents, sont choisis parmi l'hydrogène ou un radical alkyle ou hydroalkyle possédant 1 à 6 atomes de carbone, et B représente H ou Q.In the particular case where the use of a single amphiphilic type compound is preferred according to the invention, those corresponding to the general formula I are particularly suitable:

in which :
? 2 represents an alkyl or alkenyl radical comprising 7 to 22 carbon atoms, Ri represents a hydrogen atom or an alkyl radical comprising 1 to 6 carbon atoms, A represents a group (CO) or (OCH2CH2), n is 0 or 1, x is 2 or 3, y is 0 to 4, Q represents a radical -R3-COOM with R2 representing an alkyl radical comprising 1 to 6 carbon atoms, M represents a hydrogen atom, an alkali metal, a metal alkaline earth or also a quaternary ammonium group in which the radicals linked to the nitrogen atom, identical or different, are chosen from hydrogen or an alkyl or hydroalkyl radical having 1 to 6 carbon atoms, and B represents H or Q.

 Preferably, M represents a hydrogen atom, sodium, potassium and an NH4 group.

 Among these surfactants corresponding to formula 1, use is more particularly made of amphoteric derivatives of alkyl polyamines such as amphionic XL @, Mirataine H2C-HA marketed by Rhodia Chimie as well as Ampholac 7T / Xs marketed by Berol Nobel.

 It is also possible to use a main nonionic surfactant, the hydrophilic part of which contains one or more saccharide unit (s).

Said saccharide units generally contain from 5 to 6 carbon atoms. These can be derived from sugars such as fructose, glucose, mannose, galactose, talose, gulose, allose, altose, idose, arabinose, xylose, lyxose and / or ribose.

Among these surfactants with a saccharide structure, mention may be made of alkylpolyglycosides. These can be obtained by condensation (for example by acid catalysis) of glucose with primary fatty alcohols (US-A-3,598,865; US-A-4,565,647; EP-A-132,043; EP-A- 132 046; Tenside Surf. Det. 28,419,1991,3; Langmuir 1993,9,3375-3384) having a C4-C20 alkyl group, preferably of the order of 1.1 to 1.8 per mole of alkylpolyglycoside (APG); mention may be made in particular of those marketed respectively under the names GLUCOPON 600 EC @,
GLUCOPON 650 Etcs, GLUCOPON 225 CSUP @, by HENKEL.

 By way of illustration, the concentration of amphiphilic surfactant system can be between approximately 1% and 10% by weight relative to the organosoluble phase.

 According to a preferred embodiment of the invention, the surfactant incorporated at the level of the second generally organosoluble phase, is a sorbitan ester and more preferably sorbitan sesquioleate. As for the compound incorporated in the liquid medium, it is preferably a cellulose derivative and more particularly cellulose hydroxyethyte.

 According to a preferred embodiment of the invention, the material of the mineral crust derives from the hydrolysis and polycondensation of one or more alkoxides of formula 11.

M (R) n (P) m 11 in which: -M represents an element chosen from titanium, manganese, iron, cobalt,
nickel, silicon, aluminum or zirconium, -R is a hydrolyzable substituent, -n is an integer between 1 and 6, -P is a non-hydrolyzable substituent and -m is an integer between 0 and 6.

According to a preferred embodiment of the invention: -M is chosen from silica, aluminum, titanium and zirconium, -R is a group chosen from alkoxy and / or aryloxy groups in
Ci to de and preferably in Cz to C8 and n is an integer between 2 and 4
and -P is a group chosen from C1 to Ce alkyl groups,
C2 to C8 aryls or alkenyls.

 As far as R is concerned, it is preferably a C, to C6 and more preferably C 2 to C 4 alkoxy group. This alkoxy group can optionally be substituted by a C 1 -C 4 alkyl or alkoxy group or a halogen atom. In the general formula 11, R can represent identical or different alkoxy groups.

 Of course, several compounds of formula 11 can be used.

 As previously discussed, it is possible through the choice of this mineral hydrolyzable and polycondensable compound to adjust the porosity and the size of the capsules.

 Likewise, the capsule can be given a more or less hydrophobic character by varying the nature and for example the length of the alkyl and / or alkoxy chains constituting this mineral hydrolyzable and polycondensable compound.

Generally, the hydrolysis and polycondensation of this mineral precursor are carried out either spontaneously by bringing it into contact with the emulsion or are initiated by adjusting the pH and / or the temperature of the emulsion to a suitable value. at their manifestation. This adjustment may in particular arise from the presence in the ion emulsion
water-soluble like NH40H, NaOH or HCI or organosoluble like amines. These adjustments fall within the competence of a person skilled in the art.

 According to a preferred variant of the present invention, the mineral shell obtained according to the invention is based on silicon oxide. It is derived from the precipitation of at least one silicate.

 As silicate suitable for the present invention, mention may more particularly be made of tetramethylorthosilicate, TMOS, tetraethylo

 As a second phase which is very particularly suitable for the invention, mention may in particular be made of the solvent Isopar $ which is an isoparrafine sold by the company Exxon Chemicals.

 This second phase comprises at least one generally organosoluble surfactant which is preferably chosen from sorbitan esters and more preferably is represented by sorbitan sesquioleate.

 As regards the generally aqueous liquid medium, it comprises at least one hydrocolloid, optionally the hydrolysis and polycondensation agent of the mineral hydrolyzable and polycondensable precursor.

 As regards the hydrocolloid, it is preferably a cellulose derivative and more preferably hydroxyethylcellulose.

 The mineral capsules according to the invention are particularly advantageous for uses in the fields of biomedical fermentation, food and in the chemical industry.

 It is thus possible to envisage immobilizing, within the capsules claimed, cells having an activity which is of interest for the production of pharmaceutical products, metabolites and / or reagents such as chemical or pharmaceutical synthesis intermediates or else biodegradable polymers.

 The immobilization according to the invention of yeast or bacteria type microorganisms is also particularly advantageous for the food industry and especially for the dairy and wine industries.

 Enzymes immobilized according to the invention can represent, for their part, reagents of choice in numerous industrial manufacturing processes for catalytic or analytical methods.

 Likewise, it is possible to envisage using capsules according to the invention based on living cells or enzymes in the treatment of waste water or waste.

 The examples and the figure which follow are presented by way of illustration and without limitation of the subject of the present invention.

Figures:
-Figure 1 Photograph by scanning microscopy (SEM) of capsules according to the invention incorporating E. coli.

 -Figure 2 Visualization of E. coli encapsulated by transmission electron microscopy (TEM).

Raw materials: Isopar Ms (EXXON) Density at 15 C: 0.786 Arlacel 83m (HERE) Sorbitan sesquioleate
HEC (SIGMA ALDRICH) Hydroxyethylcellulose
Aqueous ammonia solution (NH40H) Density at 20 C: 0.880
Concentration in Nudes: 20%
Methyl silicate: Si (OMe) 4 Molar mass: 156 g
Density at 20 C: 1.032
Sodium phosphate buffer at pH 7.

Escherichia Coli K12 expressing an ss-galactosidase.

Example 1: Encapsulation of E. Coli
Overall composition of the reaction medium:
Aqueous phase: H20 43.40 g
HEC 2.61 g (6% / water)
NH3 5.00 g (1 molli)
E. Coli 4 g (i.e. 1 g of dry cells)
Organic phase Arlacel 83 @ 17.35 g isopar M 850 g tetramethylorthosilanes TMOS 28.5 g
Preparation of the aqueous phase:
The HEC is homogenized in purified water in a water bath at 40 ° C. for about 20 minutes. A clear yellow viscous mixture is thus obtained. The cells are then added thereto, followed by the aqueous ammonia solution.

Preparation of the organic phase:
The Arcacel 83 is dissolved in Isopar Mm.

Preparation of the emulsion:
The organic phase and the aqueous phase are emulsified using an Ultraturrax, a water / oil emulsion having a stability of several hours is thus obtained. The size of the droplets is close to ten microns.

Summary of capsules:
In the 2-liter three-necked flask fitted with a magnetic bar, the emulsion prepared previously is introduced.

 Tetramethylorthosilane TMOS (28.5 g) is added thereto with a flow rate of 0.5 ml / min. (duration of introduction of TMOS = 1 hour) at 25 C.

 The particles obtained are separated and washed with methanol then 2 times in phosphate buffer by centrifugation and then dried at room temperature overnight.

Characterizations:
The size of the silica particles which constitute the shell is close to a few nanometers.

 The capsules have a size of the order of ten pm (SEM). The encapsulation of bacteria is perfectly visualized by TEM (transmission electron microscopy), as well as the porosity of the capsule.

 The photographs presented in Figures 1 and 2 reflect the appearance of these capsules.

Example 2: Determination of the enzymatic activity of the biocapsules
The enzymatic activity of the E. coli ss-galactosidase is determined after enzymatic hydrolysis of para-nitrophenyl-ss-D-galactoside (p-NPG) to p-nitrophenol. The quantitative measurement of p-nitrophenol by spectrophotometry makes it possible to deduce therefrom the enzymatic activity of the biocapsules.

 The enzymatic activity of the biocapsules of Example 1 is expressed in umole / h / mg of CS (dry cells), this compared to the bioactivity of the starting cells. The enzymatic activity of the starting cells is 0.2 pmol / h / mg of CS, the activity yield of the biocapsules is approximately 50%. The activity of the cells is therefore well preserved.

Example 3: Obtaining silica capsules. with a particle size constituting the bark of a few nanometers.

Overall composition of the reaction medium:
Aqueous phase: H20 43.40 g
HEC 2, 61 g (6% / water)
NH3 5.0g (1 mol / 1)
E. Coli 4 g (i.e. 1 g of dry cells)
Organic phase: Arlacel 83 17.35 g (2.04% / lsopar) Isopar M @ 850 9
TEOS 39 g
Preparation of the aqueous phase:
The HEC is homogenized in the purified water in a water bath at 40 ° C. for about 20 minutes. A clear yellow viscous mixture is thus obtained. The cells are then added thereto, followed by the aqueous ammonia solution.

Preparation of the organic phase:
We solubilize the Arcacel 83s in the Isopar M
Preparation of the emulsion:
The organic phase and the aqueous phase are emulsified using an ultraturrax, thus obtaining a water / oil dispersion having a stability of several hours. The size of the droplets is close to ten microns.

Summary of:
In a 2 liter three-necked flask from a magnetic bar, the emulsion prepared previously is introduced.

 Ethyl silicate (39 g) is added thereto with a flow rate of 0.8 mt / min (duration of introduction of TEOS = 1 hour) at 25 C.

 The capsules obtained are separated and washed 1 time with ethanol then 2 times with phosphate buffer and then dried at room temperature overnight.

Characterizations:
The size of the capsules is between 1 and ten pm (SEM).

 The size of the silica particles constituting the shell is a few nanometers. The observations made by MET clearly show the encapsulation of bacteria by the silica capsule.

Example 4: Obtaining capsules with TMOS and co-alkoxide (fNHal = 0.1 mollo.

Overall composition of the reaction medium:
Aqueous phase: H20 43.40 g
HEC 2, 61 g (6% / water)
NH3 0.37 g or [NH3] = 0.1 mol / l
E. Coli 4 g (i.e. 1 g of dry cells)
Organic phase: Arlacel 83 @ 17, 35 9 (2.04% Ilsopar) Isopar M 850 9
Organic phase 2: TMOS 22.8 g
0-TMOS 5.7 g
Preparation of the aqueous phase:
The HEC is homogenized in purified water in a water bath at 40 ° C. for about 20 minutes. A clear yellow viscous mixture is thus obtained. The cells are then added thereto, followed by the aqueous ammonia solution (0.37 g).

Preparation of the organic phase:
The Arcacel 83 is dissolved in the isopar MI.

Preparation of the emulsion:
The organic phase 1 and the aqueous phase are emulsified using an ultraturrax, an oil-oil dispersion thus obtaining a stability of several hours. The size of the droplets is close to ten microns.

Procedure:
In the 2-liter three-necked flask fitted with a magnetic bar, the emulsion prepared previously is introduced.

 The mixture of alkoxides (organic phase 2) is added thereto with a flow rate of 0.5 mi / min (the duration of the introduction is 1 hour) at 25 C.

 The particles obtained are separated and washed with methanol and then dried at room temperature overnight. The particles thus obtained are hydrophobic.

Characterizations:
The particle size is between 1 and ten pm (SEM).

 The size of the silica particles constituting the shell is a few nanometers (TEM).

Claims (23)

 1. Mineral capsule consisting of a mineral shell and a liquid core in which at least one biological active material is immobilized.  2. Capsule according to claim 1, characterized in that the mineral shell consists of at least one oxide and / or hydroxide of aluminum, silicon, zirconium and / or a transition metal.  3. Capsule according to claim 1 or 2, characterized in that the mineral shell consists of at least one oxide and / or hydroxide of silicon, aluminum, titanium and zirconium.  4. Capsule according to claim 3, characterized in that the mineral shell comprises at least one silicon oxide.  5. Capsule according to one of the preceding claims, characterized in that the liquid medium constituting the core of said capsule corresponds to the natural biological medium of the immobilized biological active material.  6. Capsule according to one of the preceding claims, characterized in that the liquid medium constituting the core of said capsule is conducive to the conservation of the structure and the activity of the immobilized biological active material.  7. Capsule according to one of claims 1 to 6, characterized in that the liquid medium constituting the core of said capsule is conducive to the internal development of the immobilized biological active material.  8. Capsule according to one of the preceding claims, characterized in that the biological active material is chosen from cellular organisms such as microorganisms such as bacteria, yeasts, fungi, algae, cells of animal or vegetable origin, enzymes or proteins .  9. Capsule according to claim 8, characterized in that they are living cells or organisms.  10. Capsule according to one of the preceding claims, characterized in that it has a size between 1 and several ten micrometers.  11. Capsule according to one of the preceding claims, characterized in that its mineral shell has a thickness varying between 1 and 200 nm.  12. Method for the preparation of capsules according to one of claims 1 to 11, said method comprising  1) the emulsification of a liquid medium containing at least one biological active material within a second phase immiscible with said liquid medium so as to disperse it therein in the form of droplets,  2) bringing into contact, within the emulsion obtained, obtained, minus a hydrolyzable and polycondensable compound of zirconium, silicon, aluminum and / or a transition metal under conditions of temperature and pH conducive to the formation of a precipitate consisting of the corresponding oxide or hydroxide and  3) the recovery of the minor capsules thus formed and, where appropriate, their purification, said process being characterized in that the formation of the mineral precipitate in the second step is carried out in the presence of an amphiphilic surfactant system, present at the level of the emulsion and capable of concentrating the deposit of mineral particles of said precipitate formed at the interface of the droplets and of the second phase and of effectively blocking their diffusion within said droplets.  13. The method of claim 12, characterized in that the amphiphilic surfactant system results from the in situ interaction between a first compound present in the second phase and a compound present in the liquid medium.  14. Method according to claim 12 or 13, characterized in that the surfactant system comprises at least one surfactant with a value HLB less than 7.  15. Method according to one of claims 12 to 14, characterized in that the surfactant present in the second phase is a sorbitan ester.  16. Method according to one of claims 12 to 15, characterized in that the compound present in the liquid medium is a cellulose derivative.  17. Method according to one of claims 12 to 16, characterized in that the emulsion is obtained mechanically and / or by sonication.  18. Method according to one of claims 12 to 17, characterized in that the hydrolyzable and polycondensable compound corresponds to the general formula II:  M (R) n (P) m 11 in which: -M represents an element chosen from titanium, manganese, iron, cobalt,  nickel, silicon, aluminum or zirconium, -R is a hydrolyzable substituent, -n is an integer between 1 and 6, -P is a non-hydrolyzable substituent and -m is an integer between 0 and 6.  19. The method of claim 18, characterized in that it is preferably a silicon alkoxide.  20. The method of claim 19 characterized in that the silicon alkoxide is chosen from tetramethylorthosilicate, tetraethylorthosilicate and tetrapropylorthosilicate.  21. Method according to one of claims 12 to 20, characterized in that the formation of the precipitate is carried out in the presence of a hydrolysis and condensation agent of said compound.  22. Method according to claim 21, characterized in that the hydrolysis and polycondensation agent is ammonia.  23. Use of the capsules according to one of claims 1 to 11 in the fields of fermentation, biomedical, food and in the chemical industry.