WO2003095085A1 - Colloidal dispersion of calcium phosphate nanoparticles and at least one protein, and corresponding redispersible and calcined nanoparticles, preparation method and uses - Google Patents

Abstract

The invention concerns colloidal dispersions of calcium phosphate nanoparticles and at least one protein, the size of said nanoparticles ranging between 50 and 300 nm, and the morphology of said nanoparticles being spherical. Said dispersions are prepared by a method characterized in that it comprises the following steps: forming a mixture comprising the calcium complexing agent and a calcium source, then adding to said medium at least one protein, thereafter adding thereto a phosphorus source and heating the medium. The invention also concerns nanoparticles obtained by freeze-drying said dispersion, and the particles obtained by calcining the freeze-dried nanoparticles. The invention can be used in the food, cosmetic, pharmacological industries.

Description

 Colloidal dispersion of calcium phosphate nanoparticles and at least one protein, as well as the redispersible nanoparticles and the corresponding calcined particles, process for preparation and uses. The present invention relates to colloidal dispersions of calcium phosphate nanoparticles, and of at least one protein. It also relates to the nanoparticles obtained by lyophilization of the preceding dispersion, as well as the particles obtained by calcination of the lyophilized nanoparticles.

Finally, the invention also relates to the process for the preparation and the uses of these dispersions and of these nanoparticles.

In many products, it is useful to add a source of calcium compound to enrich this product with calcium according to the needs of consumers. This is particularly the case in the food sector where it is sought to supplement calcium with food, drinks or food preparations. This is also the case in the pharmacological field where it is useful to add a source of calcium compound in tablets or solutions for the purpose, for example, of preventing osteoporosis or for treating bone diseases.

The known techniques for supplementing a product with calcium consist in adding soluble calcium salts to it, for example calcium gluconate. It is also known to use calcium phosphate as a source of calcium, in particular in the form of a dispersion.

However, in the case of the use of calcium phosphate, the dispersions obtained are not stable.

In addition, it is difficult to obtain calcium phosphate powders which are precursors of hydroxyapatites with an isotropic morphology. In fact, hydroxyapatite conventionally develops an anisotropic morphology.

However, current techniques do not make it possible to obtain compounds which are stable and whose morphology is controlled.

In order to meet the requirements of manufacturers, it has become necessary to find other means of providing stable compounds whose morphology is controlled and of small size.

Also the problem which the invention proposes to solve is to provide a compound, in particular in the form of a dispersion, stable, of nanometric size, and with controlled morphology. For this purpose, the invention proposes, according to a first embodiment, colloidal dispersions of calcium phosphate nanoparticles and of at least one protein, and the size of which said nanoparticles is between 50 and 300 nm, and the morphology of which said nanoparticles is spherical. The invention also provides, according to a second embodiment, nanoparticles characterized in that they are obtained by lyophilization of the colloidal dispersions described above.

The invention proposes according to a third embodiment of the particles characterized in that they are obtained by calcination of the nanoparticles described above.

The invention also relates to the process for the preparation of colloidal dispersions of calcium phosphate nanoparticles and of at least one protein, characterized in that it comprises the following stages: (a) a mixture comprising the complexing agent is formed calcium and a source of calcium,

(b) at least one protein is added to the medium resulting from step (a),

(c) a source of phosphorus is added to the medium from step (b),

(d) the medium from step (c) is heated. Another subject of the invention is also the use of colloidal dispersions of calcium phosphate nanoparticles and of at least one protein in the food, cosmetic and pharmacological industries. .

Another subject of the invention is also the use of nanoparticles or calcined nanoparticles in the food, cosmetic and pharmacological industries.

The invention has the advantage of providing a source of calcium which can be a precursor of hydroxyapatite [Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ ] which is the mineral substance which is deposited on the bone matrix during of building the skeleton.

The invention also has the advantage of providing colloidal dispersions and / or calcium phosphate nanoparticles, which can be used in the dental field in order to avoid demineralization, in particular in toothpastes or mouthwashes.

The invention also has the advantage of not using caseins. Another advantage of the invention is to provide colloidal dispersions and / or nanoparticles which can provide high concentrations of calcium to the products to which they are added.

The invention also has the advantage of proposing calcined particles, such as for example a hydroxyapatite, with spherical isotropic morphology which can, for example, be of great interest in chromatography. Other advantages and characteristics of the present invention will become clear on reading the description and examples given for purely illustrative and nonlimiting purposes, which will follow. The invention relates first of all to a colloidal dispersion of calcium phosphate nanoparticles and at least one protein, and the size of said nanoparticles being between 50 and 300 nm, and the morphology of said nanoparticles is spherical. The colloidal dispersion of nanoparticles according to the invention comprises at least one protein. Advantageously, it is at least one soluble protein, preferably in an aqueous phase, in particular water.

By at least one protein is also meant according to the invention at least one polypeptide. The proteins according to the invention can be in denatured form.

Preferably, proteins of natural origin, proteins of natural origin modified to improve their solubility in an aqueous phase, natural and / or modified polypeptides, excluding caseins, will be used. These proteins may or may not contain phosphorus-based groups. As proteins, mention may in particular be made of at least one protein chosen from the group consisting of lysozymes, soy proteins, proteins from the soluble phase of milk, proteins from different types of whey.

As an example of vegetable origone proteins, mention may be made of proteins originating from peas, faba beans, lupins, beans, lentils, wheat, barley, rye, corn, rice, oats, and millet, soy, peanut, sunflower, rapeseed, mustard (especially mustard), coconut (copra), alfalfa, nettles, potato , and beetroot.

As proteins of animal origin, non-limiting mention may be made of milk proteins such as, for example, egg proteins such as ovalbumin, proteins derived from the bones and / or the skin of cattle, pigs or fish, like gelatin.

As proteins of modified natural origin, mention may be made of soy proteins, such as those sold by the company Protein Technologies International or Dupont de Nemours. The colloidal dispersions of nanoparticles according to the invention do not contain caseins. This means that there are no added caseins, or no casein salts added, during the preparation of these colloidal dispersions of nanoparticles according to the invention. On the other hand there may be traces of caseins or their salts present in the colloidal dispersions of nanoparticles, due to the use of at least one protein derived from the soluble phase of milk, or from the different types of whey, or from all other fractions.

The colloidal dispersions of nanoparticles according to the invention can also contain at least one calcium complexing agent. This calcium complexing agent can advantageously have at least one carboxylate function, preferably at least three carboxylate functions, and even more preferably at least four carboxylate functions.

The complexing agent advantageously has various acid functions characterized by pKa values, co-logarithm of the acid constants between 1.5 and 8, and dissociation constants of complexes with the calcium ion, characterized by values of pKc, co-logarithm of the complexing constants between 2 and 8, preferably between 2 and 6.

The preferred calcium complexing agent according to the invention is the sodium salt of glutamic acid N, N diacetic of formula (NaCOO) CH ₂ CH ₂ - CH (COONa) N (CH ₂ COONa) ₂ .

The calcium complexing agent may in certain cases be found inside and / or on the surface of the nanoparticles constituting the colloidal dispersion, but also in the continuous phase of the dispersion, or in both. The nanoparticles of the colloidal dispersion according to the invention are generally formed of elementary crystallites with a diameter between 3 and 20 nm.

By elementary crystallites is meant the crystallites constituting the nanoparticles. These elementary crystallites are advantageously of isotropic morphology with a size between 3 and 20 nm, visible by transmission electron microscopy.

The structure of calcium phosphate contained in the nanoparticles of the colloidal dispersion according to the invention is generally amorphous by X-ray diffraction. This characterization by X-ray diffraction is carried out on the powders obtained by lyophilization of the colloidal dispersions according to the invention. By solid NMR spectroscopy of P ³¹ , these calcium phosphates are characterized by a majority line with a chemical displacement of between 2.5 and 4 ppm, preferably around 3.4 ppm and a complementary line with a chemical displacement of between 5 and 7 ppm, preferably around 6.4 ppm. The line around 3.4 ppm is generally attributed to a carbonated hydroxyapatite. According to a preferred embodiment, the nanoparticles of the colloidal dispersion according to the invention mainly have a spherical morphology. They advantageously have a nanometric size, with a diameter between 50 and 300 nm.

By spherical morphology is meant a morphology characterized by a shape index defined by the ratio R = large diameter / small diameter.

In the case of the invention, this ratio R, advantageously tends to 1 to 1.5, preferably from 1 to 1.25 and even more preferably from 1 to 1.1. Advantageously, these nanoparticles of the colloidal dispersion according to the invention are individualized with a percentage of nanoparticles individualized in number greater than or equal to 60%, preferably greater than or equal to 80%, and even more preferably greater than or equal to 90%. The size distribution is generally monodisperse.

By monodisperse is meant nanoparticles which are homogeneous in size and characterized by a ratio r = (d ₉₀ -d ₁₀ ) / 2 d ₅₀ ) of between 0.05 and 0.5, preferably between 0.05 and 0.25 and advantageously between 0.05 and 0.125.

One can use the technique of cryo MET to determine the size, the monodispersity and the state of aggregation of the elementary particles. It allows observation by transmission electron microscopy (TEM) of samples kept frozen in their natural environment which is water. Freezing is carried out on thin films about 50 to 100 nm thick in liquid ethane. By cryo MET, the dispersion state of the particles is well preserved and representative of that present in the real environment.

The nanoparticles of the colloidal dispersion according to the invention have a calcium phosphate / protein ratio, expressed by mass of calcium phosphate / mass of proteins between 0.5 / 1 and 6/1.

The nanoparticles of the colloidal dispersion according to the invention sometimes have a homogeneous composition of calcium phosphate and proteins. That is to say that the nanoparticles are for example constituted by an assembly of elementary crystallites of calcium phosphates and proteins produced in a homogeneous manner or by a heterogeneous assembly of core-shell type, with a higher concentration of crystallites of phosphates of calcium around the nanoparticles. The degree of homogeneity of the assembly can be demonstrated by cryo transmission microscopy (Dubochet method). A stronger contrast is observed for calcium phosphate compared to the protein parts.

The colloidal dispersions of nanoparticles according to the invention advantageously have a pH of between 4 to 8, preferably a pH of between 5 to 7.5.

The surface charge of the nanoparticles of the colloidal dispersion can be positive or negative depending on the nature of the proteins. Thus, at a pH close to pH 6, the nanoparticles of the colloidal dispersion develop a surface charge identical to the charge developed by the protein at the same pH. The nanoparticles of the colloidal dispersion thus develop negative charges when the proteins are proteins of the protein type in the soluble phase of milk or of the soy protein type. They develop positive charges when the protein used is a protein of lysozyme type. These surface charges can be determined for example by drawing the curves of electrophoretic potentials as a function of pH.

The colloidal dispersions of nanoparticles according to the invention advantageously have a calcium phosphate concentration expressed in equivalent calcium concentration of between 0.2 and 2 M in calcium.

The colloidal dispersions according to the invention can be concentrated by postconcentration or by ultrafiltration. Colloidal dispersions then have calcium concentrations greater than 1M calcium.

The colloidal dispersions according to the invention may contain residual salts formed from the anions and associated cations contained in the calcium precursor and phosphate solutions of the type, for example, NaCl, NH ₄ Cl, NaNO ₃ , NH ₄ NO ₃ , KCI

KNO ₃ . They may also contain complexing agents, proteins, calcium ions and / or residual phosphate ions in free form.

Compounds initially present in the solutions of proteins can also be present within colloidal dispersions such as for example lactose.

These residual concentrations of previously defined compounds can be minimized by purification of the colloidal dispersions by ultrafiltration.

The invention relates, according to a second object, to the nanoparticles obtained by lyophilization of the colloidal dispersions described above. They are sometimes called freeze-dried nanoparticles, in the remainder of the text.

These nanoparticles according to the invention are produced according to conventional lyophilization techniques, preferably after washing by ultrafiltration of the colloidal dispersion to be lyophilized.

These nanoparticles according to the invention are redispersible in water to give colloidal dispersions.

Indeed, from the nanoparticles according to the invention, it is possible to produce colloidal dispersions advantageously having the characteristics of the dispersions previously described.

The colloidal stability of the dispersions obtained by simple redispersion in water of these lyophilized nanoparticles is advantageously greater than one day, preferably greater than one week, preferably perfectly stable over time, provided that the dispersion is kept at safe from bacteria. These nanoparticles comprise at least one protein having the same characteristics as those defined above, in the case of dispersions. The structure of calcium phosphate contained in the nanoparticles according to the second object of the invention is identical to that described for the first object of the invention.

The lyophilized nanoparticles can also contain salts of the NaCI, NaNO ₃ , NH ₄ NO ₃ , NH ₄ CI, KNO ₃ , KCI type.

The lyophilized nanoparticles may also contain water. The amount of water is then between 0.1 and 10%, preferably between 1 and 8%, percentage expressed by weight.

According to a preferred embodiment, the nanoparticles according to the invention mainly have a spherical morphology, a nanometric size, a monodisperse distribution, and sometimes have a composition of calcium phosphate and proteins which is homogeneous, as already described above. which concerns the nanoparticles of the dispersion.

The calcium phosphate / protein ratio is the same as that described for the nanoparticles of the dispersion.

Finally, the invention also relates to colloidal dispersions characterized in that they are obtained by resuspending the lyophilized nanoparticles.

The invention relates, according to a third object, to particles which are obtained by calcination of the nanoparticles described above and which themselves were obtained by lyophilization of the colloidal dispersions previously described.

The calcination is preferably carried out either in air, or in an inert atmosphere, then in air and preferably with a temperature gradient greater than or equal to 400 ° C./min. Preferably, a so-called "flash" calcination should be carried out, that is to say with introduction into an oven previously heated to the calcination temperature and maintained at this calcination temperature for a time varying from 2 min to 1 hour.

The calcination can take place at temperatures advantageously between 200 ° C and 1000 ° C, and preferably between 300 ° C and 800 ° C.

Preferably, with regard to obtaining calcined powders with isotropic morphology, redispersible powders characterized by a high “calcium phosphate: protein” mass ratio, that is to say a mass ratio varying from 2: 1 to 6: 1, are used. . It is preferable to calcine lyophilized nanoparticles having a calcium phosphate / protein ratio, greater than or equal to 3.

The calcined particles according to the invention advantageously have an isotropic morphology, possibly agglomerated and are preferably of nanometric size. These calcined particles have, after calcination, an apatite-type structure, also called hydroxyapatite, preferably of formula of the Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ , or Ca ₂ P ₂ O ₇ , or CaHPO ₄ , or Ca type. ₃ (PO ₄ ) ₂ , or also amorphous calcium phosphate, alone or as a mixture. The calcined particles according to the invention may optionally contain the presence of salts of the NaCl, NaNO ₃ , NH ₄ NO ₃ , NH ₄ CI type.

The calcined particles according to the invention may optionally and in very rare cases, contain the presence of traces of proteins or polypeptides or of complexing agent or their residues, originating from the pyrolysis of proteins or polypeptides or of complexing agent .

The calcined particles according to the invention are generally anhydrous. The calcined particles according to the invention advantageously have an isotropy, with a ratio R, (with R = large diameter (L) / small diameter (I)) tending advantageously towards 1 to 1, 5, preferably from 1 to 1, 3 .

The invention relates, according to a fourth object, to a process for preparing the colloidal dispersions according to the invention which comprises the following steps:

(a) a mixture comprising the calcium complexing agent and a source of calcium is formed, (b) at least one protein is added to the medium resulting from step (a),

(c) a source of phosphorus is added to the medium from step (b),

(d) the medium from step (c) is heated

The source of calcium used in step (a) is advantageously a calcium salt, or a calcium hydroxide or a calcium carbonate. Mention may in particular be made of calcium dichloride, calcium nitrate, hydrated lime, calcium carbonate.

The calcium concentration is advantageously at most 1 M, preferably at most 0.5 M in the mixture formed in step (a).

Step (a) of the preparation process according to the invention is characterized in that the pH of the medium resulting from step (a) is advantageously adjusted so as to obtain a pH of between 5 and 7. The adjustment of pH is conventionally achieved by the addition of HCI or HNO ₃ .

Step (a) of the process is carried out with a molar ratio Ra, for which Ra = calcium / calcium complexing agent, advantageously between 0.1 and 3. In step b), the protein solution used is generally obtained by simple dispersion of the protein in demineralized water.

The source of phosphorus used in step (c) is advantageously an alkali metal or ammonium phosphate salt. We can cite in particular the sodium dihydrogen phosphate (NaH ₂ PO ₄ ) or sodium monohydrogen phosphate (Na ₂ HPO ₄ ), or ammonium dihydrogen phosphate (NH ₄ (H ₂ PO), or ammonium monohydrogen phosphate (NH ₄ ) ₂ (HPO ₄ ).

During the preparation process according to the invention and before the addition of the phosphorus source according to step (c), the pH of the medium is advantageously adjusted to a value between 4.5 and 8.

Step (c) of the process is carried out with a mass ratio Rb, for which Rb = calcium phosphate / protein (s), advantageously between 0.3 and 6.

Step (c) of the process is carried out with a molar ratio Rc, for which Rc = moles of calcium / mole of phosphorus, advantageously between 1 and 4.

The various additions of reagents are carried out at a controlled speed or preferably instantaneously and at room temperature.

During step (d) of the process, the medium from step (c) is heated to a temperature advantageously between 20 ° C. and 90 ° C. This step is carried out according to conventional heat treatment methods.

Preferably, the medium from step (c) is introduced into an oven previously heated to the temperature of the heat treatment. The duration of the heat treatment is generally between 2 hours and 24 hours, preferably between 6 hours and 20 hours. The colloidal dispersion can then be purified by washing with demineralized water by ultrafiltration. Washing by ultrafiltration makes it possible to remove the residual salts and free complexing agents. Colloidal dispersions can also be concentrated by ultrafiltration.

It is also possible to add an anti-bacterial agent to the colloidal dispersion obtained.

The invention relates, according to a fifth object, to the use of the colloidal dispersions according to the invention or of the colloidal dispersions obtained by the preparation process according to the invention in the food, cosmetic and pharmacological industries.

The invention also relates to the use of freeze-dried nanoparticles according to the invention and calcined nanoparticles according to the invention in the food, cosmetic and pharmacological industries.

It also relates to the use of colloidal dispersions or freeze-dried nanoparticles or calcined particles for the conditioning and / or storage of proteins, polypeptides or any other molecules.

It also relates to the use of calcined nanoparticles as a support for chromatography. The following examples illustrate the invention without, however, limiting its scope.

EXAMPLES

1 / Preparation of colloidal dispersions

1-1 / Example 1-1: Colloidal dispersion of calcium phosphate, complexing agent and soy proteins

Step (a): A solution containing Ca ⁺ ions is obtained by dissolving 2.75 g of CaCl ₂ , 2H ₂ O (MW = 147.02 g, or 18.7 millimoles of Ca) in demineralized water and addition of 26.5 cm ³ of GBS 5 complexing agent with a structural formula NaCOO (CH ₂ CH ₂ ) - CH- (COONa) N (CH ₂ COONa) ₂ at 1.4 M, or 37.4 millimoles of GBS 5. The mixture has a pH of 13. It is adjusted to pH 6 with 1 M HCl. The final volume is 35 cm ³ . Step (b): A solution B containing soy proteins (marketed by Protein Technologies International, DuPont) Quality FP 940 is obtained by adding 1.87 g of proteins to demineralized water. It is made up to 25 cm ³ with demineralized water. Solution B is instantly poured into freshly prepared solution A.

Step (c): A solution C containing sodium phosphate ions is obtained by adding 0.88 g of Na ₂ HPO ₄ (MW = 141.96 g, or 6.23 millimoles of P) in 25 cm ³ . It is adjusted to pH 6 with 1 M HCl. The solution C is added to the preceding mixture instantaneously. The reaction mixture is readjusted to pH 6. The mixture is left under stirring for 5 min.

Step (d): The reaction unit is brought to an oven previously heated to 50 ° C. The dispersion has a colloidal appearance after 16 hours at 50 ° C.

1-2 / Example 1-2: Colloidal dispersion of calcium phosphate, complexing agent and lysozyme proteins

Step (a): A solution containing Ca ²⁺ ions is obtained by dissolving 2.75 g of CaCl ₂ , 2H ₂ O (MW = 147.02 g, or 18.7 millimoles of Ca) in water demineralized and addition of 26.5 cm ³ of GBS 5 complexing agent with structural formula NaCOO (CH ₂ CH ₂ ) - CH- (COONa) N (CH ₂ COONa) ₂ to 1.4 M, or 37.4 millimoles of GBS 5. The mixture has a pH of 13. It is adjusted to pH 6 by HCI 1 M. The final volume is 35 cm ³ . Step (b): A solution B containing lysozyme is obtained by adding 1.87 g of lysozyme to demineralized water. It is made up to 25 cm ³ with demineralized water. Solution B is instantly poured into freshly prepared solution A. Step (c): A solution C containing sodium phosphate ions is obtained by adding 0.88 g of Na ₂ HPO ₄ (MW = 141.96 g, or 6.23 millimoles of P) in 25 cm ³ . It is adjusted to pH 6 by 1M HCl. solution C is added to the preceding mixture instantaneously. The reaction unit is readjusted to pH 6. The whole is left under stirring for 5 min.

Step (d): The reaction unit is brought to an oven previously heated to 50 ° C. The dispersion has a colloidal appearance after 16 hours at 50 ° C.

1-3 / Example 1-3: Colloidal dispersion of calcium phosphate, complexing agent and milk proteins

Step (a): A solution containing Ca ²⁺ ions is obtained by dissolving 2.75 g of CaCl ₂ , 2H ₂ O (MW = 147.02 g, or 18.7 millimoles of Ca) in water demineralized and addition of 26.5 cm ³ of GBS 5 complexing agent with structural formula NaCOO (CH ₂ CH ₂ ) -

CH- (COONa) N (CH ₂ COONa) ₂ to 1, 4 M, ie 37.4 millimoles of GBS 5. The mixture has a pH of 13. It is adjusted to pH 6 with 1M HCl. The final volume is 35 cm ³ .

Step (b): A solution B containing milk proteins obtained from whey is obtained by adding 1.87 g of milk proteins to demineralized water. It is made up to 25 cm ³ with demineralized water. Solution B is instantly poured into freshly prepared solution A.

Step (c): A solution C containing sodium phosphate ions is obtained by adding 0.88 g of Na ₂ HPO ₄ (MW = 141.96 g, or 6.23 millimoles of P) in 25 cm ³ . It is adjusted to pH 6 with 1 M HCl. Solution C is added to the preceding mixture instantaneously.

The reaction assembly is readjusted to pH 6.

The whole is left under stirring for 5 min.

Step (d): The reaction assembly is brought to an oven previously brought to 50 ° C. The dispersion has a colloidal appearance after 16 hours at 50 ° C. 2 / Preparation of lyophilized nanoparticles

2-1 / Example 2-1: (PAH: Soy protein) = (1: 1) by weight, pH 6

The colloidal dispersion obtained in Example 1-1 is lyophilized.

By solid ³¹ P NMR, a peak is observed corresponding to a chemical shift of 3.4 ppm.

The lyophilized powder is redispersible in demineralized water. By transmission electronics we observe by cryo-microscopy (Dubochet method), perfectly individualized objects, with a monodisperse particle size distribution having a spherical morphology of size about 150 nm.

The electrophoretic potential curves showed an isoelectric point pH 4 and a negative charge at the pH of the dispersions of approximately -19 mV

2-2 / Example 2-2: (PAH: Lyzozyme protein) = (1: 1) by weight, pH 6

The colloidal dispersion obtained in Example 1-2 is lyophilized. By solid ³¹ P NMR, a peak is observed corresponding to a chemical shift of 3.4 ppm.

The lyophilized powder is redispersible in demineralized water. By transmission electronics we observe by cryo-microscopy (Dubochet method), perfectly individualized objects, with a monodisperse particle size distribution having a spherical morphology of size about 150 nm.

The electrophoretic potential curves showed an isoelectric point at pH 9 and a positive charge at the pH of the dispersions of approximately + 5 mV.

2-3 / Example 2-3: (PAH: WPC protein) = (1: 1) by weight, pH 6

The colloidal dispersion obtained in Example 1-3 is lyophilized.

By solid ³¹ P NMR, a peak is observed corresponding to a chemical shift of 3.4 ppm.

The lyophilized powder is redispersible in demineralized water. By transmission electronics we observe by cryo-microscopy (Dubochet method), objects perfectly individualized, with a monodisperse particle size distribution having a spherical morphology with a size of approximately 150 nm.

The electrophoretic potential curves showed an isoelectric point at pH 4 and a negative charge at the pH of the dispersions of approximately - 18 mV.

3 / Preparation of calcined particles

3-1 / Example 3-1: Test at (PAH: WPC protein) = (3: 1) by weight, pH 6 The colloidal dispersion is prepared as in Example 1-3 but using 0.62 g of proteins dairy. This dispersion is lyophilized. Then we perform a flash calcination at 900 ° C

The powder obtained from calcined particles consists of calcium phosphate and has an isotropic morphology in TEM, the ratio R of which is 1.4. By RX, a structure is observed with a mixture of hydroxyapatite, Ca ₃ (PO ₄ ) ₂ and Ca ₂ P ₂ O ₇ .

Claims

Colloidal dispersion of calcium phosphate nanoparticles and at least one protein, and whose size of said nanoparticles is between 50 and 300 nm, and whose morphology of said nanoparticles is spherical. Colloidal dispersion according to claim 1, characterized in that it comprises at least one calcium complexing agent. Colloidal dispersion according to one of the preceding claims, characterized in that it comprises at least one calcium complexing agent having at least one carboxylate function. Colloidal dispersion according to one of the preceding claims, characterized in that it comprises at least one calcium complexing agent having at least three carboxylate functions. Colloidal dispersion according to one of the preceding claims, characterized in that it comprises at least one calcium complexing agent having at least four carboxylate functions. Colloidal dispersion according to one of the preceding claims, characterized in that they comprise at least one soluble protein. Colloidal dispersion according to claim 6, characterized in that they comprise at least one protein chosen from the group consisting of lysozymes, soy proteins, proteins from the soluble phase of milk, proteins from different types of whey. Colloidal dispersion according to one of the preceding claims, characterized in that the nanoparticles are formed from elementary crystallites with a diameter between 3 and 20 nm. Nanoparticles characterized in that they are obtained by lyophilization of the colloidal dispersions according to claims 1 to 8. Nanoparticles according to claim 9, characterized in that they are redispersible in water to give the colloidal dispersions according to claims 1 to 8. Colloidal dispersion characterized in that they are obtained by resuspending the nanoparticles according to claims 9 to 10. Calcined particles characterized in that they are obtained by calcination of the nanoparticles according to claim 9 or 10. Calcined particles according to claim 12, characterized in that they have an isotropy of between 1 and 1.5, measured by the ratio R = L / 1. Process for the preparation of the product according to Claims 1 to 8, characterized in that it comprises the following stages: (a) forming a mixture comprising the calcium complexing agent and a source of calcium, (b) at least one protein is added to the medium resulting from step (a), (c) adding a medium of phosphorus to the medium from step (b), (d) heating the medium from step (c) Process according to Claim 14, characterized in that the pH of the medium resulting from stage (a) is adjusted so as to obtain a pH of between 5 and 7. Method according to one of claims 14 to 15 characterized in that before the addition of the phosphorus source according to step (c), the pH of the medium is adjusted to a value between 4.5 and 8. Method according to one of claims 14 to 16 characterized in that step (a) is carried out with a molar ratio Ra = calcium complexing agent / calcium of between 0.1 and 3. Method according to one of claims 14 to 17 characterized in that step (c) is carried out with a molar ratio Rb = calcium phosphate / protein (s) of between 0.3 and 6. Method according to one of claims 14 to 18 characterized in that step (c) is carried out with a mass ratio Rc = moles of calcium / mole of phosphorus, between 1 and 4. Use of the colloidal dispersions obtained according to one of claims 1 to 8 or of the colloidal dispersions obtained by the process according to one of claims 14 to 19 in the food, cosmetic, pharmacological industries. Use of nanoparticles according to claims 9 or 10, or particles according to claims 12 or 13 in the food, cosmetic, pharmacological industries.