FR3113837A1 - Hydrogel based on alginate and exopolysaccharide, process for obtaining and cosmetic or dermatological use. - Google Patents

Abstract

The invention relates to cross-linked polysaccharide compositions, in the form of bulk millimetric matrices or in spherical form, allowing the gradual release of at least one component of interest. The invention also relates to cosmetic or dermatological compositions comprising such polysaccharide matrices. The invention also relates to the use of such compositions by topical application and to a method for obtaining such cross-linked polysaccharide compositions. Figure for the abstract: Figure 2

Description

Hydrogel based on alginate and exopolysaccharide, process for obtaining and cosmetic or dermatological use.

The present invention belongs to the technical field of cosmetic formulation.

More particularly, the invention relates to a polysaccharide composition comprising alginate, an exopolysaccharide produced by the marine bacterium strain CNCM I-5035 and calcium. The invention also relates to a hydrogel comprising such a composition, in particular in the form of hydrogel spheres, allowing the progressive and controlled release of at least one active ingredient. Thus, the invention also relates to the cosmetic or dermatological use of such a hydrogel.

Technology background

The progressive and controlled release of compounds is a technique which has many advantages, because it makes it possible, for example, to target a particular area or to extend the duration of administration of a compound, in particular when the latter is irritating or has a certain toxicity at high doses. Different systems are used, which are based on diffusion, dissolution, osmosis, ion exchange resins, flotation, bio-adhesion, matrices, stimulus-induced release.

One of the most widely used techniques for the sustained and controlled release of compounds is encapsulation. It has become essential in many fields of industry such as agrifood, pharmaceuticals, textiles, or even detergents. Applied to cosmetics, this type of technique offers several advantages such as the protection of fragile molecules against hydrolysis or oxidation phenomena, the masking of odors, the reduction of toxicity, the controlled release allowing prolonging the activity of an encapsulated compound over time, or better vectorization depending on the affinity of the carrier developed.

Commonly used encapsulation processes fall into two main categories: liquid-air type processes and liquid-liquid type processes.

In liquid-air encapsulation processes, a liquid phase is extruded through a nozzle and drops are dispersed in the air, for example by dripping, jet or spray. These processes have the disadvantage of generating spheres whose size is difficult to control and covers a very wide size range.

In liquid-liquid type encapsulation processes, two immiscible liquids are brought into contact and form spheres, using two techniques: dispersion and micro/milli-fluidics. The dispersion technique consists of agitating together two immiscible liquids (aqueous phase/organic phase) to generate an oil-in-water or water-in-oil emulsion. This technique also does not allow precise control of the size of the spheres. Conversely, micro/milli-fluidics uses capillary tubes to bring two immiscible liquids into contact. Depending on the diameter of these, in particular, it is possible to generate droplets whose size is defined and homogeneous throughout the process. By the action of the shear forces exerted between these two liquids, and depending on the geometry of the capillaries, droplets of a first liquid phase (dispersed phase) are formed within a second liquid phase (continuous phase). The size of the beads generated, their composition and their structure can thus be controlled, depending on the choice of liquid phases, their flow rates and the architecture of the micro/milli-fluidic system.

Among the gradual and controlled release techniques, hydrogels are also known, which differ from gels in their ability to absorb large amounts of water and swell, while maintaining their three-dimensional structure. These hydrogels can for example be used in the form of dressings.

However, in the field of cosmetology and dermatology, consumers are very keen on using products that include natural extracts. Alginate is a well-known polymer, produced from brown algae, mainly kelp and fucus. Alginate is thus a polymer used in controlled and gradual release techniques, in particular the encapsulation of oils, in view of its low cost, ease of use, biodegradability and non-toxicity. It also has the property of gelling rapidly in the presence of divalent ions, such as Ca2+ calcium ions. However, these systems have the disadvantage of not being stable in cosmetic formulations due to the presence of surfactants and solubilizers which destabilize ionic alginate gels.

Thus, there is a need for compositions which are as natural as possible, allowing the gradual and controlled release of compounds, and suitable for topical application. In particular, there is a need for such compositions which remain stable in cosmetic formulations. There is thus also a need for new processes making it possible to obtain such compositions.

The invention relates, according to a first object, to a composition comprising alginate, exopolysaccharides produced by the marine bacterium of strain CNCM I-5035, and calcium.

Thus, the invention is based on an entirely new and original approach of proposing, according to a first object, a polysaccharide composition of natural origin, which is biocompatible and non-toxic.

According to one embodiment, the calcium is in the form of calcium carbonate.

Such a composition is particularly advantageous for obtaining a composition in which the alginate and the EPS are crosslinked in an essentially spherical form, and is very particularly suitable as a dispersed phase in a method comprising a step implementing a technique of milli - fluidics.

According to another embodiment, the alginate and the EPS are cross-linked.

This composition has the particularity of being degraded by the skin microbiota, and is therefore suitable for use for a gradual and controlled release of active ingredients. Finally, this composition has shown an action on the bacteria of the skin microbiota, favoring the growth of beneficial bacteria of this microbiota.

Preferably, the alginate is present at a content of between 20% and 80% by weight of the total dry mass of polysaccharides and the exopolysaccharides are present at a content of between 20% and 80% by weight of the total dry mass of polysaccharides.

These particular levels make it possible to adjust the hardness of the composition for optimal use on the skin.

According to a particular embodiment, the composition also comprises at least one component of interest.

Such a composition makes it possible, for example, to protect sensitive components, such as certain active molecules which degrade rapidly in the presence of oxygen or humidity. As it also allows a gradual release of the components of interest, it can be used to achieve various cosmetic or dermatological objectives. It also provides a protective environment for whole living cells. Thus, such a composition can advantageously be used in the evaluation of the efficacy and/or the toxicity of active principles of interest on particular cells or tissues.

According to a particular embodiment, the composition is in an essentially spherical form with a diameter of 200 μm to 2000 μm.

Thus, the composition takes the form of millimetric spheres, which are particularly appreciated for topical cosmetic application. In addition, it is possible to observe the manufacturing process of such millimetric spheres with the naked eye, which facilitates adjustments, if necessary, during their design.

Another object according to the invention relates to a cosmetic or dermatological composition comprising a composition as described above as well as a cosmetically or dermatologically acceptable medium, preferably demineralized water and/or a vegetable gum.

This composition makes it possible to preserve the crosslinked composition in fragmented form or in the form of spheres.

Another object according to the invention relates to the use of a composition or of a cosmetic or dermatological composition for topical application.

According to a particular embodiment, such a use allows a gradual and prolonged release of at least one compound of interest.

This gradual and controlled release is particularly advantageous when the at least one compound of interest is an active principle which has an irritating nature. Indeed, a slower administration reduces, or even cancels, the irritating effects that such an active ingredient would produce if it were applied at the same dosage directly to the skin.

Another object according to the invention relates to a process for obtaining a composition according to the invention, which comprises the following steps:

- a step of preparing a solution comprising alginate, exopolysaccharides produced by the marine bacterium strain CNCM I-5035 and, where appropriate, at least one compound of interest, then

- a cross-linking step of said alginate in the presence of Ca2+ ions, then

- a cross-linking step of said exopolysaccharides in the presence of a cross-linking agent.

This process is very simple to implement.

According to one embodiment, the method makes it possible to obtain a composition in an essentially spherical form. According to this embodiment, the solution prepared during the preparation step further comprises calcium carbonate. In addition, such a method comprises, after said preparation step:

- a step implementing a millifluidic technique, in which the composition prepared in the previous step is used as an aqueous dispersed phase, and is brought into contact with an acidic organic continuous phase, so that the dispersed phase forms essentially spherical droplets.

This process is advantageous because it allows the solution to begin to crosslink in the form of a droplet, by the release of Ca ²⁺ ions by acid hydrolysis of the calcium carbonate contained in the dispersed phase, under the action of acidic compounds of the continuous phase. Such a method is simple to implement, and economical because it requires the use of inexpensive materials. It also advantageously makes it possible to obtain the composition in the form of spheres of homogeneous size.

Preferably, the acid organic continuous phase comprises i) at least one compound chosen from organic solvents and vegetable or mineral oils, preferably Caprylic/Capric Triglyceride and ii) at least one acid molecule chosen from lactic acid , citric acid and acetic acid.

According to a particular embodiment, the crosslinking agent suitable for the process according to the invention is chosen from the group consisting of 1,2bis-(2,3-epoxypropoxy)ethylene, 1-(2,3-epoxypropyl)-2 ,3-epoxycyclohexane and 1,4-butanediol diglycidylether (BDDE).

According to one embodiment, the Ca ²⁺ ions suitable for the method according to the invention come from a solution of calcium salt, such as a solution of calcium chloride or calcium sulphate.

Brief description of figures

There schematically presents the crosslinking of alginate with Ca2+ ions.

There presents an assembly for the implementation of a milli-fluidic technique, allowing the composition to be obtained in an essentially spherical form.

There is a set of photographs taken under a microscope, which shows the evolution of the integrity of the spheres over time, and in the presence or absence of four bacterial strains representative of the skin microbiota.

There is a schematic representation of the process steps according to the invention.

Detailed description of the invention

The general principle of the invention is based on a particular mixture of polysaccharides, of natural origin, making it possible to obtain compositions for the gradual and controlled release of these encapsulated active ingredients, thanks to the action of bacteria in the skin microbiota.

Compositions according to the invention

The compositions according to the invention comprise alginate, exopolysaccharides produced by the marine bacterium of strain CNCM I-5035 and calcium.

For the purposes of the invention, the term "exopolysaccharide" or "exopolysaccharides" or "EPS" means one or more polysaccharide compounds produced by Vibrio alginolyticus , which is a planktonic marine bacterium, living in particular in Aber Benoit, in Finistère North, in Brittany (France). This bacterial strain has been the subject of a deposit according to the Budapest Treaty, at the National Collection of Cultures of Microorganisms (CNCM), Institut Pasteur, 28 rue du Docteur Roux, 75724 Paris Cedex 15, France, deposit made in the name of of POLYMARIS BIOTECHNOLOGY, based in Brest. The strain reference is CNCM I-5035.

The exopolysaccharide produced by the CNCM I-5035 strain is composed of repeating units of three sugars: D-galactose, D-N-acetylglucosamine and D-L-acetylguluronic acid, part of which is acetylated at position 3. It can be obtained by conventional culture and extraction methods, known to those skilled in the art. Patent document FR2981847B1 describes such methods. In particular, the native exopolysaccharide can be obtained by the following process: the CNCM I-5035 bacteria are cultured in nutrient medium. The medium is recovered at the end of the exponential growth phase of the bacteria. The bacteria are eliminated by any method well known to those skilled in the art (filtration, centrifugation, etc.). The exopolysaccharides are then extracted from the medium by membrane separation, by ultrafiltration with a cut-off threshold of between around 50 kDa and 600 kDa, preferably around 300 kDa. The exopolysaccharides are then purified, then optionally dried by freeze-drying.

The exopolysaccharide is present in the compositions according to the invention at a content of between 20% and 80% by weight of the total dry mass of polysaccharides. Preferably, the exopolysaccharide content is approximately 20%, approximately 30%, approximately 40%, approximately 50%, approximately 60%, approximately 70% or approximately 80% of the total dry mass of polysaccharides.

Alginate is a polymer obtained from brown algae, formed from two monomers bonded together via a β-1-4 bond: mannuronate (ManA), some of which are acetylated, and guluronate (GulA). The physical and chemical properties of alginate are in particular a function of the proportion and distribution of these two monomers in the polysaccharide.

Alginate is present in the polysaccharide composition at a content of between 20% and 80% by weight of the total dry mass of polysaccharides. Preferably, the alginate content is approximately 20%, approximately 30%, approximately 40%, approximately 50%, approximately 60%, approximately 70% or approximately 80% by weight of the total dry mass of polysaccharides.

According to one embodiment, the calcium is present in the form of calcium carbonate. In this particular embodiment, the alginate in the composition is not crosslinked, and such a composition is particularly suitable as a dispersed phase in a method implementing a milli-fluidic technique. Indeed, by using an acidic continuous phase, the acid will allow the release of the Ca ²⁺ ions from the calcium carbonate and the alginate will crosslink inside the composition. Thus, this composition makes it possible to obtain compositions in which the alginate and the EPS are crosslinked in an essentially spherical form.

Indeed, according to another embodiment, the alginate and the EPS are crosslinked in the composition. In particular, alginate is cross-linked by the formation of ionic bonds with Ca ²⁺ ions. These ions are preferably provided by a solution comprising calcium chloride or else calcium sulphate. Thus, the compositions comprising alginate and crosslinked EPS also comprise calcium in a form trapped inside a structure of the "egg box" or "egg box" type in English, as illustrated in .

When the alginate and the EPS are cross-linked, the composition has the advantage of being able to form small polysaccharide matrices, the size of which is from a few tenths of a millimeter to a few millimeters, brought together in a flexible structure with the appearance of a hydrogel. , or in the form of individualized spheres.

In the context of the invention, the term "sphere" means a product having an essentially spherical shape. Thus, according to one embodiment, the composition in which the alginate and the EPS are crosslinked is in a spherical form, that is to say in the form of one or a plurality of spheres formed from this composition. Such spheres fall within a diameter of between 200 µm and 2000 µm.

This diameter makes it possible to more or less clearly distinguish the spheres, which can be visually attractive for the user of the composition. In addition, the sensation caused by the application of millimetric spheres, formed of a flexible composition close to gel, on the skin, can be pleasant and also sought after by the end user. By integrating components such as pigments into these spheres, and by varying the colors of these, the invention makes it possible to obtain compositions that are particularly attractive and suitable for cosmetic uses.

The matrices formed from the compositions according to the invention are capable of containing one or more components of interest, and of protecting them from external conditions to improve their conservation or allow their cultivation. The inventors have shown that, surprisingly, these polysaccharide matrices can be consumed by the skin microbiota, allowing their content to be released in a controlled and progressive manner, which is particularly advantageous when it comes to an active ingredient.

Thus, according to a particular embodiment, the invention relates to a composition comprising alginate, EPS produced by the CNCM I-5035 strain, calcium and at least one component of interest. Within the meaning of the invention, the term "component of interest" means any component capable of being integrated into the compositions in which the alginate and the EPS are crosslinked, according to the invention. Thus, a component of interest must be compatible with the presence of alginate, EPS, calcium, as well as a size smaller than that of the millimetric matrices formed.

Components of interest suitable for the invention may be one or more compounds having biological properties (active principles) or not (pigments, perfumes), chosen from organic particles, mineral particles, whole living cells, polysaccharides, oligosaccharides, proteins, peptides, polypeptides, phenolic compounds, raw materials produced by bees, synthetic materials or any other polymer larger than 2000 Daltons.

Components of interest suitable for the invention include, for example, moisturizing, pigmenting, depigmenting, soothing, oxygenating, regenerating, anti-aging, anti-wrinkle, anti-redness, anti-acne active products, and combinations thereof.

In particular, the components of interest suitable for the invention can be chosen from:

- mineral particles, in particular mineral oxides (natural pigments), such as in particular iron oxides, zinc oxides, titanium oxides, and all other known sunscreens, as well as clays and calcium carbonate;

- polysaccharides or oligosaccharides, chosen among others from homogeneous polysaccharides or dietary fibers, in particular fructans such as inulin fructans of Dicotyledones ( Asteraceae , Boraginaceae , Campanulaceae ) or phlein-type fructans of Monocotyledones ( Poaceae , Liliaceae ); heterogeneous polysaccharides, such as gums and mucilages (gum arabic, gum tragacanth, gum ghatti), polysaccharides derived from mannose, such as carob, guar, konjac, fenugreek, acid heterogeneous polysaccharides such as psyllium, ispaghul, plantain, polysaccharides from Malvales such as mallow, marshmallow, linden, mucilages such as flax, quince, but also products derived from apples such as pectins; polysaccharides resulting from bacterial fermentation, very widely used in cosmetics, such as hyaluronic acid, native or partially depolymerized;

- proteins, polypeptides or peptides, chosen among others from collagen fibers, elastin, glycosaminoglycans, such as hyaluronic acid, chitosan; natural or synthetic enzymes, among which mention may be made of enzymes isolated from papaya, pineapple, ficus; sweetening proteins, such as thaumatin, monellin and miraculin; lectins, in particular Aloe barbadensis lectins (aloctins);

- phenolic compounds, chosen among others from polyphenols such as tea, apple or grape polyphenols, phloroglucinol polymers, derivatives of cinnamic acid such as rosmarinic acid, derivatives of caffeic acid, in particular chlorogenic acid, present in extracts of arnica, artichoke, rosemary, java tea, derivatives of salicylic acid such as extracts of meadowsweet, willow, or acid derivatives coumaric, such as extracts of horse chestnut, sweet clover, angelica, Tonka bean, or even derivatives of ferulic acid; flavonoids, such as flavones, flavonols, flavanones, dihydroflavonols, biflavonoids, chalcones, aurones, flavonoid glycosides, in particular those contained in fruit extracts (Citrus), Ginkgo biloba, Passionflower, thyme , Roman chamomile, rosemary, yarrow, horsetail; isoflavonoids, such as those in soy extracts, and rotenoids; anthocyanins, such as those in blueberry, cranberry, blackcurrant, red vine, black elderberry extracts; hydrolysable tannins such as gallic or ellagic tannins, and condensed tannins such as proanthocyanidins, contained in extracts of roses, oak bark, witch hazel, hawthorn, vine, pine, cypress; orcinols and phloroglucinols such as those of hemp extracts (canabinol and canabidiol), extracts of hops (humulone), of Kamala; terpenoids, in particular monoterpenes (extract of peony for example), sesquiterpenes (extract of cotton seeds for example), diterpenes, and triterpenes, or else the carotenoids present in extracts of pepper, saffron; alkaloids, in particular caffeine, theophylline, theobromine, epigallocatechin, contained in extracts of tea tree, coffee tree, cocoa tree, cola trees, mate, guarana;

- raw materials produced by bees (wax, propolis, royal jelly, honey, pollen);

- synthetic materials, such as sun screens and filters, and preservatives.

- organic components such as organic particles or whole living cells, such as for example whole microalgae and/or fragmented algae (microalgae or macroalgae). Mention may in particular be made of cyanobacteria such as spirulina, green microalgae such as chlorella, but also plant cells of plants; bacterial, plant or animal DNA; fragments of macroalgae such as those of the red alga Jania rubens , fragments of gametophytes of the brown alga Undaria pinnatifida , coccoliths of coccolithophorids, fragments of plant cells of plants, or extracts of yeasts ( Saccharomyces for example); living whole bacterial, plant or animal cells, for cultivating cells, for example for producing tissue culture models, in particular for the purpose of evaluating the effects of pharmaceutical or cosmetic active agents.

Another object according to the invention relates to a cosmetic or dermatological composition. Such a composition contains a composition in which the alginate and the EPS are crosslinked according to the invention, and a cosmetically or dermatologically acceptable medium. The compositions in which the alginate and the EPS are crosslinked according to the invention can be used directly on the skin, but it is preferable to integrate them into a composition for cosmetic or dermatological use in order to facilitate their conservation or use. , particularly when the compositions are in the form of spheres. In this case, and when the spheres contain a component of interest, the dosage of the latter can be greatly facilitated in the cosmetic or dermatological composition. In the form of spheres, it is in fact easier to control the concentration of active ingredients in a composition, by incorporating a greater or lesser quantity of spheres in the final composition, depending on the action and the use that one wishes to confer on the cosmetic product.

When the compositions in which the alginate and the EPS are crosslinked are not in the form of a sphere, the millimetric matrices which compose them can be presented in a fragmented form in such cosmetically or dermatologically acceptable media. However, in this form it is also possible not to add a special medium and to use such compositions directly on the skin. They can then fragment into millimetric matrices during their application, which can cause a pleasant sensation for the user.

A cosmetically acceptable medium may be chosen from or comprise water or a water-soluble organic solvent, texture agents, in particular a gelling agent, perfumes, preservatives, or mixtures thereof. A water suitable for the invention can be thermal and/or mineral water, chosen in particular from Vittel water, water from the Vichy basin and water from La Roche Posay. A gelling agent suitable for the invention can be chosen from pectin, guar gum, cellulose, dextrin, maltodextrin, starch, Tara gum, locust bean gum, inulin, gum acacia, gum arabic, high fucose polymers, carrageenans, Konjac gum, xanthan gum, dextran, chitosan, Tragacanth gum, Ghatti gum, Karaya gum, tamarind, agar agar, alginate, gellan gum, and mixtures thereof. According to a preferred embodiment, the cosmetically or dermatologically acceptable medium comprises demineralised water. According to another particularly preferred embodiment, the cosmetically or dermatologically acceptable medium comprises a vegetable gum.

Use of the compositions of the invention

Another object according to the invention relates to the use of the compositions according to the invention. As indicated above, the compositions of the invention are particularly suitable for topical use.

According to one embodiment, such topical use allows the gradual and controlled release of the component of interest. The inventors have demonstrated that the skin microbiota consumes the compositions in which the alginate and the EPS are cross-linked, which allows the gradual and controlled release of the component(s) of interest that they contain.

The inventors have thus found a solution making it possible to combine the advantages of encapsulation while proposing a cosmetic composition of natural origin, in the form of a hydrogel or an encapsulation matrix, which does not form degradation products. toxic during its degradation, which is easily usable and applicable by the consumer. Such a composition exhibits perfect cutaneous and ocular tolerance, good hypoallergenicity and stability under various physico-chemical conditions (pH, temperature, etc.).

The gelled texture of the compositions according to the invention is suitable for topical application of the composition, providing a feeling of beneficial freshness for the consumer. It is also easy to spread.

Once the cosmetic composition according to the invention has been applied to the skin, the active principle or principles which it contains are released gradually and act over time, which has the advantage of not requiring the renewal of the application too often. , making use of the composition convenient for the consumer. In addition, the progressive release being done thanks to the bacteria of the cutaneous microbiota, the use of this composition can allow the microbial growth of the cutaneous microbiota in an advantageous way, in particular of the microbial populations considered beneficial. This gradual release does not depend on the temperature and pH conditions of the skin, like the known sustained release compositions. Furthermore, this gradual release kinetics makes it possible to limit the side effects linked to the application of the composition, usually due to a high concentration of active agents in free form in known cosmetic compositions. Thus, components of interest can be highly dosed in the compositions according to the invention, making them therefore as effective as a highly dosed salon treatment, without causing any discomfort for the user.

Processes for obtaining compositions in crosslinked form

Another object of the invention relates to a process making it possible to obtain the compositions according to the invention, in which the alginate and the EPS are crosslinked.

With reference to the , the method comprises the following steps:

- a step 20 of preparing a composition comprising alginate, exopolysaccharides produced by the marine bacterium of strain CNCM I-5035 and, where appropriate, at least component of interest (“PA” on the ), Then

- a step 21 of crosslinking said alginate in the presence of Ca ²⁺ ions, then

- a step 22 of crosslinking said exopolysaccharides in the presence of a crosslinking agent.

When it is present, the at least one component of interest suitable for the process according to the invention is chosen from the components of interest suitable for the compositions according to the invention.

The composition prepared during the preparation step advantageously comprises between 20% and 80% by weight of alginate relative to the total mass of polysaccharides and between 20% and 80% by weight of the exopolysaccharides produced by the marine bacterium of strain CNCM I-5035, relative to the total mass of polysaccharides.

The inventors have shown that, surprisingly, the EPS produced by the marine bacterium of strain CNCM I-5035 gave better results than the other microbial EPS tested.

The crosslinking step 21 of said alginate is an ionic crosslinking step, carried out in the presence of Ca2+ calcium ions to form a network of electrostatic bonds between the alginate chains. Its principle is presented in . Preferably, the Ca2+ ions come from a calcium salt in solution, such as for example a solution of calcium chloride or a solution of calcium sulphate. This alginate crosslinking step lasts between 12 and 36 hours.

The densification of the network of electrostatic bonds leads to the bringing together of the chains of EPS in a diameter of 300 μm to 2000 μm.

This type of electrostatic bond, unlike covalent (so-called chemical) bonds, can be destabilized in the presence of other charged compounds, thus causing a relaxation of the structure of the network formed. The stability of the structure of the compositions according to the invention therefore depends on the mode of crosslinking used. In particular, since cosmetic bases often contain destabilizing agents, such as ionic surfactants or ion chelators, it is important to guarantee the stability of the compositions according to the invention with respect to these.

Thus, the crosslinking step of the alginate is followed by a step 22 of crosslinking of the EPS, which is a chemical crosslinking, and which makes it possible to form covalent bridges between the chains of EPS. These chains have been cleverly brought together during the previous alginate cross-linking step, which facilitates the realization of this step and gives a particular structure to the final network formed. The crosslinking of EPS is carried out in the presence of a crosslinking agent. Crosslinking agents suitable for the chemical crosslinking step (22) are known in the prior art, such as 1,2bis(2,3epoxypropoxy)ethylene and 1-(2,3-epoxypropyl)-2,3- epoxycyclohexane and 1,4-butanediol diglycidylether (BDDE). Preferably, the crosslinking agent is BDDE.

According to one embodiment, the crosslinking of the EPS is also carried out in the presence of a basic catalyst, in order to accelerate the reaction. A basic catalyst suitable for the process according to the invention is, for example, sodium hydroxide or potassium hydroxide, preferably sodium hydroxide at a concentration of 0.25N.

Preferably, the EPS crosslinking step is followed by a rinsing step, to remove the crosslinking agent and the excess basic catalyst. Preferably, the rinsing is carried out with water, in particular with demineralised water.

Alternatively, the crosslinking agent and the basic catalyst can be eliminated by precipitation of the crosslinked polysaccharides with ethanol, by rinsing the precipitate with a water/ethanol solution and drying the precipitate.

According to one embodiment, the composition obtained is in bulk form 221, gelled, in which the millimetric matrices are agglomerated. These matrices can be dissociated by fragmentation 224. This fragmentation step is optional, or can be carried out subsequently, for example by the user when applying the composition to the skin.

According to a particular embodiment, the compositions are obtained in the form of spheres of a composition comprising the alginate and the crosslinked EPS. In this case, the method according to the invention further comprises the addition of calcium carbonate 201, of mineral or biomineral origin, to the composition prepared during the preparation step. The calcium ions are thus present in an insoluble form and cannot cause the crosslinking of the alginate as it is. This composition can therefore be used as a dispersed aqueous phase during an additional step, after the preparation step and before the alginate crosslinking step, implementing a millifluidic technique. Experimental setup 1 allowing the implementation of a millifluidic technique is presented in .

The additional step implementing a milli-fluidic technique is described below with reference to FIGS. 2 and 4. The dispersed phase is introduced by a syringe 10 into a first tube 12, which is extended by a capillary 13 to which it is connected by the means 14. A capillary 13 suitable for the method according to the invention is known from the prior art, such as for example a glass or PTFE tube, or even a fused silica tube. Preferably, capillary 13 is a fused silica tube. The dispersed phase is then led into a second tube 16, the diameter of which is strictly greater than the diameter of the capillary 13, coaxial with the capillary 13, and in which a solution corresponding to the continuous phase circulates co-currently. A tube 16 suitable for the method according to the invention is known from the prior art. They may in particular be tubes made of fluoropolymers, such as polytetrafluoroethylene (PTFE) or fluorinated ethylene propylene (FEP), or tubes made of polyethylene, or else tubes made of glass. Preferably, tube 16 is an FEP tube. The continuous phase 202 is an acidic organic phase, or acidic oily phase, and the dispersed and continuous phases are not miscible. Preferably, the continuous phase comprises i) at least one compound chosen from organic solvents and vegetable or mineral oils, preferably Caprylic/Capric Triglyceride (CAS No: 73398-61-5 / 65381-09-1) and ii) at least one acid molecule chosen from lactic acid, citric acid and acetic acid. More preferably, the continuous phase comprises Caprylic/Capric Triglyceride and lactic acid. This continuous phase is introduced through the syringe 11 into a tube 15. This tube is also connected to the tube 16 through a tee 17, which makes it possible to maintain the tubes 15, 16 and the capillary 13, in particular to maintain an axis of common revolution between the capillary 13 and the tube 16. According to the known technique of "flow focusing", the dispersed phase will be introduced into the flow of continuous phase in the form of droplets. The continuous phase containing acid compounds, it will lead to the release of Ca2+ ions from the calcium carbonate, which will allow the alginate present in the droplets to begin to crosslink as shown in . This primary crosslinking of the alginate takes place during the contact time between the dispersed phase and the continuous phase, from the outlet of the capillary 13 and up to the outlet of the tube 16, that is to say during its evolution in part 18.

Advantageously, the diameter of the tubes (13, 16) and the flow rate of the two phases can be controlled so as to adjust the size of the spheres to the desired size. Another advantage is that once a size is chosen, and the process parameters are defined accordingly, it is possible to obtain partially crosslinked dispersed phase droplets of homogeneous size, throughout the process. For production on an industrial scale, milli-fluidics is also interesting because it is inexpensive and simple to implement. Indeed, working at this scale (channel size of about 100 to 1500 µm) does not require the use of microscopes during production to observe the systems produced, and the flow rates can be high, unlike the technique of micro-fluidics.

Following this additional step, the dispersed phase droplets follow step 21 of alginate crosslinking as described previously. Preferably, these droplets are collected in a bath of calcium chloride. Although the alginate has started to crosslink during the additional step (milli-fluidic technique), the contact time between the dispersed phase and the continuous phase is too short to allow sufficient crosslinking of the alginate. The droplet structure would be quickly lost if the alginate crosslinking was not continued during the alginate crosslinking step. During this step, the network of ionic bonds that has begun to form will be able to densify.

It will be noted that according to this embodiment, the bath also contains part of the continuous phase. Thus, it is preferable to carry out a rinsing step 210, in particular a rinsing with demineralized water, before the next step of crosslinking the EPS, in order to remove the oil residues and the calcium ions which have not not reacted with alginate.

Step 22 of crosslinking the EPS as described above is then carried out. In particular, the spheres are immersed in a bath containing a crosslinking agent, and optionally a basic catalyst, as described above.

Preferably, the EPS crosslinking step is followed by a rinsing step 222 to remove the crosslinking agent and the excess basic catalyst. Preferably, the rinsing is carried out with water, in particular with demineralised water.

According to one embodiment, the spheres formed 223 by the composition in which the alginate and the EPS are crosslinked is mixed with a cosmetically or dermatologically acceptable medium 225 as described above, preferably demineralized water or a gum vegetable.

Examples

1-Obtaining hydrogel spheres according to the invention

The experimental setup for the implementation of a milli-fluidic technique is presented in . An aqueous dispersed phase was obtained from the mixture of a polysaccharide composition according to the invention, comprising alginate and EPS produced by the marine bacterium of strain CNCM I-5035, with a suspension of calcium carbonate. An organic continuous phase was obtained by mixing Caprylic/Capric Triglycerides and lactic acid. The dispersed phase was introduced using a syringe 10 and a tube 12 into a fused silica capillary 13, 150 μm in internal diameter and 375 μm in external diameter. This dispersed phase then joined an FEP tube 16 with an internal diameter of 500 μm, in which the continuous phase flows co-currently. The continuous phase was introduced by a syringe 11 into a tube 15 then into the tube 16. A PEEK polymer tee 17 made it possible to connect the tube 12 and the tube 14 in a sealed manner. dispersed formed droplets in the continuous phase.

The end of the FEP tube 16 was placed in a static CaCl ₂ bath, in which the spheres being formed were immersed for 1 to 2 nights, in order to continue the crosslinking of the alginate. After rinsing with demineralized water, the spheres were placed for 3 to 4 nights in a stirred bath of lactic acid in the presence of BDDE, to allow crosslinking of the EPS. After rinsing with demineralised water, the spheres were incorporated into preserved demineralised water, according to a concentration of 10% by weight.

Three other bacterial EPS were used, and the same experimental protocol was applied to create polysaccharide matrix spheres. However, none of these EPS resulted in a satisfactory matrix structure. The experiments were therefore continued with the EPS produced by the marine bacterium strain CNCM I-5035.

EPS tested from strain marine bacteria Stability of the hydrogels obtained CNCM I-5035 ++ CNCM I-4354 - CNCM I-4151 - CNCM I-4353 -

-: not satisfactory; + satisfactory; ++ very satisfactory

2-Nurturing aspect of the spheres for the skin microbiota

To find out the effects of the spheres obtained on the cutaneous microbiota, the inventors tested the ability of four bacterial strains, representative of the commensal cutaneous flora, to consume and therefore to develop on a medium comprising such spheres.

Four strains representative of the commensal cutaneous flora were selected: S. epidermidis , S. hominis , S. aureus and P. acnes .

Different culture media have been designed. The suspension at 10% by weight of milli-spheres of hydrogel produced according to example 1 was used in test media, at a concentration of 0.5%, 1% and 2% in minimal medium. A minimal medium alone, as well as a rich medium alone, and finally a medium including a preservative, were used to verify the effects of the spheres at the different concentrations. The preservative used consists of phenoxyethanol, methylparaben, ethylparaben and propylparaben, and is commercially available under the brand name Phenopip XB (Clariant International Ltd).

The study was conducted for 24 hours, with counts made at the start of the experiment, then after 3, 6, 12, 15 and 24 hours.

The influence of the presence of the suspension of spheres on bacterial growth was estimated in comparison with the minimal medium alone (minimal growth), the enriched medium (optimal growth) and the preservative (no growth).

The results obtained are shown in Table 2 below.

Sample S. aureus S. epidermidis S. hominis P.acnes Sterile suspension of spheres (10%),
in the middle
minimal ± at 0h
± at 3h
+ at 6am
+ at 12 p.m.
+ at 3 p.m.
+ at 24h ± at 0h
± at 3h
+ at 6am
+ at 12 p.m.
++ at 3 p.m.
++ at 24h
± at 0h
± at 3h
+ at 6am
+ at 12 p.m.
+ at 3 p.m.
+ at 24h ± at 0h
± at 3h
± at 6 o'clock
± at 12 o'clock
± at 3 p.m.
± at 24h Effect according to concentration (%) -Idem to the 3 concentrations
-Slightly better at 1% -Dose effect
-Best effect
at 1% Dose effect Same for the 3 concentrations

+: Growth; ++: Growth equivalent to the enriched culture medium; ±: Maintenance of the bacterial population; -: Absence of bacterial population.

Three of the four strains tested developed in 24 hours on the medium comprising the spheres in suspension, whatever the concentration tested, thus demonstrating the nourishing nature of the spheres.

The size of the population of the P. acnes strain, for its part, remained stable throughout the experiment, without influence of the concentration tested.

For the S. aureus strain, an increase in the bacterial population is observed from T=6 h compared to the minimal medium control, regardless of the concentration tested.

For the S. epidermidis strain, an increase in the bacterial population is also observed from T=6 h compared to the minimal medium control. The size of the bacterial population becomes similar to that observed in the enriched culture medium from T=15h for a concentration of spheres of 1%, and from T=12h for a concentration of spheres of 2%.

For the S. hominis strain, an increase in the bacterial population is observed from T=12h compared to the minimal medium control for a sphere concentration of 0.5%, from T=6h for a sphere concentration of 1%, and from T=3h for a concentration of spheres of 2%.

The results obtained clearly highlight the nourishing nature of the spheres. It can be observed that the triggering of bacterial growth and its intensity vary depending on the microbial strains tested. For example, S. epidermidis , one of the predominant bacteria in the skin microbiota, exhibits high intensity growth, close to that observed in the enriched culture medium, from T=12h for a sphere concentration of 2%. The use of spheres would therefore make it possible to promote the development of certain preferred bacterial strains within the skin microbiota.

These results show that the bacteria of the microbiota consume the spheres within the meaning of the invention. Thus, they would contribute to gradually releasing a component contained in such spheres.

3-Controlled release thanks to the action of the skin microbiota

To visualize the progressive release of the components of interest contained in the spheres according to the invention, spheres containing a red pigment were designed according to the method of example 1, in which a red pigment was added to the phase dispersed in upstream of the ionic crosslinking. Thanks to this pigment, it was possible to follow the release kinetics of a component contained in the spheres, in the presence or absence of bacteria from the commensal cutaneous flora.

The bacteria of the commensal cutaneous flora were obtained by taking a sample from the area of the forearms of volunteers. The suspension of spheres was sterilized by overnight treatment with hydrogen peroxide, at a concentration of 2000 ppm, then tested at a concentration of 5% by weight. The suspension was then incubated overnight, in the presence of unagitated bacteria, at 30-35° C., in a rich medium. A 100 rpm agitation is then applied to the medium to allow significant growth before the inoculation of the suspension thus prepared.

The rich medium used consisted of 9 g/L of NaCl, 1 g/L of alginate, 1 g/L of yeast, 1 g/L of peptone and 0.2 g/L of sucrose. Different Erlenmeyer flasks are prepared to constitute the following batches:

- a “test” batch (100 mL of a rich medium + skin bacteria + 10 mL of the suspension containing the pigment in each of the Erlenmeyer flasks)

- a "negative control" batch (100 mL of the rich medium + 10 mL of the suspension containing the pigment in each of the Erlenmeyer flasks)

- a “positive control” batch (100 mL of rich medium + skin bacteria in each of the Erlenmeyer flasks).

Microbial growth was estimated by reading the optical density (OD) spectrophotometer at 600 nm. The results obtained are shown in Table 3 below.

OD (600nm) T0 T3h T6h T22h T29h T48h T125h Witness - 0.0096 0.0065 0.0076 0.0047 0.0070 0.0077 0.0040 Witness + 0.1130 0.5376 0.6499 1.2280 1.3149 1.1992 0.9809 Test 0.1115 0.5713 1.0969 1.2965 1.4303 1.6638 1.6684

The ODs read in the “test” batch (spheres + bacteria) are higher than the ODs read for the “negative control” batch (without bacteria) and for the “positive control” batch (without spheres). This reflects better growth of the microbial population in the presence of a suspension of spheres according to the invention. In the absence of bacteria, the pigment was not released into the culture medium. In the absence of a sphere, the bacteria cultured in a rich medium developed less well. These results contribute to demonstrating a trophic use of alginate and EPS by bacteria.

Macroscopic observations were also made during this experiment, at the start of the experiment (T=0 h), then after 3, 6, 22, 29, 48 and 125 hours. The results thus obtained are shown in Table 4 below.

Aspect T0 T3h T6h T22h T29h T48h T125h
Witness - -Yellow medium, clear
- Integral spheres Yellow medium, clear
- Integral spheres Yellow medium, clear
- Integral spheres Yellow medium, clear
- Integral spheres Yellow medium, clear
- Integral spheres Clear yellow medium
- Integral spheres Yellow medium, clear
- Integral spheres Witness + Yellow medium, hazy Yellow medium, hazy Yellow medium, hazy Yellow medium, cloudy Yellow medium, cloudy Yellow medium, cloudy Yellow medium, hazy
Erlen test Yellow medium, cloudy
- Integral spheres Yellow medium, cloudy
- Integral spheres Yellow medium, cloudy
- Integral spheres Yellow medium, cloudy
- Integral spheres More intense yellow medium, cloudy at the start of diffusion Rosy medium during diffusion
-Less Integral Spheres Pronounced red medium during diffusion
-Absence of Integral Spheres

Macroscopic observations show that in the absence of bacteria, the medium comprising the spheres shows no change in appearance: the liquid is clear yellow and the spheres remain intact. In the absence of spheres, the medium comprising the bacteria does not change in appearance either, remaining yellow and cloudy. On the other hand, as soon as the spheres containing the pigment are brought into contact with the bacteria, a pink color appears in the medium from T=48h, which becomes red from T=125h. This is explained by the gradual release of the red pigment initially contained in the spheres when the bacteria of the skin microbiota are present. These results demonstrate that skin bacteria are indeed at the origin of the degradation of the spheres, causing the gradual release of the component initially contained within them.

Finally, microscopic observations were also carried out during this experiment, so as to assess more precisely the integrity of the spheres as a function of time and as a function of the presence or absence of bacteria from the microbiota. Photographs representative of the appearance of the spheres at different times (after 6, 22, 29, 48 and 125 hours) and according to the presence (“+”, on the left) or not (“-”, on the right) of the bacteria are presented in the .

It is observed that after 6 hours in the presence of skin bacteria (first image on the left), the spheres according to the invention do not undergo any modification in their structure. They are spherical in shape and uniformly black in color. Their average diameter is 400 µm. In the absence of skin bacteria (first image on the right), the same observations are made: spherical shape and homogeneous black color of the spheres.

After a contact time of 22 hours with the bacteria in the skin (second image from the left), a sphere is observed whose diameter has increased, reaching a value of around 615 µm. It seems degraded and its hue has become lighter, presenting a non-homogeneous greyish appearance. In the absence of skin bacteria (second image from the right), the spheres according to the invention still do not undergo any change in their structure.

After 29 hours of contact with the bacteria of the skin (third image from the left), we observe that all the spheres of the environment studied present modifications similar to those observed after 22 hours of contact with the bacteria: increase in their size, revealing a degraded aspect of the perimeter of the sphere. In the absence of bacteria (third image from the right), the spheres according to the invention still do not undergo any change in their structure.

After 48 hours of contact with skin bacteria (fourth image from left), a capsule appears to be in the process of decomposition. The destructuring of the gel making up the sphere is then clearly visible, and appears to be progressive in comparison with the previous observations. In the absence of bacteria (fourth image from the right), the spheres according to the invention still do not undergo any change in their structure.

After 125 hours of contact with skin bacteria (last image on the left), the spheres are totally degraded and seem to be dispersed in the medium. In the absence of bacteria (last image on the right), the spheres according to the invention still retain their intact structure.

All of these microscopic observations, and in particular the comparison of situations with the presence and absence of bacteria, confirm a degradation of the polysaccharide hydrogel spheres according to the invention by the skin microbiota.

The results obtained in this example corroborate those obtained in example 2.

Claims (14)

Composition comprising alginate and exopolysaccharides, characterized in that the exopolysaccharides are produced by the marine bacterium of strain CNCM I-5035, and in that the composition further comprises calcium. Composition according to Claim 1, characterized in that the calcium is in the form of calcium carbonate. Composition according to Claim 1, characterized in that the alginate and the exopolysaccharides are cross-linked. Composition according to any one of the preceding claims, characterized in that the said alginate is present at a content of between 20% and 80% by weight of the total dry mass of polysaccharides and the said exopolysaccharides are present at a content of between 20% and 80% by weight of the total dry mass in polysaccharides. Composition according to any one of the preceding claims, characterized in that it additionally comprises at least one component of interest, preferably at least one component chosen from organic particles, mineral particles, whole living cells, polysaccharides , oligosaccharides, proteins, peptides, polypeptides, phenolic compounds, raw materials produced by bees, synthetic materials or any other polymer larger than 2000 Daltons. Composition according to any one of Claims 3 to 5, characterized in that it is in an essentially spherical form with a diameter of 200 µm to 2000 µm. Cosmetic or dermatological composition, characterized in that it comprises a composition according to any one of Claims 3 to 6 and a cosmetically or dermatologically acceptable medium, preferably demineralised water and/or a vegetable gum. Use of a composition according to any one of Claims 3 to 7, for topical application with moisturizing, pigmenting, depigmenting, soothing, oxygenating, regenerating, anti-aging, anti-wrinkle, anti-redness action, and combinations thereof. Use according to claim 8, for a gradual and prolonged release of said at least one component of interest. Process for obtaining a composition according to any one of Claims 3 to 6, characterized in that it comprises:
- a step of preparing a solution comprising alginate, exopolysaccharides produced by the marine bacterium of strain CNCM I-5035 and, where appropriate, at least one component of interest, then
- a crosslinking step of said alginate in the presence of Ca ²⁺ ions, then
- a crosslinking step of said exopolysaccharides in the presence of a crosslinking agent. Process according to Claim 10, for obtaining a composition according to Claim 6, characterized in that the solution prepared during the said preparation stage also comprises calcium carbonate, and in that it comprises, after the said preparation step:
- a step implementing a millifluidic technique, in which the composition prepared in the previous step is used as an aqueous dispersed phase, and is brought into contact with an acidic organic continuous phase, so that the dispersed phase forms droplets. Process according to Claim 11, characterized in that the acidic organic continuous phase comprises i) at least one compound chosen from organic solvents and vegetable or mineral oils, preferably Caprylic/Capric Triglyceride and ii) at least one molecule acid chosen from lactic acid, citric acid and acetic acid. Process according to any one of Claims 10 to 12, characterized in that the said crosslinking agent is chosen from the group consisting of 1,2bis-(2,3-epoxypropoxy)ethylene, 1-(2,3-epoxypropyl)- 2,3-epoxycyclohexane and 1,4-butanediol diglycidylether (BDDE). Process according to any one of Claims 10 to 13, characterized in that the Ca ²⁺ ions come from a solution of calcium salt, such as a solution of calcium chloride or calcium sulphate.