FR2813885A1 - Mixture of non-sulfated fucose-based oligosaccharides, has dermatological activity, stimulates cellular communication, proliferation of keratinocytes and fibroblasts and inhibits synthesis of elastase type proteases - Google Patents

Abstract

A mixture of non-sulfated fucose-based oligosaccharides is new. A mixture of non-sulfated fucose-based oligosaccharides comprises oligosaccharides of less than 13 saccharide units (with at least one fucose unit in a non-reducing end position) and can be obtained by degradation of a polysaccharide from the micro-organism Klebsiella pneumoniae subsp. pneumoniae. An Independent claim is also included for a cosmetic or pharmaceutical composition comprising the mixture.

Description

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Title: Novel mixture of non-sulphated oligosaccharides based on fucose, cosmetic or pharmaceutical composition comprising it, and its use in cosmetics or pharmacy.

 The present invention relates to a novel mixture of non-sulphated oligosaccharides based on fucose and its use in particular in products with topical application, for which an activity on the epithelial or connective tissue is sought, in particular anti-aging products, such as pharmaceutical or veterinary products, and more particularly, in cosmetics.

 During aging, the skin thins on average about 6% every ten years (Branchet et al., Gerontology, 1990, 36: 28-35). The mechanisms underlying aging are intrinsic and extrinsic. Intrinsic mechanisms include a slowing of cell proliferation and a significant loss of cutaneous extracellular matrix (ECM). This loss is mainly the result of the decrease in the synthesis of the ECM and the increasing synthesis with age, degradation enzymes that can attack the cutaneous matrix (DL ROBERT: Aging, CNRS, Belin, 1994; ROBERT: Aging, facts and theories, DOMINOS, Flammarion, 1995). The proteases responsible are mainly matrix metalloproteases (MMPs), many of which (as well as serine proteases) can degrade most of the constituents of the cutaneous matrix and in particular the elastic fibers.

 In recent years, numerous studies have sought to obtain active compounds against certain effects of skin aging. A first goal is to slow down this process. Another objective is to obtain a result of thickening of the skin, especially the dermis.

 Saccharides and polysaccharides are substances well known in cosmetics, especially for their moisturizing properties. This is the case, for example, with fucose monosaccharides and polysaccharides containing them.

 Fucose is a deoxyhexose close to galactose, of which it has the steric conformation. However, the structure of fucose differs essentially from that of galactose in that the carbon-6 atom has a methyl group

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 (-CH3) and not a primary alcohol group (-CH20H). This methyl group indeed confers on the fucose molecule an interesting partial hydrophobic character, compensated by the other hydroxyl groups on the other four carbon atoms present.

 Fucose appears early in phylogeny, polysaccharides of some algae and fungi contain relatively large amounts, alone or in combination with other chemical compounds. It can also appear in sulphated form as in fucans. Fucose is also widespread in the plant kingdom and some bacteria synthesize too.

 However, despite the large amount of work on fucose and polysaccharides containing it, the results obtained to date were hardly satisfactory for application on an industrial scale, especially in a cosmetic product.

 In particular, in addition to the poor cost-effectiveness ratio of the monosaccharide fucose, the already known fucose-containing polysaccharides do not exhibit a significant activity making it possible to effectively combat cutaneous aging as set out above.

 Thus, for example, fucans are sulfated polymers of high molecular weight (> 20kD). WO 99/32099 (IFREMER) discloses novel uses of fucans for the repair of connective tissue lesions. However, fucans, by their size and their charge, can not interact effectively with all the cellular layers of the skin. What's more, fucans can instead activate MMP-2, unacceptable property for a cosmetic treatment of normal aging of the skin.

 FR-2,750,863 (L'OREAL) describes the use of a polyholoside, in particular a polysaccharide comprising fucose, to promote desquamation, ie to promote the elimination of dead cells located on the surface of the layer cornea of the epidermis (page 3, lines 6 and 7) and / or to stimulate the epidermal renewal, ie to cause a forced elimination of the stratum corneum accelerating the renewal (page 1, lines 13-23), these effects giving rise to an assimilated result according to FR-2 750 863 to an anti-aging skin result. However, the polysaccharide as described in FR-2,750,863, because of its molecular weight and its

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 structure, does not allow to interact satisfactorily with the different constituents of the skin.

 It has now been found, quite surprisingly and unexpectedly, that a novel mixture of oligosaccharides containing fucose, obtainable by a specific treatment of a selected microorganism, has significant activities on various skin components, resulting in by a real anti-aging result, clearly visible, especially by a spectacular thickening of the skin.

 The subject of the present invention is thus a mixture of non-sulphated oligosaccharides based on fucose, characterized in that it comprises oligosaccharides of less than 13 saccharide units comprising at least one non-reducing terminal fucose unit, and in that it can be obtained by a process comprising at least one step of degradation of a polysaccharide derived from a microorganism of the genus Klebsiella pneumoniae subsp. pneumoniae.

 According to the invention, the term "non-sulphated oligosaccharide based on fucose" means, in accordance with the general knowledge of a person skilled in the art, an oligosaccharide containing at least one saccharide unit fucose and having no sulfate group -O (SO3 ) -. Fucans are in particular excluded from this definition.

 By "oligosaccharide comprising at least one non-reducing terminal fucose unit" is meant according to the invention, in agreement with the general knowledge of a person skilled in the art, an oligosaccharide containing at least one saccharide unit fucose in the terminal position of the chain. of the oligosaccharide, this fucose unit being connected to the following saccharide unit of the rest of the oligosaccharide by an acetal-type bond.

 The numbers of saccharide units can be measured using techniques well known to those skilled in the art, in particular using the HPLC chromatography technique as described in the examples below.

 Preferably, the mixture of oligosaccharides according to the invention comprises, relative to the total weight of the mixture, at least 15% by weight, and more particularly preferably from 20 to 50% by weight, of oligosaccharides of less than 13 units. saccharides comprising at least one terminal non-reducing terminal fucose unit.

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 More particularly, the mixture of oligosaccharides according to the invention is characterized in that it further comprises, relative to the total weight of the mixture, from 25 to 45% by weight of oligosaccharides having from 13 to 24 saccharide units comprising at least a fucose unit in a non-reducing end position.

 More particularly, the mixture of oligosaccharides according to the invention is characterized in that it further comprises, relative to the total weight of the mixture, from 15 to 35% by weight of oligosaccharides of more than 54 saccharide units comprising at least a fucose unit in a non-reducing end position.

 The oligosaccharide mixture being capable of being obtained by a process comprising at least one step of degradation of a polysaccharide derived from a microorganism of the genus Klebsiella pneumoniae subsp. pneumoniae, the oligosaccharides preferably comprise, at least in part, the fucose-galactose-galacturonic acid repeating unit.

 In particular, the oligosaccharide mixture according to the invention can be obtained by the process comprising the steps of: a) growing the microorganism of the genus Klebsiella pneumoniae subsp. pneumoniae in an aqueous nutrient medium by aerobic fermentation of an assimilable carbohydrate source; b) recovering the polysaccharide formed from the fermentation must; c) subjecting the polysaccharide to moderate hydrolysis; d) subjecting the hydrolysis product of step c) to enzymatic hydrolysis; and e) deactivating the enzyme and then recovering the oligosaccharide mixture thus formed.

 More particularly, these steps a) to e) can be described as follows.

Etapea)
The microorganism Klebsiella pneumoniae subsp. pneumoniae is the microorganism deposited in the National Collection of Cultures of Microorganisms under the number 1-1507, or a mutant of the latter. This microorganism is also described in detail in the application WO 96/23057.

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 The aqueous nutrient medium can be any aqueous medium known to those skilled in the art, containing sources of carbon, nitrogen and inorganic salts such as those described in application WO 96/23057.

 The fermentation can be carried out at temperatures of the order of 25 to 35 C, at a pH of about 6.0 to 7.5, under conditions of aeration and stirring, for durations of the order from 2 to 4 days.

 The fermentation can be carried out in a conventional fermenter by inoculating sterilized nutrient medium, for example by heating to a temperature of about 120 C or by sterilizing filtration.

Step b)
At the end of the fermentation period, the fermentation broth is recovered from which a polysaccharide rich in fucose is isolated in the following manner.

 The fermentation broth is subjected to a heat treatment at a temperature in particular of between approximately 100 and approximately 130 ° C., preferably between approximately 115 and approximately 125 ° C. for a period of time in particular of approximately 30 minutes to approximately 2 hours and preferably of about 40 minutes to about 1 hour and a pH in particular between about 2 and about 5.5 and preferably between about 3 and about 5.5.

 The heat treatment product is filtered by conventional means such as a plate filter press.

 A clear, viscous and cell-free polysaccharide is thus obtained.

 A precipitation is then carried out in an alcohol solvent, preferably an alcohol solvent selected from ethanol, isopropanol and mixtures thereof. In particular, about 1 to about 3.0 volumes of solvent per 1 volume of polysaccharide and preferably about 1.3 to about 2.0 volumes of solvent per 1 volume of polysaccharide are used.

 Vacuum drying is then carried out at a temperature in particular of between approximately 20 and approximately 60 [deg.] C. and preferably between approximately 30 and approximately 50 [deg.] C. until a powder is obtained.

Steps c) and d): hydrolysis of the polysaccharide
This is an essential combination of steps. It has surprisingly and unexpectedly been found that the combination of a moderate hydrolysis step, preferably by gamma irradiation and / or protolysis, with a step

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 Enzymatic hydrolysis, advantageously provides a sufficient overall hydrolysis yield, close to that of a conventional hydrolysis such as acid hydrolysis, but with the advantage of specific cuts of enzymatic hydrolysis. In particular, a conventional acidic hydrolysis does not make it possible to obtain the mixture of specific oligosaccharides according to the invention insofar as it leads to the obtaining of statistical cuts, prohibitive as to their randomness. The compounds resulting from a conventional acid hydrolysis have also proved to be biologically inactive.

Step c): Moderate polysaccharide
Moderate hydrolysis is carried out by a gamma ray treatment, a protolysis treatment or by these two successive treatments. Preferably, a gamma ray treatment is carried out successively and then a protolysis treatment.

 The treatment with gamma rays proves to cause a significant decrease in the viscosity due to a limited degradation, attributable to the action of free radicals. It can be achieved with irradiation means well known to those skilled in the art.

 This treatment with gamma rays, very penetrating rays, has the further advantage of sterilizing the polysaccharide by killing the present germs that could induce inflammation or even cause a granuloma. Thus, any bacterial attack is prevented without having to add antiseptic products to the medium which could undesirably interfere with the biological activities of the final product.

 The polysaccharide powder obtained in step b), optionally irradiated with gamma rays, can therefore also be subjected to a protolysis treatment. For this purpose, it is dissolved in aqueous solution in particular from 1 to 20% by weight and preferably from 2 to 10% by weight, relative to the total weight of the aqueous solution.

 The aqueous solution is subjected to a heat treatment, that is to say to a heating at a temperature in particular between about 75 and about 120 C and preferably between about 90 and about 100 C, for a time in particular between 1 and 6 hours, in the presence of a proton generating resin such as those marketed and well known to those skilled in the art, that is to say a resin generating protons which result in cutting of glycosidic bonds with fixation of a molecule of water.

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Step d): enzymatic hydrolysis
An acidic buffer such as citric acid buffer (4.15 g / kg) disodium hydrogen phosphate (about 10.75 g / kg) is introduced into the hydrolyzate obtained in step b). The temperature of the solution is adjusted to a temperature in particular of between approximately 25 and approximately 45 ° C. and preferably between approximately 30 and approximately 40 ° C.

 An enzyme preparation is introduced comprising at least one endofucosidase, preferably Fermizyme HCP as marketed by the company Gist Brocades, in a content of, in particular, between about 2 and about 20% by weight, and preferably between about 5 and about 15%. by weight, relative to the initial weight of polysaccharide powder used.

 The mixture thus obtained is kept under agitation for a time, in particular between about 8 and about 24 hours and preferably between about 10 and about 20 hours, at a temperature in particular between about 25 and about 45 ° C. and preferably between about 30 and about 30 ° C. and about 40 ° C., the pH being adjusted to 6 by the presence of the buffer mixture.

step e)
The hydrolysis product obtained after step d) is filtered by conventional means such as a filter press with plates.

 The solution collected is then heat-treated at a temperature in particular of between approximately 75 and approximately 120 [deg.] C. and preferably between approximately 90 and approximately 105 [deg.] C. for a time in particular of between approximately 10 and approximately 45 minutes and preferably between approximately 20 and approximately 35 minutes, in order to deactivate the enzyme and more particularly the fucosidase activity of this specific enzyme.

 It is allowed to cool to a temperature in particular of between approximately 20 and approximately 40 ° C.

 During cooling, preservatives can be added to the solution.

 The whole is then filtered under sterile conditions and then the conditioning is carried out.

 The oligosaccharide mixture according to the invention thus obtained can be characterized using techniques well known to those skilled in the art, in particular by HPLC, thin layer chromatography and other chemical methods and assays.

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 It can thus be observed that the oligosaccharides of the mixture according to the invention are such that, as fucose is found mostly at the end of the chain, in a non-reducing terminal position.

 The oligosaccharide mixture is particularly suitable as an active ingredient in a cosmetic composition, especially anti-aging, or in a pharmaceutical composition, especially a dermatological composition (topical application). In particular, the observed biological effects of this mixture of oligosaccharides are comparable or even superior to those of the monosaccharide fucose.

 Thus, the subject of the present invention is also a cosmetic or pharmaceutical composition, characterized in that it comprises, as a cosmetically or pharmaceutically active agent, at least one mixture of oligosaccharides as described above and at least one a cosmetically or pharmaceutically acceptable excipient.

 When the composition is a pharmaceutical composition, it is preferably a dermatological composition, for topical application, with of course at least one pharmaceutical excipient suitable for such a dermatological application.

 The cosmetically or pharmaceutically acceptable excipient may be any excipient among those known to those skilled in the art in order to obtain a composition according to the invention in the form of a cream, a lotion, a gel, an ointment, etc., optionally in the form of an emulsion with further components known to those skilled in the art, for improving, modifying or stabilizing the composition from a cosmetic or pharmaceutical point of view.

 The term pharmaceutically acceptable excipient includes excipients suitable for veterinary use of the composition according to the invention.

 The composition according to the invention may in particular contain other additives and formulation aids, such as antioxidants to combat free radicals. It may be mentioned in particular pure vitamin E or di-alpha-tocopherol and its derivatives, and 2,6-di-tert-buthyl-p-cresol (BHT).

 Advantageously, the composition according to the invention may in particular also comprise at least one additive chosen from the group consisting of structuring agents of the skin (such as squalane and sphingolipids), humectants

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 (such as glycerin and hydroxy prosilan C), emollients (such as butylene glycol and ethyl lactate), silicones (such as cyclomethicone), sunscreens (such as Parsol 1789 and Eusolex 6300), emulsifiers (in particular Carbopol 1342 combined with triethanolamine and soy lecithin), thickeners (in particular xanthan gum), sequestering agents (in particular EDTA), antioxidants (such as the BHT described above). above) fragrances, preservatives, water and mixtures thereof.

 Of course, the operating conditions for preparing the cosmetic or pharmaceutical composition according to the invention are part of the general knowledge of those skilled in the art.

 Preferably, the mixture of oligosaccharides is present in a proportion of between about 0.001 and about 20% by weight, and more preferably between about 0.1 and about 10% by weight, relative to the total weight of this cosmetic composition or pharmaceutical.

 Without wishing, however, to be bound by any theory, the advantageous activity of the oligosaccharide mixture according to the invention, illustrated by the examples below, would result in particular from its very great capacity to interact with cell membranes, via the hydrophobic functions of the highly available methyl groups of fucose, and its interaction with the fucose-mannose receptor of cutaneous cells, macrophages or Langerhans cells (Condaminet et al: epidermal Langerhans cells expressing the mannose-fucose binding receptor, Eur J. Immunol 1998. 28: 3541-3551). All of these effects can be characterized as a stimulatory effect of cellular communication.

 Thus, the present invention also relates to the use of the oligosaccharide mixture as described above, for the preparation of a composition intended to stimulate cellular communication between the cells of the skin.

 Furthermore, the subject of the present invention is also the use of the oligosaccharide mixture as described above for the preparation of a composition intended to stimulate the cellular proliferation of keratinocytes of the skin.

 In addition, the subject of the present invention is also the use of the oligosaccharide mixture as described above for the preparation of a composition intended to stimulate the cellular proliferation of skin fibroblasts.

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 Finally, the subject of the present invention is also the use of the oligosaccharide mixture as described above, for the preparation of a composition intended to inhibit the synthesis of elastase proteases by skin fibroblasts.

 The fibroblasts of human skin synthesize several proteases, some of which exhibit elastase-like activity. This is particularly true for MMP-2 and MMP-9 and for membrane MMPs.

 Hyaluronan added to fibroblasts at low concentrations (1 to 2 mg / ml) significantly increases elastase activity. However, we have observed surprisingly and unexpectedly that the mixture of oligosaccharides according to the invention inhibits this stimulation. Since hyaluronan is constantly present in the pericellular environment, this inhibitory activity can be considered as reducing the degradation of the cutaneous matrix with age.

 Thus, the subject of the present invention is also the use of the oligosaccharide mixture as described above, for the preparation of a composition intended to inhibit the overexpression of hyaluronan-induced MMP-2 and MMP-9 proteases. .

 More particularly, the subject of the present invention is also the use of the oligosaccharide mixture as described above, for the preparation of a composition intended to inhibit the synthesis of MMP-2 and MMP-9 proteases by fibroblasts of the skin.

 Moreover, it has recently been shown that the activity of MMPs is critical in the case of contact sensitization (M. C Lebre et al., Arcg Dermatol Res., 1999, 291: 447-452). In the presence of MMP inhibitors, Langerhans cells of the epidermis can not migrate through the dermis as is the case during contact sensitization. Inhibition of the MMP activity by the oligosaccharide mixture according to the invention thus provides a true effect of lowering the sensitivity of the skin to irritation.

 The present invention therefore also relates to the use of the oligosaccharide mixture as described above, for the preparation of a composition intended to reduce the sensitivity of the skin to irritation.

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 The oligosaccharide mixture according to the invention makes it possible to obtain effects of increasing the thickness of the epidermis and the dermis. Both effects are statistically highly significant. As the thickness of the skin decreases with age, this trophic effect of the oligosaccharide mixture according to the invention is extremely important in the prevention of cutaneous aging.

 More particularly, it is possible to observe a strengthening of the collagen bundles of the dermis of a skin treated with the oligosaccharide mixture according to the invention. Given the importance of these collagen bundles in the firmness of the skin, this is a major anti-aging result.

 The present invention therefore also relates to the use of the oligosaccharide mixture as described above, for the preparation of a composition intended to stimulate the deposition of collagen fibers in the dermis.

 The oligosaccharide mixture is used as described above preferably in a proportion of from about 0.001 to about 20% by weight, and more preferably from about 0.1 to about 10% by weight, based on the total weight of the oligosaccharide mixture. the composition.

 The composition thus prepared further comprises a cosmetically or pharmaceutically (including dermatologically) acceptable excipient, such as those known to those skilled in the art.

 Finally, the subject of the present invention is also a method for cosmetic treatment of the skin, characterized in that a cosmetic composition comprising at least one mixture of oligosaccharides as described above is applied to the skin. The cosmetic composition further comprises a cosmetically acceptable excipient, such as those known to those skilled in the art.

 The following examples are intended to illustrate the present invention and should in no way be interpreted as limiting its scope.

 Figure 1 is a histogram showing the results presented in Example 3.b.l, in terms of percentage efficiency for the reduction of the active form / inactive form ratio of MMP-2 and MMP-9 secreted by skin explants.

 Figure 2 is a histogram showing the results presented in Example 3.b.l, in terms of percentage of efficiency for the reduction of the ratio form

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 active / inactive form of MMP-2 and MMP-9 secreted by skin explants stimulated by hyaluronan.

 Figure 3 is a histogram showing the results presented in Example 3, b.2, in terms of percent efficiency for the reduction of MMP-2 expression of skin explants.

Example 1 Preparation of an Oligosaccharide Mixture According to the Invention a) Fermentation
The microorganism Klebsiella pneumoniae subsp. pneumoniae is the microorganism deposited in the National Collection of Cultures of Microorganisms under number 1-1507. The nutrient medium and other conditions of fermentation are as follows.

Preparation of inoculums Culture medium: Neosorb # 70-07 (sorbitol content: 70% MS; sold by ROQUETTE FRERES, Lille / France): 17.90 g / 1 (ie 12.5 g / 1 of sorbitol) Peptone Biokar 104003 (protein hydrolyzate, sold by SOLABIA-BIOKAR, Pantin, France): 4.50 g / l Yeast extract: 0.05 g / l KH 2 PO 4: 1.50 g / l K 2 HPO 4: 4.50 g / l MGSO 4, 7H20: 0.20 g / 1 Pluronic # PE 61000 (antifoaming agent, sold by BASF, D-6700 Ludwigshafen, Germany): 0.50 g / 1 Solution dissolved in water.

Culture condition: Sterilization at 121 C for 30 minutes Culture temperature: 30 C Inoculation rate: 5 to 10% Aeration: 1 VVM unregulated PH (pH around 7.00) Culture time: 24 hours

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 Production medium Culture medium: Neosorb 70-07: 54.00 g / 1 (ie 38 g / l sorbitol) Peptone Biokar #b 104003: 4.50 g / 1 Yeast extract: 0.05 g / l KH2PO4 4 : 1.50 g / I MgSO4, 7H2O: 0.20 g / 1 Pluronic # PE 61000: 0.50 g / 1 Solution dissolved in water.

Culture conditions (Chemap fermenter with a useful volume of 350 liters): Sterilization at 120 ° C. for 45 minutes Culture temperature: 30 ° C Inoculation rate: about 5% Agitation: 300 rpm (Rushton type stirrer) Aeration: 1 VVM PH regulated at 7.0 with 7 N NaOH Pressure: 100 to 200mbars Crop time: 60 to 65 hours Average values obtained in production: Viscosity at the end of the cycle: 40000 Mpa. s (Brookfield DV-II + model LV viscometers, mobile SP 31, chamber SC4-34 / 13R, 30 C) Concentration of the polysaccharide produced in the medium, calculated in L-fucose: 2 g / l (Dische and Shettles method) Sorbitol consumed:> 35 g / l (in sorbitol) 20% NaOH in weight consumed: 15 liters / m3 Start of pH regulation: 16 to 17 hours after fermentor inoculation Final dry extract of the fermentation medium: -20 g / 1 b ) Recovery of formed polysacharide
The fermentation must is subjected to a heat treatment at a temperature of 120 ° C. for 45 minutes and at a pH of 5.5. The product of the heat treatment is filtered using a filter press with Seitz type plates. A clear, viscous and cell-free polysaccharide is thus obtained. Precipitation is then carried out in a 1.5 volume of ethanol per 1 volume of polysaccharide. Vacuum drying is then carried out at a temperature of 25.degree.

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c) Hydrolyse modérée du polysaccharide
La poudre de polysaccharide est mise en solution aqueuse à raison de 5 % en poids, par rapport au poids total de la solution aqueuse. La solution aqueuse est soumise à un traitement thermique, c'est à dire à un chauffage à 100 C, pendant 3 heures, en présence d'une résine génératrice de protons. d) Hydrolyse enzymatique
On introduit le mélange tampon acide citrique (4,15g/kg)-hydrogénophosphate disodique (environ 10,75 g/kg) dans l'hydrolysat. On règle la température de la solution à 37 C. On introduit la préparation enzymatique Fermizyme HCP telle que commercialisée par la société Gist Brocades, selon une teneur de 10 % en poids, par rapport au poids initial de poudre de polysaccharide utilisée, c'est à dire 0,05% en poids par rapport à la masse totale de la solution aqueuse après remise en eau de la poudre comme décrit ci-dessus pour l'hydrolyse modérée par protolyse. a powder. In view of the microorganism used, this polysaccharide is composed of fucose-galactose-galacturonic acid trisaccharide repeating units and thus has the following structure:

c) Moderate hydrolysis of the polysaccharide
The polysaccharide powder is dissolved in aqueous solution at a rate of 5% by weight, relative to the total weight of the aqueous solution. The aqueous solution is subjected to a heat treatment, that is to say to a heating at 100 C, for 3 hours, in the presence of a proton generating resin. d) Enzymatic hydrolysis
The citric acid (4.15 g / kg) disodium hydrogen phosphate (about 10.75 g / kg) buffer mixture is introduced into the hydrolyzate. The temperature of the solution is adjusted to 37 ° C. The fermizyme HCP enzyme preparation is introduced, as sold by the company Gist Brocades, at a content of 10% by weight, relative to the initial weight of polysaccharide powder used. ie 0.05% by weight based on the total mass of the aqueous solution after re-watering the powder as described above for moderate hydrolysis by protolysis.

The mixture thus obtained is stirred for 15 hours at a temperature of 37 ° C., the pH being adjusted to 6 by the presence of the buffer mixture. e) Deactivation of the enzyme and recovery of the mixture of dry arid oligos
The hydrolysis product is filtered with a press filter with Seitz type plates.

The collected solution is then heat-treated at 100 ° C. for 30 minutes to deactivate the enzyme. It is allowed to cool to a temperature of 25 ° C. During cooling, the preservatives phenoxy ethanol (1% by weight) and phenonip (0.3% by weight) are added to the solution. The whole is then filtered under sterile conditions.

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 The oligosaccharide mixture thus obtained is referred to as "Mixture-1".

Example 2: HPLC Characterization of Mixture-1
Fractionation of the Mixture-1 obtained in Example 1 was carried out in the drawing to determine the proportion of oligo- and polysaccharides in its composition. a) Fractionation by preparative exclusion chromatography on the column "Xk 50/60 Superdex 75 prepgrade"
We passed 50ml of the Concentrated-1 Blend at 50mg / ml, on a preparative column "XK 50/60 Superdex 75 prepgrade" (exclusion chromatography) and we collected 95 fractions, then passed in HPLC.

Table 1: techniques concerning the preparative column
XK 50/60 Superdex 75 prepgrade

<Tb>
<tb> Filling <SEP> Superdex <SEP> 75 <SEP> prepgrade <SEP> (34 <SEP> m)
<tb> Size <SEP> of <SEP><SEP> column <SEP> Height <SEP>: <SEP> cm
<tb> Diameter <SEP>: <SEP> 60 <SEP> mm
<tb> Type <SEP> of <SEP> column <SEP> XK <SEP> 50/60
<tb> Usable <SEP> Interval <SEP> of <SEP> 5 <SEP> x <SEP> 102 <SEP> Da <SEP> - <SEP> 3 <SEP> x <SEP> 104 <SEP> Da
<tb> splitting <SEP> ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
<tb> Sample <SEP> Injection <SEP>: <SEP> 50 <SEP> ml <SEP> of <SEP> mixture <SEP> - <SEP> 1 <SEP> to <SEP><SEP> concentration <SEP > from
<tb> 50 <SEP> mg / ml. <SEP> (at <SEP> total <SEP>: <SEP> = <SEP> 2.50 <SEP> g)
<tb> Elution rate <SEP><SEP> 1 <SEP> ml / <SEP> minute
<tb> Number <SEP> of <SEP> fractions <SEP> 95 <SEP> fractions <SEP> of <SEP> 12.5 <SEP> ml <SEP> each
<tb> collected
<tb> Mobile <SEP> Phase <SEP> PBS
<Tb>

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After fractionation of the 1-Mixture on the "XK 50/60 Superdex 75 prepgrade column, 95 fractions are collected, 45 of which contain oside b) Characterization of the fractions obtained by HPLC (exclusion chromatography) columns ultrahydrogel 120 and ultrahydrogel 250
The objective of this second part of our study was to pass on an HPLC exclusion column (Utrahydrogel columns 120 and 250) every 95 fractions of Mixture-1 to analyze the molecular weights and the concentration of the components of these fractions.

 We worked for this study with a Waters HPLC system, the description of which follows.

Table 2: Technical characteristics of the HPLC chromatography system used

<Tb>
<tb> Device <SEP> HPLC <SEP> Waters <SEP> 600
<tb> Columns <SEP> Ultrahydrogel <SEP> 120 <SEP> (size <SEP> of <SEP> pores <SEP>: <SEP> 120 <SEP> Â) <SEP> and <SEP> Ultrahydrogel <SEP> 2
<tb> (size <SEP> of <SEP> pores <SEP>: <SEP> 250 <SEP>) <SEP> of <SEP> Waters. <SEP> Size <SEP>: <SEP> 7.8 <SEP> mm <SEP> x <SEP> 300 <SEP> m
<tb> containing <SEP> the <SEP> gel <SEP> of <SEP> polymethacrylate <SEP> hydroxylated
<tb> Samples <SEP> injected <SEP> 20 <SEP> (it <SEP> by <SEP> automatic <SEP> injector
<tb> Elution <SEP> 0.10 <SEP> M <SEP> NaN03 <SEP>
<tb> Time <SEP> of <SEP> elution <SEP> 50 <SEP> minutes / <SEP> samples
<tb> Speed <SEP> of <SEP> elution <SEP> 0.5 <SEP> ml / <SEP> minute.
<Tb>

Detection <SEP> By <SEP> measurement <SEP> of <SEP><SEP> index <SEP> refraction <SEP> with <SEP> a <SEP> refractometer
<tb> Waters <SEP> 410.
<Tb>

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Table 3: Molecular Weight of Polyethylene Glycol (Fluka) Used for HPLC Exclusion Chromatography

<Tb>
<tb> Weight <SEP> molecular <SEP> SEP <SEP> TIME <SEP> (MINUTES)
<tb> 400 <SEP> 37,892
<tb> 600 <SEP> 36,026
<tb> 1000 <SEP> 33,940
<tb> 2000 <SEP> 31,154
<tb> 4000 <SEP> 28,896
<tb> 6000 <SEP> 27,868
<tb> 8000 <SEP> 26.946
<tb> 12000 <SEP> 26,192
<tb> 20000 <SEP> 25.308
<tb> 35000 <SEP> 24,216
<Tb>
c) Results
The molecular weights of the components of the fractions studied were calculated using the following equation, obtained with the Fluka molecular weight standards described in Table 3:
Molecular weight of the components: = 55290000 * 10 ^ (- 0.13942 * x) R '= 0.982 x = elution time (minutes)

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The first fraction containing components of Mixture-1 is fraction No. 44 and the last fraction is No. 89, which means that even the component of the lowest molecular weight is released after 89 fractions collected (after an elution of 89 × 12.5 ml = 1112.5 ml). The collected fractions contain 184Da mono-, oligo- and polysaccharides (mixture of monosaccharides) up to about 21 kDa. This latter fraction therefore contains polysaccharides composed on average of 117 monosaccharide units or 39 trisaccharide units.

 The majority of the fractions, except fractions n 77, 78, 79, 81, 82, 83, 84, 85 and 86, contain a single saccharide peak (separation limited by the sensitivity of the separation method used).

 The approximate concentration of the components of the different fractions could be determined using a fucose standard range at increasing concentrations. This kind of "mono-compositional" standard range could be used thanks to the detection system (measurement of the refractive index with a refractometer).

According to these results, a solution of 1 g / ml of fucose gives on average a 29409 surface peak (arbitrary units of the system). By knowing the surfaces of the peaks analyzed, we were able to calculate their apparent concentrations.

 The results obtained show that the mixture contains about 26% small osids (up to 2 Kda, about 4 trisaccharide units), about 36% oligosaccharides (up to 4 Kda, 8 trisaccharide units) and about 23% polysaccharides. molecular weight greater than 10 kDa (18 trisaccharide units).

 In view of the microorganism and the specific enzyme (endo-fucosidase) used for the preparation of the Mixture-1, it appears that the oligosaccharides of the mixture comprise a non-reducing reducing terminal unit fucose.

Example 3 Activity of Fucose and Mixture-1 on the Activity of Matrix Metalloproteinases of Human Skin
It is a question of studying the effect of the monosaccharide fucose and the mixture of oligosaccharides "Mixture-1" as prepared in Example 1 above on the activity of the matrix metalloproteases (or MMPs) of the fibroblasts at 1 quiescent state or stimulated by hyaluronan. We performed these tests from cell cultures of

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Human skin fibroblasts grown in monolayers but also from explant culture to study the effect of different treatments in a cell environment closer to that of the skin. Indeed, the dermal fibroblasts are surrounded by an abundant extracellular matrix in vivo, the explant culture is a type of culture reconstituting the natural environment of the cells and thus making it possible to study the effect of the treatments on the activity. MMP fibroblasts under conditions close to physiological conditions. a) Method al) Skin explant culture a. 1.1) MMP activity of unstimulated skin explants
Skin explants from a belly skin of a 54-year-old woman are cultured in a DMEM medium without phenol red (GIBCO) with shaking at 37 ° C. for 48 hours with the various treatments. The samples are tested at a concentration of 10 μg / ml.

Sample Recovery: Extracellular medium: 1 ml of medium Intracellular medium: explant milled with ultraturax in 1 ml of MMP buffer Composition of MMP buffer: 0.1M Tris-HCl, pH 7.5, 1.0 M NaCl, 10 mM CaCl 2, 1 mM zinc acetate
0.01% Brij 35, 0.01% NaN 3 Zymography: Gelatin, MMP-s substrate, is copolymerized at 1 mg / ml final with a 10% polyacrylamide gel. Extracellular media from skin explant cultures are deposited on the top of the gel, electrophoresis is done at 150 volts. The gels are then incubated in MMP buffer for 24 hours with stirring at 37 ° C. The gels are then stained with Coomassie blue and then decolorized with distilled water. The lysis ranges, corresponding to an MMP activity, appear as white while the bottom of the gel is blue. Depending on the migration distance of

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proteins, we can determine the molecular weights of the enzymes that digested gelatin. The MMP activity is quantified by morphometric analysis using the Visiolab software. a.1.2) MMP activity of skin explants stimulated with 1 mg / ml of hyaluronan
The study protocol is the same as that used for the study of MMP activity of unstimulated skin explants but with hyaluronan at a concentration of 1 mg / ml and oligosaccharide samples at a concentration of 10 g / ml. ml, added in the culture medium. at. 2) Culture of skin fibroblasts a. 2.1) MMP activity of unstimulated skin fibroblasts
Skin fibroblasts from a belly skin of a 45-year-old woman are cultured until the 7th cell passage in DMEM Glutamax (GIBCO) containing 1% antibiotics and fungicide and 10% calf serum f # tal and are used for experimentation. The fibroblasts are seeded in 6-well plates at a density of 5 × 10 4 cells per well. When they are at the confluence, the culture medium is replaced by a DMEM medium without phenol red containing 0.1% BSA and containing the various substances to be treated at a concentration of 10 g / ml. After 24 hours of culture at 37 C (5% (v / v) CO2, 95% (v / v) air) the samples are recovered.

Extracellular medium: 1 ml of medium + 2 rinses with 0.25 ml of PBS Intracellular medium: sonicated cellular carpet with 1 ml of MMP buffer Composition of MMP buffer: 0.1 M Tris-HCl, pH 7.5.0.1 M NaCl, 10 mM CaCl 2, 1 mM zinc acetate
0.01% Brij 35, 0.01% NaN3 As in the case of skin explant cultures, the fibroblast culture media are studied in zymography.

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at. 2.2) MMP activity of skin fibroblasts stimulated with lmg / ml of hyaluronan
The study protocol is the same as that used for the study of MMP activity of unstimulated skin fibroblasts but with hyaluronan at a concentration of 1 mg / ml and oligosaccharide samples at a concentration of 10 g / ml. ml, added in the culture medium. b) Results bl) MMP activity of skin explants
MMP-s are enzymes that are secreted as inactive zymogens. Their activation is by proteolytic cleavage, the active form of the enzyme can then degrade the extracellular matrix compounds. It is therefore important to know not only the level of expression of MMP-s but also the proportion of MMP present in active form, only capable in vivo of degrading the extracellular matrix, as well as enzymes capable of activating latent forms. The results are therefore presented as the ratio of the active form / inactive form of the enzyme and the percentages relative to the control are calculated to show the effectiveness of fucose and Mix-1 on their ability to reduce the active proportion of MMPs. and optionally inhibiting the activation of the inactive form.

Table 4: MMP activity of unstimulated skin explants Ratio of the active form of the enzyme / inactive form% of treatment efficacy: reduction of this ratio

<Tb>
<tb> MMP-9 <SEP>% <SEP> MMP-2 <SEP>%
<tb> Active / Inactive <SEP> Efficiency <SEP> Active / Inactiv <SEP> Efficiency
<tb> e
<tb> Control <SEP> 5.48 <SEP> 0.85
<tb> Fucose <SEP> 10 <SEP> g / ml <SEP> 2.38 <SEP> 57 <SEP>% <SEP> 0.80 <SEP> 6 <SEP>%
<tb> Mixture-1 <SEP> 10 <SEP> g / ml <SEP> 3.09 <SEP> 46 <SEP>% <SEP> 0.67 <SEP> 21 <SEP>%
<Tb>
These results are also presented in the form of the histogram in Figure 1.

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 Fucose and Mixture-1 are able to decrease the ratio of the active form of the enzyme / inactive form. This phenomenon is more important for MMP-9 than for MMP-2. This decrease in this ratio implies that the amount of active enzyme, thus capable of degrading the extracellular matrix in vivo, is less important when the skin explants are cultured in the presence of fucose or of Mixture-1.

Table 5: MMP activity of skin explants stimulated with 1 mg / ml of hyaluronan Ratio of the active form of the enzyme / inactive form% of treatment efficacy: reduction of this ratio

<Tb>
<tb> MMP-9 <SEP>% <SEP> MMP-2 <SEP>%
<tb> Active / Inactive <SEP> Efficiency <SEP> Active / Inactive <SEP> Efficiency
<tb> Control <SEP> 7.08 <SEP> - <SEP> 1.58 <SEP> Fucose <SEP> 10 <SEP> g / ml <SEP> 3.54 <SEP> 50% <SEP> 1, 65 <SEP> Ns
<tb> Mixture <SEP> 1 <SEP> 10 <SEP> g / ml <SEP> 1.98 <SEP> 72 <SEP>% <SEP> 0.98 <SEP> 38 <SEP>% <SEP>
<Tb>
These results are also presented in the form of the histogram in Figure 2.

The presence of hyaluronan in the culture medium of the skin explants causes an increase in the active form of the MMP-s. Fucose and Mixture-1 reduce this increase and are therefore able to inhibit this stimulation of active enzyme release. b. 2) MMP activity of fibroblasts
When fibroblasts are cultured in monolayer, only the largely secreted MMP-2 is visible with a very small amount of MMP-9 nevertheless present. Only the inactive form of the MMP-2 enzyme is released.

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Table 6: MMP activity of unstimulated skin explants% of treatment efficacy: reduction of MMP-2 expression

<Tb>
<Tb>
<tb> efficiency
<tb> Control
<tb> Fucose <SEP> 10 <SEP> g / ml <SEP> 6 <SEP>% <SEP>
<tb> Mixture-1 <SEP> 10 <SEP> g / ml <SEP> 22 <SEP>%
<Tb>
These results are also presented in the form of the histogram in Figure 3.

 It can be seen that fucose and Mix-1 decrease the expression of MMP-2. Mixture-1 seems to be more effective than fucose to diminish this expression.

Table 7: MMP activity of skin explants stimulated with 1 mg / ml of hyaluronan% of treatment efficacy: reduction of MMP-2 expression compared to control

<Tb>
<Tb>
<tb> efficiency
<tb> Control
<tb> Fucose <SEP> 10 <SEP> g / ml <SEP> 11 <SEP>%
<tb> Mixture-1 <SEP> $ 10 <SEP> g / ml <SEP> 30 <SEP>%
<Tb>
c) Conclusion
It emerges from these experiments that fucose and Mixture-1 are able to reduce the synthesis as well as the activation of MMP-2 and 9 by fibroblasts of

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 human skin. Similarly, fucose and Mixture-1 are able to effectively curb overexpression of metalloproteases in the presence of hyaluronan. Given the important role of these metallo-endopeptidases in the degradation of the cutaneous extracellular matrix during aging, this is a major "anti-aging" effect.

Example 4 Activity of Fucose and Mixture-1 on Human Skin Fibroblasts
The objective of this study was to analyze the action of the mixture-1 of Example 1 on one of the characteristic parameters of cellular communication and the action of preventing the aging of human skin fibroblasts, knowledge, stimulation of cell proliferation of human skin fibroblasts a) Methodology: study of cell proliferation
The human skin fibroblasts used in this study come from a skin sample of a 20-year-old woman (26th passage). The cells are cultured in 12-well plates, in a DMEM culture medium with 10% fetal calf serum (FCS), 1% antibiotics and antifungals (FFP), and luCi / ml of [ 3H] thymidine (ICN) for 72 hours in the presence of the test products at 2 final concentrations for each sample: 1 μg / ml and 10 μg / ml.

After 72 hours of incubation (5% (v / v) CO2, 95% (v / v) air) at 37 ° C. in the presence of the samples, the cells are washed four times with PBS, and then the cell mat detached by 0.05% trypsin. Three ml of scintillation fluid is then added per sample, and the radioactivity incorporated into the cells is read in a scintillation counter. b) Results: action on cell proliferation
The 72 hour incubation of fibroblasts with the two samples tested (lug / ml and 10 μg / ml concentrations) significantly stimulated cell proliferation compared to untreated cells (see Table 8 below).

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Table 8: Different Concentrations of Mixture-1 on the Cellular Proliferation of Human Skin Fibroblasts (Proliferation versus Control)

<Tb>
<tb> Efficiency <SEP> by
<tb> report <SEP> of <SEP> p <SEP> by <SEP> report
<tb> Product <SEP> Concentration <SEP> control <SEP> at <SEP> control
<tb> Control
<tb> ***
<tb> Mixture-1 <SEP> 1 <SEP> g / ml <SEP> + 48.2% <SEP> 0.000
<tb> **
<tb> Mixture-1 <SEP> 10 <SEP> g / ml <SEP> + 30.8% <SEP> 0.002
<Tb>
Example 5: Age Cream

<Tb>
<tb>% <SEP> (in <SEP> weight)
<tb> Component
<tb> Water <SEP> qsp <SEP> 100%
<tb> Sodium <SEP> benzoate <SEP> 0.2
<tb> Disodium <SEP> EDTA <SEP> 0.08
<tb> Glycerin <SEP> 2.00
<tb> Butylene <SEP> Glycol <SEP> 4.00
<tb> Carbomer <SEP> ETD <SEP> 2020 <SEP> 0.20
<tb> Ceteareth-20 <SEP> 1.00
<tb> Mineral <SEP> Oil <SEP> 3.00
<tb> Squalane <SEP> 2.00
<tb> Octyl <SEP> Palmitate <SEP> 6,00
<tb> Shea <SEP> Butter <SEP> 2.50
<tb> Cetearyl <SEP> Alcohol <SEP> 1.00
<tb> Rosa <SEP> SEA <SEP> Rubiginosa <SEP> Seed <SEA> Oil <SEP> 0.20
<tb> Decyl <SEP> Oleate <SEP> 0.50
<tb> Octyl <SEP> Methoxycinnamate <SEP> 5.00
<tb> Butyl <SEP> Methoxy-dibenzoylmethane <SEP> 0.50
<tb> BHA <SEP> 0.01
<tb> Cyclomethicone <SEP> 5.00
<tb> Cyclomethicone <SEP>&<SEP> Dimethiconol <SEP> 2.00
<tb> Dimethicone <SEP> 2.00
<tb> Fragrance <SEP> (Crematest <SEP> Feno) <SEP> 0.09
<tb> Fragrance <SEP> (Chemoderm) <SEP> 0.09
<tb> Triethanolamine <SEP> 0.30
<tb> 2-Bromo-2-Nitropropane-1,3-Diol <SEP> 0.02
<tb> Mixture-1 <SEP> 0.50
<Tb>

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Example 6: anti-aging cream

<Tb>
<tb>% <SEP> (in <SEP> weight)
<tb> Component
<tb> Water <SEP> qsp <SEP> 100%
<tb> Sodium <SEP> benzoate <SEP> 0.2
<tb> Disodium <SEP> EDTA <SEP> 0.08
<tb> Glycerin <SEP> 2.00
<tb> Butylene <SEP> Glycol <SEP> 4.00
<tb> Carbomer <SEP> ETD <SEP> 2020 <SEP> 0.20
<tb> Ceteareth-20 <SEP> 1.00
<tb> Mineral <SEP> Oil <SEP> 3.00
<tb> Squalane <SEP> 2.00
<tb> Octyl <SEP> Palmitate <SEP> 6,00
<tb> Shea <SEP> Butter <SEP> 2.50
<tb> Cetearyl <SEP> Alcohol <SEP> 1.00
<tb> Rosa <SEP> SEA <SEP> Rubiginosa <SEP> Seed <SEA> Oil <SEP> 0.20
<tb> Decyl <SEP> Oleate <SEP> 0.50
<tb> BHA <SEP> 0.01
<tb> Cyclomethicone <SEP> 5.00
<tb> Cyclomethicone <SEP>&<SEP> Dimethiconol <SEP> 2.00
<tb> Dimethicone <SEP> 2.00
<tb> Fragrance <SEP> (Crematest <SEP> Feno) <SEP> 0.09
<tb> Fragrance <SEP> (Chemoderm) <SEP> 0.09
<tb> Triethanolamine <SEP> 0.30
<tb> 2-Bromo-2-Nitropropane-1,3-Diol <SEP> 0.02
<tb> Mixture <SEP> 0.50
<Tb>

Claims (26)

claims 1. A mixture of non-sulphated oligosaccharides based on fucose, characterized in that it comprises oligosaccharides of less than 13 saccharide units comprising at least one non-reducing terminal fucose unit, and in that it is capable of be obtained by a process comprising at least one step of degradation of a polysaccharide derived from a microorganism of the genus Klebsiella pneumoniae subsp. pneumoniae.  2. Oligosaccharide mixture according to claim 1, characterized in that it comprises, relative to the total weight of the mixture, at least 15% by weight of oligosaccharides of less than 13 saccharide units comprising at least one terminal fucose unit. non-reducing 3. Mixture of oligosaccharides according to claim 1 or 2, characterized in that it comprises, relative to the total weight of the mixture, from 20 to 50% by weight of oligosaccharides of less than 13 saccharide units comprising at least one fucose unit in a non-reducing end position.  4. Mixture of oligosaccharides according to any one of the preceding claims, characterized in that it further comprises, relative to the total weight of the mixture, from 25 to 45% by weight of oligosaccharides having from 13 to 24 saccharide units comprising at least one non-reducing terminal fucose unit.  5. Mixture of oligosaccharides according to any one of the preceding claims, characterized in that it further comprises, relative to the total weight of the mixture, from 15 to 35% by weight of oligosaccharides of more than 54 saccharide units comprising at least one unit fucose terminal non-reducing.  6. oligosaccharide mixture according to any one of the preceding claims, characterized in that the oligosaccharides comprise, at least in part, the fucose-galactose-galacturonic acid repeating unit.  7. Oligosaccharide mixture according to any one of the preceding claims, characterized in that it is obtainable by the process comprising the steps of: a) growing the microorganism of the genus Klebsiella pneumoniae subsp. pneumoniae in an aqueous nutrient medium by aerobic fermentation of an assimilable carbohydrate source; <Desc / Clms Page number 28>  b) recovering the polysaccharide formed from the fermentation must; c) subjecting the polysaccharide to moderate hydrolysis; d) subjecting the hydrolysis product of step c) to enzymatic hydrolysis; and e) deactivating the enzyme and then recovering the oligosaccharide mixture thus formed.  8. Oligosaccharide mixture according to any one of the preceding claims, characterized in that the microorganism Klebsiella pneumoniae subsp. pneumoniae is the microorganism deposited in the National Collection of Cultures of Microorganisms under the number 1-1507, or a mutant of the latter.  9. A mixture of oligosaccharides according to claim 7 or 8, characterized in that the moderate hydrolysis is carried out by a treatment selected from the group consisting of gamma ray treatments, protolysis treatments and combinations of these treatments.  10. Mixture of oligosaccharides according to any one of claims 7 to 9, characterized in that the enzymatic hydrolysis is carried out with at least one endofucosidase 11. Mixture according to claim 10, characterized in that the endo-fucosidase is Fermizyme HCP.  12. Cosmetic or pharmaceutical composition, characterized in that it comprises, as cosmetically or pharmaceutically active agent, at least one mixture of oligosaccharides according to any one of the preceding claims and at least one cosmetically or pharmaceutically acceptable excipient.  13. Composition according to claim 12, characterized in that it is a dermatological composition.  14. Composition according to claim 12 or 13, characterized in that the mixture of oligosaccharides is present in a proportion of between about 0.001 and about 20% by weight, relative to the total weight of the composition.  15. Use of the oligosaccharide mixture according to any one of claims 1 to 11, for the preparation of a composition for stimulating cellular communication between the skin cells. <Desc / Clms Page number 29>  16. Use of the oligosaccharide mixture according to any one of claims 1 to 11, for the preparation of a composition for stimulating the cellular proliferation of keratinocytes of the skin.  17. Use of the oligosaccharide mixture according to any one of claims 1 to 11, for the preparation of a composition for stimulating cell proliferation of skin fibroblasts.  18. Use of the oligosaccharide mixture according to any one of claims 1 to 11, for the preparation of a composition for inhibiting the synthesis of elastase proteases by skin fibroblasts.  19. Use of the oligosaccharide mixture according to any one of claims 1 to 11, for the preparation of a composition for inhibiting the overexpression of hyaluronan-induced MMP-2 and MMP-9 proteases.  20. Use according to claim 18 or 19, characterized in that the composition is intended to inhibit the synthesis of proteases MMP-2 and MMP-9 by the fibroblasts of the skin.  21. Use according to claim 19 or 20, characterized in that the composition is intended to reduce the sensitivity of the skin to irritation.  22. Use of the oligosaccharide mixture according to any one of claims 1 to 11, for the preparation of a composition for stimulating the deposition of collagen fibers in the dermis.  23. Use according to any one of claims 15 to 22, characterized in that the mixture of oligosaccharides is used in a proportion of between about 0.001 and about 20% by weight, relative to the total weight of the composition.  24. Use according to any one of claims 15 to 23, characterized in that the composition prepared further comprises a cosmetically or pharmaceutically acceptable excipient.  25. A method of cosmetic treatment of the skin, characterized in that a cosmetic composition comprising at least one oligosaccharide mixture as defined in any one of Claims 1 to 11 is applied to the skin.  26. Cosmetic treatment method according to claim 25, characterized in that the cosmetic composition further comprises a cosmetically acceptable excipient.