WO2015044892A1

WO2015044892A1 - Method for preparing cleaved bioactive oligosaccharides, micro-organism for its implementation, prepared oligosaccharides and cosmetic use thereof

Info

Publication number: WO2015044892A1
Application number: PCT/IB2014/064830
Authority: WO
Inventors: Frederic De Baene; Marion FRANCOIS
Original assignee: Sederma
Priority date: 2013-09-27
Filing date: 2014-09-25
Publication date: 2015-04-02
Also published as: FR3011248B1; FR3011248A1

Abstract

The method consists of providing a micro-organism able to produce at least one osidic cleavage lyase enzyme; contact green algae polysaccharides, preferably of the Ulva genus, with said lyase enzyme to induce said cleavage; and to recover said cleaved oligosaccharides. According to the invention, the micro-organism is selected from the bacteria group of the Flavobacteriaceae family consisting of the Jejuia pallidilutea strain, the Flavobacteriaceae bacterium strain, the Gaetbulibacter marinus strain and the strain referenced CNCM I-4797. The mixture of cleaved oligosaccharides obtained has a cosmetic activity on the skin cells, including anti-inflammatory, anti-radical, de-pigmenting effects and effects on molecules of the dermal extracellular matrix (such as collagen 1, collagen 4 and fibronectin), on molecules of the epidermal extracellular matrix (hyaluronic acid).

Description

METHOD FOR PREPARING CLEAVED BIOACTIVE OLIGOSACCHARIDES, MICROORGANISM FOR ITS IMPLEMENTATION, PREPARED OLIGOSACCHARIDES

AND COSMETIC USE THEREOF

TECHNICAL FIELD

The invention relates to a method for preparing cleaved bioactive polysaccharides obtained by enzymatic cleavage of polysaccharides from green algae, to a new micro-organism for implementing said method, to cleaved oligosaccharides obtained according to this method and their cosmetic use. The present invention thus concerns in particular the cosmetics industry that manufactures and/or uses products for the treatment of the skin, scalp, mucous membranes and appendages (such as hair, eyelashes, eyebrows, nails, body hairs) of mammals, animals or humans, to improve their appearance and/or global condition.

The cosmetics industry is increasing demand for new products, particularly for new active ingredients that are derived from plant material to be part of a sustainable development approach in comparison with a synthetic or chemical way of manufacturing.

Algae are thus an abundant source of raw material valued in many fields such as food, pharmaceuticals and cosmetics.

In cosmetics, algae are already widely used but mostly for the thickening or gelling properties of their extracts which contain mainly polysaccharides.

Polysaccharides form also a particularly interesting molecule class especially because they contain sequences similar to some bioactive oligomers.

The Applicant has found particular interest in green algae and more particularly in ulva genus, and in the ulva lactuca species. It is known that polysaccharides from algae of ulva genus include in particular osidic units of iduronic acid (IdopA) and rhamnose 3 sulfate (Rhap3 sulfate) having an interesting potential for therapeutic or cosmetic applications (WO2009/016275). The Applicant has found particular interest in the two types of ulva polysaccharides that can be extracted from green algae walls of the ulva genus and containing such osidic units:

1) Ulvan polysaccharide comprising two main sequences, referred to as " aldobiuronic acid sequences ":

- A first sequence

and

- A second sequence [->4)-a-L-IdopA-( l ->4)- a-L-Rhap3 sulfate-(l ->]_m.

In these sequences, the « GlcpA » units correspond to glucuronic acid units.

Within this polysaccharide, glucuronan type sequences can also be found, but in minority.

These sequences contain enchainments of glucuronic acid units:

[^4)- -D-GlcpA-(1 ^4)- -D-GlcpA-(l ^]x- 2) Glucuronan polysaccharide comprising in majority glucuronan osidic sequences containing enchainments of glucuronic acid units

[^4)- -D-GlcpA-(1 ^4)- -D-GlcpA-( l ^]x, as disclosed above. An enzyme from the lyase class is capable of causing cleavage of the glycosidic bond between the osidic units according to a β elimination reaction (or transelimination). An ulvan-lyase enzyme is adapted to cleave more particularly the osidic bond between GlcpA and Rhap3 sulfate and between IdopA and Rhap3, whereas a glucuronan-lyase enzyme is more particularly adapted to cleave the osidic bond between lowing:

This cleavage processus shows saccharidic molecule fragments, namely the oligosaccharides, having at the created non-reducing terminal end, a heterocycle having an ethylenic bond between carbon 4 and carbon 5, this residue corresponding to a 4-deoxy-(hex-4-ene) pyranosyluronic acid. The apparition of this ethylene bond and the dosage of the formed products can be monitored by UV spectroscopy at 235nm.

Cleaved oligosaccharides consist of oligo-ulvans and oligo-glucuronans. The proportion of each depends on the proportion of each of the starting glucuronan and ulvan polysaccharides contacted with the enzymatic mixture.

Cleavage processes of ulva polysaccharides are described consisting of:

- Providing a microorganism capable of producing an enzymatic mixture of lyases;

- Contacting the polysaccharides extracted from ulva genus algae with said enzymatic lyase mixture to cause cleavage of the osidic bonds, and

- Recovering the cleaved oligosaccharides.

In the above mentioned WO2009/016275 document, a microorganism of Ochrobactrum triciti type and filed under the reference 1-3776 at the CNCM (National Collection of Micro-organism cultures, Institut Pasteur, Paris) is used to produce an enzyme mixture having a double enzymatic activity, ulvan lyase and glucuronan lyase, allowing thus to obtain a mixture of oligo-glucuronans and oligo- ulvans having a polymerisation degree (DP) inferior to 25 and with good yields for industrial production. According to the present application, a DP corresponds to a diholoside unit (containing two oses). For example, a sequence [->4)- -D-GlcpA-(l->4)-a-L-Rhap3 sulfate-(l->]_n corresponds to a DP of 1, comprising one glucose/rhamnose diholoside unit.

The marine bacterium Nonlabens ulvanivorans of the flavobacteriaceae family filed under number I- 4324 at the CNCM has also been proposed for such a glycoside cleavage.

The aim of the present invention is to propose an alternative method for preparing cleaved bioactive oligosaccharide mixtures obtained by enzymatic cleavage of polysaccharides from green algae.

SUMMARY OF THE INVENTION

The present invention thus provides a method comprising the following steps:

- Providing a micro-organism able to produce at least one osidic cleavage lyase enzyme; - Contact said polysaccharides with said enzyme in order to induce said cleavage; and

- Recover said cleaved oligosaccharides,

wherein said micro-organism is chosen in the Flavobacteriaceae family bacteria group consisting of the Jejuia pallidilutea strain, the Flavobacteriaceae bacterium strain, the Gaetbulibacter marinus strain and the CNCM 1-4797 referenced strain.

These different strains are related in terms of taxonomy. Cleaved oligosaccharides can be obtained with these strains, having particularly interesting cosmetic activities as shown in particular in in vitro test results given below as example.

The present invention encompasses especially the strain CNCM 1-4797. This strain was deposited in the name of the Applicant at the National Collection of Microorganism Culture (CNCM) at the headquarters of the Institut Pasteur, 25 rue du Docteur Roux, F-75724 Paris cedex 15.

Preferably, the method according to the invention is implemented with this new strain CNCM 1-4797.

The Jejuia pallidilutea strain is for example available from the DSMZ collection (German Collection of Microorganisms and Cell Cultures, InhoffenstraBe 7B 3812 Braunschweig, Germany) under the reference DSMZ21165.

The Gaetbulibacter marinus strain is available for example from the "National Institute of Technology and Evaluation (NITE)", # 120, Kazusakamatari 2-5-8, Kisarazu-shi, Chiba 292-0818, Japan, under the reference NBRC 102040.

The microorganism according to the invention is capable of producing an ulvan lyase cleavage enzyme and/or a glucuronan lyase cleavage enzyme, preferably the two enzymes. Thus, the method according to the invention is preferably implemented to achieve the cleavage of sulfated polysaccharides derived from green algae of the Ulva genus, particularly Ulva lactuca, comprising sulfated ulvan and/or glucuronan polysaccharides.

As explained above, said polysaccharides extracted from green algae of the Ulva genus comprise:

- Aldobiuronic sequences [^4)- -D-GlcpA-(1^4)-a-L-Rhap3 sulfate-(l^]„ and [-»4)-a-

L-IdqpA-(1^4)- a-L-Rhap3 sulfate-(l^]_m; and

- Glucuronan sequences [^4)- -D-GlcpA-(1^4)- -D-GlcpA-(l ^k

Wherein in said sequences, the GlcpA units corresponding to glucuronic acid units, Rhap3 sulfate to rhamnose 3 sulfate units and IdqpA to iduronic acid units.

The ulvane-lyase enzyme according to the invention is adapted to cleave the osidic bond between

GlcpA and Rhap3 sulfate units and between IdqpA and Rhap3 sulfate units, whereas the gucuronan lyase according to the invention is adapted to cleave the osidic bond between two GlcpA units.

Preferably, according to the invention, the method is limited to the cleavage of ulvan polysaccharides priorly removed from glucuronan type polysaccharides and thus comprising mostly aldobiuronic sequences, the oligosaccharide mixture obtained being thus enriched in oligo-ulvans. The present invention also encompasses the cleaved bioactive oligosaccharides prepared according to the method of the invention, the present invention also covering the non-therapeutic cosmetic use for the skin and its appendages of said cleaved oligosaccharides. The cosmetic treatment can be selected in particular from a depigmenting, anti -radical, moisturizer (eg. via the synthesis of hyaluronic acid in epidermal matrix) or anti-aging via stimulation of molecules the dermal extracellular matrix (collagen 1, 4 collagen and fibronectin) treatments. In particular, via the stimulation of the synthesis of molecules of the dermal extracellular matrix, the loss of skin density and firmness, wrinkles and fine lines, can be treated, and via the stimulation of the molecules of the epidermal matrix, loss of hydration, in particular transepidermal water loss, can be treated. Specific skin disorders can also be aimed, such as stretch marks, redness, and reactive skin. Skin treatments can include soothing, healing, etc.

Furthermore, according to the invention, it has been shown that particularly advantageous cosmetic activities can be obtained for example with a mixture of cleaved oligosaccharides having a DP between 2 and 10 by comparison to other DPs. Other fractions of oligosaccharide mixture can be obtained belonging to a different DP interval according to the operating conditions implemented for the process. Generally, oligosaccharides having a DP less than 50, more preferably less than 20, are seeked.

According to the invention, a topical cosmetic composition is also provided comprising bioactive sulfated oligosaccharides of the invention and a physiologically acceptable medium.

The polymerization degree (DP) is measured by an ion chromatographic method. The average weight of the fraction is 6 kD measured by CPC/ELSD (centrifugal partition chromatography) with a degree of sulfation of from 13 to 20%.

According to other advantageous features, the oligosaccharides can be used in combination with one or more additional active ingredients, preferably to provide a range of wider cosmetic properties. The additional active ingredients are used either to reinforce or to supplement activity.

The additional active ingredients can be for example selected from lightening agents, anti-redness, anti-spots, calming, for treating reactive or sensitive skins, UV sunscreens, moisturizing, humectant, exfoliating, smoothing, toning, firming, volumizing, anti-aging, anti-wrinkles and fine lines, improving the mechanical and elastic properties, skin radiance, detoxifying actives, anti-hair-growth, acting on cutaneous barrier, anti -acne, action of sebum secretion, matifying, unifying, antiinflammatory, anti-oxidant, anti-radical, anti-glycation, for eye contour (dark circles and under eye bags), promoting blood circulation, peptides, vitamins etc. These active ingredients can be obtained from plant materials such as plant extracts or products of plant cell culture or fermentation.

More specifically, the oligosaccharides of the present invention can be combined advantageously with oligo-glucuronans, in particular the acetylated oligo-glucuronans disclosed in the patent application WO20120067327 and sold under the trade name Subliskin™ by Sederma. They are oligomer compounds of D-glucuronic acid or D-glucuronate with a β (1-4) sequence according to the following formula (

Preferably each ring of glucuronic acid of formula (I) comprises an acetylation degree comprised between 8,7±0,5 and 9,2±0,5% in weight of 0-CO-CH₃ groups with regard to the weight of the glucuronic acid ring and a polymerisation degree comprised between 18 and 19 ±2. These oligomer compounds act at the level of dermal and epidermal elasticity and increase the dermo-epidermal cohesion in order to fight against skin ageing, wrinkles, fine lines, tactile and/or visible skin discontinuities, loss of firmness, of elasticity and tonus, and to fight against cutaneous tissue deformability. They also act on skin hydration.

Usual cosmetic active ingredients such as B3 vitamin compounds like niacinamide and tocopherol, retinoid compounds such as retinol, hexamidine, a-lipoic acid, resveratrol or DHEA, peptides, including N-acetyl-Tyr-Arg-O-hexadecyl, Pal-VGVAPG (SEQ ID NO: 1), Pal-KTTKS (SEQ ID NO: 2), Pal-GHK, Pal-KM0₂K and Pal-GQPR (SEQ ID NO: 3) can be combined with the oligosaccharides of the present invention.

Other examples of additional ingredients are given below in the detailed description.

The present invention also aims a topical treatment method of the skin and its appendages, comprising the topical application to the skin and/or the appendages of a subject in need thereof of an effective amount of a composition according to the invention comprising the oligosaccharides as recited above. "Topical treatment" means according to the invention an application that is intended to act where it is applied: skin, mucous membranes and/or skin appendages.

Improvements in the appearance and general condition of the skin, mucous membranes, and appendages can be obtained by topical application on a regular basis such as daily.

The topical composition is preferably applied once daily for a period of at least a week, but it can be applied during periods of 2, 4, 8 or 12 weeks. The topical composition is preferably applied to the face and neck, but can be applied to any part of skin requiring an aesthetic improvement, where the composition remains on the skin area to be treated, and preferably is not removed or flushed from the skin.

It is also to be understood that, as used herein, the terms treating and treatment include and encompass the reduction, improvement, progress, relief, and/or elimination of dermatological effects, including aging. The compositions of the present invention and the methods are suitable for use to treat skin conditions of the skin in many areas of the skin, including without limitation, the face, forehead, lips, neck, neckline, arms, hands, body, legs, knees, feet, chest, back, buttocks, and others. One of the major advantages of the present invention resides in the ability whenever necessary or desirable to be able to apply local selective "gentle" treatments through this topical, non-invasive method of application. In the case of anti-wrinkle use for example it may be applied very locally using a syringe or micro-canula.

The topical composition according to the invention is generally applied twice daily, morning and evening, but can also be applied once a day, for a period of at least one week, and that can be up to 12 weeks. As an indication, for a cosmetic treatment of the face, the European standard assay for a cream is 2.72 mg/cm²/day/person, and for a cosmetic treatment of the body of 0.5 mg/cm²/day/person (according to data published by the EU Scientific Committee for Consumer Safety (SCCS); « SCCS'S NOTES OF GUIDANCE FOR THE TESTING OF COSMETIC SUBSTANCES AND THEIR SAFETY EVALUATION 8TH REVISION », 11 decembre 2012)).

According to other specific features, the cosmetic treatment method according to the invention can be combined with one or more other treatment methods targeting the skin such as lumino-therapy, heat or aromatherapy treatments.

According to the invention, devices with several compartments or kits may be proposed to achieve the method described above which may include for example and non-restrictively, a first compartment containing a composition comprising the oligosaccharides of the invention, and in a second compartment a composition containing another active ingredient and/or excipient, the compositions contained in the said first and second compartments in this case being considered to be a combination composition for simultaneous, separate or stepwise use in time, particularly in one of the treatment methods recited above.

The present invention will be better understood in the light of the following detailed description.

DETAILED DESCRIPTION

A. Preparation of the oligosaccharides of the invention

Polysaccharide forming the starting substrate

A mixture of polysaccharides comprised in the walls of ulva lactuca genus algae is heat extracted. After elimination of the insoluble elements, the pH of the extract is lowered to 2 with diluted acid in order to form a precipitate comprising glucuronic acid residues. The solution is thus recovered and brought to a neutral pH around 7. The solution is then purified, for example by ultrafiltration and precipitated with alcohol. The glucuronan polysaccharide fraction is thus eliminated and an ulvan polysaccharide is obtained comprising essentially aldobiuronic sequences, the glucuronan sequence left being in minority and the one integrated within the ulvan polysaccharide. The aim is here to subject only the ulvan polysaccharide to enzymatic cleavage in order to recover oligo-ulvans in majority (a mixture enriched in oligo-ulvan) but it goes without saying that the process can also according to a variant be achieved on an ulvan and glucuronan polysaccharide mixture. The mean molecular weight of the ulvan polysaccharide is determined by sterical exclusion chromatography coupled to a light diffusion detector in line with a refractometer (SEC-MALLS technolocy, for « Size Exclusion Chromatography Coupled to Multi-angle Laser-Light-Scattering »); this mean weight is 6.10⁵.

Preparation of the bacterial strain

From one of the bacterial strains of the invention, here the strain referenced CNCM 1-4797, an enzymatic substance is prepared having potentially a double enzymatic activity: ulvan-lyase and glucuronan-lyase .

The CNCM 1-4797 strain according to the invention has the following characteristics: it is pigmented (orange/yellow color), gram -negative and aerobic. Colonies on agar culture medium are creamy and opaque. In microscopy, medium sized non-mobile bacilli are observed. The strain is grown on a mineral minimal nutritive medium comprising: NaCl: 22g; Na₂SC>4_: 3.7g; KC1: 0.6g; KBr: O. lg;

MgCl₂,6H₂0: lOg; CaCl₂,2H₂0: 2.94g; NaHC0₃: 0.16g; NaN0₃: 25mg; NaH₃P0₄, 12H₂0: 5mg and

H₂0 QSP 1 liter; at a pH of 7.2.

The ulvan polysaccharide as prepared above (2g/L) is added to this nutrient medium as a carbone source substrate.

The strain, capable of degrading this ulvan polysaccharide, is isolated on a petri dish containing the same nutrient medium supplemented with agar. After incubation, colonies of strain, which metabolize the substrate by degrading it according by β-elimination and which are the most developed, are gathered.

Preparation of the enzyme activity

The CNCM 1-4797 strain is inoculated in a 2 liters capacity bioreactor, filled with 1.5 liters of a mineral medium: NaCl: 22g; Na₂S0₄: 3.7g; KC1 0.6g; KBr: O. lg; MgCl₂,7H₂0: 5mg; NaN0₃: 25 mg; NaH₃P0₄,12H₂0, H₂0 qs 1 liter; at pH of 7.2 and supplemented with yeast extract: 1 g/L; peptone: 5 g/L and ulvan polysaccharide of (2g/L).

Incubation is preferably carried out between 25 and 35°C under stirring for an average duration of 48 hours. During incubation, the concentration of enzyme mixture (ulvan-lyase and glucuronan-lyase) in the culture medium of the strain increases and therefore the enzymatic activity of the culture medium increases as well.

The culture medium is then cleared of the bacteria after incubation followed by addition of ammonium sulfate (60% w/v) to precipitate the proteins contained therein. This is followed by centrifugation of the precipitated culture medium and recovering of the protein base which is then reintroduced into about 10 ml of a buffer solution of Tris/HCl 20 mM at pH 7.5.

Chromatography on anion exchange resin can then retrieve only the fraction of an enzymatic substance having in majority an ulvan lyase activity and in minority a glucuronan lyase activity. An enzymatic activity of 30.10^"3 μιηοΐ/ml/min was quantified by measuring the absorbance at 235 nm. Enzymatic cleavage

The enzyme substance obtained above is applied to a substrate solution at 50g of ulvan polysaccharide for 1 liter of enzymatic solution.

The degradation experience is carried out at 20-30°C for a 3 hours incubation period (theoretically ranging from 1 to 72h).

The chromatographic profile by SEC-MALLS technology described above allows monitoring the oligo-ulvan fraction expected according to the invention (its average mass).

Analysis of the product resulting from the degradation by size exclusion chromatography on P2 Biogel (refractometry detection) highlights a major fraction analyzed by ESI-Q/TF type mass spectrometry (for electrospray-quadrupole-time of flight). The oligosaccharides have a polymerization degree DP comprised between 2 and 10, an average weight of 6kD and a sulfate ratio of 16.6%.

This method can be implemented on the three other bacterial strains according to the invention: Jejuia pallidilutea referenced DSMZ21165, the Flavobacteriaceae bacterium bacterial strain or the Gaetbulibacter marinus bacterial strain.

B. In vitro Tests

The various following tests were conducted on the oligosaccharide mixture obtained in section A above, with the CNCM 1-4797 strain, to highlight the cosmetic activity.

1) Measurement of the anti-inflammatory effect on normal human fibroblasts (NHF)

FHN are grown until confluence. At this stage, they are put in contact with the products to be tested for 24h, then the cell mat are irradiated (UVB 70mJ/cm2) and contacted again with the products for 24 hours. The amounts of synthesized PGE2, IL-6 and IL-8 are measured in the culture supematants by ELISA assay.

2) Measurement of the effect on the epidermal extracellular matrix (ECM):

Human keratinocytes (HaCat) are cultured for 24h in plates. The cells are contacted or not with the products to be tested for 3 days. The culture supematants are removed and the hyaluronic acid amount is assayed. Retinoic acid is used as a positive control.

3) Measurement of the anti-radical effect (DCFH intracellular ROS):

FHN are seeded in well plates. After 24 hours of attachment, the cells are contacted for 24h with the products. Products are removed and the mats are rinsed. Cells are loaded for 30min with a "DCFH- DA" probe which becomes fluorescent in the presence of ROS (reactive oxygen species). After rinsing, the cells were placed again in contact with the products and the stress agent (800um H₂O₂) for 2 hours. A fluorescence reading is used to estimate the amount of ROS present in the cells.

4) Measurement of the effect on dermal MEC (FHN)

FHN are grown in plate for 24 hours. The cells are contacted or not with the products to be tested for 3 days. The synthesis of collagen 1, collagen 4 and fibronectin is assessed by ELISA. 5) Measurement of the depigmenting effect:

Mouse melanoma cells (B 16) are cultured in plates in DMEMc medium (DULBECCO'S Modified Eagle Medium) + 10% FCS (fetal calf serum). After 24 hours of attachment, the cells are contacted with the products to evaluate in the same medium for 48 h. At the end of the contact, the culture media were removed and the total melanin was assayed by spectrophotometry.

Tables 1 and 2 below show the results of the different tests on the oligosaccharides of the invention:

Table 1 :

Table 2:

These results show that the oligosaccharides mixture of the invention has a cosmetic activity.

C. Preparation of a composition according to the invention

The expression "physiologically acceptable medium" means according to the present invention, without limitation, an aqueous or hydro-alcoholic solution, a water-in-oil emulsion, an oil-in-water emulsion, a micro-emulsion, an aqueous gel, an anhydrous gel, a serum, a dispersion of vesicles or a powder.

"Physiologically acceptable" means that the compositions are suitable for topical or transdermal use, in contact with mucous membranes, appendages (nails, hair, body hair), scalp and skin of mammals, particularly human, without risk of toxicity, incompatibility, instability, allergic response, and others. The "physiologically acceptable medium" forms what is commonly called the excipient of the composition.

The choice of excipient of the composition is made according to the constraints of the oligosaccharides (stability, solubility, etc.), and if necessary according to the dosage form intended further for the composition. The oligosaccharides of the invention may be incorporated into the composition by means of an aqueous solution, or be dissolved by the usual physiologically acceptable solubilizers, for example and without limiting to this list: ethanol, propanol, isopropanol, propylene glycol, glycerin, butylene glycol, or polyethylene glycol or any combination thereof. It may also be interesting to solubilize the extract with emulsifiers.

Preferably according to the invention, an aqueous or hydro-alcoholic solution is used as the physiologically acceptable medium.

According to the invention, the effective quantity of oligosaccharides, that is to say their dosage, depends on various factors such as the age, the condition of the patient, etc. An effective amount means a not toxic amount enough to achieve the desired effect, which the skilled person is able to adapt.

More specifically, the amount of oligosaccharides which can be used according to the present invention can vary over a large range and would preferably be between 0.00001% and 50%, more preferably between 0.0001% and 30%, and even more preferably between 1% and 5% by weight compared to the total weight of the composition.

The topical compositions for the present invention are generally prepared by conventional methods well known to one skilled in the art. Such methods may involve a mixture of ingredients in one or more steps to obtain a uniform state, with or without heating, cooling, etc.

The different galenic forms that may contain the oligosaccharides of the invention can be all forms i.e. creams, lotions, milks or creams ointments, gels, emulsions, dispersions, solutions, suspensions, cleansers, foundations, anhydrous preparations (sticks in particular lip balm, body and bath oils), shower and bath gels, shampoo and hair care lotions, milks or creams for skin care or hair, cleansing lotions or milks, sunscreen lotions, milks or creams, artificial tanning lotions, milks or creams, pre shave, shaving or aftershave creams, foams, gels or lotions, makeup, lipstick, mascara or nail polish, skin essences, serums, adhesive or absorbent materials, transdermal patches, or emollient powders, lotions, milks or creams, sprays, body and bath oils, foundation basis, ointment, emulsion, colloid, compact suspension or solid, pencil, sprayable formulation, brushable, blush, red, eyeliner, lipliner, lip gloss, face or body powder, styling gels or mousses, nail conditioning, lip balms, skin conditioners, moisturizers, lacquers, soaps, exfoliants, astringents, depilatories agents, permanent waving solutions, antidandruff formulations, antiperspirant or antiperspirant compositions, including sticks, "roll-on" deodorants, air fresheners, sprays for the nose and etc. These compositions may also be in the form of lipsticks intended either to color the lips or to prevent them from chapping, or makeup for eyes, eyes- shadows and foundations for the face. The compositions for the invention can include cosmetics, personal care products and pharmaceutical preparations. A composition in the form of foam or in the form of aerosol compositions also comprising a pressurized propellant can be considered.

The oligosaccharides according to the present invention may be used in the form of solution, dispersion, emulsion, paste, or powder, individually or as a premix or vehicled individually or as a premix in vectors such as macro-, micro-, or nanocapsules, macro-, micro- or , nanospheres, liposomes, oleosomes or chylomicrons, macro-, micro-, or nanoparticles or macro-, micro or nanosponges, micro- or nanoemulsions, or adsorbed on organic polymer powders, talcs, bentonites, spores or exines, and other inorganic or organic supports.

The oligosaccharides according to the present invention may be used in any form whatsoever, in a form bound to or incorporated in or absorbed in or adsorbed on macro-, micro-, and nanoparticles, or macro-, micro-, and nano-capsules, for the treatment of textiles, natural or synthetic fibers, wools, and any materials that may be used for clothing or underwear for day or night, handkerchiefs or cloths, intended to come into contact with the skin, to exert their cosmetic effect via this skin/textile contact and to permit continuous topical delivery.

Additional ingredients

The CTFA (International cosmetic ingredient dictionary & handbook (13th Ed. 2010)), published by the Cosmetic, Toiletry, and Fragrance Association, Inc., Washington, D.C.) describes a non-limited wide variety of cosmetic and pharmaceutical ingredients conventionally used in the skin care industry that can be used as additional ingredients/compounds in the compositions for the present invention as long as they physically and chemically compatibles with the other ingredients of the composition and especially with the oligosaccharides of the present invention. Also the nature of these additional ingredients should not unacceptably alter the benefits of the oligosaccharides of the invention. These additional ingredients can be synthetic or natural such as plants extracts, or come from bio- fermentation process. Further skin care and hair care active ingredients that are particularly useful combined with the composition can be found in SEDERMA commercial literature and on the website www.sederma.fr.

The following commercial actives can also be mentioned, as examples: betain, glycerol, Actimoist Bio 2™ (Active organics), AquaCacteen™ (Mibelle AG Cosmetics), Aquaphyline™ (Silab), AquaregulK™ (Solabia), Carciline™ (Greentech), Codiavelane™ (Biotech Marine), Dermaflux™ (Arch Chemicals, Inc), Hydra'Flow™ (Sochibo), Hydromoist L™ (Symrise), RenovHyal™ (Soliance), Seamoss™ (Biotech Marine), Argireline™ (trade name of the acetyl hexapeptide-3 of Lipotec), spilanthol or an extract of Acmella oleracea known under the name Gatuline Expression™, an extract of Boswellia serrata known under the name Boswellin™, Deepaline PVB™ (Seppic), Syn-AKE™ (Pentapharm), Ameliox™, Bioxilift™ (Silab), Subliskin™ (Sederma), Venuceane™ (Sederma), Moist 24™ (Sederma), Essenskin™ (Sederma), Juvinity™ (Sederma), Revidrat™ (Sederma), Resistem™ (Sederma), Chronodyn™ (Sederma), Kombuchka™ (Sederma), Chromocare™ (Sederma), Calmosensine™ (Sederma), Glycokin factor S™ (Sederma), Odawhite™ (Sederma), Lumisphere™ (Sederma) , Legance™ (Sederma), Intenslim™ (Sederma), Zingerslim™, (Sederma), Prodizia™ (Sederma), Beautifeye™ (Sederma), Meiritage™ (Sederma), Senestem™ (Sederma), Pacifeel™ (Sederma), or mixtures thereof.

Among other plant extracts which can be combined with the composition of the invention, there may more particularly be mentioned extracts of Ivy, in particular English Ivy (Hedera Helix), of Bupleurum chinensis, of Bupleurum Falcatum, of arnica {Arnica Montana L), of rosemary (Rosmarinus officinalis N , of marigold (Calendula officinalis), of sage (Salvia officinalis L), of ginseng (Panax ginseng), of Zingiber zerumbet sm., of ginko biloba, of St.-John's-Wort (Hyperycum Perforatum), of butcher's- broom (Ruscus aculeatus L), of European meadowsweet (Filipendula ulmaria L), of big- flowered Jarva tea (Orthosiphon Stamincus Benth), of algae (Fucus Vesiculosus), of birch (Betula alba), of green tea, of cola nuts (Cola Nipida), of horse-chestnut, of bamboo, of Centella asiatica, of heather, of fucus, of willow, of mouse-ear, of escine, of cangzhu, of chrysanthellum indicum, of the plants of the Armeniacea genus, Atractylodis Platicodon, Sinnomenum, Pharbitidis, Flemingia, of Coleus such as C. Forskohlii, C. blumei, C. esquirolii, C. scutellaroides, C. xanthantus and C. Barbatus, such as the extract of root of Coleus barbatus, extracts of Ballote, of Guioa, of Davallia, of Terminalia, of Barringtonia, of Trema, of antirobia, cecropia, argania, dioscoreae such as Dioscorea opposita or Mexican, extracts of Ammi visnaga, of Siegesbeckia, in particular Siegesbeckia orientalis, vegetable extracts of the family of Ericaceae, in particular bilberry extracts (Vaccinium angustifollium) or Arctostaphylos uva ursi, aloe vera, plant containing sterols (e.g., phytosterol), Manjistha (extracted from plants of the genus Rubia, particularly Rubia Cordifolia), and Guggal (extracted from plants of the genus Commiphora, particularly Commiphora Mukul), kola extract, chamomile, red clover extract, Piper methysticum extract (Kava Kava™ from SEDERMA), Bacopa monieri extract (Bacocalmine™ from SEDERMA) and sea whip extract, extracts of Glycyrrhiza glabra, of mulberry, of melaleuca (tea tree), of Larrea divaricata, of Rabdosia rubescens, of Euglena gracilis, of Fibraurea recisa Hirudinea, of Chaparral Sorghum, of sun flower extract, of Enantia chlorantha, of Mitracarpe of Spermacocea genus, of Buchu barosma, of Lawsonia inermis L., of Adiantium Capillus-Veneris L., of Chelidonium majus, of Luffa cylindrica, of Japanese Mandarin (Citrus reticulata Blanco var. unshiu), of Camelia sinensis, of Imperata cylindrica, of Glaucium Flavum, of Cupressus Sempervirens, of Polygonatum multiflorum, of loveyly hemsleya, of Sambucus Nigra, of Phaseolus lunatus, of Centaurium, of Macrocystis Pyrifera, of Turnera Diffusa, of Anemarrhena asphodeloides, of Portulaca pilosa, of Humulus lupulus, of Coffea Arabica, of Ilex Paraguariensis, or of Globularia Cordifolia, of Albizzia julibrissin, of Oxydendron arboretum, of Zingimber Zerumbet Smith, of Astragalus membranaceus, of Bupleurum falcatum of Atractylodes macrocephalae, of Plantago lanceolata, or of Mirabilis jalapa,

The compositions of the present invention may include peptides, including, without limitation, the di-, tri-, tetra-, penta-and hexapeptides and their derivatives. According to a particular embodiment, the concentration of the additional peptide, in the composition, ranges from lxl0^"7% and 20%, preferably from lxl0^"6% and 10%, preferably between lxl0^"5% and 5% by weight.

According to the present invention, the term "peptide" refers to peptides containing 10 amino acids or less, their derivatives, isomers and complexes with other species such as a metal ion (e.g. copper, zinc, manganese, magnesium, and others). The term "peptides" refers to both natural peptides and synthetic peptides. It also refers to compositions that contain peptides and which are found in nature, and/or are commercially available. Suitable dipeptides for use herein include but are not limited to Carnosine (beta-AH), YR, VW, NF, DF, KT, KC, CK, KP, KK or TT. Suitable tripeptides for use herein include, but are not limited to RKR, HGG, GHK, GKH, GGH, GHG, KFK, KPK, KMOK, KM0₂K or KAvaK. Suitable tetrapeptides for use herein include but are not limited to RSRK (SEQ ID NO: 4), GQPR (SEQ ID NO: 5) or KTFK (SEQ ID NO: 6). Suitable pentapeptides include, but are not limited to KTTKS (SEQ ID NO: 7). Suitable hexapeptides include but are not limited to GKTTKS (SEQ ID NO: 8) and VGVAPG (SEQ ID NO: 9).

Other suitable peptides for use herein include, but are not limited to: lipophilic derivatives of peptides, preferably palmitoyl derivatives, and metal complexes as aforementioned (e.g. copper complex of the tripeptide HGG). Preferred dipeptide include for example N-Palmitoyl-beta-Ala-His, N-Acetyl-Tyr- Arg-hexadecylester (Calmosensine™, Idealift™ from Sederma). Preferred tripeptide derivatives include for example N-Palmitoyl-Gly-Lys-His, and Pal-Gly-His-Ly, (Pal-GKH and Pal-GHK from Sederma), the copper derivative of HGG (Lamin™ from Sigma), Lipospondin (N-Elaidoyl-KFK) and its analogs of conservative substitution, N-Acetyl-RKR-NH₂ (Peptide CK+), N-Biot-GHK (from Sederma), Pal-KM0₂K (Matrixyl Synthe6™ from Sederma) and derivatives thereof. Suitable tetrapeptide derivatives for use according to the present invention include, but are not limited to, N- Pal-GQPR (SEQ ID NO: 3) (from Sederma), suitable pentapeptide derivatives for use herein include, but are not limited to, Pal-KTTKS (SEQ ID NO: 2) (available as Matrixyl™ from Sederma), Pal- YGGF-X (SEQ ID NO: 10) with X Met or Leu or mixtures thereof. Suitable hexapeptide derivatives for use herein include, but are not limited to, Pal-VGVAPG (SEQ ID NO: 1) and derivatives thereof. The mixture of Pal-GHK and Pal-GQPR (SEQ ID NO: 3) (Matrixyl™ 3000, Sederma) can also be mentioned.

The preferred compositions commercially available containing a tripeptide or a derivative include Biopeptide-CL™, Maxilip™, Biobustyl™, Procapil™ and Matrixyl™synthe'6™ of Sederma. The compositions commercially available preferred sources of tetrapeptides include Rigin™, Eyeliss™, Matrixyl™ Reloaded and Matrixyl 3000™ which contain between 50 and 500 ppm of Pal-GQPR (SEQ ID NO: 3) and an excipient, proposed by Sederma.

The following marketed peptides can be mentioned as well as additional active ingredients: Vialox™, Syn-ake™ or Syn-Coll™ (Pentapharm), Hydroxyprolisilane CN™ (Exsymol), Argireline™, Leuphasyl™, Aldenine™, Trylgen™, Eyeseryl™, Serilesine™ or Decorinyl™ (Lipotec), Collaxyl™ or Quintescine™ (Vincience), BONT-L-Peptide™ (lnfinitec Activos), Cytokinol™LS (Laboratoires Serobiologiques/Cognis), Kollaren™, IP2000™ or Meliprene™ (lnstitut Europeen de Biologie Cellulaire), Neutrazen™ (Innovations), ECM-Protect™ (Atrium Innovations), Timp-Peptide™ or ECM Moduline™ (lnfinitec Activos).

Claims

1. A preparation method of bioactive cleaved oligosaccharides obtained by an enzymatic cleavage of polysaccharides extracted from green algae, the method comprising the steps of:

Providing a micro-organism able to produce at least one osidic cleavage lyase enzyme;

Contact the polysaccharides with said lyase enzyme to induce said cleavage; and

Recover said cleaved oligosaccharides,

wherein said micro-organism is selected from the bacteria group of the Flavobacteriaceae family consisting of the Jejuia pallidilutea strain, the Flavobacteriaceae bacterium strain, the Gaetbulibacter marinus strain and the strain referenced CNCM 1-4797.

2. Method according to claim 1, wherein the micro-organism corresponds to the bacterial strain referenced CNCM 1-4797.

3. Method according to claim 1 or 2, wherein said micro-organism is capable to produce an ulvan lyase and/or glucuronan cleavage enzyme.

4. Method according to claim 1 or 2, wherein said polysaccharides are sufated polysaccharides.

5. Method according to claim 4, wherein the sulfated polysaccharides are ulvan and/or glucuronan polysaccharides.

6. Method according to anyone of claims 1 to 5, wherein the green algae is of the Ulva genus.

7. Method according to claim 6, wherein the green algae is of the Ulva lactuca genus.

8. Microorganism referenced CNCM 1-4797 in particular for the implanting of the method according to anyone of claims 1 to 7.

9. Cleaved bioactive oligosaccharides prepared according to the method of anyone of claims 1 to 7.

10. Topical cosmetic composition comprising oligosaccharides according to claim 9 and a physiologically acceptable medium.

11. Use of oligosaccharides according to claim 9 or of a composition according to claim 10 for a non-therapeutical cosmetic treatment of the skin and its appendages.

12. Use of sulfated bioactive oligosaccharides prepared according to the method of claim 2 and 7, the oligosaccharides having a polymerization degree (DP) ranging between 2 and 10, for a non-therapeutical cosmetic treatment.

13. Use according to claim 11 or 12, wherein the cosmetic treatment is a treatment selected from the group consisting of a depigmenting, anti-radical, hydrating and anti-age via a stimulation of the dermal extracellular matrix molecules treatment.

14. Use according to anyone of claims 11 to 13, wherein the oligosaccharides are combined with a mixture of acetylated oligo-glucuronans, oligomers of D-glucuronic acid or D-glucuronate having a β(1-4) sequence.