FR3094225A1 - Alteromonadaceae extract and its cosmetic use - Google Patents

Abstract

Title Extract of Alteromonadaceae and its cosmetic use The present invention relates to a particular extract of Alteromonada ceae and to a method of cosmetic treatment of the skin using this extract. In particular, the present invention relates to an exopolysaccharide. Figure for the abstract: none

Description

Extract of Alteromonadaceae and its cosmetic use

The present invention relates to a particular extract of Alteromonada ceae and to a method for the cosmetic treatment of the skin using this extract. In particular, the present invention relates to an exopolysaccharide included in this extract.

Exopolysaccharides are high molecular weight polymers composed of oses and secreted by a microorganism in its close environment. Due to the great diversity in composition, exopolysaccharides have found many applications in various industries.

In terms of biotechnological exploitation, bacterial exopolysaccharides are advantageous because their production and quality do not depend on climatic, ecological and political vagaries, unlike the equivalents extracted from algae or plants. Finally, these polymers have a greater degree of structural regularity and can be extracted and purified more easily.

Furthermore, there is always a need for new effective cosmetic active ingredients. Men and women, regardless of age, are concerned with the appearance of their skin, and in particular that of their face. The skin can be dry and be marked by the presence of wrinkles and fine lines, spots, hyperpigmentation, lose elasticity, but also lose luminosity, or radiance. In particular new active agents making it possible to hydrate the skin, to increase the luminosity of the skin, to improve the texture of the skin, to improve the color of the skin or even to improve the radiance of the complexion of the skin are sought.

Unexpectedly, the inventors discovered a new exopolysaccharide of a particularly large size, and very rich in uronic acid. The use of this new exopolysaccharide improves skin luminosity, evens skin tone and texture, thus improving the radiance of the complexion, and promotes hydration, generating a feeling of well-being. to be. In addition, this exopolysaccharide, by virtue of its high molecular weight, confers sensory properties on the cosmetic composition, reinforcing this feeling of well-being. In addition, the effective concentration is lower than those conventionally described for other exopolysaccharides, which is of economic interest for the formulator. This exopolysaccharides can in particular be obtained from Alteromonadaceae .

The present invention therefore relates to an extract of Alteromonadaceae comprising an exopolysaccharide with a molecular weight of between 3.10 ⁶ and 5.10 ⁶ Da which comprises the following monosaccharides: glucose, galactose, glucuronic acid and galacturonic acid.

A subject of the invention is also a cosmetic composition comprising an extract according to the invention.

Finally, the subject of the invention is a cosmetic use and a method for the cosmetic treatment of the skin using an extract according to the invention or a composition according to the invention.

Extract from Alteromonadaceae

The Alteromonadaceae extract according to the invention comprises an exopolysaccharide with a molecular weight of between ^{3 ×10} 6 and 5×10 6 Da, preferably between 3.5 and 4.5×10 ⁶ Da. The molecular weight can be determined by techniques well known to those skilled in the art and in particular by high performance steric exclusion chromatography (HPSEC).

The exopolysaccharide present in the extract according to the invention comprises the following monosaccharides: glucose, galactose, glucuronic acid and galacturonic acid.

According to a particular form of the invention, the exopolysaccharide comprised in the extract comprises by decreasing masses present galactose, then glucuronic acid, then galacturonic acid in an amount of the same order as glucose.

Uronic acids are chemical compounds obtained by oxidation of the last carbon of simple sugars. In particular, the exopolysaccharide present in the extract comprises more than 20% by weight of uronic acid, preferably more than 30% and more particularly more than 35% of uronic acid. In a preferred embodiment the exopolysaccharide comprises between 35 and 45% by weight of uronic acid.

Certain monosaccharides of the exopolysaccharide present in the extract of the invention may carry substituents, in particular lactate or acetate substituents. In particular, the exopolysaccharide present in the extract according to the invention does not include any substituents other than the lactate and acetate substituents. Preferably, the exopolysaccharide according to the invention does not comprise a sulphate group.

In one embodiment, the osidic composition of the exopolysaccharide present in the extract of the invention was determined by gas chromatography.

The exopolysaccharide present in the extract according to the invention is preferably very hygroscopic.

Preferably, the extract according to the invention comprises between 0.1 and 10% of this exopolysaccharide by weight.

Preferably, the extract according to the invention has a polydispersity index of less than 2 and more particularly between 1 and 1.5. In one embodiment the polydispersity index was determined by size exclusion chromatography.

By " Alteromonadaceae " is meant the gamma proteobacteria of this family. In particular, the extract according to the invention comes from Alteromonas sp . Preferably, the extract is obtained from the microorganism of Alteromonas sp . deposited according to the Budapest Treaty, on November 6, 2018, with the National Collection of Microorganism Cultures ((CNCM), Paris, France) under number CNCM I-5375). This microorganism normally associated with the sea anemone was collected in the lagoon of Moorea.

The invention also relates to an extract of Alteromonadaceae obtainable by the production process comprising the following steps:

(i) fermentation of Alteromonadaceae in sea water at a temperature between 25 and 40°C, for at least 12 hours with stirring,

(ii) centrifugation of the culture obtained in step (i) between 15,000 and 20,000 g,

(iii) ultrafiltration of the supernatant from step (ii),

(v) dilution of the retentate obtained in step (iii) in water,

(vi) sterilizing filtration, and

(vii) ultrafiltration.

Preferably, the process may also comprise a step (viii) of concentration and freeze-drying or atomization.

By “sea water”, we mean salt water containing 5 to 40 g of salts per liter of water and having a pH between 7.5 and 8.4. In a preferred embodiment, the seawater used in the process is seawater reconstituted by diluting synthetic salt. Preferably, the seawater used in the process is seawater reconstituted by dilution of "Instant Ocean ®" synthetic salt at a concentration of 12 g/L.

“Stirring” means a stirring speed of between 50 and 300 rpm (rotation per minute), preferably between 100 and 200 rpm.

By “ultrafiltration” is meant filtration through a filter having pores with a diameter of between 1 and 20 nm, preferably between 5 and 15 nm.

The steps of sterilizing filtration, atomization and freeze-drying are steps well known to those skilled in the art.

Preferably, the extract according to the invention is stored under vacuum, or in a dry and cold place, preferably between 0 and 25°C, more preferably between 2 and 8°C and in particular at around 4°C.

Composition

The invention also relates to a composition, in particular cosmetic, comprising an extract according to the invention.

The invention further relates to a composition, in particular cosmetic, comprising an extract obtainable by the production process according to the invention.

Preferably, the composition according to the invention comprises 0.001% and 5% of exopolysaccharide as defined above by weight relative to the weight of the composition. In a preferred embodiment, the composition according to the invention comprises between 0.002 and 0.1% of exopolysaccharide as defined above by weight relative to the weight of the composition, more particularly between 0.004 and 0.01% by weight relative to the weight of the composition. Indeed, even at very low concentrations, the exopolysaccharide retains its effectiveness.

The large size of the exopolysaccharide present in the composition gives it interesting sensory properties. In particular a feeling of softness to the touch.

The invention also relates to a non-therapeutic cosmetic use of an extract according to the invention or of a composition according to the invention for moisturizing the skin and/or improving the radiance of the complexion. In a preferred embodiment, the concentration of said exopolysaccharide in the composition or in the extract is between 0.001% and 5% by weight relative to the weight of the composition or of the extract respectively.

By “improving the radiance of the complexion”, we mean increasing the luminosity of the skin and/or improving the texture of the skin and/or homogenizing the color of the skin.

By "skin" is meant human skin and/or human scalp. Means in particular the skin of the body, in particular of the legs, arms, hands, neck and the skin of the face. Preferably, the term "skin" means the skin of the face.

The invention also relates to a non-therapeutic cosmetic use of an extract according to the invention or of a composition according to the invention to promote a feeling of well-being.

According to one embodiment, the composition according to the invention or the extract according to the invention can be used in a subject having sensitive skin and in particular dry and/or aged skin.

According to one embodiment, the composition according to the invention or the extract according to the invention can be used in a subject having dull skin and/or with an unsatisfactory texture and/or an uneven complexion and/or color spots.

According to one embodiment, the composition according to the invention or the extract according to the invention can be used in a subject having a dry scalp.

Preferably, the composition used according to the invention or the extract used according to the invention is intended for topical application.

The compositions used according to the invention preferably contain a medium that is physiologically acceptable, that is to say compatible with the skin. This physiologically acceptable medium may more particularly consist of water and optionally of a physiologically acceptable organic solvent such as, for example, propanediol.

Said composition can in particular be in the form of a cream or a lotion. The composition can be for regular application and in particular for daily or bi-daily application.

The amounts of the various constituents of the physiological medium of the composition according to the invention are those generally used in the fields considered. In addition, these compositions are prepared according to the usual methods.

Preferably, the composition is used according to the invention as active agent in an effective amount, that is to say in an amount allowing it to have the desired effect.

The composition may also comprise at least one adjuvant, in particular adjuvants usually used in the cosmetics field, such as hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic additives, preservatives, antioxidants, solvents, perfumes, fillers, filters, odor absorbers and colorants. The amounts of these various adjuvants are those conventionally used in the cosmetics field, and for example vary from about 0.01% to 10% of the total weight of the composition. These adjuvants, depending on their nature, can be introduced into the fatty phase, into the aqueous phase and/or into the lipid spherules.

The invention also relates to a cosmetic process for moisturizing and/or improving the radiance of the complexion of the skin, comprising a topical application step, of an effective amount of a composition of the invention or of an extract according to the invention.

The invention relates to a cosmetic process for promoting a feeling of well-being comprising a step of topical application of an effective amount of a composition of the invention or of an extract according to the invention.

In the description and in the following examples, unless otherwise indicated, the percentages are percentages by weight and the ranges of values denominated in the form "between ... and ...", "ranging from ... to ...", or " greater than...” include the limits specified.

Throughout the application, the wording "comprising a" or "comprising a" means "comprising at least one" or "comprising at least one" unless otherwise specified.

tricks

: Figure 1 represents the chromatogram obtained after high performance steric exclusion chromatography (HPSEC), of the extract of Alteromonadaceae obtained in example 1. Detectors: LS: Light diffusion, UV: Absorption spectrometer ultraviolet, dRI: Differential refractometer.

: Figure 2 represents the chromatogram obtained after gas chromatography (GC) of the extract of Alteromonadaceae obtained in example 1.

: Figure 3 is a graph representing the evolution between the start of the test (D0) and the end of the test (D28) of the brightness, of the homogeneity of color, and of the homogeneity of texture of the skin. These three parameters make it possible to evaluate the radiance of the complexion. Two compositions were tested, a so-called placebo and one comprising the extract according to the invention (abbreviated EPS) (see Example 4). *: statistically significant compared to D0 (p<0.05).

: Figure 4 is a graph representing the evolution between the beginning of the test (D0) and the end of the test (D28) of the hydration of the skin. Two compositions are tested, a so-called placebo and one comprising the extract according to the invention (abbreviated EPS) (see Example 4). *: statistically significant compared to D0 (p<0.05).

: Figure 5 is a graph representing the evolution between the start of the test (D0) and the end of the test (D28) of the frequency of the voice. Two compositions are tested, a so-called placebo and one comprising the extract according to the invention (abbreviated EPS) (see Example 5). +: on the verge of being statistically significant compared to the placebo.

: Figure 6 is a graph representing the evolution between the start of the test (D0) and the end of the test (D28) of the volume of the voice. Two compositions are tested, a so-called placebo and one comprising the extract according to the invention (abbreviated EPS) (see Example 5). *: statistically significant compared to placebo (p<0.05).

: Figure 7 represents the evolution between the start of the trial (D0) and the end of the trial (D28) of the words used by the volunteers to answer the question "How would you describe yourself physically?" (see example 5). The y-axis represents the number of statistical occurrences.

: Figure 8 represents the evolution between the start of the trial (D0) and the end of the trial (D28) of the words used by the volunteers to answer the question "What would be the first impression of someone who you will meet for the first time?” (see example 5). The y-axis represents the number of statistical occurrences.

The examples below are presented by way of illustration and not limitation of the field of the invention.

Example 1: preparation of an extra it's Alteromonadaceae

The strain of Alteromonas sp . deposited according to the Budapest Treaty, on November 6, 2018, with the National Collection of Microorganism Cultures ((CNCM), Paris, France) under the number CNCM I-5375 is fermented for 12 hours with stirring in seawater at 30°C. Then the culture moult was recovered and centrifuged. The supernatant was then filtered on a 100 kDa polyethersulfone (PES) membrane then the retentate was diluted in distilled water and filtered on a 0.22 μm membrane, then again filtered on a 100 kDa PES membrane against distilled water, concentrated to minimum volume and freeze-dried. Finally, the extract obtained was stored under vacuum.

Example 2: Analysis of the extra it's Alteromonadaceae

The extract obtained in Example 1 was analyzed by high performance steric exclusion chromatography (HPSEC), see FIG. 1. This figure shows that the extract obtained is of high purity. Analysis of the results made it possible to determine the polydispersity index and the weight of the molecule detected. The polydispersity index is 1.4, which confirms the purity of the extract. The weight of the molecule detected is 4.16.10 ⁶ Da.

This extract was also analyzed by gas chromatography (GC), see figure 2. The extract is composed of multiple oses, the molecule included in the extract is therefore a polysaccharide, named EPS thereafter. It includes glucose, galactose, glucuronic acid and galacturonic acid in the following proportions: 1/2/1.5/1. EPS is therefore particularly rich in uronic acid (about 45%). However, uronic acid is known for its effects on cell regeneration, modulation of inflammation and hydration.

The inventors have also determined by nuclear magnetic resonance (NMR) that the EPS comprises lactate and acetate substituents. Finally, no sulphate group was detected by Fourier transform infrared spectroscopy (FTIR).

Example 3: D determination of the interaction profile of the extract

The interaction profile of the extract was obtained by testing the ability of this extract to inhibit interactions between a lectin and the ose binding to it. 20 lectin/ose pairs (see Table 1) were tested at 3 different concentrations.

Summary of lectins tested Short name Full name of the organism from which the lectin is derived Con A Canavalia ensiformis LcH Lens culinaris FimH Escherichia coli adhesin FimH BC2L-A Burkholderia cenocepacia lectin A EPS Bauhinia purpurea HPA Helix pomatia WFA Wisteria floribunda AIA / Jacalin Artocarpus intergrifolia PA-IT Pseudomonas aeruginosa lectin A (Lec A) NAP Arachis hypogaea GSL II Griffonia simplicifolia WGA Triticum vulgare UEA I Ulex europeus BC2L-C Inter Burkholderia cenocepacia lectin C (N terminal domain) RSL Ralstonia solanacearum PAII-L Pseudomonas aeruginosa lectin B (Lec B) ANS Sambucus nigra CorM Coregonus lavaretus marenae Langerin Langerin ECD (Extra Cellular Domain) DC-SIGN DC-SIGN ECD (Extra Cellular Domain)

Each analysis was repeated twice. This innovative technique makes it possible to obtain the glycobiological function of the extract by establishing its interaction profile. In addition to confirming the residues present in the exopolysaccharide, this technique makes it possible to verify their bioavailability as well as to detect motifs which behave like residues (such as the so-called "Rhamnose like" motifs which interact in a manner similar to a rhamnose residue) .

The exopolysaccharide from the extract of example 1 had an effect on the following lectins ConA LCH, DC-Sign, BC2L-A, AIA, PNA, PA-IL, GSL-II, WGA, HPA and COR-M . This shows that the exopolysaccharide comprises glucose, galactose, glucuronic acid and galacturonic acid residues and bioavailable rhamnose-like motifs. These results confirm the osidic sequence deciphered in GC, and reveal a new motif, the rhamnose-like.

In particular DC-Sign is involved in chronic skin inflammation. In addition, its inhibition by the exopolysaccharide indicates that it could have a role in modulating the inflammatory aspects of the skin. Binding to COR-M indicates the presence of a bioavailable “rhamnose-like” motif and indicates that the exopolysaccharide could promote dermo-epidermal regeneration by activating the extracellular matrix and promoting the production of inflammation mediator gene.

Example 4: Clinical test - biometric settings

40 volunteers aged 46 to 60 considering they have skin with a lack of luminosity and an uneven complexion took part in this clinical test. They were separated into two groups which applied either a placebo solution or a solution comprising the EPS. These solutions are applied by the volunteers twice a day every day, for 28 days, on their face. The placebo solution includes a mixture of water, propanediol and pentylene glycol. The solution comprising the EPS comprises the same mixture and 0.005% by weight of EPS. This solution was obtained by diluting the extract of Example 1 to 1%.

The luminosity, the homogeneity of the color and the texture of the skin were evaluated on the first day and the last day of the test (see figure 3) by the CLBT method (Visual evaluation in vivo of 'complexion radiance' using the CLBT™ sensory methodology. Skin Research and Technology 10(1):50-6 March 2004): evaluation by an expert on a scale of 1 to 10.

Similarly, hydration was assessed from the penetration of electromagnetic fields into the skin (GreyIndex measurement) (see figure 4).

The inventors have observed that the extract makes it possible to obtain a significant gain in luminosity, in homogeneity of color and texture. The extract provides a much more satisfactory result than the placebo. Similarly, skin hydration is improved in the presence of the extract compared to the placebo solution. The extract therefore moisturizes the skin. The extract also increases skin luminosity, improves skin texture and evens out skin color, thus improving the radiance of the complexion.

Example 5: Clinical test - emotional parameters

The Emospin test was used to assess the emotional impact of using the extract.

On the one hand, the prosody of the volunteers in example 4 was studied before the test and at the end of it. In particular, the frequency (measured in Hertz (Hz)) and the volume (measured in decibels (dB)) of the voice of the volunteers of example 4 were evaluated (see figures 5 and 6). The inventors observed that the frequency and volume of the voice of the volunteers who used the extract decreased significantly compared to the volunteers who used the placebo. This decrease is a sign of relaxation and therefore of an improvement in the person's feeling of well-being.

On the other hand, the words used by the volunteers in example 4 to answer the questions “How would you describe yourself physically?” (Figure 7) and “What would be the first impression of someone meeting you for the first time?” (Figure 8) were studied before the test and at the end of it. The following words are considered positive "cheerful", "less", "likes", "listening", "honest", "friendship", "nice", "sociable", "funny", "good in her skin”, “comely”, “smiling”, and the following negative words “nothing”, “aged”, “ugly”, “bland”, “droopy”, “difficult”, “wrinkled”, “pocket under the eyes”, “still”, “average”, “unkempt” and “simple”.

The inventors observed that the frequency of positive words was increased concomitantly with the decrease in the frequency of negative words in the volunteers who used the extract. This modification of the words used is a sign of the improvement in the feeling of well-being of the volunteers who used the extract. On the contrary, the volunteers who used the placebo continued to use mainly negative words.

Claims (9)

Extract of Alteromonadaceae comprising an exopolysaccharide with a molecular weight of between 3.10 ⁶ and 5.10 ⁶ Da which comprises the following monosaccharides: glucose, galactose, glucuronic acid and galacturonic acid. Extract according to claim 1, wherein the Alteromonadaceae is Alteromonas sp . Extract according to claim 1 or 2, in which the exopolysaccharide comprises between 35 and 45% by weight of uronic acid. Extract according to any one of Claims 1 to 3, having a polydispersity index of less than 2. Extract of Alteromonadaceae obtainable by the following production process: Process for the production of an extract as defined in any one of claims 1 to 4, comprising the following steps:
(i) fermentation of Alteromonadaceae in sea water at a temperature between 25 and 40°C, for at least 12 hours with stirring,
(ii) centrifugation of the culture obtained in step (i) between 15,000 and 20,000 g,
(iii) ultrafiltration of the supernatant from step (ii),
(v) dilution of the retentate obtained in step (iii) in water,
(vi) sterilizing filtration and
(vii) ultrafiltration. Cosmetic composition comprising an extract according to any one of Claims 1 to 5. Non-therapeutic cosmetic use of an extract as defined in any one of Claims 1 to 5 or of a composition according to Claim 6 for moisturizing the skin and/or improving the radiance of the complexion of the skin, and/ or promote a feeling of well-being. Non-therapeutic cosmetic use according to claim 7, wherein the concentration of said exopolysaccharide in the composition or in the extract is between 0.001% and 5% by weight relative to the weight of the composition or the extract respectively. Cosmetic process for moisturizing and/or improving the radiance of the complexion of the skin, and/or promoting a feeling of well-being, comprising a step of topical application, of an effective amount of a composition according to Claim 6 or of an extract according to any one of Claims 1 to 5.