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Title:
OLIGOPEPTIDE FRACTION OBTAINED FROM A BIOMASS OF BACTERIUM OR BACTERIA BELONGING TO THE GENUS VITREOSCILLA SP, AS A COSMETIC ACTIVE INGREDIENT
Document Type and Number:
WIPO Patent Application WO/2018/108973
Kind Code:
A1
Abstract:
The invention relates to an oligopeptide fraction obtained from a biomass of a bacterium or bacteria belonging to the genus Vitreoscilla sp., as a cosmetic active agent, to compositions, especially cosmetic and/or dermatological compositions, that contain it, and to its cosmetic and/or dermatological use, preferably as a moisturizing, anti-age and/or hair fibre strengthening active agent.

Inventors:
TOURNIER-COUTURIER, Lucie (1 ave Eugène Schueller, BP22, AULNAY-SOUS-BOIS, 93601, FR)
YU, Peiyan (1 ave Eugène Schueller, BP22, AULNAY-SOUS-BOIS, 93601, FR)
GARCON, Romain (1 ave Eugène Schueller, BP22, AULNAY-SOUS-BOIS, 93601, FR)
Application Number:
EP2017/082540
Publication Date:
June 21, 2018
Filing Date:
December 13, 2017
Export Citation:
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Assignee:
L'OREAL (14 rue Royale, PARIS, PARIS, 75008, FR)
International Classes:
A61K8/64; C07K14/22
Attorney, Agent or Firm:
BROHMI, Karim (L'Oreal, Service DIPI, rue Pierre Dreyfus Clichy, 92110, FR)
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Claims:
CLAIMS

1. Oligopeptide fraction isolated from a lysate of a biomass of a bacterium or bacteria belonging to the genus Vitreoscilla sp.

2. Oligopeptide fraction according to Claim 1, which comprises

i) from 30 to 50% oligopeptides,

ii) between 1% and 10% amino acids,

iii) 10 to 20% of a mixture of oligosaccharides and monosaccharides.

3. Oligopeptide fraction according to any one of the preceding claims, wherein said bacterium belonging to the genus Vitreoscilla sp. is the V. filiformis strain, preferably the V. filiformis strain ATCC 15551. 4. Oligopeptide fraction according to any one of the preceding claims, characterized in that it is obtained by step of enzymatic digestion of said biomass.

5. Composition comprising, in a physiologically acceptable medium, an oligopeptide fraction according to any one of Claims 1 to 4.

6. Use of an oligopeptide fraction obtained from a biomass of a bacterium or bacteria belonging to genus Vitreoscilla sp., as a cosmetic active agent.

7. Use of an oligopeptide fraction according to Claim 6, wherein said cosmetic active agent is a moisturizing active agent.

8. Use of an oligopeptide fraction according to Claim 6, wherein said cosmetic active agent is an anti-age active agent. 9. Use of an oligopeptide fraction according to Claim 6, wherein said cosmetic active agent is an active agent for strengthening the hair fibre.

10. Use according to any one of Claims 6 to 9, wherein said oligopeptide fraction is obtained by a step of enzymatic digestion of said biomass.

11. Cosmetic process for treating a keratin material, comprising a step of application of an oligopeptide fraction according to any one of Claims 1 to 4 or of a composition containing it according to Claim 5.

12. Cosmetic process for moisturizing skin, comprising a step of topical application of an oligopeptide fraction according to any one of Claims 1 to 4 or of a composition containing it according to Claim 5.

13. Cosmetic process for treating and/or preventing the signs of ageing of the skin, comprising a step of topical application of an oligopeptide fraction according to any one of Claims 1 to 4 or of a composition containing it according to Claim 5.

14. Cosmetic process for strengthening the hair fibre, comprising a step of topical application of an oligopeptide fraction according to any one of Claims 1 to 4 or of a composition containing it according to Claim 5.

15. Process for preparing an oligopeptide fraction obtained from a lysate of a biomass of a bacterium or bacteria belonging to the genus Vitreoscilla sp., comprising the steps consisting in:

(i) providing a biomass of a bacterium or bacteria belonging to the genus Vitreoscilla sp. ;

(ii) performing double enzymatic digestion on said biomass;

(iii) inactivating the enzymatic digestion.

16. Preparation process according to Claim 15, characterized in that step (iii) is optionally followed by a step of spray drying the complete reaction medium.

17. Preparation process according to Claim 16, wherein said enzymatic digestion is performed from carbohydrase and protease enzymes.

18. Preparation process according to Claims 16 and 17, wherein said enzymatic digestion is performed from carbohydrase enzymes in a first step then from protease enzymes in a second step.

Description:
Oligopeptide fraction obtained from a biomass of bacterium or bacteria belonging to the genus Vitreoscilla sp, as a cosmetic active ingredient Field of the invention

The present invention relates to active agents intended to be used in the field of cosmetics and/or dermatology, preferably cosmetics.

More particularly, the invention relates to an oligopeptide fraction obtained from a biomass of a bacterium or bacteria belonging to the genus Vitreoscilla sp., as a cosmetic active agent, to compositions, especially cosmetic and/or dermatological compositions, that contain it, and to its cosmetic and/or dermatological use, preferably as a moisturizing, anti- age and/or hair fibre strengthening active agent.

Technological background

Over time, it appears that keratin materials, such as the skin, eyelashes, eyebrows, nails and hair are subject to ageing, reflected especially by a modification of their structures and their functions.

Endogenously, keratin materials are subject to attacks by superoxide ions, naturally produced during physiological cellular metabolic processes.

Exogenously, keratin materials are first in line for exposure to environmental factors which take part in their ageing, such as, for example, sunlight, especially via ultraviolet radiation, or else pollution. They may disturb the filaggrin cycle and therefore the production of natural moisturizing factors (NMFs). Consequently the skin loses its natural humectants and becomes sensitive to desiccation. The barrier function is then more fragile and changed; the skin is more exposed to external attacks.

The extrinsic ageing of the skin influenced by the environment, for example, supplements the intrinsic ageing linked to age, giving rise to characteristic skin damage and changes which are well described in the literature. From a cellular and molecular perspective, skin ageing is reflected by an imbalance between the production of dermal constituent elements, like collagen and fibrillin for example, a reduction in differentiation markers such as, for example, transglutaminases (TGMs) and their destruction by different metalloproteases (MMPs) or genes responsible for the synthesis of hyaluronic acid such as the gene HAS 3 (Hyaluronan synthase), causing the scales to tip in the favour of degradation of the extracellular matrix and tissue destruction.

From a macroscopic perspective, skin ageing is apparent from the appearance of wrinkles and of slackness. Older skin is thinner and more fragile than younger skin. Finally, older skin tends to lose its elasticity, its barrier function against external attacks, and its ability for repair.

Several compositions or methods for preventing and/or treating the signs of skin ageing are known in the prior art. Indeed it is known that the use of a total lysate of bacteria belonging to the genus Vitreoscilla sp. , in a complete fermentation medium, is known from the prior art as an active agent for preventing and/or treating hyperseborrheic conditions not associated with a dandruff condition of the scalp (FR 2 988 607) or else as an active agent for preventing and/or treating dandruff conditions of the scalp, especially associated with a proliferation of pathogenic microorganisms on the scalp and/or an imbalance in the scalp ecoflora (FR 2 973 700).

The lysates described in these documents thus comprise a mixture of protein, lipid and polysaccharide fractions in their culture medium and are not intended for moisturizing skin, treating and/or preventing skin ageing and/or strengthening the hair fibre.

Moreover, the oligopeptide fraction according to the invention is distinguished from the lipopoly saccharide extract described by document FR 2 838 056, especially by its oligopeptide composition.

For example, use of a non-photosynthetic, non-fruiting filamentous bacterial extract has been proposed (FR 2 719 768 and FR 2 838 056). In this case, prevention and/or treatment of the signs of skin ageing is/are especially obtained with an extract of Vitreoscilla filiformis containing lipopolysaccharides, by inducing an increase in the endogenous synthesis of superoxide dismutase (FR 2 838 056).

Moreover, it has been described in document FR 2 762 782 that a clarified and stabilized culture medium of at least one non-photosynthetic filamentous bacterium has beneficial properties from a perspective of reducing and/or delaying skin ageing.

What is more, in the hair field, users seek products that improve the quality of hair. Specifically, a growing need exists for durable hair strengthening, particularly for fine or altered hair. There is therefore a need for novel active agents or novel treatments for preventing and/or repairing damage caused to keratin materials and more particularly to dehydrated and/or aged skin or damage caused to the hair fibre.

A need exists for novel cosmetic treatments that can strengthen keratin materials and more particularly anti-age active agents that can maintain, or even strengthen, the skin's barrier properties by repairing skin damage, and active agents capable of strengthening keratin fibres. In a surprising manner, the Applicant has demonstrated that the oligopeptide fraction according to the invention promotes moisturization of the stratum corneum and consequently skin moisturization. In the same way, the oligopeptide fraction can increase the synthesis of pro-collagen 1, which demonstrates its anti-age activity. Moreover the oligopeptide fraction according to the invention increases the bending force of the hair fibre, which strengthens it.

Summary of the invention

Consequently according to a first feature, the invention relates to an oligopeptide fraction isolated from a lysate of a bio mass of a bacterium or bacteria belonging to the genus Vitreoscilla sp. According to another feature, the invention relates to a process for preparing the oligopeptide fraction isolated from a lysate of a biomass of a bacterium or bacteria belonging to the genus Vitreoscilla sp., comprising the steps consisting in:

(i) providing a biomass of a bacterium or bacteria belonging to the genus Vitreoscilla sp. ;

(ii) performing double enzymatic digestion on said biomass;

(iii) inactivating the enzymatic digestion;

- optionally clarifying the reaction medium by one of more filtration steps; and

- optionally spray drying the optionally clarified complete reaction medium. According to another feature, the invention relates to an oligopeptide fraction isolated from a lysate of a biomass of a bacterium or bacteria belonging to the genus Vitreoscilla sp., as a cosmetic active agent, preferably as an active agent for strengthening keratin materials and especially as a moisturizing, anti-age and hair fibre strengthening active agent.

Within the meaning of the invention, keratin materials are understood to mean the skin and its appendages such as keratin fibres, particularly human keratin fibres such as hair.

Within the meaning of the present invention, 'hair fibre' is understood to mean hair.

Within the meaning of the present invention, skin is understood to mean the skin of the body such as the skin of the hands, legs, arms, face, neck, and the scalp.

According to another feature, the invention relates to a composition, especially a cosmetic and/or dermatological composition comprising said oligopeptide fraction, according to the invention, obtained from a biomass of a bacterium or bacteria belonging to genus Vitreoscilla sp.

According to another feature, the invention relates to the use of said composition according to the invention obtained from a biomass of a bacterium or bacteria belonging to the genus Vitreoscilla sp., as a cosmetic (and/or dermatological) active agent, preferably as an active agent for strengthening keratin materials and especially as a moisturizing, anti-age and hair fibre strengthening active agent.

According to another feature, the invention relates to a cosmetic process for treating a keratin material, comprising a step of topical application of the oligopeptide fraction isolated from a lysate of a biomass of a bacterium or bacteria belonging to genus Vitreoscilla sp.

According to another feature, the invention relates to a cosmetic process for moisturizing skin, especially dry skin, comprising a step of topical application of the oligopeptide fraction isolated from a lysate of a biomass of a bacterium or bacteria belonging to genus Vitreoscilla sp. According to another feature, the invention relates to a cosmetic process for treating and/or preventing the signs of skin ageing, comprising a step of topical application of the oligopeptide fraction isolated from a lysate of a biomass of a bacterium or bacteria belonging to genus Vitreoscilla sp.

According to another feature, the invention relates to a cosmetic process for strengthening the keratin fibre, comprising a step of topical application of the oligopeptide fraction isolated from a lysate of a biomass of a bacterium or bacteria belonging to genus Vitreoscilla sp.

Detailed description of the invention

Oligopeptide fraction isolated from a lysate of a biomass of a bacterium or bacteria belonging to the genus Vitreoscilla sp.

"Oligopeptide fraction" is understood to be a fraction constituted of: i) 30 to 50% oligopeptides,

ii) between 1% and 10% amino acids,

iii) 10 to 20% of a mixture of oligosaccharides and monosaccharides.

Preferably the oligopeptides of the active agent have a molecular weight of between 200Da and 3 kDa, with peptide distribution centred around the 200-800 Da zone; particularly, the oligopeptides are peptides of between 2 and 7 amino acids on average.

Glutamic acid, glutamine, histidine and arginine constitute at least 15% of said 1 to 10% amino acids.

In a preferred manner, the oligosaccharides are chosen from stachyose, sucrose, glucose, fructose and trehalose. • Bacteria belonging to the genus Vitreoscilla sp. (especially species: Vitreoscilla filiformis)

As specified above, the microorganism considered according to the invention is a non-synthetic filamentous bacterium as defined according to the classification in Bergey's Manual of Systematic Bacteriology (Vol. 3, sections 22 and 23, 9 th edition, 1989), and belonging to the genus Vitreoscilla sp. (especially species: Vitreoscilla filiformis)

More particularly, it is a bacterium belonging to the genus Vitreoscilla.

According to a preferred variant of the invention, it is the bacterium Vitreoscilla filiformis.

In one particular embodiment, the bacterium is the Vitreoscilla filiformis strain, preferably the Vitreoscilla filiformis strain ATCC 15551.

• Complete fermentation medium

Within the meaning of the present invention, the expression "complete fermentation medium" is intended to denote a medium originating from a culture method which has been used for the growth and cell lysis of the microorganism, said medium moreover not having been subjected to any additional handling with the aim of separating and/or eliminating all or some of the non-aqueous constituents thereof. · Composition of fermentation medium

By definition, a fermentation or else culture medium is a support which enables the culture, and hence depending on the case, the growth, of cells, of bacteria or of yeasts.

In principle, the cells find, in this medium, the essential components for their rapid multiplication in large numbers, and also sometimes elements which will enable the growth of a specific genus of bacteria or of a particular family to be favoured, in this instance a bacterium belonging to the genus Vitreoscilla sp. (especially, species: Vitreoscilla filiformis).

The composition thereof must therefore satisfy the nutritional requirements of the microorganism considered, which are necessary for the proliferation of the latter.

More specifically, the composition of this culture medium must:

- meet the requirements for mineral ions and growth factors and provide the source of carbon and energy; - have a pH close to the optimal pH;

- have an optimal ionic strength (the medium may be isotonic, but this is not compulsory). Thus, the composition of a fermentation medium suitable for the invention comprises at least:

- a source of carbon and energy, generally represented by a sugar, and advantageously glucose;

- a source of potassium and phosphorus like, for example, K2HPO4; - a source of nitrogen and sulfur, which may be represented by the compound

(NH 4 ) 2 S0 4 ;

- a source of magnesium, such as, for example, MgCl 2 ;

- a source of calcium, such as, for example, CaCl 2 ;

- a source of iron, and more particularly iron citrate, the citrate having the role of keeping the iron in solution;

- a source of trace elements, chosen especially from Cu, Zn, Co, Ni, B or Ti salts;

- a source of water, generally sterile, essential for all lifeforms; and

- a pH buffer which may be represented by KH 2 P0 4 .

If one of these components is not present, the bacteria do not grow, since they cannot themselves compensate for the absence thereof.

By way of illustration of a fermentation medium suitable to the growth of a microorganism in accordance with the invention, mention may especially be made of the medium represented in example 1 below.

An effective amount of the microorganism considered according to the invention is introduced therein and the whole is placed under conditions conducive to the proliferation thereof.

• Digestion of the bacteria to obtain an active agent according to the invention

Consequently, the oligopeptide fraction considered according to the invention may be obtained via a process consisting of: - the culture of at least one bacterium belonging to the genus Vitreoscilla sp. (especially species: Vitreoscilla filiformis) on a fermentation medium under conditions conducive to the proliferation of said bacterium, and

- a centrifugation step, and

- optionally a pH adjustment, and

- the enzymatic digestion of the residue derived from the centrifugation of said bacteria in said fermentation medium.

The oligopeptide fraction within the meaning of the invention denotes the active agent derived from the digestion of the residue derived from the centrifugation which is optionally adjusted to a given pH and subsequently treated by a carbohydrase enzyme and a protease enzyme.

According to a first embodiment, the digestion of the residue derived from the centrifugation is, after optional adjustment of the pH to a given value, performed sequentially by the action of a carbohydrase enzyme then by the action of a protease enzyme.

According to a second embodiment, the digestion of the residue derived from the centrifugation is, after optional adjustment of the pH to a given value, performed sequentially by the action of a protease enzyme then by the action of a carbohydrase enzyme.

According to another embodiment, an inactivation step, particularly thermal inactivation, occurs between the two enzymatic digestion steps. According to a first variant of this embodiment, the residue derived from the centrifugation is optionally adjusted to a given pH then first treated with a protease enzyme, then is subjected to an inactivation step, then is treated with a carbohydrase enzyme. According to a second variant of this embodiment, the residue derived from the centrifugation is optionally adjusted to a given pH then first treated with a carbohydrase enzyme, then is subjected to an inactivation step, then is treated with a protease enzyme.

The pH adjustment that may precede the step of enzymatic digestion of the residue is optional. When it is performed, the pH is changed by adding an aqueous solution of organic or inorganic acid or base, the goal of this adjustment being to adjust the pH of the residue if need be to a value corresponding to the optimal pH for using the enzymes used in the digestion step. Preferably, the pH is adjusted. Preferably, the adjustment is performed by adding an organic or inorganic solution of acid, preferably to a pH of 3.5 to 5, more preferably of 3.8 to 4.2, particularly to a pH of 4.

Organic or inorganic acid is understood for example to mean carboxylic acids or sulfonic acids such as sulfonic, methanesulfonic, para-toluene sulfonic, camphorsulfonic acids, tartaric acid, citric acid, acetic acid, aspartic acid, glutamic acid, or inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, phosphonic acid.

Carbohydrase enzyme means for example enzymes having classification EC3.2.1.

According to a preferred form of the invention, carbohydrase enzyme means the Viscozyme® enzyme sold by the company Novozyme.

Protease enzyme means for example enzymes having classification EC3.4.

According to a preferred form of the invention, protease enzyme means the Sumizyme AP enzyme sold by the company Novozyme.

According to a preferred embodiment, the digestion of the residue derived from the centrifugation is performed sequentially by the action of Viscozyme® sold by the company Novozyme then by the action of Sumizyme AP sold by the company Novozyme.

To obtain an oligopeptide fraction obtained from a biomass of bacteria belonging to the genus Vitreoscilla sp. in a complete fermentation medium, according to a process comprising a step of culturing said bacteria, those skilled in the art may especially refer to example 1.

The invention also relates to a process for preparing an oligopeptide fraction isolated from a biomass of a bacterium or bacteria belonging to the genus Vitreoscilla sp., comprising the steps consisting in:

(i) providing a biomass of a bacterium or bacteria belonging to the genus Vitreoscilla sp. ; (ii) performing double enzymatic digestion on said biomass;

(iii) inactivating the enzymatic digestion.

In a preferred manner, step (iii) is followed by a step of spray drying the complete reaction medium.

The invention also relates to a process for preparing an oligopeptide fraction isolated from a biomass of a bacterium or bacteria belonging to the genus Vitreoscilla sp., comprising the steps consisting in:

- providing a biomass of a bacterium or bacteria belonging to the genus Vitreoscilla sp. ; then

- optionally performing centrifugation;

- optionally adjusting the pH;

- performing a double enzymatic digestion on said biomass;

- inactivating the enzymatic digestion;

- optionally adjusting the pH;

- optionally clarifying by successive filtrations;

- optionally spray drying the complete reaction medium.

"Biomass" means all or some of the mass of living or dead organisms contained in a completed culture, that is to say after disappearance of all or some of the nutritive elements constituting the initial complete fermentation medium.

In a preferred embodiment, the biomass is subjected to a centrifugation step prior to the double digestion step and the double digestion step is performed on the residue derived from the centrifugation.

The pH adjustment that may precede the step of enzymatic digestion is optional. When it is performed, the pH is changed by adding an aqueous solution of organic or inorganic acid or base, the goal of this adjustment being to adjust the pH of the residue if need be to a value corresponding to the optimal pH for using the enzymes used in the digestion step.

Preferably, the pH is adjusted. Preferably, the adjustment is performed by adding an organic or inorganic solution of acid, preferably to a pH of 3.5 to 5, more preferably of 3.8 to 4.2, particularly to a pH of 4.

Preferably the pH is adjusted with an organic acid such as citric acid.

In a specific embodiment, the double enzymatic digestion of the biomass of a bacterium or bacteria belonging to genus Vitreoscilla sp. is performed using carbohydrase and protease enzymes.

According to a first embodiment, the digestion is, after optional adjustment of the pH to a given value, performed sequentially by the action of a carbohydrase enzyme then by the action of a protease enzyme.

According to a second embodiment, the digestion is, after optional adjustment of the pH to a given value, performed sequentially by the action of a protease enzyme then by the action of a carbohydrase enzyme.

According to an embodiment, an inactivation step, particularly thermal inactivation, occurs between the two enzymatic digestion steps. According to a first variant of this embodiment, the optionally centrifuged biomass is optionally adjusted to a given pH then first treated with a protease enzyme, then is subjected to an inactivation step, then is treated with a carbohydrase enzyme. According to a second variant of this embodiment, the optionally centrifuged biomass is optionally adjusted to a given pH then first treated with a carbohydrase enzyme, then is subjected to an inactivation step, then is treated with a protease enzyme.

According to a preferred form of the invention, carbohydrase enzyme means the Viscozyme® enzyme sold by the company Novozyme. Viscozyme is a broad spectrum endoprotease, purified and isolated from Bacillus liceniformis. The quantity of carbohydrase used is from 0.05 to 1.5% by weight, more preferably 0.2%. The temperature of use is from 50°C to 60°C, preferably 55°C; the pH of use is between 4 and 5 (limits included) and the treatment duration is from 2 hours to 24 hours, preferably 3 hours.

Protease enzyme means for example enzymes having classification EC3.4.

According to a preferred form of the invention, protease enzyme means the Sumizyme AP enzyme sold by the company Novozyme. Sumizyme is a broad spectrum endoprotease, purified and isolated from Aspergillus niger. The quantity of protease used is from 0.01 to 1% by weight, more preferably 0.04%. The temperature of use is from 50°C to 60°C, preferably 55°C; the pH of use is between 3 and 5 (limits included), preferably from 3 to 4.5, and the treatment duration is from 2 hours to 24 hours, preferably 3 hours.

According to a preferred embodiment, the digestion of the biomass is performed sequentially by the action of Viscozyme® sold by the company Novozyme then by the action of Sumizyme such as Sumizyme AP sold by the company Novozyme.

According to a preferred embodiment, the inactivation of the enzymatic digestion is thermal inactivation that may be performed during a period of between 10 minutes to 30 minutes, preferably a period of 15 minutes.

In a specific embodiment, the inactivation of the enzymatic digestion is performed at a constant temperature of between 80°C and 90°C, preferably a temperature of 90°C. Approximately 15 min means a duration of 15 min ± 5 min.

In a specific embodiment, at the end of the inactivation step, the pH of the reaction medium is adjusted to 7 with an organic or inorganic base such as sodium hydroxide (NaOH).

In a specific embodiment, the reaction medium after double digestion is clarified by filtration. In a preferred manner, this clarification step consists in a double filtration, preferably using a filtration with a 10 μιη bag filter then with a K2 filter. According to a specific embodiment, the spray drying technique is employed on the complete reaction medium optionally clarified and preferably on the clarified medium. In a specific embodiment, the spray drying technique is performed with a BUCHI mini spray dryer B-290 with the following parameters: Pump 65%; Inlet temperature 110°C to 230°C and preferably 210°C; Outlet temperature of 80°C to 90°C and particularly 87°C; Aspirator 95; Q flow 45.

The conditions of the enzymatic digestion inactivation step may be adapted by those skilled in the art.

According to a specific embodiment, the process for preparing an oligopeptide fraction obtained from a biomass of a bacterium or bacteria belonging to the genus Vitreoscilla sp. comprises the steps consisting in:

- providing a biomass of a bacterium or bacteria belonging to the genus Vitreoscilla sp. ; then

- optionally performing centrifugation;

- optionally adjusting the pH;

- performing a double enzymatic digestion on said biomass;

- inactivating the enzymatic digestion;

- adjusting the pH to a physiological pH, i.e. a pH compatible with the application to keratin materials.

According to this embodiment, the oligopeptide fraction according to the invention may be packaged in sterile packaging, and/or have one or more preservatives such as phenoxyethanol added.

According to another specific embodiment, the process for preparing an oligopeptide fraction obtained from a biomass of a bacterium or bacteria belonging to the genus Vitreoscilla sp. comprises the steps consisting in:

- providing a biomass of a bacterium or bacteria belonging to the genus Vitreoscilla sp. ; then

- optionally performing centrifugation;

- optionally adjusting the pH; - performing a double enzymatic digestion on said biomass;

- inactivating the enzymatic digestion;

- optionally clarifying by successive filtrations;

- spray drying the complete reaction medium.

According to a preferred embodiment, the process for preparing an oligopeptide fraction obtained from a biomass of a bacterium or bacteria belonging to the genus Vitreoscilla sp. comprises the steps consisting in:

- providing a biomass of a bacterium or bacteria belonging to the genus Vitreoscilla sp. ; then

- performing a centrifugation; then

- performing a pH adjustment by adding an organic or inorganic acid such as hydrochloric acid or citric acid, preferably citric acid, to bring the pH to an acidic pH, preferably between 3.8 and 5; then

- performing a double enzymatic digestion on said biomass, preferably by sequentially using first a carbohydrase then a protease and more particularly using first Viscozyme® sold by Novozyme then Sumizyme AP sold by Novozyme, in the conditions mentioned previously; then

- inactivating the enzymatic digestion, preferably thermally; then

- clarifying by successive filtrations; then

- spray drying the reaction medium.

Use of the oligopeptide fraction

The inventors have observed, surprisingly, that the oligopeptide fraction obtained from a biomass of a bacterium or bacteria belonging to the genus Vitreoscilla sp. has beneficial properties from the perspective of caring for keratin materials and especially the skin and/or the hair fibre. As will emerge from the examples presented below, the inventors have discovered, unexpectedly, that the oligopeptide fraction of a biomass of such a bacterium in a complete fermentation medium exhibits unexpected cosmetic properties. The invention relates to a use of the oligopeptide fraction from a lysate of a biomass of a bacterium or bacteria belonging to the genus Vitreoscilla sp. according to the invention as a cosmetic and/or dermatological active agent, preferably as a moisturizing, anti-age and/or hair fibre strengthening active agent.

In the scope of the invention, "oligopeptide fraction obtained from a biomass of a bacterium or bacteria belonging to genus Vitreoscilla sp." , means the result of enzymatic digestion of the residue derived from the centrifugation of the fermentation medium that was used to culture the micro-organism belonging to genus Vitreoscilla sp. by a carbohydrase enzyme and by a protease enzyme such as the carbohydrase enzyme Viscozyme sold by the company Novozyme and the protease enzyme Sumizyme AP sold by the company Novozyme.

"Cosmetic and/or dermatological active agent" means an active agent having beneficial properties after application to keratin materials, particularly the skin and/or hair. In particular, application is topical.

• Composition

The invention also relates to a cosmetic and/or dermatological composition comprising the oligopeptide fraction isolated from a biomass of a bacterium or bacteria belonging to the genus Vitreoscilla sp.

Preferably the composition according to the invention further comprises a physiologically acceptable medium.

"Physiologically acceptable medium" is, according to the invention, a medium compatible with human keratin materials, in particular with human skin.

The physiologically acceptable medium will be adapted to the nature of the support onto which the composition is to be applied, and also to the form in which the composition is intended to be packaged, in particular solid or fluid at room temperature and atmospheric pressure.

The physiologically acceptable medium may particularly be a cosmetically acceptable medium.

"Cosmetically acceptable medium" means an odourless, colourless medium without unpleasant appearance, and that does not generate discomfort for the user.

A composition containing the active agent according to the invention may be administered by or topically.

"Administer" means the fact of externally (topically) ingesting or applying a composition according to the invention for aesthetic purposes, i.e. to improve the appearance and the texture of the keratin materials and more particularly of the skin, mucous membranes, scalp of the hair and keratin fibres. In other words, within the meaning of the invention, the term "administer" does not cover a treatment for medical purposes.

Preferably, the composition according to the invention is a composition intended to be applied topically to the skin, or a hair care composition.

A composition according to the invention advantageously comprises a quantity of oligopeptide fraction obtained from a biomass of a bacterium or bacteria belonging to genus Vitreoscilla sp.

In some embodiments, a composition according to the invention advantageously comprises a quantity of oligopeptide fraction according to the invention ranging from 0.01 to 10% by weight, relative to the total weight of said composition, more preferably 0.05%> to 10% by weight and more particularly from 0.06 to 5% relative to the total weight of the composition.

In other embodiments, a composition according to the invention advantageously comprises an amount of oligopeptide fraction ranging from 1% to 5% by weight relative to the total weight of said composition.

A composition according to the invention may therefore be presented in any delivery form normally available for the specific selected mode of administration.

The support may be of diverse nature according to the type of composition considered. As more particularly regards compositions intended for external topical administration, they may be aqueous, aqueous-alcoholic or oily solutions, solutions or dispersions of the lotion or serum type, emulsions of liquid or semi- liquid consistency of the milk type, obtained by dispersing a fatty phase in an aqueous phase (O/W) or vice-versa (W/O), or suspensions or emulsions, of soft, semi-solid or solid consistency, of the cream type, aqueous or anhydrous gel, microemulsions, microcapsules, microparticles, or vesicular dispersions of ionic and/or non-ionic type.

These compositions are prepared according to the usual methods.

These compositions may especially constitute cleansing, protective, treatment or care creams, lotions, gels or mousses for caring for keratin materials such as the skin, the mucous membranes and the scalp.

They may be used for the cosmetic treatment of keratin materials especially the skin, the scalp and/or keratin fibres, in the form of solutions, creams, gels, emulsions, mousses or else in the form of compositions adapted for use in an aerosol or a spray, for example also containing a pressurized propellant.

In certain embodiments, a topical composition according to the invention may advantageously be formulated in any delivery form that is suitable for haircare, especially in the form of a hair lotion, a shampoo, a conditioner, a detangler, a hair cream or gel, a styling lacquer, a hairsetting lotion, a treating lotion, a dye composition (especially for oxidation dyeing) optionally in the form of a colouring shampoo, a hair-restructuring lotion, a permanent-waving composition, a lotion or gel for combating hair loss, an antiparasitic shampoo or a medicated shampoo, especially an anti-seborrhoea shampoo, an antidandruff product, a scalp care product, which is especially anti- irritant, anti-ageing or restructuring.

When a composition of the invention is an emulsion, the proportion of the oil phase may range from 5% to 80% by weight and preferably from 10% to 50% by weight relative to the total weight of the composition. The oils, emulsifiers and coemulsifiers used in the composition in emulsion form are chosen from those conventionally used in cosmetics and/or dermatology. The emulsifier and the coemulsifier may be present in the composition in a proportion ranging from 0.3% to 30% by weight and preferably from 0.5% to 20% by weight relative to the total weight of the composition.

When the composition of the invention is an oily solution or gel, the fatty phase may represent more than 90% of the total weight of the composition. In a known manner, delivery forms intended for topical administration may also contain adjuvants that are common in the cosmetic field, such as hydrophilic or lipophilic gelling agents, hydrophilic or lipophilic active agents other than the oligopeptide fraction according to the invention, preservatives, antioxidants, solvents, fragrances, fillers, screening agents, odour absorbers and colorants.

The amounts of these various adjuvants are those conventionally used in the field under consideration, for example from 0.01% to 20% of the total weight of the composition. Depending on their nature, these adjuvants may be introduced into the fatty phase and/or into the aqueous phase.

As emulsifiers that may be used in the invention, mention may be made, for example, of glyceryl stearate, polysorbate 60, the mixture of cetylstearyl alcohol/cetylstearyl alcohol oxyethylenated with 33 mol of ethylene oxide sold under the name Sinnowax AO® by the company Henkel, the mixture of PEG-6/PEG-32/glycol stearate sold under the name Tefose® 63 by the company Gattefosse, PPG-3 myristyl ether, silicone emulsifiers such as cetyl dimethicone copolyol, and sorbitan monostearate or tristearate, PEG-40 stearate and oxyethylenated (20 EO) sorbitan monostearate.

As solvents that may be used in the invention, mention may be made of lower alcohols, for instance ethanol, isopropanol and propylene glycol.

The composition of the invention may also advantageously contain a spring and/or mineral water, chosen especially from Vittel water, the waters from the Vichy Basin, and Roche Posay water.

Hydrophilic gelling agents that may be mentioned include carboxylic polymers such as carbomer, acrylic copolymers such as acrylate/alkyl acrylate copolymers, polyacrylamides and in particular the mixture of polyacrylamide, C13-14 isoparaffm and laureth-7 sold under the name Sepigel 305® by the company SEPPIC, polysaccharides, for instance cellulose derivatives such as hydroxyalkyl celluloses and in particular hydroxypropyl cellulose and hydroxy ethyl cellulose, natural gums such as guar gum, locust bean gum and xanthan gum, and clays.

Lipophilic gelling agents that may be mentioned include modified clays, such as bentones, metal salts of fatty acids, such as aluminium stearates, and hydrophobic silica, or alternatively ethylcellulose and polyethylene. • Processes

The invention also relates to a cosmetic process for treating a keratin material, comprising a step of topical application of an oligopeptide fraction obtained from a biomass of a bacterium or bacteria belonging to genus Vitreoscilla sp.

Within the context of the invention, "treating a keratin material" means the topical application of an oligopeptide fraction according to the invention for caring for the skin, the mucous membranes, the lips, the nails, the scalp and the hair.

The invention also relates to a cosmetic process for moisturizing skin, comprising a step of topical application of an oligopeptide fraction obtained from a biomass of a bacterium or bacteria belonging to genus Vitreoscilla sp.

The invention also relates to a cosmetic process for treating and/or preventing the signs of skin ageing, comprising a step of topical application of an oligopeptide fraction obtained from a biomass of a bacterium or bacteria belonging to genus Vitreoscilla sp.

Within the context of the present invention, "treating the signs of skin ageing" means the reduction or easing of wrinkles, fine lines, withering of the skin, loss of skin elasticity, loss of skin tonicity, thinning of the dermis, degradation of collagen fibres and loss of skin firmness.

Within the context of the present invention, "preventing the signs of skin ageing" means the absence of appearance, or the delayed appearance, of wrinkles, fine lines, withering of the skin, loss of skin elasticity, loss of skin tonicity, thinning of the dermis, degradation of collagen fibres and loss of skin firmness.

The invention also relates to a cosmetic process for treating the hair fibre and especially for strengthening the hair fibre, comprising a step of application to the hair fibre of an oligopeptide fraction obtained from a biomass of a bacterium or bacteria belonging to genus Vitreoscilla sp.

FIGURES

Figure 1 : Induction of procollagen I

Figure 2 : Comparative electrophoresis between the oligopeptide fraction (according to the invention : example 1) and a Vitreoscilla filiformis lysate in a complete fermentation medium (outside the invention as described in FR3007648 and FR3007650) Example 1

I) Preparation of an oligopeptide fraction within the meaning of the invention Table 1 : composition of complete nutritive medium for the culture of V. filiformis.

To obtain a cosmetic active agent according to the invention, the following process is carried out.

The initial strain of V. filiformis is obtained from the ATCC (strain 15551). The biomass is obtained by fermentation in an industrial bioreactor of 3000 effective litres; the composition of the culture medium is given above. During the culture, the pH is kept constant (7.00), as is the temperature (26°C) and the dissolved oxygen (0.5%). The culture is stopped when the solids content reaches 0.6%> and the glucose content reaches 0.035%). The V. filiformis strain is fermented in its complete culture medium.

The whole is centrifuged and the residue is treated with enzymatic digestion.

For the enzymatic digestion, first the carbohydrase Viscozyme® sold by Novozyme is added in a quantity of 0.2%> w/w over a 3 h period at a temperature of 55°C; the pH is adjusted and maintained at 4.5 by citric acid, then a Sumizyme AP protease sold by Novozyme is added in a quantity of 0.04% w/w for over a 3 h period at a temperature of 55°C; the pH is maintained at 4.5 after adjustment by adding citric acid if necessary. After a second enzymatic digestion, the medium is neutralized at pH 7 using a sodium hydroxide solution (NaOH). The enzyme is inactivated by heating the medium at a temperature of 90°C for 15 minutes.

The neutralized and inactivated medium is clarified by successive filtrations first with a 10 μιη bag filter then a K2 filter.

A step of spray drying the complete clarified reaction medium is performed with a BUCHI mini spray dryer B-290 with the following parameters: Pump 30%; Inlet temperature 160°C; Outlet temperature 85°C; Aspirator 95; Q flow 35. The active agent is then recovered in the form of powder and is used as is.

2) Preparation of an extract of biomass of V. filiformis

The strain of Vitreoscilla filiformis (ATCC 15551) is cultured according to the process described in patent application WO- A-94-02158.

This is a continuous culture process. The culture occurs at 26°C for at least 48 hours until a suitable cell concentration is obtained corresponding to an optical density at 600 nm greater than or equal to 1.5. The strain is subcultured at 2% v/v into new medium for about 48 hours until a stable culture is obtained. A 1 litre Erlenmeyer containing 200 ml of new medium is then inoculated with 4 ml of the previous culture.

Culture in an Erlenmeyer occurs at 26°C on a culture table stirred at 100 rpm. The resulting residue in the bottom of the tank serves as inoculum in a 50 litre fermenter. Growth occurs at 26°C, pH 7, 100 rpm and p0 2 > 15%.

After 30 hours of growth, the biomass is transferred to a fermenter with a working volume of 3000 litres, to be cultured in the same conditions. After 48 hours of growth the cells are harvested continuously. The biomass is then concentrated about 50 times by centrifugation.

The cells obtained are then frozen as the culture continues. These cells may be used as is after defrosting (unstabilized cell extract) or may be stabilized by autoclaving at 121°C for 20 to

40 minutes (stabilized cell extract). The cells then burst during sterilization releasing cytosol and agglomerating the proteins and the walls. The product obtained is then biphasic.

The supernatant liquid phase can be filtered at 0.22 μιη to remove the particles

("supernatant"). The bacteria extract, in the form of a cell extract (stabilized or not) or of supernatant, can be used as is (aqueous form) or can be lyophilized according to the conventional techniques (lyophilized form).

3) Effect of an oligopeptide fraction according to the invention, example 1.1 on moisturization of the stratum corneum and comparison with Vf extract outside the invention described in example 1.2. A test was performed to evaluate the moisturizing potential of the compounds of the invention formulated in an aqueous solution at an amount of 4% by weight relative to the total weight of the composition.

The technique makes it possible to measure the dielectric capacitance of the stratum corneum (SC), which depends on the mean dielectric permittivity value of the tissue. The dielectric permittivity varies greatly with the amount of water contained in the stratum corneum (SC).

The SC samples are conditioned at 75% relative humidity and at 25°C before/during the measurements and the treatment. The capacitance measurement is performed using a Corneometer™ (Courage & Khazaka, Germany).

The test compound according to the invention and the comparison active agent (Vf) are dissolved in a water/n-propanol mixture (80/20) and the solution is deposited onto the SC at a rate of 10 μΐ/cm 2 followed by air-drying for a total duration of 1 hour 30 minutes.

Measurement is taken at TO, before the treatment, and a measurement Ttreat is taken after total drying of the treatment.

Each treatment is systematically compared with its control (carrier) and with its TO. Using at least two different batches of SC, 4 to 5 samples of SC are measured per treatment.

The variation in the corneometer signal (HCM) after treatment is calculated first for each SC sample: DHCMi = HCMi(Ttreat) - HCMi(TO). The mean of the DHCMi(carrier) variations is then calculated for the control samples (treated with the carrier); this mean value is subtracted from all the DHCMi(active agent) variations to correct the systematic bias. The following is measured for each sample i:

- For the carrier (control): DHCMi(car) = HCMi ca r(Ttreat) - HCMi ca r(T0)

- For the active agent: DHCMiactive agent = HCMi ac tive agent(Ttreat) - HCMi ac tive agent(TO)

To correct the systematic bias associated with the carrier, the corrected value DHCM . active agent is considered for the active agent according to: DHCMi CO rr. active agent = DHCMiactive agent - M, in which M corresponds to the mean of the DHCMi( ca r) variations observed on the n control samples:

M =

The mean DHCMicorr. active agent values, and also the associated standard deviations that were obtained, are reported in the table below.

Variation of corneometer

value

Product concentration

Standard

Mean (n=4)

deviation

carrier

(milliQ

water/n- propanol, 8:2

vol) 2.9 6.7

oligopeptide

fraction

according to

the invention

(example 1.1

solubilized in 4% AI 14.9 3.3 water at 4%

active

ingredient)

extract of

Vitreoscilla

filiformis

outside the

invention

(example 1.2) 4% AI 3.1 2.8

It emerges that this test shows that the dielectric capacitance of the SC is much better with the oligopeptide fraction of the invention in comparison with that obtained with the Vitreoscilla filiformis extract of the prior art described in example 1.2 at the same concentration of active ingredient.

The oligopeptide fraction according to the invention allows good moisturization of the SC and thus of the skin.

4) Effect of an oligopeptide fraction according to the invention (example 1.1), on skin ageing.

Pro-collagen I synthesis stimulation

Biological model

Normal human fibroblasts are cultured and inoculated in a 96- well microtitration plate with 3500 cells per well. The cells are cultured for 6 hours in supplemented MEM culture medium.

Treatment

The compound of the invention (example 1.1) is added to the cells with renewal of the culture medium at several concentrations. The cells are then incubated for 5 days. After incubation, the culture medium is separated from the cells. ELISA assay

The culture medium is used for the quantification of procollagen I by ELISA. The antibody used for capture will be an anti Procollagen Type I antibody supplied by US Biological (reference = P9005-74D).

The antibody used for detection will be an anti Procollagen Type I Propeptide antibody supplied by US Biological (reference = P9005-74E).

Cell counts

The number of ceils is counted by a spectrofl uorimeter after the cells are pre-treated with the reactive agent CyQuant.

Result

At 0.08 g/1, the compound of the inv ention stimulates the production of pro-collagen by factor of 2: EC2.0 = 0.08 g/1 (Figure 1).

Example 2

Comparative electrophoresis between the oligopeptide fraction (according to the invention: example 1.1) and a Vitreoscilla filiformis lysate in a complete fermentation medium (outside the invention as described in FR3007648 and FR3007650) :

A deposit of ΙΟμΙ to 40mg/ml of the compound of the invention and of a Vitreoscilla filiformis lysate in a complete fermentation medium is made on a polyacrylamide gel NuPAGE 4-12% Bis Tris gel (Invitrogen) with MES SDS running buffer. After 45min at 200V, a silver staining is used to observe a coloration of oligopeptides and/or proteins.

The results are presented in Figure 2.

The proteins profile is different between the two samples tested, in particular the oligopeptide fraction according to the invention has a lower molecular weight than the compound outside of the invention for which there are many various proteins with various molecular weights. The two compounds tested are therefore very different from each other.