WO2019158637A1 - Yeast extract compositions for treating keratin materials - Google Patents

Yeast extract compositions for treating keratin materials Download PDF

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Publication number
WO2019158637A1
WO2019158637A1 PCT/EP2019/053650 EP2019053650W WO2019158637A1 WO 2019158637 A1 WO2019158637 A1 WO 2019158637A1 EP 2019053650 W EP2019053650 W EP 2019053650W WO 2019158637 A1 WO2019158637 A1 WO 2019158637A1
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Prior art keywords
cosmetic
hair
dermatologic
yeast
cytoplasmic macromolecules
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PCT/EP2019/053650
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French (fr)
Inventor
Christian Sanchez
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Danstar Ferment Ag
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Priority to EP18156759 priority Critical
Priority to EP18156759.5 priority
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Publication of WO2019158637A1 publication Critical patent/WO2019158637A1/en

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    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61QSPECIFIC USE OF COSMETICS OR SIMILAR TOILET PREPARATIONS
    • A61Q5/00Preparations for care of the hair
    • A61Q5/002Preparations for repairing the hair, e.g. hair cure
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K8/00Cosmetics or similar toilet preparations
    • A61K8/18Cosmetics or similar toilet preparations characterised by the composition
    • A61K8/96Cosmetics or similar toilet preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/97Cosmetics or similar toilet preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
    • A61K8/9728Fungi, e.g. yeasts
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61QSPECIFIC USE OF COSMETICS OR SIMILAR TOILET PREPARATIONS
    • A61Q19/00Preparations for care of the skin

Abstract

The present invention relates to cosmetic or dermatologic compositions containing yeast cytoplasmic macromoleculesand to the use of said cosmetic or dermatologic compositions for treating keratin materials.

Description

YEAST EXTRACT COMPOSITIONS FOR TREATING KERATIN MATERIALS

DOMAIN OF THE INVENTION

The present invention relates to cosmetic or dermatologic compositions for treating keratin materials, more particularly to cosmetic or dermatologic compositions containing yeast cytoplasmic macromolecules, and to method of use of the cosmetic or dermatologic compositions for treating keratin materials.

PRIOR ART

EP 2 203 153 discloses a cosmetic or dermatological compositions containing mannoproteins as an active ingredient and/or adjuvant, to the use of said cosmetic or dermatological compositions and to cosmetic treatment methods. EP 2 203 153 discloses that the mannoproteins are extracted from the cell walls of fungi in accordance with EP 1 094 117.

FR 2 984 142 discloses a non-coloring composition for treating keratin fibers comprising an aqueous dispersion of particles of hydrophobic hybrid acrylic film- forming polymer, a linear block silicone copolymer and a pigment, and a method for treating keratinous fibers using such a composition. The composition allows to obtain a residual cladding shampoo, leaving individualized treated fibers with improved cosmetic feel, a contribution of mass and volume.

BRIEF DESCRIPTION OF THE INVENTION

The present invention provides a cosmetic or dermatologic composition, suitable for treating keratin materials, characterized in that the composition comprises at least 0.01 to 30% of yeast cytoplasmic macromolecules based on dry weight and cosmetically acceptable excipients, the yeast cytoplasmic macromolecules have a molecular weight >3 kDa, preferably >4 kDa. In an embodiment, the keratin material is skin, scalp, mucous membranes, lips and keratin fibres, such as eyelashes, eyebrows and/or hair. In another embodiment, the composition comprises at least 0.01 to 10% of yeast cytoplasmic macromolecules based on dry weight, preferably at least 0.04 to 5% of yeast cytoplasmic macromolecules based on dry weight, more preferably at least 0.04 to 2% of yeast cytoplasmic macromolecules based on dry weight or even more preferably at least 0.04 to 1%. In yet another embodiment, treating keratin material comprises improving the condition of the hair. In such embodiment, treating keratin material comprises hardening, strengthening, sealing, restructuring, repairing, stabilizing, enhancing luster, enhancing volume, enhancing combability, enhancing growth, preventing and reducing hair splitting, or increasing the tensile strength and elasticity of the hair. In a further embodiment, treating keratin material comprises treating the skin, scalp, mucous membranes and/or lips. In yet another embodiment, the skin or the scalp are sensitive skin or sensitive scalp. In a further embodiment, the yeast cytoplasmic macromolecules are free of proteases. In an embodiment, the yeast cytoplasmic macromolecules comprise ribonucleotides. In yet another embodiment, the yeast cytoplasmic macromolecules are in solution at a concentration of between 20 g/l and 400 g/l, between 50 g/l and 300 g/l, 4 between 100 g/l and 400 g/l, between 100 g/l and 300 g/l, between 125 g/l and 275 g/l, between 150 g/l, and 275 g/l, between 150 g/l and 250 g/l, between 175 g/l and 225 g/l or most preferably about 200 g/l. In still another embodiment, the yeast cytoplasmic macromolecules are from yeast strains belonging to the genus Saccharomyces. For example, Saccharomyces is Saccharomyces cerevisiae.

The present invention also provides uses of a cosmetic or dermatologic composition as described above for strengthening or even restoring a barrier function, and strengthening innate immunity of the skin, scalp, mucous membranes and/or integuments.

According to another aspect, the present invention provides the use of a cosmetic or dermatologic composition as define above for treating keratin materials. In an embodiment, the keratin material is skin, scalp, mucous membranes, lips and keratin fibres, such as eyelashes, eyebrows or/and hair. In another embodiment, treating keratin material comprises improving the condition of the hair. In such embodiment, improving the condition of the hair comprises hardening, strengthening, sealing, restructuring, repairing, stabilizing, enhancing luster, enhancing volume, enhancing combability, enhancing growth, preventing and reducing hair splitting, or increasing the tensile strength and elasticity of the hair. In another embodiment, treating keratin material comprises treating skin, scalp, mucous membranes and/or lips. In yet another embodiment, the yeast cytoplasmic macromolecules are free of proteases. In still another embodiment, the yeast cytoplasmic macromolecules comprise ribonucleotides.

The present invention further provides a method of cosmetic or dermatologic treatment for treating keratin materials, the method comprises providing a cosmetic or dermatologic composition comprising a yeast cytoplasmic macromolecules and cosmetically or dermatologically acceptable excipients as described above; and contacting the cosmetic or dermatologic composition with keratin materials of a subject in need of treatment of keratin material.

The present invention also provides a method of cosmetic or dermatologic treatment for treating or improving the condition of skin, scalp, mucous membranes and/or integuments, the method comprises providing a cosmetic or dermatologic composition comprising a yeast cytoplasmic macromolecules and cosmetically or dermatologically acceptable excipients as described above; and contacting the cosmetic or dermatologic composition with the skin, scalp, mucous membranes and/or integuments of a subject in need of strengthening or even restoring a barrier function, and strengthening innate immunity of the skin, scalp, mucous membranes and/or integuments.

The present invention also provides a method of cosmetic or dermatologic treatment for treating or improving the condition of sensitive skin or sensitive scalp, the method comprises providing a cosmetic or dermatologic composition comprising a yeast cytoplasmic macromolecules and cosmetically or dermatologically acceptable excipients as described above; and contacting the cosmetic or dermatologic composition with the sensitive skin or sensitive scalp of a subject in need of treatment or improvement of the condition of sensitive skin or sensitive scalp.

The present invention also provides a method of cosmetic or dermatologic treatment for improving the condition of hair comprising : providing a cosmetic composition comprising a yeast cytoplasmic macromolecules as described above and cosmetically acceptable excipients, the yeast cytoplasmic macromolecules have a molecular weight >3 kDa; contacting the cosmetic composition with hair of a subject in need for improving the condition of hair.

DESCRIPTION OF THE FIGURES

Figures 1A and IB show hair before treatment (Fig. 1A), wherein hair is strongly fragmented in the cuticle (white circles) and after treatment (Fig. IB).

Figures 2A to 2F show loosened scales due to the lack of ceramides before treatment (Fig. 2A, 2C and 2E) and the effect of the treatment that stick them together (Fig. 2B, 2D and 2F) .

Figures 3A to 3F shows the difference between before (Fig. 3A, 3C and 3E) and after (Fig. 3B, 3D and 3F) the application of the Yeast Cytoplasmic Macromolecules according to the present invention that tightened and smoothed hair scales and repaired cuticles.

Figure 4 shows an increase of hair mean diameter after an application of the Yeast Cytoplasmic Macromolecules of the present invention.

Figure 5 shows the decrease of hair recovered on the brush on a daily basis after use of the yeast cytoplasmic macromolecules of the present invention.

Figure 6 shows the collected hair distribution in length untreated and treated with yeast cytoplasmic macromolecules of the present invention .

Figure 7 shows volunteers' sensory evaluation of the treatment with Yeast Cytoplasmic Macromolecules of the present invention.

Figure 8 shows the distribution of peak load parameters for each treatment.

Figure 9 shows the variation in clarity after a permanent followed by a treatment with Yeast Cytoplasmic Macromolecules of the present invention. Figure 10 shows the variation in clarity after treatment with Yeast Cytoplasmic Macromolecules of the present invention followed by a permanent.

Figure 11 shows the variation in clarity after a heat treatment followed by a treatment with Yeast Cytoplasmic Macromolecules of the present invention.

Figure 12 shows the variation in clarity after treatment with Yeast Cytoplasmic Macromolecules of the present invention followed by a heat treatment.

Figure 13 shows the variation in brightness after a heat treatment and followed with a treatment of Yeast Cytoplasmic Macromolecules of the present invention. Figure 14 shows the variation in brightness after treatment with Yeast Cytoplasmic Macromolecules of the present invention followed by a heat treatment.

Figure 15 shows the effect and activity of yeast cytoplasmic macromolecules on Normal Fluman Epidermal Keratinocyte (NHEK) cells by measuring the gene expression.

Figure 16 shows the effect of yeast cytoplasmic macromolecule of the present invention on TGK expression in normal human epidermal keratinocytes.

Figure 17 shows the effect of yeast cytoplasmic macromolecule of the present invention on hBD-2 production by normal human epidermal keratinocytes.

DETAILED DESCRIPTION OF THE INVENTION

It has been surprisingly discovered that the use of a cosmetic or dermatologic composition comprising yeast cytoplasmic macromolecules in accordance with the present invention is beneficial in assisting to treating keratin materials. When used herein, the keratin materials means skin, scalp, mucous membranes, lips and keratin fibres, such as, for example, nails, eyelashes, eyebrows and hair.

The biological activity of yeast cytoplasmic macromolecules (intra-cytosolic extract (cytosol)) is based on the differentiation of the keratinocytes and their cohesion, all with the primary objective of strengthening or even restoring a barrier function essential for healthy skin and integuments. Strengthening the barrier function:

Keratinocytes are cells constituting 90% of the superficial layer of the human skin (epidermis). The epidermis has the primary function of producing the stratum corneum which is the barrier between the human being and its environment. Skin renewal, and thus the desquamation (also called peeling) that it induces, ensures its depollution on the one hand, and on the other hand it participates indirectly regulation of the bacterial population that it hosts. Its main endogenous impact is the regulation of the body's water loss by maintaining sufficient hydration, thus also participating in the thermoregulation of the body. It contains many nerve endings but no blood or lymphatic vessels.

Two groups of keratins are involved in the cohesion of keratinocytes ensuring or reinforcing the barrier function of the epidermis. These are keratin type I (KRT10) and type II (KRT1). Any increase in the synthesis of these proteins is recognized as a factor for improving the barrier function by ensuring the continuity of the link between keratinocytes.

It has been surprisingly observed that the yeast cytoplasmic macromolecules of the present invention increase very significantly the expression of KRT10 (> 5 times the normal level) in normal human epidermal keratinocytes (NHEK) as well as an even stronger increase (> 34 fold) of KRT1 in these same keratinocytes. Given the state of the art in this field, these effects are surprising, and as shown in the Examples, extracts of other strains have no effect on the expression of these same genes in the same conditions, while the yeast cytoplasmic macromolecules tested gave expression on which are dose-dependent.

Involucin (IVL) contributes to the formation of cellular envelopes that protect corneocytes in the skin. It is also seen that, unlike the other two extracts tested here, the yeast cytoplasmic macromolecules increase the expression of IVL by nearly three times with a saturated signal, which suggests a higher activity at lower doses.

SPRR2A (Small Prolin-Rich Protein 2A) is a keratinocyte protein that allows the meshing of membrane proteins through transglutaminase. This fact is reinforced by the increase of TGM1 (transglutaminase 1). Its overexpression up to 18 times, dose dependent also favors for the structuring and enhancement of the barrier effect as with SPRR1A.

In addition to the above structural elements, which are highly overexpressed, aquaporin 3 (AQP3) was also increased, suggesting active transport of water to the cells. In addition to the hydration expected by this active transport, the expected turgor pressure is an important factor in strengthening the cell structure and therefore the barrier function.

Strengthening Innate Immunity:

The reinforcement of the physical barrier of the epidermis discussed above is here supplemented by a major function of the skin : the recognition of the self and non self by the immune system of the skin (Langerhans cells for example)

The decrease of RNase7, a broad-spectrum beta defensin, favors a targeted skin defense generated by a skin response to the yeast cytoplasmic macromolecules of the present invention. The yeast cytoplasmic macromolecules of the present invention increases the synthesis of beta defensins with targeted action.

This increase in targeted action beta defensins, such as, for example, the increase of S100A7 (beta defensin), which is known to be active against Gram-negative bacteria, in particular E. coli, favors the protection of the skin against this bacterium, which is not described as commensal (normal flora of the skin). It is the same for the anti-microbial peptide SAP1 encoded by the gene DEFB4 active against Candida sp. or Pseudomonas sp. among others. Finally, the increase in PI3 results in the synthesis of recent antimicrobial peptide (SR-0379) which not only accelerates healing (in connection with the preceding paragraphs on strengthening the barrier function) but also has an antimicrobial response very interesting.

The Yeast Cytoplasmic Macromolecules of the present invention advantageously provide a better structured skin by improving the turgor pressure, the adhesion of the skin's cells, a skin that looks younger by its brightness due to desquamation (on expression of KLK7), thus which regenerates more quickly, and a skin which regulates its microbiota by increasing its innate immune defenses by the secretion of beta defensins whose spectrum of action is respectful of the commensal microbial population. The Yeast Cytoplasmic Macromolecules of the present invention also advantageously improve the softness aspect of the keratin fiber, especially the film-forming aspect especially for subject with sensitive skin, dry skin, aged skin, during shaving and for aftershave areas, depilatories, and help keeping the hands softs.

The present invention provides a cosmetic or dermatologic composition for treating keratin materials. Examples of keratin material are, for example, skin (epidermis), scalp, mucous membranes, lips and keratin fibres, such as, for example, nails, eyelashes, eyebrows and hair. The cosmetic or dermatologic composition of the invention comprises at least 0.01 to 30% of yeast cytoplasmic macromolecules based on dry weight, and cosmetically acceptable excipients. The yeast cytoplasmic macromolecules of the present invention have a molecular weight >3 kDa. In an embodiment of the invention, the yeast cytoplasmic macromolecules of the present invention have a molecular weight >4 kDa.

In an embodiment of the present invention, the cosmetic or dermatologic composition comprises at least 0.01 to 10% of yeast cytoplasmic macromolecules based on dry weight. In another embodiment of the invention, the cosmetic or dermatologic composition comprises at least 0.04 to 5% of yeast cytoplasmic macromolecules based on dry weight. In a further embodiment of the invention, the cosmetic or dermatologic composition comprises at least 0.04 to 3% of yeast cytoplasmic macromolecules based on dry weight. In yet a further embodiment of the invention, the cosmetic or dermatologic composition comprises at least 0.04 to 2% of yeast cytoplasmic macromolecules based on dry weight. In another embodiment of the invention, the cosmetic or dermatologic composition comprises at least 0.04 to 1% of yeast cytoplasmic macromolecules based on dry weight.

In an embodiment of the present invention, the cosmetic or dermatologic composition suitable for treating keratin materials comprises at least 0.01 to 30% of yeast cytoplasmic macromolecules based on dry weight, and cosmetically acceptable excipients, the yeast cytoplasmic macromolecules have a molecular weight >3 kDa.

When used herein the expression « yeast cytoplasmic macromolecules » refers to cytoplasmic macromolecules obtained by subjecting a suspension of yeast cells to lysis before or after inactivation of native yeast enzymes, whereby the yeast cells are degraded and cytoplasmic macromolecules are solubilized and released from the degraded yeast cells. The solubilized cytoplasmic macromolecules are recovered as a solution. The yeast cytoplasmic macromolecules solution can further comprises ribonucleotides. The yeast cytoplasmic macromolecules solution can further comprises mannoproteins from the periplasm of the cell and inside the yeast cell, predominantly from the vacuoles. The yeast cytoplasmic macromolecules solution is free of protease activity.

In yet a further embodiment, the yeast cytoplasmic macromolecules of the present invention are free of protease activity and comprise ribonucleotides and/or intra- cytosolic mannoproteins.

In an embodiment, the yeast cytoplasmic macromolecules of the present invention are in a solution which has a turbidity, when measured by nephelometry at a concentration of 200 g/l of yeast cytoplasmic macromolecules and at a pH in the range of pH 4 to pH 8 lower than 70 NTU.

In an embodiment, the cosmetic or dermatologic composition is suitable for the improvement of the condition of the skin, the scalp, lips and/or mucous membranes, such as, for example, strengthening or even restoring a barrier function, and strengthening innate immunity. Improving the condition of the skin, scalp, lips and/or mucous membranes also includes improving the softness aspect of the fiber, especially the film-forming aspect especially for subject with sensitive skin, dry skin, aged skin, during shaving and for aftershave areas, depilatories, and help in keeping the hands softs.

The use of yeast cytoplasmic macromolecules was also found to be beneficial in assisting to improving the condition of hair, particularly for sealing, hardening, luster improvement, strengthening, protecting as well as improving the structure (restructuring) of human hair, and preferably damaged human hair.

That human hair can be damaged by environmental factors or as a result of physiological status or mechanical and chemical effects is known. The damage results in deleterious mechanical, chemical and physical properties of the affected hair

In the case of human hair, such effects manifest themselves particularly as loss of luster, reduced tear strength and poor combability. These effects are a result of aging processes brought about primarily physiologically or induced by physical (weathering), and mechanical factors (combing, brushing, rubbing). In long hair, these effects become apparent particularly at the hair tips (splits).

Commercial rinses and conditioners contain as active substances mainly cationic surfactants or polymers, waxes and/or oils. The more damaged the hair, the greater is the amount of anionic groups present on the surface. Cationic compounds are attracted by said surface electrostatically, because said surface bears an electric charge of opposite sign, whereas oils and waxes interact with the hydrophobic groups of the keratin. Hence, an improvement in the internal hair structure or an effective sealing of the hair cannot be achieved with these hair-care products.

In an embodiment the cosmetic or dermatologic composition is suitable for the improvement of the condition of hair and for providing effective protection or sealing so as to eliminate the afore-indicated drawbacks.

According to the invention, these objectives are reached by the use of yeast cytoplasmic macromolecules in a cosmetic composition for improving the condition of hair.

The improvement in the condition of hair consists preferably of hardening, strengthening, sealing, restructuring, repairing, stabilizing, enhancing luster, enhancing volume, enhancing combability, enhancing growth, preventing and reducing hair splitting, or increasing the tensile strength and elasticity of the hair. Surprisingly, the Applicant have found that by use of yeast cytoplasmic macromolecules described herein below improve the hair structure overall, but particularly the surface of the hair, is changed so that, as a result, protection, hardening, strengthening, sealing, restructuring, repairing, stabilizing, enhancing luster, enhancing volume, enhancing combability, enhancing growth, preventing and reducing hair splitting, or increasing the tensile strength and elasticity of the hair is achieved. Improvement of the hair structure overall, but particularly the surface of the hair, providing protection, hardening, sealing and strengthening as well as an increase in breaking strength, tear strength is also achieved, particularly on dry, weakened and damaged hair.

In addition to the hair-care action described herein above and resulting from an effect on the hair surface (cuticula), a repairing action is also achieved. Said action is due to changes within the hair (cortex).

By "improving the structure (restructuring) of human hair", is meant obtaining a smooth appearance of the hair. The outer layer of a hair, called the cuticle, is composed of scales which overlap with each other. A reconstructive effect results in a smooth relief.

The yeast cytoplasmic macromolecules of the present description can be produced in accordance with the following method. The method comprises a) subjecting a suspension of yeast cells to enzymatic lysis, preferably under the action of exogenous enzymes and after inactivation of native yeast enzymes, whereby said yeast cells are degraded and yeast cytoplasmic macromolecules are solubilized and released from the degraded yeast cells; b) recovering the solubilized yeast cytoplasmic macromolecules as a solution, preferably using membrane filtration; and e) inactivating protease by subjecting the yeast cytoplasmic macromolecules solution obtained after step b) heat treatment at a temperature of 70°C or higher.

In step a) the yeast cytoplasmic macromolecules is extracted from an aqueous suspension of yeast cells by subjecting the suspension of yeast cells to enzymatic lysis whereby said yeast cells are degraded and yeast cytoplasmic macromolecules are solubilized and released from the degraded yeast cells.

Any type of yeast can be used in the process of the invention. In particular, yeast strains belonging to the genera Saccharomyces, Kluyveromyces or Candida may be suitably used. Yeast strains belonging to the genus Saccharomyces, for example the strain Saccharomyces cerevisiae, are preferred.

The process of the present invention may start with a suspension of yeast cells in an aqueous liquid, for instance a fermentation broth of the yeast cells in question . Suitable fermentation processes leading to suspensions of yeast cells are known in the art. In some cases the fermentation broth can be concentrated before use in the present process, for example by centrifugation or filtration. For example, cream yeast (Baker's yeast which has been concentrated to 15-27% w/w of dry matter content) may be used :

In step a) the enzymatic lysis of the suspension of yeast cells may be performed by subjecting said suspension to the action of native yeast enzymes and/or added exogenous enzymes.

The conditions used to perform the enzymatic lysis are dependent on the type of enzyme used and can be easily determined by those skilled in the art. Generally, enzymatic lysis will be performed at a pH between 4 and 10 and at a temperature between 40°C and 70°C degrees. Generally the enzymatic lysis will be performed for a time comprising between 1 and 24 hours.

In a preferred embodiment of the invention native yeast enzymes are first inactivated and subsequently exogenous enzymes are added to the suspension. Those skilled in the art know how to inactivate native yeast enzymes. Inactivation may for example be affected by a pH treatment or a heat shock, the latter method being preferred. A heat shock can be suitably performed by treating the yeast cell suspension at a temperature of 80-97°C for 5 to 10 minutes. Once the native yeast enzymes have been inactivated, exogenous enzymes can be added to the suspension of yeast cells to perform the enzymatic lysis. Preferably a protease, more preferably an endoprotease, is used for this purpose. Preferably the exogenous enzymes used to perform enzymatic lysis comprise an enzyme to degrade RNA into ribonucleotides. Any RNase can be used at this regard, for instance 5'-Fdase (5'-phosphodiesterase) . The enzyme used to degrade RNA into ribonucleotides can be added together with or subsequently to the treatment with the above-mentioned enzymes, for instance together with or subsequently to the treatment with protease. Optionally, a deaminase, for instance adenylic deaminase, is also used together with, or subsequently to, the treatment with the above- mentioned enzymes. In all embodiments, the exogenous enzymes used for the enzymatic lysis in the present description does not include a yeast cell wall lytic enzyme low in protease such as those produced by Arthrobacter or Oerskovia. More specifically, the exogenous enzymes used for the enzymatic lysis in the present description does not include a cell wall lytic enzyme produce by Arthrobacter luteus ATCC 21606

Prior to step b), the enzyme(s) used in step a) may be inactivated for instance by using methods as mentioned above.

In step b) of the process the yeast cytoplasmic macromolecules are recovered as a solution, preferably using membrane filtration. The solution is optionally concentrated up to the desired final concentration. The yeast cytoplasmic macromolecules solution can further comprises mannoproteins from the periplasm of the cell and inside the yeast cell, predominantly from the vacuoles.

Insoluble material, for instance derived from yeast cell walls, is removed prior to recovery of the yeast cytoplasmic macromolecules in step b), generally by a solid- liquid separation method, preferably by centrifugation or filtration. The yeast cytoplasmic macromolecules may be recovered by any method suitable thereto.

In a preferred embodiment in step b) the yeast cytoplasmic macromolecules are recovered as a solution using membrane filtration, preferably using ultrafiltration (UF) or diafiltration. Membrane filtration methods, in particular ultrafiltration or diafiltration are known to those skilled in the art. Preferably, the yeast cytoplasmic macromolecules are recovered by ultrafiltration (UF) or diafiltration. In cases where UF or diafiltration is used to recover yeast cytoplasmic macromolecules, filters with a molecular weight cut-off of at least 3kDa or higher, preferably 4kDa or higher, can be used. The yeast cytoplasmic macromolecules fraction remains in the retentate resulting from the ultrafiltration or diafiltration step. When the insoluble material is not removed prior to ultrafiltration or diafiltration, the retentate comprising yeast cytoplasmic macromolecules and the insoluble material can be resuspended (in solution) and the insoluble material is preferably removed, for instance using common solid-liquid separation techniques as mentioned above. Preferably, after recovering the yeast cytoplasmic macromolecules as a solution the recovered yeast cytoplasmic macromolecules can be treated in step c) of the process of the invention, with an RNase, for instance Fdase (phosphodiesterase), under conditions sufficient to convert RNA residues present in the recovered yeast cytoplasmic macromolecules solution into ribonucleotides. The treatment can be performed using methods known in the art. This treatment is optionally followed by one or more membrane filtration steps for instance ultrafiltration or diafiltration, (step d) of the process of the invention), in order to remove impurities of low molecular weight using membranes as those indicated above. The yeast cytoplasmic macromolecules solution is recovered in the retentate.

The yeast cytoplasmic macromolecules solution can undergo pH adjustment and/or concentration at any suitable step in the production process using methods known to those skilled in the art.

The yeast cytoplasmic macromolecules solution produced by the process of the invention is free of protease and optionally b-glucanase activity. The latter result is achieved by step e) subjecting the yeast cytoplasmic macromolecules solution obtained after step b), c) or d) to a treatment suitable to inactivate protease and optionally b-glucanase, by subjecting the solution to heat treatment at a temperature of 70°C or higher, preferably at a temperature comprised between 80°C and 140°C, wherein the heat treatment is preferably performed for a time comprised between 2 seconds and 60 minutes. Preferably step e) will be performed after step d) or after sterilization, for instance after sterile filtration as mentioned hereafter.

The temperature used in the heat treatment is preferably between 80°C and 140°C. The duration of the heat treatment will be such that preferably all enzyme activity will be removed (and proteins are generally denatured) from the yeast cytoplasmic macromolecules solution. The duration of the heat treatment will depend on the temperature used. For example lower temperatures will require a longer duration of the heat treatment while higher temperatures will require only a short time. Therefore a treatment at 80°C for 30 minutes will fall under the scope of the claim as well as a treatment at 130°C for 7 seconds. The heat treatment is preferably performed by using a UHT treatment using conditions which are commonly used in the food or beverage industry. A UHT treatment may typically be effected at a temperature of 130-145°C for a duration of time of 2-10 seconds. With a heating treatment performed at 130°C or higher for sufficient time, for example a heating treatment at 130°C for 7 seconds, a solution will be obtained which is free from protease and b-glucanase activity, and in general free of any enzymatic activity.

By using a heating step generally at least part of micro-organisms present in the yeast cytoplasmic macromolecules solution will be deactivated or killed. Preferably the heating treatment is a UHT treatment as indicated above because in the latter case all microorganisms or microbial spores which might be present in the solution will be killed or deactivated.

The process of the invention may comprise subjecting the yeast cytoplasmic macromolecules obtained after step b), c), d) or e) to sterilization, preferably to sterile filtration (step f) in order to remove all microorganisms present in the solution. The latter can be achieved by using specific filters known to those skilled in the art. Preferably sterile filtration is performed after step d) or e).

At the end of the production process the yeast cytoplasmic macromolecules solution can be aseptically packed according to methods known to those skilled in the art. The sterile containers which can be used can be produced of several materials. Suitable containers may be sterile bags within a cardboard box. The bags may be provided with a gland which can be perforated by a screw-on tap.

It will be understood by those skilled in the art that the process of the present invention may encompass a step in which the yeast cytoplasmic macromolecules solution is dried by methods known to those skilled in the art, for instance by lyophilisation or spray drying, to yield a yeast cytoplasmic macromolecules in solid form, for Instance in the form of a powder or granulate. Said yeast cytoplasmic macromolecules in solid form is also encompassed by the present invention.

In one embodiment the yeast cytoplasmic macromolecules can be in the form of granulate, powder or in the form of a solution. In one embodiment, the yeast cytoplasmic macromolecules granulate is a micro-granulate. In an alternative embodiment, the yeast cytoplasmic macromolecules solution according to the present disclosure is preferably an aqueous solution. The concentration of yeast cytoplasmic macromolecules in the solution is preferably between 20 g/l and 400 g/l, between 50 g/l and 300 g/l, 4 between 100 g/l and 400 g/l, between 100 g/l and 300 g/l, between 125 g/l and 275 g/l, between 150 g/l, and 275 g/l, between 150 g/l and 250 g/l, between 175 g/l and 225 g/l or most preferably about 200 g/l. In one embodiment, the yeast cytoplasmic macromolecules solution which has a turbidity, when measured by nephelometry at a concentration of 200 g/l of yeast cytoplasmic macromolecules and at a pH in the range of pH 4 to pH 8, lower than 70 NTU, preferably lower than 60 NTU, more preferably lower than 50 NTU, most preferably lower than 40 NTU.

Because the turbidity of a yeast cytoplasmic macromolecules solution is dependent on the concentration and on the pH, the values of turbidity are measured at a concentration of 200 g/l of the solution. However this should not be interpreted as meaning that the solution of the present disclosure has a fixed concentration of yeast cytoplasmic macromolecules of 200 g/l, but that the turbidity thereof is measured at a concentration of 200 g/l. Therefore depending on its initial concentration the yeast cytoplasmic macromolecules solution can be diluted or concentrated up to 200 g/l prior to measurement of turbidity. At a pH of 5.5 the turbidity of the yeast cytoplasmic macromolecules solution, measured by nephelometry at a concentration of 200 g/l yeast cytoplasmic macromolecules, is preferably 100 NTU or lower, more preferably 70 NTU or lower, more preferably 50 NTU or lower, even more preferably 40 NTU or lower, most preferably 30 NTU or lower. The pH of the solution is preferably such that the stability of the yeast cytoplasmic macromolecules is preserved in time and such as to avoid turbidity of the solution. Therefore the pH of the yeast cytoplasmic macromolecules solution is preferably comprised between 3 and 8, more preferably between 4 and 8, more preferably between 4 and 7, or most preferably between 5 and 6.

In an embodiment, the yeast cytoplasmic macromolecules have a molecular weight >3 kDa, preferably a molecular weight >4 kDa.

The cosmetic composition in accordance with the present description also includes cosmetically or dermatologically acceptable excipients, but does not contain no polyphenols or ovomucin.

By "cosmetically or dermatologically acceptable excipient", is meant hydrophilic compounds constituting an aqueous phase, hydrophobic compounds constituting an oil phase or surfactants. The hydrophilic compounds of the aqueous phase are in particular chosen from water, alcohols and polyols. The hydrophobic compounds of the oil phase are chosen from the hydrocarbons, fatty acids, fatty alcohols, esters, glycerides, cerides, phosphatides and silicones. The surfactants are amphiphilic molecules capable of keeping together two media which are normally immiscible with each other by reducing the interfacial tensions. The surfactants are ionic (anionic, cationic or amphoteric) or non-ionic.

Examples of cosmetically or dermatologically acceptable excipients, are but not limited to:

Desquamation agents (or peeling agents) such as, for example, lactic acid, jasmonic acid, gentisic acid, a derivative thereof, and salicylic acid derivatives;

essential oils alone or in combinations such as, for example, mint, chrysantellum, eucalyptus, lavandin, lavender, vetiver, litsea cubeba, lemon, sandalwood, rosemary, chamomile, savory, nutmeg , cinnamon, hyssop, caraway, orange, geraniol, cade and bergamot;

Natural vegetable waxes such as, for example, Carnauba wax, Candelila wax, olive wax, rice wax, hydrogenated jojoba wax or absolute waxes of flowers such as blackcurrant essential wax, waxes Marines and their associations;

Synthetic silicones; Ceramides such as, for example, - 2-N-linoleoylamino-octadecane-l,3-diol, - 2-N - oleoylamino-octadecane-l,3-diol, 2-N-palmitoylamino-octadecane-l,3-diol, 2-N- stearoylamino-octadecane-l,3-diol, 2-N-behenoylamino octadecane-l,3-diol, -2-N- [2-hydroxy-palmitoyl] -aminooctadecane-l,3-diol; 2-N-stearoyl aminooctadecane- 1,3,4-triol and especially N-stearoyl phytosphingosine, 2-N-palmitoylamino- hexadecane-l,3-diol (bis- (N-hydroxyethyl-N-cetyl) malonamide), N- (2- hydroxyethyl) -N Cetyl acid (3-cetyloxy-2-hydroxypropyl) amide;

Esters such as, for example, methyl palmitate and isopropyl palmitate, isopropyl butyl myristate, butyl stearate, isobutyl stearate, dioctyl maleate;

Pigments such as, for example : titanium dioxide, optionally surface-treated, zirconium or cerium oxides, as well as oxides of zinc, iron or chromium, manganese violet, ultramarine blue and ferric blue, carbon black, and lacquers based on carmine cochineal, barium, strontium, calcium, aluminum, white pearlescent pigments such as mica coated with titanium, or bismuth oxychloride, colored pearlescent pigments such as mica titanium with iron oxides, titanium mica with ferric blue or chromium oxide, titanium mica; and

Inorganic or organic, lamellar or spherical fillers such as, for example, talc, mica, silica, kaolin, nylon (D, poly-ss-alanine and polyethylene powders, Teflon®, lauroyl- lysine, starch, boron nitride, tetrafluoroethylene polymer powders, hollow microspheres and silicone resin microbeads, precipitated calcium carbonate, magnesium carbonate and hydrocyanate, hollow silica microspheres, microcapsules of glass or ceramic.

The cosmetically or dermatologically acceptable excipients of the present invention can be any or any combinations of the above. Other excipients not listed above but suitable for the compositions of the present invention are also incorporated.

Without any limitations, the cosmetic or dermatologic composition comprising yeast cytoplasmic macromolecules of the present invention can be formulated as compositions for cleaning and / or removing makeup from the face, facial care creams, body lotions, shampoos, rinses, conditioners, foams, firming compositions, hair gels, shower gels, bath gels, permanent hair deformation compositions, hair coloring compositions, hair sprays, fixatives or hair-smoothing agents. Other formulations of the cosmetic or dermatologic composition of the present invention and suitable for treating the keratin materials are also encompassed .

The present disclosure also provides a method of cosmetic or dermatologic treatment for treating keratin materials, the method comprises providing a cosmetic or dermatologic composition comprising a yeast cytoplasmic macromolecules and cosmetically or dermatologically acceptable excipients as described above; and contacting the cosmetic or dermatologic composition with keratin materials of a subject in need of treatment of keratin material .

The present disclosure also provides a method of cosmetic or dermatologic treatment for treating or improving the condition of skin, scalp, mucous membranes and/or integuments, the method comprises providing a cosmetic or dermatologic composition comprising a yeast cytoplasmic macromolecules and cosmetically or dermatologically acceptable excipients as described above; and contacting the cosmetic or dermatologic composition with the skin, scalp, mucous membranes and/or integuments of a subject in need of strengthening or even restoring a barrier function, and strengthening innate immunity of the skin, scalp, mucous membranes and/or integuments.

The present disclosure also provides a method of cosmetic or dermatologic treatment for treating or improving the condition of sensitive skin or sensitive scalp, the method comprises providing a cosmetic or dermatologic composition comprising a yeast cytoplasmic macromolecules and cosmetically or dermatologically acceptable excipients as described above; and contacting the cosmetic or dermatologic composition with the sensitive skin or sensitive scalp of a subject in need of treatment or improvement of the condition of sensitive skin or sensitive scalp.

The present disclosure also provide a method of cosmetic or dermatologic treatment for improving the condition of hair comprising : providing a cosmetic composition comprising a yeast cytoplasmic macromolecules and cosmetically acceptable excipients, the yeast cytoplasmic macromolecules have a molecular weight >3 kDa; contacting the cosmetic composition with hair of a subject in need for improving the condition of hair.

EXAMPLES:

Example 1: Use of yeast cytoplasmic macromolecules on dry air

Cream yeast from a strain of Saccharomyces cerevisiae was heated to at least 50°C. Subsequently, protease was added and the mixture was incubated for 15 to 20 hours at a pH of above 5 for autolysis. Next, the mixture or hydrolysate was heated for 1 hour at a temperature above 70°C to inactivate all enzyme activity. The pH of the mixture or hydrolysate is then adjusted with NaOH to about 6.0, heated to 75°C for 60 seconds. The extract (soluble fraction) was separated from the insoluble cell walls by means of centrifugation. The soluble fraction was treated with 5'-phosphodiesterase at a temperature of 55°C. Finally, the yeast cytoplasmic macromolecules having a molecular weight >3 kDa are separated from other soluble microbial cell material by ultrafiltration.

Yeast Cytoplasmic Macromolecules of the present invention were tested on volunteers having dry hair. The protocol consisted of comparing the hair of each volunteer before and after the application of the yeast cytoplasmic macromolecules according to the present invention. The goal was to show the effects of the yeast cytoplasmic macromolecules on the hair.

Protocol:

During the three (3) first days, the volunteers brushed their hair and collected the hair left on the brush. The hair were analyzed by weight, number and quality and observed with scanning electronic microscopy. During the following four (4) days, the volunteers applied, after brushing and collecting, a spray lotion* containing 2% of Yeast Cytoplasmic Macromolecules in accordance with the present invention. The effect of the lotion was evaluated after one day.

Table 1 : Cosmetic or dermatologic composition in accordance with the invention

Figure imgf000021_0001
Figure imgf000022_0001

*(The total volume of the composition of Table 1 is 300ml; 2% of a yeast cytoplasmic macromolecules in solution at a concentration of about 200g/L represent 0.4% w/w of the composition)

Results

Scanning electronic microscopy analysis of hair collected

Dry hair were characterized according to several damage types: large fractures in the cuticle, unstuck scales, lack of ceramides (cement between scales), which weakens hair through hydration and mechanical resistance and make it more porous and thus more sensitive to external aggressions. The hair collected were observed with scanning electronic microscopy (SEM) before and after one (1) day of the treatment with the yeast cytoplasmic macromolecules.

Fig. 1A correspond to hair before treatment and shows that hair is strongly fragmented in the cuticle (white circles). These damages mean weakness to mechanical efforts. After treatment, as shown in Fig. IB, hair scales were repaired (no fractures of the curticule), which helps hair to retain moisture and to gain in softness and resistance.

Sticking scales to each other

Fig. 2A to 2F show loosened scales due to the lack of ceramides and the treatment can stick them together.

Dry hair present porous and damaged cuticles as shown on the Fig. 2A to 2F. Superficial defaults (white circles) are clearly sawn as rough and uneven cuticle. When applied on dry hair, YCM can sooth the cuticles and fill the gap. After the treatment, the hair were smoother and silky without any defaults.

As shown in Figs. 3B, 3D and 3F, the application of the Yeast Cytoplasmic Macromolecules according to the present invention tightened and smoothed hair scales and repaired cuticles. Flair were more structured, more resistant and they reacted more easily against external aggressions. Effect on hair resistance

The hair collected were analyzed according the following criterias in order to evaluate its strength and resistance:

- Hair mean diameter (by SEM)

- Amount of hair collected

- Distribution in length of collected hair.

As shown in Fig. 4, the hair mean diameter was increased by 54% after an application with the yeast cytoplasmic macromolecules. The hair were visibly strengthened.

Amount of collected hair

As shown in Fig. 5, the number of hair recovered on the brush on a daily basis is 18.44% lower after use of the yeast cytoplasmic macromolecules of the present description.

Collected hair distribution in length

As shown in Fig. 6, the hair distribution was directly correlated to the product efficacy and thus very important for the consumer. The small segments come from brushing, especially at the hair end, which was particularly old, thin and fragile. That is what consumers expect most frequently: to protect and maintain the hair quality especially at the hair ends.

Sensory evaluation by the volunteers

The volunteers performed a sensory evaluation of the treatment with Yeast Cytoplasmic Macromolecules (see Fig. 7). Different parameters were studied : softness, radiance, ease to style and untangling, which indicates the quality and the structure of hair surface and particularly the cuticle integrity.

The yeast cytoplasmic macromolecules were able to coat hair, reinforce the cuticle, tighten the scales, which can be perceived by improving these parameters. This study shows that the volunteers felt an improvement of all parameters after the treatment:

- 66% of the volunteers observed more radiant hair with a mean increase of 25% - 77% of the volunteers felt their hair softer with a mean increase of 44%

- 67% of the volunteers find their hair easier to style

- 100% of the volunteers observed an untangling effect at the 1st day of treatment.

In addition to this, radiance, softness and untangling effect were perceived right after the first application.

Yeast Cytoplasmic Macromolecules were able to interact with hair, repair, protect and reinforce its structure, as shown with SEM analysis. Yeast Cytoplasmic Macromolecules can be used for hair care, especially for dry and damaged hair.

Example 2: Expression analysis

The yeast cytoplasmic macromolecules of the invention were tested, by evaluating by RT-qPCR their activity on a model of normal human epidermal keratinocytes (NHEK), by expression analysis (mRNA) of 64 markers (including 3 reference genes) selected for their importance in epidermal differentiation, barrier function and inflammation.

Material and Methods

• Biological model :

- Cell type: Normal human epidermal keratinocytes (NHEK), Reference Bioalternatives K341, used in 3rd pass

- Culture conditions: 37 °C, 5% C02

- Culture medium : Keratinocyte-SFM supplemented with Epidermal Growth Factor (EGF) 0.25 ng / ml Pituitary extract (EP) 25 pg / mIGentamycin 25 pg / ml

- Test environment: Keratinocyte-SFM supplemented with Gentamycin 25 pg / ml

• Compound tested :

Cytoplasmic macromolecules Storage: + 4 ° C / concentration 0.6% and 3%

Preparation of the stock solution : Suspension at 50 g / L, vortexing and stirring for at least 30 minutes (rehydration phase) at RT, heating in a water bath at 100 ° C for 1 hour allowing 'get the' yeast cream ', corresponding to the stock solution to be considered as 100%. • Preliminary cytotoxicity test:

-Cell type: NHEK in a test environment

Incubation time: 24 hours

Evaluation parameters: MTT reduction and morphological observations under the microscope. At the end of the treatment, the cells were incubated in the presence of MTT (tetrazolium salt) whose transformation into blue crystals of formazan is proportional to the activity of succinate dehydrogenase (mitochondrial enzyme). After dissociation of the cells and solubilization of formazan by addition of DMSO, the optical density (OD), representative of the number of living cells and their metabolic activity, was measured with a spectrophotometer at 540 nm (VERSAmax, Molecular Devices).

• Culture and treatment:

The keratinocytes were inoculated into 24 well plates and cultured in culture medium for 24 hours and then in test medium. The medium was then replaced with test medium with or without (control) test compounds or references (1.5 mM CaCI 2 and 10-7 M retinoic acid) and the cells were incubated for 24 hours. All conditions were realized in triplicates. At the end of the incubation, the culture supernatants were removed and the cell mats were rinsed with PBS solution. The plates were immediately frozen dry at -80 °C.

• Differential expression analysis - PCR array:

The expression of the markers was evaluated by RT-qPCR on the total RNA extracted from the cell mats of each experimental condition (the replicates of the same experimental condition were pooled before the RNA extraction). The analysis of the transcripts was carried out in duplicates using a "PCR array" dedicated to research and adapted to the "screening" format (mQPA produced by BioAlternatives) and targeting 64 genes selected for their importance in the epidermal differentiation, barrier function and inflammation (mQPA-NHEK-64).

• Reverse transcription :

The total RNAs of each sample were extracted using TriPure Isolation Reagent® according to the techniques known in the art. The quantity and quality of the RNAs were evaluated by capillary electrophoresis (Bioanalyzer 2100, Agilent). Complementary DNAs (cDNA) were synthesized by reverse transcription of total RNA (or mRNA) in the presence of oligo (dT) and the enzyme "Transcriptor Reverse Transcriptase" (Roche). The quantities of cDNA were then adjusted before the PCR step

• Quantitative PCR:

PCR reactions (polymerase chain reactions) were performed by quantitative PCR (Roche Molecular Systems Inc.) and according to known procedures. The reaction mixture (10 pi final) for each sample contained : 2.5 mI of cDNA, the primers of the different markers used, the reaction mixture containing the enzyme Taq DNA polymerase, the SYBR Green I marker and MgCb.

• Processing quantitative PCR data :

The raw data has been transferred and processed using Microsoft Excel™ software. Fluorescence incorporation into the amplified DNA is measured continuously during PCR cycles. These measurements make it possible to obtain fluorescence intensity curves as a function of PCR cycles and thus to evaluate a relative expression value for each marker. The number of cycles is determined from the "exit" points of the fluorescence curves. For the same marker analyzed, the longer a sample leaves (high number of cycles), the lower the initial number of copies of the mRNA. The relative expression value is determined according to the formula : (1/2 number of cycles) x 106

• Data processing :

The raw data has been transferred and processed using Microsoft Excel software. Formulas used in this report:

Standard error of the mean :

Percentage of viability:

The standard error of the mean (SEM) represents the deviation of the sample mean from the average of the true population. The SEM is calculated by dividing the Sd by the square root of the sample size.

Viability (%) = (compound OD / control OD) x 100 Results and conclusion

Preliminary cytotoxicity test

Table 2 : Effect of Cytoplasmic Macromolecules on the Viability of Keratinocytes after 24 Hours of Incubation

Figure imgf000027_0001

Legend :

+ : normal population

g : grains of compound

+/- : growth reduction

Op : opacity due to the compound

- : toxicity

* : morphological changes

0 : cellular mortality

ag : agglomerated cells The results of the MTT viability test and the observation of the cell mats led to the selection of the concentrations to be tested in the rest of the study.

Effect and activity of yeast cytoplasmic macromolecules on Normal Human Epidermal Keratinocyte (NHEK) cells by measuring the gene expression is shown in Figure 15.

· Validation The treatment of normal human epidermal keratinocytes (NHEK) with the reference CaCb, tested at 1.5 mM, induced, in a manner expected cell differentiation mainly stimulating the expression of genes coding for the markers involved in the differentiation processes (IVL , KRT1, KRT10, SPRR1A, SPRR1B, SPRR2A and TGM 1), desquamation (KLK7), lipid synthesis (FABP5, SULT2B1) and innate immunity (DEFB4A and PI3) . These results were expected and validated the trial. In parallel, the expression of markers of cell-cell interactions (CLDN1, DSG1 and EVPL) was stimulated.

The reference retinoic acid, tested at 10 7 M, induced a dedifferentiating effect, by inhibiting the expression of genes coding for markers involved in the keratinocyte differentiation processes (KRT1, KRT10, TGM1, SPRR1A and IB, and, to a lesser extent, CALML5 and FLG) as well as in cell-cell interactions (CDSN, DSG1 and CLDN1). In parallel, an increase in the expression of genes involved in proliferation (KRT19), desquamation (KLK5 and 7) and in innate immunity (S100A7) was observed.

All of these results were expected to validate the trial.

• Effect of Cytoplasmic Macromolecules:

The Cytoplasmic Macromolecules compound, tested at 0.6% and 3%, strongly modulated the expression of the genes analyzed, in a dose-dependent manner. This compound strongly stimulated the expression of genes coding for differentiation of keratinocytes (CASP14, IVL, KRT1, KRT10, KRT2, SPRR2A, TGM1, and to a lesser extent SFN, SPRR1A and IB), lipid synthesis (FABP5, GBA and SULT2B1), desquamation (KLK7) cell-cell interactions (CLDN1, DSG1, SDC1 and EVPL), the extracellular matrix (MMP9), innate immunity (DEFB4A, PI3 and S100A7). Surprisingly, the antimicrobial peptide RNASE7 was modulated in the opposite direction and exhibited a strong inhibition of its expression. At the same time, this compound strongly inhibited the expression of genes coding for the dermo- epidermal junction (COL4A1, LAMC2 and COL7A1), cell-matrix interactions (ITGA2, SPARC), inflammation (CXCL5 and IL8) and proliferation (KRT19). This example showed that the Yeast Cytoplasmic Macromolecules of the present invention demonstrated a pro-differentiating effect.

Example 3: Effects of cytoplasmic macromolecule compound on TGK expression and b-defensin 2 production by normal human epidermal keratinocytes

In this example, the effect of cytoplasmic macromolecule compound was investigated at the protein level in normal human epidermal keratinocytes (NHEK) on epidermal differentiation and innate immunity. More specifically, the potential effect has been evaluated on : the expression of Transglutaminase 1 (TGK) by in situ immunofluorescence and image analysis, and the production of b-defensin 2 (hBD-2) by ELISA assay.

Material and methods

• Cell type:

Normal human epidermal keratinocytes (NHEK), Reference Bioalternatives K341, used in 3rd pass

- Culture conditions: 37 ° C, 5% CO2

- Culture medium : Keratinocyte-SFM supplemented with Epidermal Growth Factor (EGF) 0.25 ng / ml Pituitary extract (EP) 25 pg/ml Gentamycin 25 pg/ml

- Test Medium: Keratinocyte-SFM supplemented with Gentamycin 25 pg / ml

• Tested compound:

Culture and treatment:

The keratinocytes were inoculated into 96-well plates and cultured in the culture medium for 24 hours. The culture medium was then replaced with the test medium (for the TGK expression test) or the culture medium (for the production test of hBD-2) containing or not (control) the test compound, or a reference compound (1.5 mM CaCh for the TGK expression test and a mixture of cytokines (IL-17 + OSM + TNF-a at 5 ng / ml each) for the hBD-2 production test) then the cells were incubated for 72 hours.

All the experimental conditions were made with n = 5. • Expression of TGK in NHEK -Insitu Immunolabeling :

After incubation, the medium was removed and the cells were rinsed, fixed and permeabilized. The cells were then labeled with the primary antibody (see Table 3) directed against the protein of interest (TGK). This antibody was then revealed by a secondary antibody coupled to a fluorochrome (see Table 3). In parallel, the nuclei of the cells were stained with Hoechst 33258 (bis-benzimide).

Table 3 : Primary and secondary antibodies

Figure imgf000030_0001

The labels were quantified by measuring the fluorescence intensity of the protein relative to the number of cells identified by Hoechst (digital data integration by the Developer Toolbox 1.5 software, GE Healthcare).

• ELISA assay:

Effects of cytoplasmic macromolecule compound on TGK expression and b-defensin 2 production by normal human epidermal keratinocytes after incubation, the amount of hBD-2 present in the culture supernatants was measured using the

ELISA assay kit.

Table 4: ELISA assay kit used in the study

Figure imgf000030_0002

• Data processing:

The raw data has been transferred and processed using Microsoft Excel software. Intergroup comparisons were performed using the unpaired bilateral Student t test. Formula used in this report: Standard error of the mean : SEM = Standard deviation (Sd)/Vn

The standard error of the mean (SEM) represents the deviation of the sample mean from the average of the true population. The SEM is calculated by dividing the Sd by the square root of the sample size.

Stimulation percentage: Stimulation (%) = ((Value / Mean control)* 100) - 100

Results

• Effect on TGK expression :

As shown in Figure 16, with the control condition, TGK was weakly expressed and restricted to a small number of keratinocytes. Treatment of the cells with the 1.5 mM calcium chloride reference greatly increased the overall expression of TGK as well as the number of cells expressing this marker. These results were expected and validated the trial.

As shown in Figure 16, under experimental conditions of this example, the Yeast Cytoplasmic Macromolecules of the present invention, tested at 0.6%, 1.4%, 2.2% and 3%, had a very significant stimulating effect on the expression of TGK, in a dose-dependent manner (respectively, 410%, 969%, 1387% and 1819% of the control).

• Effect on hBD-2 production :

As shown in Figure 17 with the control condition, the amount of hBD-2 produced by the keratinocytes (total, intracellular + extracellular fraction) was negligible. This production was greatly increased by the treatment with the combination of the 3 cytokines (IL-17 + OSM + TNF-a at 5 ng / ml each) (approximately 54,000% of the control). These results were expected and validated the test

Still in Figure 17, but under experimental conditions, the yeast cytoplasmic macromolecule of the present invention, tested at 0.6%, 1.4% and 2.2%, stimulated the production of hBD-2 by keratinocytes in a dose-dependent manner (respectively 790%, 1945%, and 2038% of the control). At a highest concentration of 3%, although a stimulating effect of the compound was observed, it was less marked than that observed at lower concentrations (1807% of the control).

Conclusion: The stimulating effect of the observed yeast cytoplasmic macromolecule of the present invention on the expression of the genes associated with epidermal differentiation and innate immunity was confirmed at the protein level. The compound exhibited a stimulating effect on the expression of TGK (epidermal differentiation marker) and hBD2 (innate immunity marker) .

Example 4:

The objective of this study was to evaluate the repairing and protective conditioning and/or detangling effect by measures by combing resistance test on dry hair using the MTT 175 (Dia-Stron®) .

Yeast Cytoplasmic Macromolecules of the present invention were tested on volunteers. The different biomechanical properties of hair, as tensile strength and hair combing force were evaluated with the MTT 175 of Dia-Stron® which is a traction device. It is supplied as a complete system, comprising the UV1000 control unit, MTT 175 mechanical test module and UvWin PC applications software.

The following treatments were selected for the study: (1) negative control, not stressed, not treated ("T"); (2) treated with Yeast Cytoplasmic Macromolecules (via spray), not rinsed ("PI"); (3) stressed via oxidative solution ("S"); (4) stressed via oxidative solution, treated with Yeast Cytoplasmic Macromolecules (via spray), not rinsed ("SP1"); (5) stressed via oxidative solution, treated with Yeast Cytoplasmic Macromolecules (via shampoo xlO) ("SP2"); (6) treated with Yeast Cytoplasmic Macromolecules (via spray), non-rinsed, stressed via oxidative solution ("PIS"); and (7) treated with Yeast Cytoplasmic Macromolecules (via shampoo xlO), stressed via oxidative solution ("P2S") .

The MTT 175 of Dia-Stron® is a traction device for measurement of different biomechanical properties of hair as tensile strength and hair combing force. The use of the MTT 175 device in "combing" type allows to evaluate, at the speed of 2000 mm/min, the strength requires to comb hair locks. This is a quantitative, sensitive and reproducible in vitro method of measuring the force required to pull a comb through a tress of hair. Combing facility of hair is then exactly measured and the effect of a treatment can be highlighted. The lower the force is, the better the combing property is.

A better combing efficacy on hair is observed if:

- the peak load, i.e. the maximum load (force), is less important,

- the total work, i.e. the sum of the load, is low.

The results are not significantly different. However, the results by combing resistance test on dry hair showed : (1) a repairing effect of the studied product with Yeast Cytoplasmic Macromolecules used in the form of a spray considering the combing force required which is lower (peak load : 115 grams force) compared to that of stressed non-treated hair (peak load 127.2 grams force); and (2) a protective effect on the product used in the form of shampoo 10X considering the combing force required which is lower (peak load 81.6 grams force) compared to that of stressed non-treated hair (peak load 127.2 grams force) (see Figure 8).

Example 5:

The objective of this study was to evaluate the protective and repairing activity of the hair fiber of a product from hair strands ex vivo.

The purpose of the study was to evaluate the action of a product comprising Yeast Cytoplasmic Macromolecules on the protection and repair of hair fiber after stress (permanent or heat). To evaluate this action, color and gloss measurements (brightness index or "glow") were performed by the goniospectrocolorimeter (GP150 SEELAB®, France). After aggression of the hair strands and/or treatment with the products, the strands were analyzed.

The following treatments were selected for the study: (1) negative control (spray) ("R"); (2) Yeast Cytoplasmic Macromolecules via spray ("PI"); and (3) Yeast Cytoplasmic Macromolecules via shampoo ("P2").

Forty-two strands (per donor) of hair were weighted and measured.

The hair strands were treated as follows: Table 5: Stress 1 - Permanent

Figure imgf000034_0001

Table 6: Stress 2 - Hair straightened by heat

Figure imgf000034_0002
Prior to the study, the strands were washed with shampoo (1 g of shampoo per strand), rinsed in a reproducible way (water between 30-40°C, constant flow controlled, for 20 sec) and gently dried (heat 40°C, for 20 min, suspended strands). The strands of treatment T did not receive any treatment.

The strands of the SI treatment were permed according to the following protocol :

1. 20 min incubation in reductive solution;

2. Rinsing : water with controlled temperature (between 30-40°C), constant flow, controlled time: 20 sec;

3. 7 min incubation in oxidizing solution SIB; and

4. Shampoo (1 g of shampoo per strand), rinsed in a way that reproducible (water between 30-40°C, constant flow controlled, for 20 sec), drying gently (oven 40°C, for 20 min).

The strands of treatment S2 were subjected to 5 heat treatments at 210°C with pliers flat with the following standardised protocol : 5 passes on each drill bit with a slow movement of 3 to 4 seconds per passage.

The strands of treatment R, once dry, were treated with the reference spray R according to the following standardised protocol : 1 g of product per strands or 0.5 g (2 sprays) on one side and 0.5 g (2 sprays) on the other. The product then has been evenly distributed over the strands with a single comb stroke.

The strands of treatment PI, once dry, were treated with the PI spray according to the following standardised protocol : 1 g of product per strands or 0.5 g (2 sprays) on one side and 0.5 g (2 sprays) on the other. Then the product has been evenly distributed over the strands with a single comb stroke.

The strands of treatment Plr, once dry, were treated with the PI spray according to the following standardised protocol : 1 g of product per wick or 0.5 g (2 sprays) on one side and 0.5 g (2 sprays) on the other. The product then has been evenly distributed over the strands with a single comb stroke. Then, the strands were rinsed according to the following standard protocol: water from controlled temperature (between 30-40°C), controlled constant flow, and controlled time: 20 sec after receiving the product to be tested. Finally, the strands were dried according to the following standardised protocol : oven 50°C, drying time 45 min, suspended.

The strands of treatment P2, once dry, were treated with P2 shampoo. Ten shampoos were made. After each shampoo, the strands were rinsed reproducibly (water between 30-40°C, constant flow controlled, for 20 sec) and dried according to the following standard protocol : 50°C oven, drying time 45 min, suspended strands.

• Measurement of colour and brightness index by the goniospectrocolorimeter: The technique developed for the goniospectrocolorimeter device allows an optical characterization by giving access to the 3D nuances of the 3 components of the aspect: brightness/matity, colour and surface finish .

Measurements were made at different times:

- TO : before stress occurs or the product is applied,

- TS : after application of stress,

- TP: after application of the product.

Results:

First, the effect of each stress was characterized, and then the effect of the treatments has been evaluated.

• Evaluation of the clarity and shine/glow of hair strands exposed to a permanent (stress 1) :

The first aspect evaluated was the curative effect, i.e. application of the perm and then the treatments. As shown in Figure 9, the curative application of the product P2 makes it possible to compensate for the loss of clarity induced by the permanent. The second aspect evaluated was the preventive effect, i.e. the application of treatments and then of the perm. As illustrated in Figure 10, the preventive application of the products PI and P2 partially reduces the loss of clarity caused by the perm.

• Evaluation of the clarity and shine/glow of hair strands exposed to a heat treatment (stress 2) :

Straightened the hair by heat resulted in increased clarity and shine. The first aspect evaluated was the curative effect, i.e. application of heat and then the treatments. As shown in Figure 11, the curative application of P2 partially reduces the increase in clarity induced by the heat treatment. The second aspect evaluated was the preventive effect, i.e. the application of treatments and then of the heat treatment. As illustrated in Figure 12, the preventive application of the product P2 does not reduce the increase in clarity induced by the heat treatment. Next, the effect of the treatments on glow was evaluated. The first aspect evaluated was the curative effect, i.e. application of heat and then the treatment of the present invention. As shown in Figure 13, the curative application of P2 slightly reduces the increase in gloss induced by the heat treatment. On the other hand, the curative application of the product PI totally protects against the increase in gloss induced by the smoothing. The second aspect evaluated was the preventive effect, i.e. the application of the heat treatment and then of heat treatment. As illustrated in Figure 14, the preventive application of the product PI totally reduces the increase in gloss induced by the heat treatment while the preventive application of P2 reduces the increase in gloss induced by smoothing.

While the invention has been described in connection with specific embodiments thereof, it will be understood that the scope of the claims should not be limited by the preferred embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole.

Abbreviations

DMSO Dimethyl sulfoxide

DNA Deoxyribonucleic acid

DNAc Complementary deoxyribonucleic acid

ECM extra-cellular matrix

EGF Epidermal growth factor

ELISA Enzyme-linked immunosorben