Use of Lactobacillus crispatus strains and / or compositions containing same in cosmetics, dietary supplements or dermatology

Abstract

The present invention relates to the cosmetic, nutraceutical and / or pharmaceutical, in particular dermatological, use of a Lactobacillus crispatus strain and / or a composition comprising same, for maintaining and / or increasing the expression of molecules of the extracellular matrix of the skin and / or mucosa, in particular for maintaining and / or increasing the biomechanical properties of healthy skin and / or healthy mucosa, preferentially the hardness and / or elasticity and / or density of healthy skin and / or healthy mucosa.

Description

[Technical field]

[0001] The present invention relates to the cosmetic, nutraceutical and / or pharmaceutical, in particular dermatological, use of a strain of Lactobacillus crispatus and / or a composition comprising same, for maintaining and / or increasing the expression of extracellular matrix molecules of the skin and / or mucosa, in particular for maintaining and / or increasing the biomechanical properties of healthy skin and / or healthy mucosa, preferentially the hardness and / or elasticity and / or density of healthy skin and / or healthy mucosa, even more preferentially the hardness and / or elasticity of healthy skin and / or healthy mucosa. [Background technology]

[0002] The structure and properties of the skin change as a result of complex biological, physical and biomechanical processes, causing a decrease in the elasticity, firmness and density of the dermis, in particular as a result of changes in the molecules of the extracellular matrix.

[0003] The extracellular matrix of skin and mucous membranes, known as the ECM, is composed of a combination of three types of molecules: - Fibers: collagen and elastin, - glycoproteins: less abundant than fibers such as fibronectin and laminin, but have a more important role in cell adhesion; - Highly hydrated polysaccharides that constitute a gel that fills the matrix: this matrix plays a major role in maintaining the structure and properties of the skin, in particular the elasticity, hardness and density of the dermis.

[0004] As these ECM molecules are synthesised in particular by fibroblasts and keratinocytes, the renewal of said cells is essential to maintain the biomechanical properties of the skin.

[0005] Elastin is a protein that, in combination with other molecules such as fibrillin and MAGP (microfibril associated glycoprotein), constitutes elastic fibers.

[0006] Elastin is synthesized in the form of soluble tropoelastin, which acquires its physicochemical properties (insolubility, elasticity) after its intra- and intermolecular cross-linking through lysyl oxidase (LOX) and its deposition on microfibrils.

[0007] In the skin, functional elastic fibers are formed by dermal fibroblasts, but some of the components necessary to generate elastic fibers are also found within epidermal cells.

[0008] Although the turnover rate of elastic fibers is very low in adulthood, the total elastin content of the skin can increase. In newborns, the microfibrils are not completely covered with elastin at all, and this occurs around puberty. From the age of 40, inclusions on the fibers are more frequently observed in women, followed by fragmentation of the elastic fibers and their disappearance under the dermo-epidermal junction (DEJ). This fragmentation and / or disappearance under the DEJ is indicated by a decrease in skin elasticity and the formation of wrinkles. The synthesis of non-functional elastic fibers is observed during photoaging, but this increase is accompanied by an accelerated decrease in elastic fibers under the DEJ.

[0009] Collagen is a component protein of the extracellular matrix (ECM) that is abundant in vertebrate tissues. It is a large family that includes 29 different types.

[0010] Among the various types of collagen, in particular type I collagen is present in many human tissues such as tendons, ligaments, the cornea and the skin, more precisely located in the dermis. Type I collagen is the main collagen of the skin.

[0011] Fibrillar collagen is formed from procollagen fibers that are then organized into a network and stabilized by cross-links. It is the collagen that gives tissues their mechanical strength and, as a result, helps maintain their rigidity.

[0012] During skin aging, the rate of collagen synthesis decreases overall and its degradation increases, which causes a decrease in firmness. This phenomenon is even more evident when the skin is especially exposed to UV light. This is called photobiological aging.

[0013] Therefore, the molecules of the extracellular matrix are the subject of numerous studies and innovations in the cosmetics field for the development and improvement of cosmetic active ingredients aimed at maintaining the biomechanical properties of the skin.

[0014] In recent years, advanced genome sequencing techniques have revealed that millions of microorganisms (bacteria, viruses, fungi) live on the skin and form the skin microbiome, also known as the skin flora. Most of these microorganisms (this microbiome) are commensals and are beneficial to the skin. They live and interact with skin cells, and in part, they give rise to a symbiosis with the skin. They are involved in defending the skin by synthesizing or stimulating the synthesis of skin defense molecules. They are involved in repairing the skin in case of injury and in dealing with inflammation. These beneficial commensal microorganisms secrete molecules and metabolites that are involved in the homeostasis and equilibrium of the skin.

[0015] The composition and quality of the skin microbiota changes with ageing. Summary of the Invention [Problem to be solved by the invention]

[0016] Surprisingly, the Applicant has shown that with age, certain bacteria become less prevalent on the skin and decrease in abundance, this is in particular the case for the bacterium Lactobacillus crispatus.

[0017] The majority of solutions found on the market address the visible consequences of ageing and do not take into account the skin microbiome, some of which live in symbiosis with skin cells and are involved in their balance and appearance.

[0018] The invention has the advantage of being more complete in the sense that it rebalances the microflora of aging skin by supplementing it with bacteria of the skin microflora that decrease over time, which then contribute to the rejuvenation and sustained protection of the skin against external aggressions by reactivating biological processes that decrease with age.

[0019] Probiotic bacteria and their derivatives are already used to treat skin aging problems, but in most cases the bacteria used do not originate from the skin. The bacteria according to the invention have the advantage that they occur naturally on young skin.

[0020] Patent application WO 2009 / 031106 discloses the use of hesperidin in combination with probiotics to prevent or strengthen the reduction of the barrier function of the skin. The application does not disclose the use of Lactobacillus crispatus strains to maintain and / or increase the expression of extracellular matrix molecules of the skin and / or mucous membranes.

[0021] Patent application WO 2019 / 111189 discloses the probiotic-containing use of Lactobacillus bacteria for preventing UV radiation-induced damage. The application does not disclose the use of Lactobacillus crispatus strains for maintaining and / or increasing the expression of extracellular matrix molecules in the skin and / or mucous membranes.

[0022] Thus, to the Applicant's knowledge, there is no prior art disclosing or suggesting the cosmetic, nutraceutical and / or pharmaceutical use of strains of Lactobacillus crispatus and / or compositions comprising same for maintaining and / or increasing the expression of extracellular matrix molecules in the skin and / or mucosa, or for maintaining and / or increasing the biomechanical properties of the skin and / or mucosa, or for maintaining and / or increasing the expression of collagen in the skin and / or mucosa and / or elastic fibres in the skin and / or mucosa, and in particular for the dermatological use. [Means for solving the problem]

[0023] A first subject of the present invention is the use of a strain of Lactobacillus crispatus in a cosmetic and / or dietary supplement to maintain and / or increase the expression of extracellular matrix molecules in healthy skin and / or healthy mucosa.

[0024] A subject of the present invention is also a cosmetic care process, characterized in that it comprises the topical application of a strain of Lactobacillus crispatus according to the invention or a cosmetic composition comprising it to at least one area of ​​healthy skin and / or healthy mucosa, in order to maintain and / or increase the expression of extracellular matrix molecules of healthy skin and / or healthy mucosa, preferably of collagen of healthy skin and / or healthy mucosa and / or of elastic fibers of healthy skin and / or healthy mucosa, and / or to maintain and / or increase the biomechanical properties, in particular the hardness and / or elasticity and / or density, preferably the hardness, of healthy skin and / or healthy mucosa.

[0025] A subject of the present invention is also a Lactobacillus crispatus strain according to the invention or a dermatological composition comprising it, for the treatment and / or prevention of skin and / or mucosal pathologies involving a decrease in the amount and / or expression of extracellular matrix molecules in the skin and / or mucosa, in particular a decrease in the amount and / or expression of collagen in the skin and / or mucosa and / or elastic fibres in the skin and / or mucosa and / or a decrease in the biomechanical properties of the skin and / or mucosa, in particular a decrease in the hardness and / or elasticity and / or density of the skin and / or mucosa.

[0026] The present invention also relates to a strain of Lactobacillus crispatus deposited under the Budapest Treaty at the Institute Pasteur, 28 rue du Docteur Roux, F-75024 Paris cedex 15, on September 9, 2020 under the name CNCM I-5579, or a cosmetic composition comprising same, for maintaining and / or increasing the expression of extracellular matrix molecules in healthy skin and / or healthy mucosa, in particular for maintaining and / or increasing the expression of collagen in healthy skin and / or healthy mucosa and / or elastic fibres in healthy skin and / or healthy mucosa, and / or for maintaining and / or increasing the biomechanical properties, in particular the hardness and / or elasticity and / or density, preferentially the hardness, of healthy skin and / or healthy mucosa.

[0027] Finally, the present invention relates to a strain of Lactobacillus crispatus deposited under the Budapest Treaty at the Institute Pasteur, 28 rue du Docteur Roux, F-75024 Paris cedex 15, under the name CNCM I-5579 on September 9, 2020, or a dermatological composition comprising same, for the treatment and / or prevention of pathologies of the skin and / or mucosa which are accompanied by a decrease in the amount and / or expression of extracellular matrix molecules of the skin and / or mucosa, in particular a decrease in the amount and / or expression of collagen and / or elastic fibres of the skin and / or mucosa and / or a decrease in the amount and / or biomechanical properties of the skin and / or mucosa, in particular a decrease in the hardness of the skin and / or mucosa and / or a decrease in the elasticity of the skin and / or mucosa and / or a decrease in the density of the skin and / or mucosa. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] For the purposes of the present invention, the term "cosmetic uses and / or compositions" refers to non-pharmaceutical uses and / or compositions, i.e. those which are not intended for therapeutic use and which are applied to "healthy" parts of the body, in particular to "healthy" areas of the skin and / or mucous membranes.

[0029] For the purposes of the present invention, the expression "nutraceutical uses and / or nutraceutical compositions" refers to uses and / or compositions for non-pharmaceutical oral administration, i.e. those not requiring therapeutic treatment.

[0030] For the purposes of the present invention, the term "healthy skin" or "healthy mucosa" refers to an area of ​​the skin or mucosa to which a strain according to the invention is applied, which is said by a dermatologist to be "non-pathological", i.e. free of infection, scarring, candidiasis, impetigo, psoriasis, eczema, acne or dermatitis, or a disease or skin disorder, or a wound or injury.

[0031] According to the invention, "mucous membrane" means the ocular, nasal, auricular, urogenital and / or oral mucosa, in particular the oral, labial and / or gingival mucosa, preferentially the ocular and / or oral mucosa and more preferentially the labial and / or ocular mucosa.

[0032] Advantageously, the term does not include the vaginal mucosa.

[0033] For the purposes of the present invention, the term "maintaining and / or increasing the expression of extracellular matrix molecules" means preventing the decrease and / or increasing the level of gene expression, i.e. mRNA (messenger RNA) and / or protein synthesis, of extracellular matrix molecules in healthy skin and / or healthy mucosa treated with a strain according to the invention, relative to the level of gene expression and / or protein synthesis detected in the absence of the strain according to the invention. Preferentially, it is maintaining and / or increasing the protein expression of extracellular matrix molecules.

[0034] According to the present invention, the term "ECM molecules" refers to organic molecules that constitute and / or are contained within the extracellular matrix, of human or animal origin, and that are synthesised by cells.

[0035] These are, in particular: - The ECM collagen family, - The family of ECM elastic fibers: elastin, tropoelastin, elastin-associated proteins, especially fibrillin 1, lysyl oxidase, especially LOX and LOXL, EBP (elastin-binding protein), fibulin 3 and 5, emilin 1 and 2 In particular, the protein is a component of the ECM, selected from the group consisting of:

[0036] These are in particular secreted proteoglycans selected from proteoglycan components of the ECM, in particular perlecan, versican, leucine-rich proteoglycans (SLRPs), in particular decorin, biglycan and lumican.

[0037] It also includes the glycoprotein components of the ECM, in particular fibronectin, laminin, SPARC (secreted protein rich in cysteine) and tenascin.

[0038] It also contains the glycosaminoglycans (GAGs) that make up the ECM, in particular hyaluronic acid, heparan sulfate, dermatan sulfate and chondroitin sulfate.

[0039] It also includes ECM growth factors, which are proteins contained in the ECM, particularly VEGF, PDGF, HGF and FGF, particularly FGF2 and FGF7.

[0040] The term "ECM molecule" refers to molecules in the ECM or other compartments in the medium, optionally in precursor form, in particular the intracellular and extracellular compartments.

[0041] According to the present invention, the term "ECM molecule precursor" refers to native and / or intermediate forms of ECM molecules synthesized by cells prior to constituting or being contained within the ECM. These forms are often named by the prefixes "Pro-" or "Tropo-" because they undergo a maturation event prior to being present in the ECM. They are, for example, procollagen, tropocollagen, proelastin, and tropoelastin.

[0042] Preferentially, the extracellular matrix molecules are chosen from collagens of healthy skin and / or healthy mucosa and / or elastic fibres of healthy skin and / or healthy mucosa.

[0043] For the purposes of the present invention, the term "maintaining and / or increasing the expression of collagen of healthy skin and / or healthy mucosa and / or elastic fibers of healthy skin and / or healthy mucosa" means preventing the decrease and / or increasing the level of gene expression, i.e. mRNA (messenger RNA) expression and / or protein synthesis, of collagen of healthy skin and / or healthy mucosa and / or elastic fibers of healthy skin and / or healthy mucosa treated with a strain according to the invention, relative to the gene expression and / or protein synthesis measured in the absence of the strain according to the invention.

[0044] In particular, it is an increase of at least 20%, preferentially at least 40% and even more preferentially at least 50% in the level of collagen and / or elastic fiber gene expression and / or protein synthesis relative to the level of collagen and / or elastic fiber gene and / or protein expression measured in the absence of a Lactobacillus crispatus strain according to the invention.

[0045] In a preferred embodiment of the invention, it is an increase in the level of protein expression of fibulin-5 and / or emilin-1 by at least 20%, preferentially at least 40% and even more preferentially at least 50% relative to the level of protein expression measured in skin fibroblasts cultured in vitro in the absence of the strain, as described, for example, in Example 3.

[0046] Advantageously, the measurement of the increase in protein expression of fibulin-5 and / or emilin-1 is preferentially carried out by in vitro measurement, after analysis by capillary electrophoresis, for example according to the method presented in Example 3.

[0047] For the purposes of the present invention, the term "capillary electrophoretic analysis" refers to a technique comprising the binding of antibodies specific for the test proteins fibulin-5 and / or emilin-1 to said proteins conjugated to peroxidase and a chemiluminescent substrate, with the aim of detecting the proteins by measuring a chemiluminescent signal.

[0048] For the purposes of the present invention, the term "collagen" refers to collagen proteins of types I, III, IV, V, VI, VII, XII, XIII, XIV, XVI, XVII, XXIV, XXIX present in healthy skin and / or healthy mucosa. According to an advantageous embodiment, the collagen is selected from collagen types I, III, IV, V and / or XIV, preferentially collagen types I, IV and / or V, more preferentially collagen type I.

[0049] For the purposes of the present invention, the term "type I collagen protein" refers to a collagen protein present in the skin, cornea, tendons, ligaments and bones, more preferentially in the skin. Thus, in a preferred embodiment of the present invention, a Lactobacillus crispatus strain according to the invention is used to maintain and / or increase the protein expression of type I collagen in healthy skin and / or healthy mucosa.

[0050] According to an advantageous embodiment of the invention, this is an increase relative to the level of collagen gene and / or protein expression measured in skin fibroblasts cultured in vitro in the absence of a strain of Lactobacillus crispatus according to the invention.

[0051] In a preferred embodiment of the invention, this is an increase in the level of protein expression of type I collagen of at least 10%, preferentially at least 50% and even more preferentially at least 20% relative to the level of protein expression measured in dermal fibroblasts cultured in vitro in the absence of the strain, for example according to a protocol as described in Example 2.

[0052] Advantageously, the measurement of the increase in protein expression is preferentially carried out by in vitro measurement after enzyme-linked immunosorbent assay or ELISA, for example according to the method as presented in Example 2.

[0053] For the purposes of the present invention, the term "enzyme immunoassay" refers to a technique that involves immobilizing an antibody specific for a given collagen protein, preferentially type I collagen protein, on said protein, for its fluorometric detection. Advantageously, the detection is carried out by fluorescence.

[0054] According to another advantageous embodiment, the measurement of the increase in gene expression is preferentially carried out by in vitro measurement after RT-qPCR.

[0055] In a preferred embodiment, the strains according to the invention are used to maintain and / or increase the biomechanical properties of healthy skin and / or healthy mucosa, preferentially the firmness and / or elasticity and / or density of healthy skin and / or healthy mucosa, more preferentially the firmness of healthy skin and / or mucosa, in particular healthy dermis and / or the elasticity of healthy skin and / or mucosa, in particular healthy dermis.

[0056] For the purposes of the present invention, the term "biomechanical properties of healthy skin and / or healthy mucosa" means the hardness and / or elasticity and / or density of healthy skin and / or healthy mucosa and / or the resistance to compression and / or the resistance to stretching and / or the flexibility and / or extensibility and / or the ability to resist deformation. Preferentially, it is the hardness and / or elasticity and / or density of healthy skin and / or healthy mucosa, in particular the healthy dermis, and even more preferentially, it is the hardness of healthy skin and / or healthy mucosa, in particular the healthy dermis and / or the elasticity of healthy skin and / or healthy mucosa, in particular the healthy dermis.

[0057] The biomechanical properties of healthy skin and / or healthy mucosa can be tested by measurement techniques known to the person skilled in the art, in particular pulling, twisting, suction, indentation, revatometry, ballistometry, high-resolution ultrasound imaging or elastography, preferentially indentation or high-resolution ultrasound imaging.

[0058] For the purposes of the present invention, the term "maintaining and / or increasing the biomechanical properties of healthy skin and / or healthy mucosa" means preventing a decrease and / or increasing the biomechanical properties of healthy skin and / or healthy mucosa, preferentially the hardness of the healthy dermis and / or the elasticity of the healthy skin and / or healthy mucosa, in particular the healthy dermis, and / or the density of the healthy skin and / or healthy mucosa, in particular the healthy dermis, treated with a strain according to the invention, measured in the absence of the strain according to the invention.

[0059] Preferentially, it is an increase of at least 5%, preferentially 10%, of the biomechanical properties of healthy skin and / or healthy mucosa treated with a strain of Lactobacillus crispatus according to the invention relative to the biomechanical properties of healthy skin and / or healthy mucosa measured in the absence of a strain of Lactobacillus crispatus according to the invention.

[0060] For the purposes of the present invention, from a cosmetic point of view, the term "maintaining and / or increasing the firmness of healthy skin and / or healthy mucosa, in particular healthy dermis" means, for cosmetic purposes, preventing the loss of firmness and / or increasing the firmness of healthy skin and / or healthy mucosa, in particular those that have lost a significant amount of firmness as a result of intrinsic factors. The intrinsic factors may be tissue ageing of the skin and / or mucosa, i.e. ageing induced over time, which can be found, for example, in "mature" skin, i.e. skin of persons over 40 years of age. It may be due to non-pathological physiological changes, such as cell stress, dietary changes or hormonal fluctuations, in particular during puberty, pregnancy, menopause and / or andropause. This decrease in firmness may also occur as a result of extrinsic factors, such as aggressive environmental factors, for example pollution, smoke, tobacco, toxins or climatic and / or mechanical aggressive factors.

[0061] According to a preferential embodiment, the decrease in hardness is not UV radiation induced.

[0062] Preferentially, it is an increase of at least 1%, preferentially at least 3% and more advantageously at least 5% of the firmness of healthy skin and / or healthy mucosa, in particular healthy dermis, in the presence of a strain of Lactobacillus crispatus according to the invention. In one advantageous embodiment of the invention, it is preferentially an increase measured in vivo on human facial skin.

[0063] For the purposes of the present invention, from a cosmetic point of view, the term "maintaining and / or increasing the elasticity of healthy skin and / or healthy mucosa, in particular healthy dermis" means, for cosmetic purposes, preventing the decrease in elasticity and / or increasing the elasticity of healthy skin and / or mucosa, in particular tissue ageing of the skin and / or mucosa, i.e. healthy skin and / or healthy mucosa, which have significantly lost elasticity as a result of intrinsic factors such as non-pathological physiological changes such as ageing induced over time, cellular stress, dietary changes, or hormonal fluctuations, in particular during puberty, pregnancy, menopause and / or andropause, as found, for example, in "mature" skin, i.e. skin of persons over 40 years of age, in particular over 50 years of age. This decrease in elasticity can also occur as a result of extrinsic factors such as aggressive environmental factors, for example pollution, smoke, tobacco, toxins or climatic and / or mechanical aggressive factors.

[0064] According to a preferential embodiment, the decrease in elasticity is not UV radiation induced.

[0065] Furthermore, the term "increases elasticity" of the skin and / or mucous membranes means an increase of at least 2%, advantageously at least 4%, and more advantageously at least 6% in the elasticity measured in vivo in the presence of the strain according to the invention compared to the elasticity detected in the absence of the strain. In a preferred embodiment of the invention, this measurement is taken on human facial skin. In a preferred aspect, this increase is evaluated after at least 10 days, preferentially 28 days, more preferentially 30 days and advantageously at least 56 days of treatment in the presence of the strain according to the invention.

[0066] More advantageously, the elasticity is assessed by measuring its various elastic components, in particular the net elasticity, the elastic recovery and / or the total elasticity, in particular as described in Example 4.

[0067] For the purposes of the present invention, from a cosmetic point of view, the term "maintaining and / or increasing the density of healthy skin and / or healthy mucosa, in particular healthy dermis" means, for cosmetic purposes, preventing the loss of density and / or increasing the density of healthy skin and / or healthy mucosa, in particular those that have lost a significant amount of density as a result of intrinsic factors. The intrinsic factors may be tissue aging of the skin and / or mucosa, i.e. aging induced over time, which can be found, for example, in "mature" skin, i.e. skin of persons over 40 years of age. It may be due to non-pathological physiological changes, such as cell stress, dietary changes or hormonal fluctuations, in particular during puberty, pregnancy, menopause and / or andropause. This decrease in density may also occur as a result of extrinsic factors, such as aggressive environmental factors, for example pollution, smoke, tobacco, toxins or climatic and / or mechanical aggressive factors.

[0068] According to a preferential embodiment, the decrease in density is not UV radiation induced.

[0069] Preferentially, it is an increase of at least 1%, preferentially at least 3% and more advantageously at least 5% in the density of healthy skin and / or healthy mucosa, in particular healthy dermis, in the presence of a strain of Lactobacillus crispatus according to the invention. In an advantageous embodiment of the invention, this is preferentially an increase measured in vivo on human facial skin.

[0070] In vivo measurements of hardness and / or elasticity and / or density may be performed according to conventional methods known to those skilled in the art, in particular by measurements using a Cutometer, a Tonoderm™, an ultrasound scanner or a DynaSKIN® with a dermaTOP.

[0071] According to the invention, the Lactobacillus crispatus strain may be used in its complete form, in particular live and / or inactivated, in particular killed, and / or in the form of a lysate and / or in the form of one or more of its fractions and / or in the form of one or more of its metabolic products, preferentially in the form of its secretome.

[0072] In each of its forms, the strain according to the invention may be isolated or combined with the fermentation and / or culture medium.

[0073] Preferentially, the strain according to the invention is combined with the fermentation and / or culture medium.

[0074] For purposes of the present invention, the term "isolated" means that the compound or compounds are not mixed with the compound or compounds that may be associated with it in the fermentation and / or growth medium.

[0075] For the purposes of the present invention, the term "intact form" refers to its native form, in which the bacterial envelope is intact, as opposed to a lysed form. When used in intact form, the Lactobacillus crispatus strain may be viable and / or inactive and / or dead.

[0076] For the purposes of the present invention, the term "viable" refers to a Lactobacillus crispatus strain according to the invention that is capable of forming colonies in culture.

[0077] The production of a strain of Lactobacillus crispatus in viable intact form can be carried out according to any method conventionally known to the skilled artisan, advantageously according to the protocols as described, for example, in examples 1.a or 1.f.

[0078] For the purposes of the present invention, the term "inactivated" refers to a strain of Lactobacillus crispatus according to the invention that is no longer capable of forming colonies in culture, either temporarily or definitively.

[0079] The inactivation of the strain of Lactobacillus crispatus can be carried out by any method conventionally known to the person skilled in the art. It can in particular be inactivated by irradiation, heat treatment or freeze-drying, high pressure treatment or release under certain conditions. Advantageously, it is carried out by heat treatment, more advantageously according to a protocol such as that described in example 1.b.

[0080] Heat inactivation may be carried out by incubating the Lactobacillus crispatus strain at a temperature advantageously between about 60° C. and 150° C. for a period of time between about 10 seconds and 90 minutes, preferentially between about 15 minutes and 1 hour. Preferentially, it is incubated at about 80° C. for about 30 minutes.

[0081] For the purposes of the present invention, the term "dead" refers to a strain of Lactobacillus crispatus according to the invention that is ultimately no longer capable of forming colonies in culture.

[0082] For the purposes of the present invention, the term "lysate" refers to the material obtained after disruption or lysis of living cells, using a phenomenon known as cell lysis, which causes the release of intracellular biological components naturally contained within the cells of the microorganism under consideration, as well as fragments of cell membrane components. The lysate used is therefore formed from all the intracellular biological components as well as cell wall and membrane components of the strain of Lactobacillus crispatus according to the invention.

[0083] The lysate can be obtained via any method conventionally known to the person skilled in the art, in particular by osmotic shock, heat shock, ultrasound, enzymatic lysis, tindalization, or under mechanical stress of centrifugation type, or by increasing the pressure, or by a combination of these various techniques.

[0084] Preferentially, the lysate is obtained by a combination of mechanical action and increased pressure. More preferentially, the lysate is obtained according to the protocol as described for example in example 1.b.

[0085] In a preferred embodiment of the invention, the lysate is combined with a bacterial culture and / or a fermentation medium.

[0086] For the purposes of the present invention, the term "fraction" refers to a fragment of a Lactobacillus crispatus strain according to the invention that is effective in maintaining and / or increasing the expression of extracellular matrix molecules of healthy skin and / or healthy mucosa and / or for maintaining and / or increasing the biomechanical properties of healthy skin and / or healthy mucosa. This fraction corresponds to a non-total part of the intracellular biological components and cell wall and membrane components obtained by lysis of a Lactobacillus crispatus strain according to the invention.

[0087] According to certain embodiments of the invention, the fraction may be bacterial cytoplasm.

[0088] In a preferred embodiment of the invention, the fraction is combined with the bacterial culture and / or fermentation medium.

[0089] For the purposes of the present invention, the term "metabolites" refers to one or more organic and / or inorganic molecules obtained from the metabolism of a Lactobacillus crispatus strain according to the invention and which are efficient for maintaining and / or increasing the expression of extracellular matrix molecules of healthy skin and / or healthy mucosa and / or for maintaining and / or increasing the biomechanical properties of healthy skin and / or healthy mucosa. Preferentially, this is the secretome of a Lactobacillus crispatus strain according to the invention.

[0090] In certain embodiments of the invention, the metabolites are combined with the bacterial culture and / or fermentation medium.

[0091] For the purposes of the present invention, the term "secretome" means all the organic and / or inorganic molecules secreted by the strain of Lactobacillus crispatus according to the invention that are efficient to maintain and / or increase the expression of extracellular matrix molecules of healthy skin and / or healthy mucosa and / or to maintain and / or increase the biomechanical properties of healthy skin and / or healthy mucosa.

[0092] The secretome can be obtained via any method conventionally known to the person skilled in the art. Advantageously, it is obtained, for example, according to a protocol such as that described in Example 1.d or 1e.

[0093] In a preferred embodiment of the invention, the secretome is combined with a bacterial culture and / or a fermentation medium.

[0094] According to a preferential embodiment of the invention, the Lactobacillus crispatus strain according to the invention is used in viable whole form and / or in lysate form.

[0095] The strain of Lactobacillus crispatus according to the invention may be derived from any known species of Lactobacillus crispatus. Preferentially, the strain of Lactobacillus crispatus is the species deposited under the Budapest Treaty on September 9, 2020 at the Institute Pasteur (28 rue du Docteur Roux, F-75024 Paris cedex 15) under the name CNCM I-5579.

[0096] The advantage of this particular strain of Lactobacillus crispatus is that it is the first Lactobacillus crispatus species found naturally on healthy skin.

[0097] According to the invention, the strains of Lactobacillus crispatus according to the invention may be used alone, in the form of active ingredients and / or in cosmetic and / or dietary supplement and / or pharmaceutical compositions, in particular dermatological compositions, preferentially intended for topical and / or oral application, more preferentially for topical application.

[0098] When it is used in a living whole form according to the invention and when it is used alone in the form of an active ingredient, the strain according to the invention may be in dry form, i.e. in powder form, advantageously in maltodextrin, preferably with a content of 10% to 80% (w / w) by weight of the strain, preferentially 30% to 70% (w / w) by weight of the strain and more advantageously 40% to 60% (w / w) by weight of the strain relative to the total weight of the powder.

[0099] Alternatively, the living whole form according to the invention can be used for dissolving and / or diluting with a solvent. Preferentially, this solvent contains less than 20% by volume (v / v) of water, more preferentially less than 5% by volume (v / v) of water; more preferentially, the solvent does not contain any water. Very preferentially, this solvent is a cosmetically acceptable oil.

[0100] When it is used in its total inactivated, especially killed, form, and / or in the form of a lysate and / or in the form of one or more of its fractions and / or in the form of one or more of its metabolites, especially its secretome, and when it is used alone in the form of an active ingredient, the strain according to the invention may be in dry form, i.e. advantageously in the form of a powder of maltodextrin, preferentially with a strain content of between 5% and 80% (w / w) by weight of the strain relative to the total weight of the powder, preferentially between 10% and 70% (w / w) by weight of the strain, very preferentially between about 20% and 50% (w / w) by weight of the strain and more advantageously between 10% and 50% (w / w) by weight of the strain relative to the total weight of the powder.

[0101] Alternatively, according to the present invention, the total inactivation, in particular the killed forms, and / or the lysates, and / or the fractions and / or the metabolites, in particular the secretome, may be used for solubilization and / or dilution with a solvent, in particular a polar solvent, such as water, alcohol, polyol, glycols such as pentylene glycol and / or hexylene glycol and / or caprylyl glycol or mixtures thereof, preferentially comprising water-glycols or aqueous alcoholic mixtures, more preferentially glycols selected from hexylene glycol, caprylyl glycol and mixtures thereof.

[0102] In another embodiment, the strain according to the invention may be incorporated into a cosmetic and / or nutraceutical composition comprising at least one cosmetically acceptable excipient and / or nutraceutical acceptable excipient.Preferentially, it is incorporated into a cosmetic composition comprising at least one cosmetically acceptable excipient.

[0103] For the purposes of the present invention, the term "cosmetically acceptable excipient" means a compound and / or solvent that is topically acceptable, i.e., one that does not induce an allergic response on contact with the skin and / or mucous membranes, including the human scalp, and is non-toxic and not chemically unstable.

[0104] In a preferred embodiment of the invention, the strain according to the invention is present in a cosmetic and / or dermatological composition in a concentration of 1×10 by volume relative to the total volume of the composition, which also contains at least one cosmetically acceptable excipient. -4 % to 10% (v / v), preferentially 1 × 10 by volume -4 % to 5% (v / v), more preferably 1 × 10 by volume -3 % to 3% (v / v), more preferentially at a content of 0.1% to 2% (v / v) by volume.

[0105] Advantageously, the strain according to the invention is present in the cosmetic and / or dermatological composition in a concentration of 1×10 relative to the total weight of the composition, also including at least one cosmetically acceptable excipient. -4 % by weight to 10% by weight, preferentially 1×10 -4 % by weight to 5% by weight and more preferably 1×10 -3 It is present in concentrations of 0.5% by weight to 0.5% by weight.

[0106] In a particularly advantageous manner, when the strain according to the invention is used in the form of one or more of its metabolites, in particular its secretome, it is present in the cosmetic and / or dermatological composition at a concentration between 0.05% and 0.5% by weight, more preferentially at a concentration of about 0.1% by weight, relative to the total weight of the composition, also including at least one cosmetically acceptable excipient.

[0107] Particularly advantageously, when the strain according to the invention is used in its viable intact form, it is present in the cosmetic and / or dermatological composition at a concentration of between 0.01% and 0.1% by weight, more preferentially at a concentration of about 0.025% by weight, relative to the total weight of the composition, also including at least one cosmetically acceptable excipient.

[0108] The cosmetic and / or dermatological composition of the present invention may be selected from aqueous or oily solutions, creams or aqueous or oily gels, in particular shower gels, shampoos; milks; emulsions, microemulsions or nanoemulsions, in particular oil-in-water or water-in-oil or multiple or silicone-based emulsions; masks; serums; lotions; liquid soaps; dermatological bars; ointments; balms; butters; mousses; patches; anhydrous products, preferably in the form of liquids, pasties or solids, such as makeup powders, wands or sticks, in particular in the form of lipsticks.

[0109] It can also be a make-up product or a make-up removal product.

[0110] Advantageously, the Lactobacillus crispatus strain or composition of the invention is intended to be applied to all or part of the body and / or face and / or scalp, preferably to the lower legs, thighs, arms, stomach, neckline, neck, armpits or lips, more preferentially to all or part of the face and preferentially to the cheeks, forehead, chin, lips or eye contour.

[0111] Advantageously, the strain and / or a composition comprising it is intended to be applied to areas of healthy skin exhibiting sagging and / or areas of breakdown of healthy skin and / or areas of healthy skin in a reduced state of tone.

[0112] Advantageously, the cosmetic composition is therefore intended for topical application to healthy skin and / or healthy mucous membranes.

[0113] Preferentially, the strains of Lactobacillus crispatus according to the invention are suitable for the formulation of neutral and mild compositions, in particular with a focus on the skin, including the sebaceous glands, in particular the scalp and / or mucous membranes.

[0114] Alternatively, the Lactobacillus crispatus strain according to the invention may be in any presentation form conventionally used for oral application, in particular in the form of a nutraceutical active ingredient and / or a nutraceutical composition.

[0115] The compositions according to the invention may contain any suitable solvent, and / or any suitable vehicle, and / or any suitable excipient, optionally in combination with other compounds of interest.

[0116] As a result, for these compositions, the excipient contains at least one compound selected from the group including, for example, protectants, emollients, emulsifiers, surfactants, moisturizers, thickeners, conditioners, mattifying agents, stabilizers, antioxidants, texturing agents, gloss agents, film formers, solubilizers, pigments, colorants, fragrances, and sunscreens. These excipients are preferably selected from the group consisting of amino acids and derivatives thereof, polyglycerols, esters, cellulose polymers and derivatives, lanolin derivatives, phospholipids, lactoferrin, lactoperoxidase, sucrose-based stabilizers, vitamin E and derivatives thereof, natural and synthetic waxes, vegetable oils, triglycerides, unsaponifiable matter, phytosterols, vegetable esters, silicones and derivatives thereof, protein hydrolysates, jojoba oil and derivatives thereof, fat-soluble / water-soluble esters, betaines, amine oxides, plant extracts, sucrose esters, titanium dioxide, glycine and parabens, and more preferably butylene glycol, steareth-2, steareth-21, glycol-15 stearyl ether, cetearyl alcohol, phenoxyethanol, methylparaben, ethylparaben, propylparaben, butylparaben, butylene glycol, natural tocopherols, glycerol, sodium dihydroxycetyl phosphate, isopropyl hydroxycetyl ether. Ingredients include: ethylhexyl ether, glycol stearate, triisononanoin, octyl cocoate, polyacrylamide, isoparaffin, laureth-7, carbomer, propylene glycol, glycerol, bisabolol, dimethicone, sodium hydroxide, PEG-30 dipolyhydroxystearic acid, capric / caprylic triglyceride, cetearyl octanoate, dibutyl adipate, grapeseed oil, jojoba oil, magnesium sulfate, EDTA, cyclomethicone, xanthan gum. , citric acid, sodium lauryl sulfate, mineral waxes and oils, isostearyl isostearate, propylene glycol dipelargonate, propylene glycol isostearate, PEG8, beeswax, glycerides from hydrogenated palm kernel oil, glycerides from hydrogenated palm oil, lanolin oil, sesame oil, cetyl lactate, lanolin alcohol, castor oil, titanium dioxide, lactose, sucrose, low density polyethylene, isotonic saline solution, and mixtures thereof.

[0117] Numerous cosmetic active ingredients are known to those skilled in the art to improve the health and / or physical appearance of the skin (including the scalp) and / or mucous membranes. Those skilled in the art understand how to formulate cosmetic, nutraceutical or dermatological compositions to obtain the best effect. Furthermore, these compounds may have synergistic effects when combined with each other. These combinations are also encompassed by the present invention. The CTFA Cosmetic Ingredient Handbook, Second Edition (1992) describes various cosmetic and pharmaceutical ingredients commonly used in the cosmetics and pharmaceutical industries, particularly suitable for topical use. Examples of these classes of ingredients include, but are not limited to, the following compounds: abrasives, absorbents, compounds for cosmetic purposes such as fragrances, pigments, dyes, essential oils, astringents and the like (e.g., clove oil, menthol, camphor, eucalyptus oil, eugenol, menthyl lactate, witch hazel distillate), anti-acne agents, anti-flocculants, anti-foaming agents, antibacterial agents (e.g., iodide propyl butylcarbamate), antioxidants, binders, biological additives, buffers, swelling agents, chelating agents, additives, biocides, denaturants, thickeners and vitamins and derivatives or equivalents thereof, film-forming materials, polymers, opacifiers, pH adjusters, reducing agents, depigmenting or whitening agents (e.g., hydroquinone, kojic acid, ascorbic acid, magnesium ascorbyl phosphate, ascorbyl glucosamine), conditioning agents (e.g., moisturizers).

[0118] The cosmetic composition may further comprise other cosmetic and / or nutraceutical agents having the same properties as the strain of Lactobacillus crispatus according to the invention and inducing a synergistic or non-synergistic effect therewith, or cosmetic agents having a complementary effect.

[0119] These may be, for example, anti-wrinkle ingredients, agents that stimulate fibroblast activity or proliferation, agents that stimulate molecules of the extracellular matrix.

[0120] They may furthermore be anti-aging active ingredients and / or tensioning agents, due to their synergistic effect with the Lactobacillus crispatus strains. Advantageously, they are active ingredients that increase the gene and / or protein expression of active ingredients that prevent the degradation of collagen, such as collagen or retinol, vitamin C, the extract of Davilla rugosa sold under the name Collguard™ by BASF Beauty Care Solutions, the extract of Hibiscus abelmoschus seeds sold under the name Linefactor™, peptides sold under the name Dermican™ by the Applicant, or the products sold under the name Matrixyl™, Matrixyl3000™ and Regestril™ by the company Sederma, or Neuroguard by the company Codif, or Eternaline™ by the company Silab.

[0121] Tensioning agents that may be used in the present invention may be selected from synthetic polymers such as polyurethane latexes or acrylic latexes, polymers of natural origin, in particular polyholosides in the form of starch or in the form of carrageenan, alginates, agar, gellan, cellulose-based polymers and pectins; vegetable proteins and protein hydrolysates; mixed silicates; wax microparticles; colloidal particles of inorganic fillers, for example selected from silica, silica-alumina complexes; and mixtures thereof.

[0122] Finally, they may be cosmetic ingredients, such as antibacterial agents, free radical scavengers, soothing agents, soothing or relaxing agents, agents acting on the microcirculation to improve the complexion, in particular the radiance of the face, healing or slimming agents. Advantageously, the cosmetic and / or dermatological composition of the invention also contains one or more tensioning agents, and / or one or more antibacterial agents, and / or one or more free radical scavengers, and / or one or more soothing agents, and / or one or more slimming agents, and / or one or more agents acting on the microcirculation.

[0123] Among the antibacterial agents that may be combined with the Lactobacillus crispatus strain in a preferred embodiment of the invention are 2,4,4'-trichloro-2'-hydroxydiphenyl ether (or triclosan), 3,4,4'-trichlorovanillide, phenoxyethanol, phenoxypropanol, phenoxyisopropanol, hexamidine isethionate, metronidazole and its salts, miconazole and its salts, itraconazole, terconazole, econazole, ketoconazole, saperconazole, fluconazole, clotrimazole, buta ... Mention may be made of benzoyl peroxide, 3-hydroxybenzoic acid, 4-hydroxybenzoic acid, phytic acid, N-acetyl-L-cysteine, lipoic acid, azelaic acid and its salts, arachidonic acid, resorcinol, octoxyglycerol, octanoylglycine, caprylyl glycol, 10-hydroxy-2-decanoic acid, farnesol, phytosphingosine and mixtures thereof.

[0124] The free radical scavengers may be vitamin C and its derivatives, such as ascorbyl glucoside, phenols and polyphenols, in particular tannins, ellagic acid and tannic acid; epigallocatechin and natural extracts containing it, in particular green tea extract; anthocyanins; phenolic acids, stilbenes; monocyclic or polycyclic aromatic compound scavengers, tannins, such as ellagic acid and indole derivatives and / or heavy metal scavengers, such as EDTA, free radical scavengers, such as vitamin E and its derivatives, such as tocopherol acetate; bioflavonoids; coenzyme Q10 or ubiquinone.

[0125] As soothing agents contained in the compositions according to the invention, there may be used pentacyclic triterpenes, ursolic acid and its salts, oleanolic acid and its salts, betulinic acid and its salts, salts of salicylic acid and in particular zinc salicylate, bisabolol, allantoin, omega-3 unsaturated oils, cortisone, hydrocortisone, indomethacin and betamethasone, anti-inflammatory active agents and in particular those described in patent FR 2 847 267, in particular the root extract of Pueraria lobata sold by the Applicant under the name Inhipase™ and the extract of Theobroma cacao.

[0126] The active ingredients acting on the microcirculation, vasoprotectants or vasodilators may be selected from flavonoids, ruscogenin, nicotinic acid and essential oils.

[0127] Slimming active agents may in particular be selected from lipoprotein lipase inhibitors, such as those described in US 2003 / 086949 (Coletica) and in particular cat's claw extract (Uncaria tomentosa); excretory active ingredients, in particular hesperitin laurate (Flavagrum™) or quercitin caprylate (Flavenger™); phosphodiesterase enzyme inhibitors, adenylate cyclase activators, cAMP and / or active agents capable of removing spermine and / or spermidine. Mention may be made of extracts of Coleus forskohlii root, extracts of Cecropia obtusa, Uva lactuca, caffeine, forskolin, theophylline, theobromine and / or derivatives thereof, the hydrolyzed kappa carrageenan product under the name Slimexcess™ sold by the Applicant and / or mixtures thereof.

[0128] A subject of the present invention is also a cosmetic care process comprising the topical application to at least one area of ​​healthy skin and / or healthy mucosa of a strain of Lactobacillus crispatus according to the invention or a cosmetic composition comprising it, in order to maintain and / or increase the expression of extracellular matrix molecules of healthy skin and / or healthy mucosa, preferably of collagen of healthy skin and / or healthy mucosa and / or of elastic fibres of healthy skin and / or healthy mucosa, and / or to maintain and / or increase the biomechanical properties, in particular the hardness and / or elasticity and / or density, preferably the hardness, of healthy skin and / or healthy mucosa.

[0129] Advantageously, the strain of Lactobacillus crispatus according to the invention, preferentially in the form of a cosmetic composition according to the invention, is used by regular topical application and preferentially at least once a day, advantageously twice a day, for at least 10 days, preferentially 20 days and more preferentially at least 28 days.

[0130] Advantageously, the topical application of the strain of Lactobacillus crispatus according to the invention or of a cosmetic composition comprising it is carried out on all or part of the body and / or face and / or scalp, preferentially on the legs, thighs, arms, stomach, neckline, neck, armpits or lips, more preferentially on all or part of the face and preferentially on the cheeks, forehead, chin, lips or eye contour.

[0131] A cosmetic care process according to the invention for maintaining and / or increasing the expression of extracellular matrix molecules of healthy skin and / or healthy mucosa, preferentially for maintaining and / or increasing the expression of collagen of healthy skin and / or healthy mucosa and / or elastic fibres of healthy skin and / or healthy mucosa, and / or for maintaining and / or increasing the biomechanical properties, in particular the hardness and / or elasticity and / or density, preferentially the hardness, of healthy skin and / or healthy mucosa, advantageously comprises the following steps: - identification for each of the areas of healthy skin and / or healthy mucosa where it is desired to maintain and / or increase the expression of extracellular matrix molecules of healthy skin and / or healthy mucosa, preferentially to maintain and / or increase the expression of collagen of healthy skin and / or healthy mucosa and / or elastic fibres of healthy skin and / or healthy mucosa, and / or to maintain and / or increase the biomechanical properties of healthy skin and / or healthy mucosa, in particular the hardness and / or elasticity and / or density, preferentially the hardness, and - an amount of a cosmetic composition comprising a strain of Lactobacillus crispatus according to the invention effective to maintain and / or increase the expression of extracellular matrix molecules of healthy skin and / or healthy mucosa, preferentially to maintain and / or increase the expression of collagen of healthy skin and / or healthy mucosa and / or elastic fibres of healthy skin and / or healthy mucosa, and / or to maintain and / or increase the biomechanical properties, in particular the hardness and / or elasticity and / or density, preferentially the hardness, of healthy skin and / or healthy mucosa, i.e. - 1 x 10 by volume relative to the total volume of the composition -4 % to 10% (v / v), preferentially 1 × 10 by volume -4 % to 5% (v / v), more preferably 1 × 10 by volume -3 % to 3% (v / v), more preferentially 0.1% to 2% (v / v) by volume, and / or - 1 x 10 by weight relative to the total weight of the composition -4 % to 10% (w / w), preferentially 1×10 by weight -4 % to 5% (w / w), more preferably 1×10 -3 %~0.5%(w / w) topical application to this area of ​​healthy skin and / or healthy mucosa with a Lactobacillus crispatus strain of Includes.

[0132] For the purposes of the present invention, the term "topical route" refers to the direct topical administration and / or spraying of a Lactobacillus crispatus strain according to the invention or a composition thereof onto the surface of an area of ​​healthy skin and / or healthy mucosa.

[0133] For purposes of the present invention, the term "topically and / or orally acceptable" means an ingredient that is non-toxic and non-irritating to the skin, including the scalp, does not induce an allergic response, and is not chemically unstable, suitable for topical and / or oral application, respectively.

[0134] A subject of the present invention is a strain of Lactobacillus crispatus according to the invention, alone or in a dermatological composition comprising it for its use via the topical route, for the treatment and / or prevention of pathologies of the skin and / or mucosa which are accompanied by a decrease in the amount and / or expression of extracellular matrix molecules of the skin and / or mucosa and / or a decrease in the amount and / or expression of collagen and / or elastic fibres of the skin and / or mucosa and / or a decrease in the amount and / or biomechanical properties of the skin and / or mucosa, in particular a decrease in the hardness of the skin and / or mucosa and / or a decrease in the elasticity of the skin and / or mucosa and / or a decrease in the density of the skin and / or mucosa.

[0135] For the purposes of the present invention, from a dermatological point of view, the term "treating and / or preventing a pathology of the skin and / or mucosa accompanied by a decrease in the firmness of the skin and / or mucosa" means, for therapeutic purposes, an increase in the firmness of the skin and / or mucosa which has lost firmness as a result of a skin and / or mucosa pathology, for example rosacea or telangiectasia.

[0136] For the purposes of the present invention, from a dermatological point of view, the term "treating and / or preventing a pathology of the skin and / or mucosa accompanied by a decrease in the elasticity of the skin and / or mucosa" means, for therapeutic purposes, an increase in the elasticity of the skin and / or mucosa which has lost its elasticity as a result of a skin and / or mucosa pathology, such as an actinic elastosis and / or cutis laxa pathology.

[0137] For the purposes of the present invention, from a dermatological point of view, the term "treating and / or preventing a disease of the skin and / or mucosa accompanied by a decrease in the density of the skin and / or mucosa" means, for therapeutic purposes, an increase in the density of the skin and / or mucosa which has lost its density as a result of a disease of the skin and / or mucosa, such as Ehlers-Danlos syndrome and / or dermatoporosis.

[0138] The Lactobacillus crispatus strain according to the invention may be in the form of a pharmaceutical composition, preferentially a dermatological composition, comprising at least one pharma- ceutically and / or dermatologically acceptable excipient. In one embodiment of the invention, said composition is applied topically and / or orally, preferentially topically.

[0139] Advantageously, in a pharmaceutical composition, and preferentially in a dermatological composition, it is present in an amount of 1×10 relative to the total weight of the composition, also including at least one pharma- ceutically acceptable excipient. -4 % by weight to 10% by weight, preferentially 1×10 -4 % by weight to 5% by weight, more preferably 1×10 -3 It is present in concentrations of 0.5% by weight to 0.5% by weight.

[0140] Advantageously, the strain of Lactobacillus crispatus according to the invention is present in a pharmaceutical composition, preferentially a dermatological composition, in a concentration of 1×10 by volume relative to the total volume of the composition, which also contains at least one pharma- ceutically acceptable excipient. -4 % to 10% (v / v), preferentially 1 × 10 by volume -4 % to 5% (v / v), more preferably 1 × 10 by volume -3 % to 0.5% (v / v), more preferentially at a concentration of 0.1% to 2% (v / v) by volume.

[0141] The invention will be understood in more detail on reading the following figures and the description of the examples.

[0142] Examples are presented below with reference to the description of the present invention. These examples are given for illustrative purposes and are not intended to limit the scope of the present invention in any way. Each of the examples has a general scope.

[0143] The examples form an integral part of the present invention, and any features which are recognized as novel over any prior art from the description taken as a whole, including the examples, form an integral part of the present invention.

[0144] Additionally, all percentages in the examples are given on a weight basis unless otherwise indicated, temperatures are expressed in degrees Celsius unless otherwise indicated, and pressures are at atmospheric pressure unless otherwise indicated. EXAMPLES

[0145] Example 1. Preparation of different forms of Lactobacillus crispatus Example 1.a: Production of viable intact forms of L. crispatus Isolated L. crispatus bacteria (CNCM I-5579) were inoculated into MRS medium and incubated anaerobically at 37° C. The pH was maintained at a value of pH=6 and the medium was incubated for 24 hours. At the end of the incubation, the medium was centrifuged and the cell concentrate was resuspended in fermentation medium and freeze-dried in the presence of maltodextrin with a final maltodextrin amount of 70% by weight (w / w) relative to the total weight of the components.

[0146] Example 1.b: Production of an inactivated whole form of L. crispatus Isolated L. crispatus bacteria (CNCM I-5579) were inoculated into MRS medium and incubated anaerobically at 37° C. The pH was maintained at a value of pH=6 and the medium was incubated for 24 hours. At the end of the incubation, the medium was centrifuged and the cell concentrate was inactivated by heating at 80° C. for 30 minutes. The cell concentrate was then resuspended in glycerol.

[0147] Example 1.c: Production of L. crispatus bacterial lysate The isolated L. crispatus bacteria (CNCM I-5579) were inoculated into MRS medium and incubated anaerobically at 37°C. The pH was maintained at a value of pH=6 and the medium was incubated for 24 hours. At the end of the incubation, the whole was treated with a high-pressure homogenizer. The lysate was then resuspended in glycerol.

[0148] Example 1.d: Generation of the L. crispatus bacterial secretome Isolated L. crispatus bacteria (CNCM I-5579) were inoculated into MRS medium and incubated anaerobically at 37°C. The pH was maintained at a value of pH=6 and the medium was incubated for 24 hours. At the end of the incubation, the medium was filtered and the bacterial secretome was collected. The secretome was then resuspended in glycerol.

[0149] Example 1.e: Production of L. crispatus bacterial secretome in powder form Isolated L. crispatus bacteria (CNCM I-5579) were inoculated into MRS medium and incubated anaerobically at 37°C. The pH was maintained at a value of pH=6 and the medium was incubated for 24 hours. At the end of the incubation, the medium was filtered and the bacterial secretome was collected. The secretome was then sprayed with maltodextrin with a final maltodextrin amount of 90% (w / w) relative to the total weight of the components.

[0150] Example 1.f: Production of viable intact forms of L. crispatus Isolated L. crispatus bacteria (CNCM I-5579) were inoculated into MRS medium and incubated anaerobically at 37° C. The pH was maintained at a value of pH=6 and the medium was incubated for 24 hours. At the end of the incubation, the medium was centrifuged and the cell concentrate was resuspended in fermentation medium and freeze-dried in the presence of maltodextrin with a final maltodextrin amount of 50% by weight (w / w) with respect to the total weight of the components.

[0151] Example 2: Increased expression of type I collagen by fibroblasts in the presence of L. crispatus strains according to the invention "Normal" human fibroblasts, i.e., free of pathology, obtained from a healthy 34-year-old donor were seeded in 24-well plates and then cultured to confluence in DMEM medium.

[0152] Incubation was continued for 10 6 The incubation period lasted 48 hours in the presence of L. crispatus bacteria according to Example 1.a, inoculated in gondolas on the culture wells in MRS medium diluted with PBS to a concentration of CFU / mL. The same medium without the addition of the strain according to the invention was used as a control.

[0153] At the end of the incubation, the bacteria were removed and the cells were lysed with ammonium hydroxide buffer, which was then removed and the wells saturated with a mixture of PBS / BSA (phosphate-buffered saline / bovine serum albumin).

[0154] After saturation, anti-type I collagen primary antibody (Interchim / Acris, Montlucon, France, LH0284 / R1038) is incubated for several minutes, followed by europium-conjugated secondary antibody (Perkin Elmer), and then fluorescence is measured (λexc. 340 nm / λem. 615 nm) using an EnVision® multilabel plate reader (Perkin Elmer).

[0155] The fluorescence results were normalized to the fluorescence obtained in the same cell culture medium in the absence of the L. crispatus strain according to the invention (control) and related to the amount of DNA obtained under each condition. The results presented correspond to the average of six assays (n=6).

[0156] result

[0157] [Table 1]

[0158] The product of the invention, the L. crispatus strain according to Example 1.a, showed a significant stimulation of collagen type I by human dermal fibroblasts cultured in vitro and can therefore be used to increase the firmness of healthy skin and / or healthy mucosa.

[0159] Example 3: Increased expression of the proteins fibulin-5 and emilin-1 involved in the formation of elastic fibres by fibroblasts in the presence of a strain of L. crispatus according to the invention "Normal" human fibroblasts, i.e. without pathology, obtained from a healthy 19-year-old donor, were cultured in FGM (Fibroblast Growth Medium). Once cultured, incubation was continued for 48 hours in the presence of L. crispatus bacterial secretome obtained according to Example 1.e, at a concentration of 0.1% (w / w) relative to the total weight of medium and secretome. The same medium without the addition of the strain according to the invention was used as a control.

[0160] At the end of the incubation, the medium was removed and the cells were lysed with lysis buffer.

[0161] Fibulin-5 and emilin-1 were identified and quantified using a protein analysis system based on capillary electrophoresis. The amount of protein obtained from the cell lysate, i.e. 0.2 mg / mL of lysate for fibulin-5 and 0.2 mg / mL of lysate supernatant for emilin-1, was precipitated in a capillary to which the primary anti-fibulin-5 or anti-emilin-1 antibody was added, followed by the secondary antibody conjugated to horseradish peroxidase and a chemiluminescent substrate. The whole was incubated for 30 minutes, and then the chemiluminescent signal obtained from the detection of fibulin-5 protein was quantified by capillary electrophoresis using a suitable software.

[0162] Results are the mean ± standard deviation of the assays performed expressed as a percentage relative to the untreated control normalized to 100%. Statistical analysis of the results was performed relative to the untreated control using a one-way ANOVA test. The significance level was set at 5%.

[0163] result

[0164] [Table 2]

[0165] The secretome of the Lactobacillus crispatus strain according to example 1.e of the present invention showed a significant 50% increase in the protein expression of fibulin-5 by human skin fibroblasts cultured in vitro. Thus, the Lactobacillus crispatus strain and in particular its secretome can be used to increase the elasticity of healthy skin and / or healthy mucosa.

[0166] [Table 3]

[0167] The secretome of the Lactobacillus crispatus strain according to Example 1.e of the present invention showed a significant 53% increase in the protein expression of emilin-1 by human skin fibroblasts cultured in vitro. Thus, the Lactobacillus crispatus strain and in particular its secretome can be used to increase the elasticity of healthy skin and / or healthy mucosa.

[0168] Example 4: In vivo measurement of the increase in skin elasticity in the presence of a strain of L. crispatus according to the invention A group of 30 women aged 40-50 years, exhibiting a decrease in skin elasticity on the face, was administered a cream in the form of an emulsion as described in Example 7b) containing a secretome of an L. crispatus strain according to Example 1.e, in a final weight concentration of 1% (w / w) relative to the total weight of the cream, or in the form of the same "placebo" emulsion, in which the secretome was replaced by water, at the rate of two applications per day on one side of the face for a period of two months.

[0169] The evaluation of the biomechanical properties of the skin was carried out using a Cutometer®. This clinical evaluation instrument has the ability to measure three different parameters: total elasticity of the skin, instantaneous elasticity and elastic recovery, all of which are indicative of the elasticity of the skin. From these measurements, different parameters were deduced that are indicative of the state of the elasticity of the skin.

[0170] The total and immediate elasticity of the skin as well as the elastic recovery were measured using a Cutometer® at the start of the study (D0), one month after application (D28) and finally two months after application (D56).

[0171] The efficacy of the secretome of a Lactobacillus crispatus strain according to example 1.e was compared with that obtained with a placebo emulsion.

[0172] result

[0173] [Table 4]

[0174] After 2 months of use, the preparation containing the secretome of the Lactobacillus crispatus strain according to Example 1.e induced a significant increase in all elasticity parameters, an increase that is significant not only relative to the start of the study at D0, but also relative to the placebo.

[0175] These results show a beneficial effect of formulated Lactobacillus crispatus strains, in particular their secretome, for maintaining and / or increasing the biomechanical properties of healthy skin and / or healthy mucosa in vivo, in particular the elasticity of healthy skin and / or healthy mucosa.

[0176] Example 5: In vivo measurement of increased skin density in the presence of a strain of L. crispatus according to the invention A group of 31 women aged 45 to 65 years, presenting wrinkles on the forehead and a decrease in the biomechanical properties of the skin on the face, was administered a cream in the form of an emulsion as described in the formulation presented in Example 7.c, containing the L. crispatus strain obtained according to Example 1.f, at a final weight concentration of 0.05% (w / w) relative to the total weight of the cream, or in the form of the same "placebo" emulsion, in which said strain was replaced by water, on one side of the face at a rate of two applications per day for a period of two months.

[0177] Assessment of skin density was performed using a scanner equipped with an ultrasound probe, which allows visualization of skin density, indicative of the synthesis of extracellular matrix compounds.

[0178] The improvement in skin density and thickness was measured at the start of the study (D0), after one month of application (D28) and finally after two months of application (D56).

[0179] The efficacy of the Lactobacillus crispatus strain according to Example 1.f was compared with that of a placebo emulsion.

[0180] result

[0181] [Table 5]

[0182] After 2 months of use, the preparation of Lactobacillus crispatus strain according to Example 1.f induced a significant increase in skin density, which is significant not only relative to the start of the study at D0 but also relative to the placebo.

[0183] These results show a beneficial effect of formulated Lactobacillus crispatus strains for maintaining and / or increasing the biomechanical properties of healthy skin and / or mucosa, in particular the density of healthy skin and / or mucosa in vivo.

[0184] Example 6: Examples of cosmetic compositions containing Lactobacillus crispatus strains according to the invention

[0185] [Table 6]

[0186] Example 7: Examples of cosmetic compositions containing strains of Lactobacillus crispatus according to the invention Example 7.a: Example of a cosmetic composition containing a Lactobacillus crispatus strain according to Example 1.c Using methods known to those skilled in the art, the following various phases A, B, C and D are mixed together to prepare a composition according to the invention. Proportions are expressed as percentages and the capitalized names correspond to the INCI names of the ingredients. Phase A Glyceryl stearate and PEG-100 stearate 4.00 Pentaerythrityl distearate 1.50 Cetearyl Isononanoate 3.00 Propylheptyl caprylate 5.00 Cocoyl Caprylate 2.00 Dicaprylyl Carbonate 3.00 Dimethicone 1.00 B phase Water 64.43 Propylene glycol, phenoxyethanol, chlorphenesin, methylparaben 1.00 Glycerol 1.57 Xanthan gum 0.20 Butylene Glycol 2.00 Sodium hydroxide, water 0.15 C phase Carbomer 0.15 water 10 D phase Extract of Lactobacillus crispatus obtained according to Example 1c) 1.00

[0187] Example 7.b: Examples of cosmetic compositions containing Lactobacillus crispatus strains according to Example 1.e Ratios are expressed as percentages and the capitalized names correspond to the INCI names of the ingredients. Phase A Glycerin 5.00 Butylene glycol 10.00 Xanthan gum 2.00 B phase Wednesday 58.95 Sodium benzoate 0.25 Glycerin, Water, Sodium Levulinate, Sodium Anisate 1.00 C phase Polyglyceryl-2 Dipolyhydroxystearate 1.00 Dipropylheptyl carbonate 3.00 Dicaprylyl Carbonate 3.00 Caprylic Acid / Caprylyl Caprate 10.00 Laureth-7 Citric Acid 0.50 D phase Citric acid 0.30 E phase Extract of Lactobacillus crispatus obtained according to Example 1e 1.00 Water 4.00

[0188] Using methods known to those skilled in the art, the various phases A, B and C are mixed together by adding B to A followed by adding C to AB under stirring at room temperature. The pH is adjusted to 4.9 with D. Phase E is then added. Finally, homogenization is performed using an Ultra-Turrax blender to prepare the composition according to the invention.

[0189] Example 7.c: Examples of cosmetic compositions containing Lactobacillus crispatus strains according to Example 1.f The Lactobacillus crispatus strain obtained according to example 1.f is added at a final concentration of 0.05% (w / w) relative to the total weight of the formulation immediately prior to application to the skin in the formulation as defined below according to table 8.

[0190] [Table 7]

[0191] Example 8: Examples of dermatological compositions Example 8a): Ointment containing a viable whole strain of L. crispatus according to the invention The compositions below are prepared according to methods known to those skilled in the art, particularly with regard to the various phases that are to be mixed together.

[0192] The amounts listed are based on weight percentages based on the total weight of the composition.

[0193] [Table 8]

[0194] Example 8b): Cream containing a strain of Lactobacillus crispatus according to example 1c)

[0195] [Table 9]

[0196] Phases A and B are heated to 75°C and then mixed with stirring. When the mixture has cooled to room temperature, phases C and D are added with stirring.

Claims

1. A cosmetic and / or nutraceutical composition for maintaining and / or increasing the expression of extracellular matrix molecules in healthy skin and / or healthy mucosa, comprising a strain of Lactobacillus crispatus, preferentially the species deposited under the designation CNCM I-5579.

2. The composition according to claim 1 , wherein the extracellular matrix molecules of healthy skin and / or healthy mucosa are selected from collagen fibers of healthy skin and / or healthy mucosa and / or elastic fibers of healthy skin and / or healthy mucosa.

3. 3. The composition according to claim 1 or 2 for maintaining and / or increasing the expression of type I, III, IV, V, VI, VII, XII, XIII, XIV, XVI, XVII, XXIV and / or XXIX collagens, preferentially type I, III, IV, V and / or XIV, preferentially type I, IV and / or V collagen fibres, more preferentially type I collagen fibres.

4. A composition according to any one of claims 1 to 3 for maintaining and / or increasing the biomechanical properties of healthy skin and / or healthy mucosa, preferentially the hardness and / or elasticity and / or density of healthy skin and / or healthy mucosa, more preferentially the hardness and / or elasticity of healthy skin and / or healthy mucosa.

5. The composition according to any one of claims 1 to 4, wherein said strain is used in its complete form, in particular live, and / or inactivated, in particular dead, and / or in the form of a lysate, and / or in the form of one or more of its fractions, and / or in the form of one or more of its metabolic products, preferentially in the form of its secretome.

6. 6. The composition according to any one of claims 1 to 5, wherein the strain is used in viable whole form and / or in lysate form and / or the strain used is the Lactobacillus crispatus strain deposited under the designation CNCM I-5579.

7. The composition according to any one of claims 1 to 6, wherein the Lactobacillus crispatus strain is combined with the fermentation and / or culture medium.

8. A composition described in any one of claims 1 to 7, which is applied topically to healthy skin and / or healthy mucosa.

9. The strain of Lactobacillus crispatus is present in the cosmetic composition in an amount of 1×10 based on the total weight of the composition, which also contains at least one cosmetically acceptable excipient. －４ % by weight to 10% by weight, preferentially 1×10 －４ % to 5% by weight, more preferably 1×10 －３ The composition of any one of claims 1 to 8, which is present in a concentration of from 0.5% by weight to 0.5% by weight.

10. 10. A cosmetic care composition for maintaining and / or increasing the expression of extracellular matrix molecules in healthy skin and / or healthy mucosa, preferably for maintaining and / or increasing the expression of collagen in healthy skin and / or healthy mucosa and / or elastic fibres in healthy skin and / or healthy mucosa, and / or for maintaining and / or increasing the biomechanical properties, in particular the hardness and / or elasticity and / or density, preferably the hardness, of healthy skin and / or healthy mucosa, comprising a strain of Lactobacillus crispatus as defined in any one of claims 5 to 7 or a cosmetic composition according to claim 9, which is applied topically to at least one area of ​​healthy skin and / or healthy mucosa.

11. The composition described in claim 10, wherein at least one area of ​​healthy skin and / or healthy mucosa is all or part of the body and / or face and / or scalp, preferentially the lower legs, thighs, arms, stomach, neckline, neck, armpits or lips, more preferentially all or part of the face, preferentially the cheeks, forehead, chin, lips or eye contour.

12. The strain of Lactobacillus crispatus is present in the composition in an amount of 1 x 10 based on the total weight of the composition, which also includes at least one cosmetically acceptable excipient. －４ % by weight to 10% by weight, preferentially 1×10 －４ % to 5% by weight, more preferably 1×10 －３ 12. The composition of claim 10 or 11, which is present in a concentration of from 0.5% by weight to 0.5% by weight.

13. A reduction in the amount and / or expression of extracellular matrix molecules in the skin and / or mucosa. and / or a decrease in the amount and / or expression of collagen and / or elastic fibers in the skin and / or mucous membranes, and / or a decrease in the biomechanical properties of the skin and / or mucosa, in particular a decrease in the hardness of the skin and / or mucosa and / or a decrease in the elasticity of the skin and / or mucosa and / or a decrease in the density of the skin and / or mucosa. Dermatological composition for treating and / or preventing skin and / or mucosal pathologies associated with said strain is used in its complete form, in particular live, and / or inactivated, in particular dead, and / or in the form of a lysate and / or in the form of one or more of its fractions and / or in the form of one or more of its metabolic products, preferentially its secretome, and / or the strain is combined with the fermentation and / or culture medium; Dermatological composition.

14. The dermatological composition of claim 13, comprising said strain of Lactobacillus crispatus and at least one dermatologically acceptable excipient.

15. The strain is present in the composition in an amount of 1 x 10 based on the total weight of the composition, which also includes at least one pharma- ceutically acceptable excipient. －４ % by weight to 10% by weight, preferentially 1×10 －４ % to 5% by weight, more preferably 1×10 －３ Dermatological composition according to claim 13 or 14, characterized in that it is present in a concentration of between 0.5% by weight and 0.5% by weight.

16. A dermatological composition described in any one of claims 13 to 15, characterized in that the strain is as defined in any one of claims 5 to 7.