Cosmetic, nutraceutical or dermatological use of a lactobacillus crispatus strain and / or of a composition comprising same

Abstract

The present invention relates to the cosmetic, nutraceutical use of a Lactobacillus crispatus strain and / or of a composition comprising same to decrease the pigmentation of the skin and / or of the mucous membranes and / or prevent the increase thereof, in particular to preserve and / or improve the complexion of the skin and / or of the mucous membranes, preferably to preserve and / or improve the radiance and / or the brightness and / or the uniformity of the complexion of the skin and / or of the mucous membranes, and / or to prevent the appearance and / or reduce the presence of pigmentary spots on the skin and / or mucous membranes and / or detoxify the skin and / or mucous membranes. The invention also relates to the Lactobacillus crispatus strain, or to a dermatological composition comprising same, for use in the treatment and / or prevention of diseases of the skin and / or mucous membranes associated with an increase in the pigmentation of the skin and / or mucous membranes, in particular in depigmenting and / or preventing hyperpigmentation of diseased skin and / or diseased mucous membranes.

Description

[0001] Cosmetic, nutraceutical and / or dermatological use of a strain of Lactobacillus crispatus and / or a composition comprising it

[0002] The present invention relates to the cosmetic, nutraceutical and / or pharmaceutical use, in particular dermatological use, of a strain of Lactobacillus crispatus and / or a composition comprising it for reducing the pigmentation of the skin and / or mucous membranes and / or preventing the increase in the pigmentation of the skin and / or mucous membranes.

[0003] The structure and appearance of the skin change with age and / or under the influence of extrinsic and / or intrinsic factors. These changes are numerous and include changes in the pigmentation of the skin and / or mucous membranes. The skin becomes dull, less luminous, less radiant, its radiance diminishes and the complexion is less even, with the appearance of pigment spots.

[0004] With chronological aging or accelerated aging, particularly under the effect of radiation, typically ultraviolet or pollution, the appearance of these changes such as pigment spots is accelerated. They are linked to the presence of melanin and lipofuscin, the excess and abnormal distribution of which in the skin cause the appearance of so-called senile or pigmented brown spots, also called "aged spots". These can appear as a marker of an acceleration of melanogenesis and are often considered unsightly. They make the complexion uneven and dull.

[0005] Melanogenesis and oxidative stress are the subject of numerous studies and innovations in the field of cosmetics for the development and improvement of cosmetic active ingredients aimed at reducing and / or preventing the increase in pigmentation of healthy skin and / or healthy mucous membranes.

[0006] There is a need in the field of cosmetics and dermatology to develop alternative natural active ingredients, capable of reducing the pigmentation of the skin and / or mucous membranes and / or preventing the increase in pigmentation of the skin and / or mucous membranes, in particular of preserving and / or improving the complexion of the skin and / or mucous membranes, in particular its radiance and / or its luminosity and / or its homogeneity, in particular within a population more prone to pigment spots, in particular skin phototypes IV to VI as defined by the Fitzpatrick classification method.

[0007] There is an ongoing need to identify agents with one or more of these properties, including those with proven efficacy and safety.

[0008] Particularly surprisingly, the Applicant has discovered that Lactobacillus crispatus, a beneficial commensal bacterium of the skin and / or mucous membranes, has the property of reducing the pigmentation of the skin and / or mucous membranes and / or preventing its increase, in particular to preserve and / or improve the complexion of the skin and / or mucous membranes, preferably to preserve and / or improve the radiance and / or luminosity and / or homogeneity of the complexion of the skin and / or mucous membranes, and / or to prevent the appearance and / or reduce the presence of pigment spots on the skin and / or mucous membranes.

[0009] The Applicant also unexpectedly discovered that this bacteria also has the property of detoxifying the skin and / or mucous membranes.

[0010] The Applicant's invention is all the more interesting since the bacterium Lactobacillus crispatus, and in particular that filed under number CNCM I-5579, has the advantage of being naturally present on young skin, unlike the bacteria used until now in cosmetic and / or dermatological treatments.

[0011] This invention is even more interesting as the Applicant has demonstrated that with age and / or under the influence of intrinsic and / or extrinsic factors, the bacterium Lactobacillus crispatus is less present and less abundant on the skin. Thus, the subject of the present invention has the advantage of rebalancing and / or supplementing the microbiota of the skin and / or mucous membranes, in particular with a bacterium of the skin flora which decreases and becomes rarer with time and / or exposure to intrinsic and / or extrinsic factors.

[0012] The subject of the present invention is thus particularly suitable for the treatment of so-called mature skin, in particular of people over 50 years of age. The strain according to the invention also has the advantage of being an ingredient that is easily formulable and can be easily manufactured on an industrial scale. It is an active ingredient that is topically acceptable for the skin and mucous membranes and does not present any risk of allergy.

[0013] Applications have already described the use of Lactobacillus crispatus in cosmetics. Application WO9822082 discloses the use of sphingomyelinase that can be extracted from lactic acid bacteria for topical application to increase ceramide levels in the skin and mucous membranes, for the prevention or therapeutic treatment of several pathologies.

[0014] Application WO2009031099 discloses the use of a probiotic to prevent the appearance and / or to treat the manifestation of feelings of discomfort and / or cutaneous signs associated with a surface skin treatment or an invasive treatment, for aesthetic purposes.

[0015] Application WO2019111189 describes the use comprising a probiotic in particular Lactobacillus paracasei for the prevention of inflammatory damage induced by UV radiation.

[0016] However, none of these applications discloses or suggests the cosmetic, nutraceutical and / or pharmaceutical, in particular dermatological, use of a strain of Lactobacillus crispatus and / or a composition comprising it to reduce the pigmentation of the skin and / or mucous membranes, and / or to prevent its increase, in particular to preserve and / or improve the complexion of the skin and / or mucous membranes, preferably to preserve and / or improve the radiance and / or luminosity and / or homogeneity of the complexion of the skin and / or mucous membranes, and / or to prevent the appearance and / or reduce the presence of pigment spots of the skin and / or mucous membranes.

[0017] Furthermore, none of these applications discloses or suggests the use of a strain of Lactobacillus crispatus and / or a composition comprising it for detoxifying the skin and / or mucous membranes.

[0018] The present invention relates firstly to the cosmetic and / or nutraceutical use of a strain of Lactobacillus crispatus, preferably the strain of Lactobacillus crispatus deposited under number CNCM I-5579, for reducing pigmentation of the skin and / or mucous membranes and / or preventing its increase, in particular for preserving and / or improving the complexion of healthy skin and / or healthy mucous membranes, preferably for preserving and / or improving the radiance and / or brightness and / or homogeneity of the complexion of healthy skin and / or healthy mucous membranes, and / or for preventing the appearance and / or reducing the presence of pigment spots on healthy skin and / or healthy mucous membranes, preferably brown spots and / or age spots, and / or detoxifying healthy skin and / or healthy mucous membranes, in particular skin of phototype IV to VI as defined by the Fitzpatrick classification method.In the context of the invention, the strain of Lactobacillus crispatus according to the invention is in particular used as a cosmetic and / or nutraceutical active ingredient.

[0019] According to a particular embodiment of this use, the strain according to the invention decreases and / or inhibits melanogenesis, in particular by acting on the reduction and / or inhibition of melanin, preferentially on the reduction and / or inhibition of the synthesis and / or the quantity of melanin, and / or by decreasing and / or inhibiting the activity of tyrosinase, and / or by detoxifying healthy skin and / or healthy mucous membranes.

[0020] According to one aspect of the invention, the strain according to the invention can be used to preserve and / or improve the complexion of healthy skin and / or healthy mucous membranes. The strain according to the invention can also be used to preserve and / or improve the radiance and / or luminosity and / or homogeneity of the complexion, of healthy skin and / or healthy mucous membranes. The strain according to the invention can further be used to prevent the appearance and / or reduce the presence of pigment spots on healthy skin and / or healthy mucous membranes, preferably brown spots and / or age spots. The strain according to the invention can also be used to detoxify healthy skin and / or healthy mucous membranes, in particular by an antioxidant action and / or on lipid peroxidation and / or by an increase and / or stimulation of glutathione synthesis induced by oxidative stress.

[0021] It also relates to a cosmetic care method characterized in that it comprises the topical application to at least one area of ​​healthy skin and / or healthy mucous membrane of a strain of Lactobacillus crispatus according to the invention, preferably the strain of Lactobacillus crispatus CNCM I-5579, or of a cosmetic composition comprising it, to reduce the pigmentation of the skin and / or mucous membranes and / or to prevent its increase, in particular to preserve and / or improve the complexion of healthy skin and / or healthy mucous membranes, preferably to preserve and / or improve the radiance and / or luminosity and / or homogeneity of the complexion of healthy skin and / or healthy mucous membranes, and / or to prevent the appearance and / or reduce the presence of pigment spots on healthy skin and / or healthy mucous membranes, and / or to detoxify the skin and / or mucous membranes.In the context of the invention, the strain of Lactobacillus crispatus according to the invention is in particular used as a cosmetic and / or nutraceutical active ingredient.

[0022] The invention also relates to a strain of Lactobacillus crispatus according to the invention, preferably the strain of Lactobacillus crispatus CNCM I-5579, or a pharmaceutical composition, in particular a dermatological composition comprising it, for the prevention and / or treatment of pathologies of the skin and / or mucous membranes associated with an increase in the pigmentation of the skin and / or mucous membranes, in particular for depigmenting and / or preventing hyperpigmentation of pathological skin and / or pathological mucous membranes, for reducing the pigmentation of the skin and / or mucous membranes and / or preventing its increase, in particular for preserving and / or improving the complexion of pathological skin and / or pathological mucous membranes, preferably for preserving and / or improving the radiance and / or luminosity and / or homogeneity of the complexion of pathological skin and / or pathological mucous membranes,and / or to prevent the appearance and / or reduce the presence of pigment spots in pathological skin and / or pathological mucous membranes and / or to detoxify pathological skin and / or pathological mucous membranes. In the context of the invention, the strain of Lactobacillus crispatus according to the invention is in particular used as an active ingredient.,

[0023] For the purposes of the present invention, the term "cosmetic use" means a non-pharmaceutical use, i.e. one which is not intended for therapeutic use and is applied to a so-called healthy part of the body, in particular to a so-called healthy area of ​​the skin and / or mucous membranes.

[0024] For the purposes of the present invention, "nutraceutical use" means a use intended for oral non-pharmaceutical use, i.e. which does not require or involve therapeutic treatment. "Skin" means any area of ​​skin on any part of the body and / or face, including the scalp.

[0025] For the purposes of the present invention, the term "skin of phototype IV to VI" means particular skin types classified according to the Fitzpatrick scale which makes it possible to classify individuals according to the reaction of their skin and hair during exposure to UV rays, and / or classified as such by a dermatologist according to the skin tone, which depends in particular on the level of activity of the melanocytes, the creation of these cells and their groupings. Preferably, these dark skins of phototype IV to VI come from characteristic ethnic origins, respectively from India, Asia and / or Africa. In particular,

[0026] - Phototype IV (Mediterranean type) corresponds to light, matte skin and brown or black hair and eyes. Sunburn is rare, the skin tans well, a dark brown;

[0027] - Phototype V (Latino type) corresponds to light, matte skin, black hair and eyes. Sunburn is rare, the skin tans well, a dark brown;

[0028] - Phototype VI (African type) corresponds to very dark skin, black hair and eyes. Sunburn is very rare.

[0029] According to the invention, the term "mucous membrane(s)" denotes the ocular mucosa, the nasal mucosa, the auricular mucosa, the vaginal mucosa, the urogenital mucosa and / or the oral mucosa, in particular the buccal, labial and / or gingival mucosa, preferably the ocular and / or buccal mucosa, and even more preferably the labial and / or ocular mucosa. Advantageously, it does not include the vaginal mucosa.

[0030] For the purposes of the present invention, the term "healthy skin" or "healthy mucosa" means an area of ​​skin or mucosa to which the strain according to the invention is applied and deemed "non-pathological" by a dermatologist, i.e. not presenting any infection, scar, disease, inflammation or skin condition such as candidiasis, impetigo, psoriasis, eczema, acne or dermatitis, or wounds or injuries. For the purposes of the present invention, the term "Lactobacillus crispatus strain" means the bacterium Lactobacillus crispatus in whole form or in partial form, in particular in the form of a lysate, in the form of a fraction or in the form of metabolites. Advantageously, the strain of Lactobacillus crispatus is not genetically modified.

[0031] The strain of Lactobacillus crispatus according to the invention may be derived from any known species of Lactobacillus crispatus. Preferably, the strain of Lactobacillus crispatus is the species deposited in accordance with the Budapest Treaty at the Pasteur Institute (28 rue du Docteur Roux, F -75024 Paris cedex 15) on 09 / 09 / 2020 under the designation CNCM I-5579.

[0032] The advantage of this particular strain of Lactobacillus crispatus is that it is the first species of Lactobacillus crispatus found naturally on the skin.

[0033] For the purposes of the present invention, the term "reducing the pigmentation of the skin and / or mucous membranes and / or preventing its increase" means depigmenting, whitening, lightening, preventing the increase, maintaining and / or preserving and / or improving the complexion and / or skin tone of the skin and / or mucous membranes, and preferably reducing and / or preventing the increase in melanogenesis, and even more preferably reducing and / or inhibiting totally or partially gene and / or protein expression and / or tyrosinase activity and / or reducing and / or inhibiting the synthesis and / or quantity of melanin.

[0034] The process of skin pigmentation is also called "melanogenesis" and involves many steps, from the synthesis of melanin in melanocytes to its transport to the keratinocytes of the epidermis via melanosomes. Several proteins are involved in this process. Tyrosinase is the enzyme that intervenes at key stages of melanin synthesis; it cooperates with other proteins to transform melanin into eumelanin and phaeomelanin, the melanins responsible for the pigmentation process.

[0035] “Decrease and / or inhibit melanogenesis” means totally or partially reducing and / or inhibiting gene and / or protein expression and / or tyrosinase activity and / or totally or partially reducing and / or inhibiting the synthesis and / or quantity of melanin.

[0036] According to the invention, the term "totally or partially reducing and / or inhibiting the activity of tyrosinase" means causing a reduction of at least 25%, preferably at least 50%, more preferably at least 75% of the activity of tyrosinase in the presence of the strain of Lactobacillus crispatus according to the invention, compared to the level of activity measured in the absence of the extract.

[0037] Preferably, the measurement of the tyrosinase activity is carried out on the tyrosinase extracted from so-called normal human melanocytes, still preferentially by measuring the production of melanin by photometrically measuring the absorbance at 540 nm from the tyrosinase extracted from so-called normal human melanocytes, still preferentially in the presence of the strain of Lactobacillus crispatus prepared according to example 1.e, advantageously under the conditions described in example 3.

[0038] According to the invention, the term "totally or partially reducing and / or inhibiting the synthesis and / or quantity of melanin" means a reduction in melanin of at least 10%, preferably at least 20%, more preferably at least 30% of the synthesis and / or quantity of melanin in the presence of the strain of Lactobacillus crispatus according to the invention compared to the synthesis and / or quantity of melanin detected in the absence of the strain. In a preferred embodiment of the invention, this is a reduction in the quantity of melanin in the presence of the strain of Lactobacillus crispatus according to the invention prepared according to example 1.a or according to example 1.e. Preferably again, the quantity of melanin is measured by optical density at 475 nm from B16 melanocytes cultured in vitro in a culture medium comprising fetal calf serum, advantageously under the conditions described in example 2.In another preferred embodiment, the quantity of melanin is measured by optical density at 475 nm from a coculture of melanocytes and so-called normal human keratinocytes, cultured in vitro in a culture medium comprising growth factors, advantageously under the conditions described in example 4.

[0039] For the purposes of the present invention, the term "preserve and / or improve the complexion" means maintaining and / or preventing the reduction and / or increasing the radiance and / or luminosity and / or radiance and / or shine of the complexion and / or the homogeneity of the complexion and / or diminishing and / or reducing the dull and / or earthy complexion of the skin and / or mucous membranes.

[0040] The measurement of the radiance of the complexion, the brightness and the homogeneity of the complexion of the skin and / or mucous membranes can be studied by the measurement techniques known to those skilled in the art, in particular can for example be carried out in vivo, by chromametry or by image analysis. This last in vivo measurement method consists of taking high-resolution photographs in crossed polarized configuration of the face of volunteers taken at 45° before and after application of the tested product. On the basis of these digital photographs, an image analysis makes it possible to extract and quantify specific parameters (for example: L*, a*, b*, C, h°) related to the color, radiance and homogeneity of the skin.

[0041] For the purposes of the present invention, the term "preserve and / or improve the radiance of the complexion" means maintaining and / or preventing the reduction and / or increasing the luminosity and / or radiance and / or shine and / or clarity, and / or diminishing and / or reducing the dull and / or earthy complexion of the skin and / or mucous membranes.

[0042] For the purposes of the present invention, the term "preserve and / or improve the homogeneity of the complexion" means maintaining and / or preventing the increase and / or reduction of the quantity and / or extent of imperfections and / or irregularities in the color of the complexion, in particular chosen from redness, pigment spots, comedones, freckles and / or visible pores.

[0043] The measurement of skin tone homogeneity and the amount of melanin in the skin can be measured by Mexametry. This in vivo measurement method uses the absorption / reflection principle to deduce a melanin index that reflects the amount of melanin present in the skin.

[0044] Advantageously, the measurement of the improvement in complexion is carried out on a population of women having pigment spots on the face, preferably by applying a cream containing the strain of Lactobacillus crispatus prepared according to example 1.e, under the conditions described in example 7. The term "maintain and / or prevent the increase and / or decrease the imperfections and / or irregularities of the color of the complexion" means preventing the increase and / or decrease the quantity of melanin in the skin, by at least 2%, advantageously by at least 3% in the presence of the strain according to the invention during a 28-day application on the skin compared to the quantity of melanin detected in the absence of the strain and / or by at least 2%, advantageously by at least 5% in the presence of the strain according to the invention during a 56-day application on the skin compared to the quantity of melanin detected in the absence of the strain.

[0045] “Preventing the appearance and / or reducing the presence of pigment spots” means maintaining and / or preventing the increase and / or reducing the pigmentation of localized pigment spots, in particular the number and / or intensity of pigment spots typically on the areas of the face, décolleté, neck, back, shoulders and / or hands, particularly spots on the top of the hands.

[0046] Pigment spots are in particular brown and / or dark and / or white spots and / or "aged spots" when they are located on a part of the body exposed to UV radiation and / or linked to chronological or UV-induced aging, for example during sun exposure. Pigment spots also include freckles and / or moles and / or post-inflammatory spots and / or spots that appear in response to aggressions, and / or of hormonal origin, in particular in the context of chloasma or melasma, otherwise known as pregnancy mask, and / or of drug origin, and / or to prevent and / or combat unsightly pigmentary manifestations of the skin and / or mucous membranes accompanying a non-cutaneous pathology.

[0047] The Applicant has also discovered that the strain of Lactobacillus crispatus, in particular that deposited under the designation CNCM I-5579, can be used to detoxify the skin and / or mucous membranes, in particular healthy skin and / or mucous membranes.

[0048] The term "detoxifying the skin and / or mucous membranes" means the prevention and / or total and / or partial inhibition of oxidative stress, in particular by the prevention and / or total and / or partial inhibition of the formation of free radicals, and / or of lipid peroxidation and / or by increasing and / or stimulating the synthesis of glutathione which has antioxidant and detoxifying properties.

[0049] Detoxification helps protect the skin against both external and internal aggressions that cause oxidative stress that can affect its proper functioning and appearance, in particular a means capable of stimulating the natural protection and repair systems of skin cells and limiting the induction of cellular damage.

[0050] Oxidative stress refers to the formation of excessive reactive species, including reactive oxygen species and reactive nitrogen species, which are formed when the skin is subjected to oxidative stress caused by various factors such as solar ultraviolet, infrared, and visible light, environmental pollution, including ozone and particulate matter, and psychological stress.

[0051] In addition to impacting the effectiveness of the skin's detoxification systems, oxidative stress can also exacerbate pigmentation, particularly by inducing uneven skin tone or even pigmentation disorders.

[0052] In a preferred embodiment of the invention, it is a question of reducing and / or inhibiting the formation of DPPH° (2,2-diphenyl-1-picrylhydrazyl) free radicals by at least 10%, preferably at least 30%, in the presence of the strain of Lactobacillus crispatus according to the invention compared to the formation of DPPH° free radicals detected in the absence of the strain, and / or reducing and / or inhibiting the release of intracellular free radicals induced by oxidative stress, preferably by UVA, by at least 20%, preferably at least 50%, in the presence of the strain of Lactobacillus crispatus according to the invention compared to the release of intracellular free radicals detected in the absence of the strain.

[0053] In a preferred embodiment of the invention, it is a question of reducing and / or inhibiting the spontaneous formation of DPPH° free radicals, in the presence of the strain of Lactobacillus crispatus according to the invention prepared according to example 1.a and / or 1.e.

[0054] Preferably again, the formation of DPPH° free radicals is measured in tubo by optical density at 530 nm, under the conditions described in example 5.a.

[0055] In a preferred embodiment of the invention, it is a question of reducing and / or inhibiting the release of intracellular free radicals induced by oxidative stress, preferentially by UVA, in the presence of the strain of Lactobacillus crispatus according to the invention prepared according to example 1.a.

[0056] Preferably again, the release of intracellular free radicals induced by oxidative stress, preferentially by UVA, is measured by fluorescence emitted by the formation of DCF (2',7-dichlorofluorescein) from the probe DCFH-DA (2',7'-dichlorodihydrofluorescein diacetate) at wavelengths of 485 nm excitation and 538 nm emission from normal human fibroblasts, under the conditions described in example 5. b.

[0057] In another embodiment, it is a question of "preventing and / or inhibiting the peroxidation of membrane lipids", that is to say of preventing and / or inhibiting totally or partially the oxidation of lipids induced by oxidative stress, preferably by UVA of at least 10%, preferably of at least 30% in the presence of the strain of Lactobacillus crispatus according to the invention compared to the lipid peroxidation detected in the absence of the strain.

[0058] In a preferred embodiment of the invention, it is a question of preventing and / or inhibiting the peroxidation of membrane lipids induced by oxidative stress, preferentially by UVA, in the presence of the strain of Lactobacillus crispatus according to the invention prepared according to example 1.a.

[0059] Preferably again, the peroxidation of membrane lipids induced by oxidative stress, preferentially by UVA, is measured by fluorescence of the formation of MalonDiAldehde (MDA) at wavelengths of 532 nm excitation and 560 nm emission from normal human fibroblasts, under the conditions described in example 5.c.

[0060] According to another embodiment, it is a question of maintaining and / or stimulating the synthesis of glutathione, that is to say of preventing the reduction and / or increasing the expression and / or the quantity of glutathione in the presence of oxidative stress, preferentially induced by UVA, of at least 20%, preferentially at least 50%, in the presence of the strain of Lactobacillus crispatus according to the invention compared to the synthesis of glutathione detected in the absence of the strain.

[0061] In a preferred embodiment of the invention, it is a question of maintaining and / or stimulating the synthesis of glutathione induced by oxidative stress, preferentially by UVA, in the presence of the strain of Lactobacillus crispatus according to the invention prepared according to example 1.a.

[0062] Preferably again, glutathione synthesis is measured in vitro by fluorescence at wavelengths of 355 nm excitation and 430 nm emission from normal human fibroblasts, under the conditions described in example 6.

[0063] According to the present invention, the strain of Lactobacillus crispatus can be used in whole form, in particular viable and / or inactivated, in particular dead, and / or in lysate form, and / or in the form of one or more of its fractions and / or in the form of one or more of its metabolites, preferably its secretome.

[0064] For each of its forms, the strain according to the invention can be isolated or associated with its culture medium.

[0065] Preferably, the strain according to the invention is used associated with its culture medium, that is to say contained in its culture medium.

[0066] For the purposes of the present invention, the term "culture medium" means a support containing the nutrients allowing the culture and / or multiplication of the strain of Lactobacillus crispatus according to the invention. Preferably, it is an MRS medium (Man, Rogosa and Sharp agar).

[0067] For the purposes of the present invention, the term “isolated” means not mixed with one or more compounds capable of being associated with it in its culture medium.

[0068] For the purposes of the present invention, "whole form" means its native form, a form in which the bacterial envelope is intact, as opposed to a lysed form. When used in whole form, the Lactobacillus crispatus strain may be viable and / or inactivated and / or dead.

[0069] For the purposes of the present invention, the term “viable” means a strain of Lactobacillus crispatus according to the present invention capable of forming colonies in culture.

[0070] The production of the Lactobacillus crispatus strain in viable whole form may be carried out according to any method conventionally known to those skilled in the art. Advantageously, it will be carried out according to the protocol as described for example in example 1.a.

[0071] For the purposes of the invention, "inactivated" means a strain of Lactobacillus crispatus according to the present invention which is no longer capable, temporarily or permanently, of forming colonies in culture.

[0072] The inactivation of the Lactobacillus crispatus strain may be carried out according to any method conventionally known to those skilled in the art. It may in particular be inactivated by irradiation, heat treatment or, under certain conditions, by high-pressure treatment or by extrusion; advantageously, it will be carried out by heat treatment, again advantageously according to the protocol as described for example in example 1.b.

[0073] Thermal inactivation can be carried out by incubating the Lactobacillus crispatus strain for a given period of time, advantageously from about 10 s to 90 min, preferably from about 15 minutes to one hour, and at a temperature advantageously from about 60°C to 150°C. Preferably, it will be incubated for about 30 minutes at about 80°C.

[0074] For the purposes of the invention, "dead" means a strain of Lactobacillus crispatus according to the present invention which is no longer capable, definitively, of forming colonies in culture.

[0075] For the purposes of the invention, the term "lysate" means a material obtained following the destruction or dissolution of biological cells by a phenomenon known as cell lysis, thus causing the release of intracellular biological constituents naturally contained in the cells of the microorganism in question and fragments of cell membrane components. The lysate used is therefore formed from all the intracellular biological constituents and the constituents of the cell walls and membranes of the strain of Lactobacillus crispatus according to the invention. The lysate may be obtained by any method conventionally known to those skilled in the art. It may in particular be obtained by osmotic shock, thermal shock, ultrasound, enzymatic lysis, tyndallization, or even under mechanical stress such as centrifugation, or by increasing the pressure or combinations of these different technologies.

[0076] Preferably, the lysate will be obtained by a combination of mechanical action and increased pressure. Even more preferably, the lysate will be obtained according to the protocol as described for example in Example 1.c.

[0077] In a preferred embodiment of the invention, the lysate will be associated with the culture medium of the bacteria.

[0078] Preferably, the lysate obtained does not contain any active enzyme, in particular active sphingomyelinase.

[0079] For the purposes of the present invention, the term "fractions" means a fragment of the Lactobacillus crispatus strain according to the present invention having an efficacy for reducing the pigmentation of the skin and / or mucous membranes and / or preventing its increase, in particular for preserving and / or improving the complexion of the skin and / or mucous membranes, preferably for preserving and / or improving the radiance and / or brightness and / or homogeneity of the complexion of the skin and / or mucous membranes, and / or for preventing the appearance and / or reducing the presence of pigment spots of the skin and / or mucous membranes and / or detoxifying the skin and / or mucous membranes. This fraction corresponds to a non-total part of the intracellular biological constituents and of the constituents of the cell walls and membranes obtained by lysis of the Lactobacillus crispatus strain according to the invention.

[0080] According to a particular embodiment of the invention, the fractions may be the protoplasm of the bacterium. According to a particular embodiment of the invention, the fraction corresponds to all the intracellular biological constituents, but does not contain constituents of the cell walls and membranes of the Lactobacillus crispatus strain. According to a particular embodiment of the invention, the fraction obtained does not contain any active enzyme, in particular active sphingomyelinase.

[0081] In a preferred embodiment of the invention, the fractions will be associated with the culture medium of the bacteria.

[0082] For the purposes of the present invention, the term "metabolites" means one or more organic and / or inorganic molecules resulting from the metabolism of the strain of Lactobacillus crispatus according to the present invention and also having an efficacy for reducing the pigmentation of the skin and / or mucous membranes and / or preventing its increase, in particular for preserving and / or improving the complexion of the skin and / or mucous membranes, preferably for preserving and / or improving the radiance and / or luminosity and / or homogeneity of the complexion of the skin and / or mucous membranes, and / or for preventing the appearance and / or reducing the presence of pigment spots on the skin and / or mucous membranes and / or detoxifying the skin and / or mucous membranes. Preferably, it is the secretome of the strain of Lactobacillus crispatus according to the invention.

[0083] In a particular embodiment of the invention, the metabolites will be associated with the culture medium of the bacteria.

[0084] For the purposes of the present invention, the term "secretome" means all the secreted organic and / or inorganic molecules secreted by the strain of Lactobacillus crispatus according to the present invention having an effectiveness in reducing the pigmentation of the skin and / or mucous membranes and / or preventing its increase, in particular to preserve and / or improve the complexion of the skin and / or mucous membranes, preferably to preserve and / or improve the radiance and / or luminosity and / or homogeneity of the complexion of the skin and / or mucous membranes, and / or to prevent the appearance and / or reduce the presence of pigment spots on the skin and / or mucous membranes and / or detoxify the skin and / or mucous membranes.

[0085] The secretome may be obtained by any method conventionally known to those skilled in the art. Advantageously, it will be obtained according to the protocol as described for example in example 1.e.

[0086] In a preferred embodiment of the invention, the secretome will be associated with the culture medium of the bacteria. According to a particular embodiment of the invention, the metabolites and / or the secretome do not contain an active enzyme, in particular active sphingomyelinase.

[0087] According to a preferred embodiment of the invention, the strain of Lactobacillus crispatus according to the invention will be used in whole viable form and / or in the form of one or more of its metabolites, preferably its secretome.

[0088] Preferably, the strain of Lactobacillus crispatus according to the invention will be used in an effective quantity to reduce the pigmentation of the skin and / or mucous membranes and / or prevent the increase in the pigmentation of the skin and / or mucous membranes.

[0089] According to an advantageous embodiment, the strain of Lactobacillus crispatus according to the invention does not contain sphingomyelinase.

[0090] According to an advantageous embodiment of the invention, the strain of Lactobacillus crispatus according to the invention is applied topically to healthy skin and / or healthy mucous membranes. Preferably, the skin and / or mucous membranes are of phototype IV to VI. Phototypes IV to VI are as defined by the Fitzpatrick classification method.

[0091] For the purposes of the present invention, the term "topical route" means the direct local application and / or vaporization of the strain of Lactobacillus crispatus according to the invention or of the composition comprising it according to the invention on the surface of the area of ​​skin and / or mucous membrane concerned.

[0092] According to the invention, the strain of Lactobacillus crispatus according to the invention can be used alone, in the form of an active ingredient and / or in a cosmetic and / or nutraceutical, and / or pharmaceutical, in particular dermatological, composition, preferably intended for topical and / or oral application, even more preferably for topical application.

[0093] When used in whole live form according to the invention, and when used alone, as an active ingredient, the strain according to the invention may be in dry form, i.e. in powder form, advantageously in maltodextrin, preferably in a strain content of 10% to 80% (w / w) by weight of the strain, preferably 30% to 70% (w / w), still advantageously 40 to 60% (w / w) by weight of the strain relative to the total weight of powder.

[0094] Alternatively, the whole living form according to the invention can be used dispersed and / or diluted in a solvent. Preferably, this solvent contains less than 20% (v / v) by volume of water, even more preferably less than 5% (v / v) by volume of water, even more preferably the solvent does not contain water.

[0095] According to an alternative embodiment, this solvent contains less than 20% (w / w) by weight of water, even more preferably less than 5% (w / w) by weight of water, even more preferably the solvent does not contain water. Very preferably, this solvent is a cosmetically acceptable oil.

[0096] When used in whole inactivated form, in particular dead, and / or in lysate form, and / or in the form of one or more of its fractions and / or in the form of one or more of its metabolites, in particular its secretome, and when used alone in the form of active ingredient, the strain according to the invention may be in dry form, i.e. in powder form, advantageously in maltodextrin, preferably in a strain content of 1% to 80% (w / w) by weight of the strain, preferably of 3% to 50% (w / w) by weight of the strain, very preferably of approximately 5% or 20% (w / w) by weight of the strain relative to the total weight of powder, still preferably approximately 10% (w / w), still preferably 10% (w / w) by weight of the strain relative to the total weight of powder.

[0097] Alternatively, the inactivated whole forms, in particular dead, and / or the lysates, and / or the fractions, and / or the metabolites, in particular the secretome according to the invention, can be used soluble and / or diluted and / or dispersed in a solvent, in particular polar, such as water, an alcohol, a polyol, a glycol such as pentylene glycol and / or hexylene glycol and / or caprylyl glycol and / or butylene glycol, or one of their mixtures, preferably a hydroglycolic or hydroalcoholic mixture, still preferably comprising a glycol chosen from hexylene glycol, caprylyl glycol and their mixtures. In another embodiment, the strain according to the invention can be incorporated into a cosmetic and / or nutraceutical composition comprising at least one cosmetically acceptable and / or nutraceutically acceptable excipient. Preferably, it is incorporated into a cosmetic composition comprising at least one cosmetically acceptable excipient.

[0098] For the purposes of the present invention, the term “cosmetically acceptable” excipient means a topically acceptable compound and / or solvent, i.e. one that does not induce an allergic response upon contact with the skin, including the human scalp, and / or mucous membranes, that is non-toxic, non-unstable, chemically.

[0099] In a preferred embodiment, the strain according to the invention will be incorporated into a cosmetic composition alone, that is to say it will not be used to ferment plant extracts. In other words, the cosmetic composition comprising the strain according to the invention does not comprise a fermentate of plant extracts by the strain.

[0100] In a preferred embodiment of the invention, the strain according to the invention is present in the cosmetic composition in an effective amount, namely a concentration of 1x10' 4 % to 10% (w / w) by weight, preferably between 1x10' 4% and 5% (w / w) by weight, still advantageously between 1x10' 3 % and 0.5% (w / w) by weight, relative to the total weight of the composition, said composition further comprising at least one cosmetically acceptable excipient.

[0101] Particularly advantageously, 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 composition in an amount effective for reducing the pigmentation of the skin and / or mucous membranes and / or preventing the increase in the pigmentation of the skin and / or mucous membranes, namely a concentration of 0.05 to 0.5% (w / w) by weight, more preferably at a concentration of approximately 0.1% (w / w) by weight, relative to the total weight of the composition, said composition further comprising at least one cosmetically acceptable excipient.Particularly advantageously, when the strain according to the invention is used in whole viable form, it is present in the cosmetic composition, in an amount effective for reducing the pigmentation of the skin and / or mucous membranes and / or preventing the increase in the pigmentation of the skin and / or mucous membranes, namely a concentration of 0.01 to 0.1% (w / w) by weight, more preferably at a concentration of approximately 0.025% (w / w) by weight, relative to the total weight of the composition, said composition further comprising at least one cosmetically acceptable excipient.

[0102] The cosmetic composition of the invention may be chosen from a suspension or a solution, aqueous or oily, a cream or an aqueous gel or an oily gel, in particular a shower gel, a shampoo; a milk; an emulsion, a microemulsion or a nanoemulsion, in particular oil-in-water or water-in-oil or multiple or silicone; a mask; a serum; a lotion; a liquid soap; a dermatological bar; an ointment; a balm; a butter; a mousse; a patch; an anhydrous product, preferably liquid, pasty or solid, for example in the form of makeup powders, sticks or sticks, in particular in the form of lipstick.

[0103] It can also be a makeup product or a makeup remover.

[0104] Advantageously, the strain of Lactobacillus crispatus or the composition of the invention is intended to be applied to healthy skin and / or healthy mucous membranes of all or part of the body and / or the face and / or the scalp, preferably the legs, thighs, arms, stomach, décolleté, neck, armpits, lips, still preferably all or part of the face, and preferably the cheeks, forehead, chin, lips, and the area around the eyes.

[0105] Advantageously, the strain and / or the composition comprising it is intended to be applied to an area of ​​healthy skin having a dull complexion and / or an area of ​​non-homogeneous healthy skin and / or an area of ​​healthy skin with the appearance of pigment spots.

[0106] Preferably, the strain of Lactobacillus crispatus according to the invention is particularly suitable for the formulation of so-called neutral and gentle compositions to respect the sebaceous gland, in particular the skin including the scalp and / or the mucous membranes.

[0107] Alternatively, the strain of Lactobacillus crispatus according to the invention can be presented in all the galenic forms conventionally used for oral application, in particular in the form of a nutraceutical active ingredient and / or nutraceutical composition.

[0108] 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.

[0109] Therefore, for these compositions, the excipient contains, for example, at least one compound chosen from the group comprising preservatives, emollients, emulsifiers, surfactants, moisturizers, thickeners, conditioners, matting agents, stabilizers, antioxidants, texturizing agents, shine agents, film-forming agents, solubilizers, pigments, colorants, perfumes and sunscreens. These excipients are preferably chosen from the group consisting of amino acids and their derivatives, polyglycerols, esters, cellulose polymers and derivatives, lanolin derivatives, phospholipids, lactoferrins, lactoperoxidases, sucrose-based stabilizers, vitamins E and its derivatives, natural and synthetic waxes, vegetable oils, triglycerides, unsaponifiables, phytosterols, vegetable esters, silicones and its derivatives, protein hydrolysates,Jojoba oil and its derivatives, lipo / water-soluble esters, betaines, amino oxides, plant extracts, Sucrose esters, Titanium dioxides, glycines, and parabens, and more preferably from the group consisting of butylene glycol, steareth-2, steareth-21, glycol-15 stearyl ether, cetearyl alcohol, phenoxyethanol, methylparaben, ethylparaben, propylparaben, butylparaben, butylene glycol, natural tocopherols, glycerin, dihydroxycetyl sodium phosphate, isopropyl hydroxycetyl ether, glycol stearate, triisononanoin, octyl cocoate, polyacrylamide, isoparaffin, laureth-7, a carbomer, Propylene glycol, glycerol, bisabolol, dimethicone, sodium hydroxide, PEG 30-dipolyhydroxysterate, capric / caprylic triglycerides, cetearyl octanoate, dibutyl adipate, grape seed 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, PEG 8, beeswax, hydrogenated palm kernel oil glycerides, hydrogenated palm oil glycerides, lanolin oil, sesame oil, cetyl lactate, lanolin alcohol, castor oil, titanium dioxide, lactose, sucrose, low density polyethylene, isotonic saline, and mixtures thereof.,

[0110] Many cosmetically 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 know how to formulate cosmetic, nutraceutical or dermatological compositions to achieve the best effects. Furthermore, these compounds may have a synergistic effect when combined with each other. These combinations are also covered by the present invention. The CTFA Cosmetic Ingredient Handbook, Second Edition (1992) describes various cosmetic and pharmaceutical ingredients commonly used in the cosmetic and pharmaceutical industry, which are particularly suitable for topical use. Examples of these classes of ingredients include, but are not limited to, the following compounds: abrasives, absorbents, compounds for aesthetic purposes such as perfumes, pigments, dyes, essential oils, astringents, etc.(e.g., clove oil, menthol, camphor, eucalyptus oil, eugenol, menthyl lactate, witch hazel distillate), anti-acne agents, anti-flocculating agents, anti-foaming agents, antimicrobial agents (e.g., iodopropyl butylcarbamate), antioxidants, binders, biological additives, buffering agents, bulking agents, chelating agents, additives, biocidal agents, denaturants, thickeners, and vitamins, and derivatives or equivalents thereof, film-forming materials, polymers, opacifying agents, pH adjusters, reducing agents, depigmenting or lightening agents (e.g., hydroquinone, kojic acid, ascorbic acid, magnesium ascorbyl phosphate, ascorbyl glucosamine), conditioning agents (for example: humectants).

[0111] The cosmetic composition may also include other cosmetic and / or nutraceutical agents having the same properties and inducing a synergistic effect or not with the strain of Lactobacillus crispatus according to the invention, or cosmetic agents with complementary effects.

[0112] These may be, for example, depigmenting and / or lightening ingredients and / or ingredients intended to reduce pigment spots on the skin, such as niacinamide or vitamin B3, arbutin, azelaic acid, ascorbic acid or its derivatives, a combination of the plant extracts Saxifraga sarmentosa, Psidium guajava and Carica papaya, marketed by BASF Beauty Care Solutions France under the name Dermawhite™ WF, a combination of sulfites and extracts of Camellia sinensis, Scutellaria baicalensis, Cucumis sativus, Pyrus malus, marketed under the name Phytolight™ BG, a standardized extract of Lansium domesticum leaves marketed under the name DN-Aura™, a combination of a pea extract and sucrose dilaurate marketed under the name Actiwhite™, derivatives of 4-hydroxyphenoxy acetic acid, in particular 2-(4-hydroxyphenoxy)-propionic acid marketed under the name Radianskin™ by BASF Beauty Care Solutions France,or an extract of purified polysaccharides from Cassia angustifolia seeds marketed under the name Hyalurosmooth™, an extract of stabilized proteases isolated from Carica papaya latex marketed under the name X-Pressin™, a cationic liposome encapsulating ferulic acid marketed under the name Cytovector ferulic™, or a complex of glycolic acid and L-arginine marketed under the name AH-care™ by BASF Beauty Care Solutions France.,

[0113] Among other active ingredients that may be combined with the strain according to the invention, it may be a plant extract having similar properties of preventing the appearance of pigmentation and / or increasing the radiance of the complexion, in particular an extract of the mushroom Inonotus obliquus marketed under the name Inolixir™ by the applicant, an extract of Argania spinosa oil marketed under the name Arganyl™ by the applicant, an extract of Moringa oleifera seeds marketed under the name Purisoft™ by the applicant, a combination of an extract of Salvia miltiorrhiza and niacinamide marketed under the name ColIRepair™ by the applicant, an extract of Achillea millefolium marketed under the name Neurobiox™ by the applicant, an extract of Cassia alata leaves marketed under the name DN-Age™ and / or an extract of lychee marketed under the name Litchiderm™ as anti-oxidant active ingredients,of a chicory extract marketed under the name Lox-Age™, a yeast extract marketed under the name Vitacell™, a Polygonum bistorta extract marketed under the name Perlaura™, a galangal extract marketed under the name Hyalufix™, a corn extract marketed under the name Deliner™ or a Voandzeia subterranea extract marketed under the name Epigenist™ by the applicant or active ingredients promoting skin firmness such as a synthetic tetrapeptide marketed under the name Dermican™, a Hibiscus abelmoschus extract marketed under the name Linefactor™, a purified pea extract marketed under the name Proteasyl™,an extract of Manilkara multinervis marketed under the name Elestan™ by the applicant. An extract of the plant Origanum majorana marketed under the name Dermagenist™ and / or an extract of Khaya senegalensis marketed under the name Collalift®18 and / or an extract of Eperua falcata marketed under the name Eperuline™ or an extract comprising phytosterols, in particular [3-sitosterol and / or campesterol and / or brassicasterol from rapeseed oil and marketed by the applicant under the name Phytosoothe™, may also be added to the cosmetic composition.,

[0114] These may also include anti-aging active ingredients and / or firming agents for a synergistic effect with the Lactobacillus crispatus strain. Advantageously, these are active ingredients increasing the gene and / or protein expression of collagen or active ingredients preventing the degradation of collagen such as retinol, vitamin C, an extract of Davilla rugosa marketed under the name Collguard™ by BASF Beauty Care Solutions, an extract of Hibiscus abelmoschus seed marketed under the name Linefactor™, an extract of soy protein hydrolyzate marketed under the name Phytokine™, a peptide marketed under the name Dermican™ by the applicant or the products marketed under the names Matrixyl™, Matrixyl 3000™ and Regestril™ by the company Sederma or Neuroguard by the company Codif, or Eternaline™ by the company Silab.

[0115] The tensioning agents that can be used in the invention may be chosen 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 carrageenans, alginates, agars, gellans, cellulose polymers and pectins; vegetable proteins and protein hydrolysates; mixed silicates; wax microparticles; colloidal particles of inorganic filler chosen for example from silica, silica-alumina composites; as well as their mixtures.

[0116] Advantageously, the Lactobacillus crispatus strain according to the invention may be combined with a cosmetic agent chosen from benzoic acid and / or its salts, levulinic acid and / or its salts such as sodium levulinate, sorbic acid and / or its salts, anisic acid and / or its salts, and their mixture.

[0117] Finally, it may be cosmetic ingredients such as, for example, antimicrobial agents, anti-free radical agents, soothing, calming or relaxing agents, agents acting on microcirculation to improve the radiance of the complexion, in particular of the face, healing agents or slimming agents. Advantageously, the cosmetic and / or dermatological composition of the present invention also contains one or more tightening agents and / or one or more antimicrobial agents and / or one or more anti-free radical agents and / or one or more soothing agents and / or one or more slimming agents and / or one or more agents active on microcirculation.

[0118] Among the antimicrobial agents associated with the strain of Lactobacillus crispatus in non-viable form in a preferred embodiment of the present invention, mention may be made of 2,4,4'-trichloro-2'-hydroxy diphenyl ether (or triclosan), 3,4,4'-trichlorobanilide, phenoxyethanol, phenoxypropanol, phenoxyisopropanol, hexamidine isethionate, metronidazole and its salts, miconazole and its salts, itraconazole, terconazole, econazole, ketoconazole, saperconazole, fluconazole, clotrimazole, butoconazole, oxiconazole, sulfaconazole, sulconazole, terbinafine, undecylenic acid and its salts, benzoyl peroxide, 3- hydroxy benzoic acid, 4-hydroxy benzoic acid, phytic acid, N-acetyl-L-cysteine ​​acid, lipoic acid, azelaic acid and its salts, arachidonic acid, resorcinol, octoxyglycerin, octanoylglycine, caprylyl glycol, 10-hydroxy-2-decanoic acid, farnesol, phytosphingosines and mixtures thereof.

[0119] The free-radical scavengers may be vitamin C and its derivatives including ascorbyl glucoside, phenols and polyphenols, in particular tannins, ellagic acid and tannic acid; epigallocatechin and natural extracts containing them, in particular green tea extracts; anthocyanins; phenol acids, stilbenes; active ingredients for trapping mono- or polycyclic aromatic compounds, tannins such as ellagic acid and indole derivatives and / or active ingredients for trapping heavy metals such as EDTA, free-radical scavengers such as vitamin E and its derivatives such as tocopheryl acetate; bioflavonoids; coenzyme Q10 or ubiquinone.

[0120] As soothing agents included in the composition of the invention, it is possible to use 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 beta methasone, anti-inflammatory active ingredients, and in particular those described in application FR2847267, in particular the extract of Pueraria lobata root marketed under the name Inhipase™ by the applicant, extracts of Theobroma cacao.

[0121] Active ingredients acting on microcirculation, vasoprotectors or vasodilators, can be chosen from flavonoids, ruscogenins, nicotinates, essential oils.

[0122] The slimming active ingredients may be chosen in particular from lipoprotein lipase inhibitory agents such as those described in patent US2003086949 (Coletica) and in particular an extract of Peruvian liana (Uncaria tomentosa), draining active ingredients, in particular hesperitin laurate (Flavagrum™), or quercitin caprylate (Flavenger™); phosphodiesterase enzyme inhibitory agents, adenylate cyclase activating agents, cAMP and / or active ingredients capable of trapping spermine and / or spermidine. Examples include Coleus Forskohlii root extract, Cecropia obtusa extract, Uva lactuca extract, caffeine, forskolin, theophylline, theobromine and / or their derivatives, a hydrolyzed kappa carrageenan product called Slimexcess™ marketed by the applicant and / or their mixtures.

[0123] The present invention also relates to a cosmetic care method comprising the topical application to at least one area of ​​healthy skin and / or healthy mucous membrane of a strain of Lactobacillus crispatus according to the invention, or of a cosmetic composition comprising it, to reduce the pigmentation of the skin and / or mucous membranes and / or prevent its increase, in particular to preserve and / or improve the complexion of healthy skin and / or healthy mucous membranes, preferably to preserve and / or improve the radiance and / or luminosity and / or homogeneity of the complexion of healthy skin and / or healthy mucous membranes, and / or to prevent the appearance and / or reduce the presence of pigment spots on healthy skin and / or healthy mucous membranes and / or detoxify the skin and / or mucous membranes.

[0124] Advantageously, the strain of Lactobacillus crispatus according to the invention, preferably in the form of a cosmetic composition according to the invention, is used in regular topical application and preferably at least once a day, advantageously twice a day, for at least 10 days, preferably for 20 days, and even more preferably for at least 28 days.

[0125] Advantageously, the topical application of a strain of Lactobacillus crispatus according to the invention or of a cosmetic composition comprising it, is carried out on healthy skin and / or healthy mucous membranes of all or part of the body and / or the face and / or the scalp, preferably the legs, thighs, arms, stomach, décolleté, neck, armpits, lips, still preferably all or part of the face, and preferably the cheeks, forehead, chin, lips, and the eye contour.

[0126] In an advantageous embodiment of the cosmetic process according to the invention, the cosmetic composition comprises the strain of Lactobacillus crispatus according to the invention at a concentration of between 1x10' 4 % and 10% (w / w) by weight, preferably between 1x10' 4 % and 5% (w / w) by weight, still advantageously between 1x10' 3 % and 0.5% (w / w) by weight, relative to the total weight of the composition, said composition further comprising a cosmetically acceptable excipient.

[0127] The cosmetic care method according to the invention aims to reduce the pigmentation of the skin and / or mucous membranes and / or to prevent its increase, in particular to preserve and / or improve the complexion of healthy skin and / or healthy mucous membranes, preferably to preserve and / or improve the radiance and / or brightness and / or homogeneity of the complexion of healthy skin and / or healthy mucous membranes, and / or to prevent the appearance and / or reduce the presence of pigment spots on healthy skin and / or healthy mucous membranes, advantageously comprises the following steps: - The identification on the individual of an area of ​​healthy skin and / or healthy mucous membrane, for which it is desired to reduce the pigmentation of the skin and / or mucous membranes and / or prevent its increase, in particular to preserve and / or improve the complexion of healthy skin and / or healthy mucous membranes,preferentially to preserve and / or improve the radiance and / or luminosity and / or uniformity of the complexion of healthy skin and / or healthy mucous membranes, and / or to prevent the appearance and / or reduce the presence of pigment spots on healthy skin and / or healthy mucous membranes, and,

[0128] - The topical application to this area of ​​healthy skin and / or healthy mucous membrane, of a cosmetic composition comprising the strain of Lactobacillus crispatus according to the invention, in an amount effective for reducing the pigmentation of the skin and / or mucous membranes and / or preventing its increase, in particular for preserving and / or improving the complexion of healthy skin and / or healthy mucous membranes, preferably for preserving and / or improving the radiance and / or luminosity and / or homogeneity of the complexion of healthy skin and / or healthy mucous membranes, and / or for preventing the appearance and / or reducing the presence of pigment spots on healthy skin and / or healthy mucous membranes, namely in a content of strain of Lactobacillus crispatus advantageously between 1x10' 4 % and 10% (w / w) by weight, preferably between 1x10' 4 % and 5% (w / w) by weight, still advantageously between 1x10' 3 % and 0.5% (w / w) by weight, relative to the total weight of the composition.

[0129] For the purposes of the present invention, the term "topically and / or orally acceptable" means an ingredient suitable for application respectively by topical and / or oral route, non-toxic, non-irritating to the skin including the scalp and not inducing an allergic response, and which is not chemically unstable.

[0130] The invention also relates to the strain of Lactobacillus crispatus according to the invention, alone or in a pharmaceutical composition, preferably dermatological, comprising it, for its use, advantageously by topical route for the treatment and / or prevention of pathologies of the skin and / or mucous membranes involving hyperpigmentation and / or an increase in pigmentation, in particular a reduction in the radiance and / or brightness and / or homogeneity of the complexion of the pathological skin and / or pathological mucous membranes, and / or an increase in the appearance of pigment spots of the pathological skin and / or pathological mucous membranes, and / or an increase in the activity of melanogenesis, in particular by increasing melanin and / or tyrosinase activity, and / or an increase in oxidative stress of the pathological skin and / or pathological mucous membranes.

[0131] In particular, the subject of the invention is the strain of Lactobacillus crispatus according to the invention or a pharmaceutical composition, preferably dermatological, comprising it, for its use, advantageously by topical route, for the treatment and / or prevention of pathologies of the skin and / or mucous membranes associated with an increase in the pigmentation of the skin and / or mucous membranes, in particular for depigmenting and / or preventing hyperpigmentation of pathological skin and / or pathological mucous membranes, and / or for the treatment and / or prevention of pathologies of the skin and / or mucous membranes associated with oxidative stress.

[0132] The invention also relates to a method for the treatment and / or prevention of pathologies of the skin and / or mucous membranes involving hyperpigmentation and / or an increase in pigmentation, in particular a reduction in the radiance and / or brightness and / or homogeneity of the complexion of the pathological skin and / or pathological mucous membranes, and / or an increase in the appearance of pigment spots of the pathological skin and / or pathological mucous membranes, and / or an increase in the activity of melanogenesis, in particular by means of an increase in melanin and / or tyrosinase activity, and / or an increase in oxidative stress of the pathological skin and / or pathological mucous membranes, said method comprising the administration, advantageously by topical route, of the strain of Lactobacillus crispatus according to the invention, alone or in a pharmaceutical composition, preferably a dermatological composition comprising it,in a pharmaceutically effective amount in a subject in need thereof.,

[0133] In particular, the subject of the invention is a method for the treatment and / or prevention of pathologies of the skin and / or mucous membranes associated with an increase in pigmentation of the skin and / or mucous membranes, in particular for depigmenting and / or preventing hyperpigmentation of pathological skin and / or pathological mucous membranes, and / or for the treatment and / or prevention of pathologies of the skin and / or mucous membranes associated with oxidative stress, said method comprising the administration, advantageously by topical route, of the strain of Lactobacillus crispatus according to the invention or a pharmaceutical composition, preferably dermatological, comprising it, in a pharmaceutically effective amount in a subject in need thereof.

[0134] The invention also relates to the use of the strain of Lactobacillus crispatus according to the invention, alone or in a pharmaceutical composition, preferably dermatological, comprising it, for the preparation of a medicament, advantageously for topical administration, for the treatment and / or prevention of pathologies of the skin and / or mucous membranes involving hyperpigmentation and / or an increase in pigmentation, in particular a reduction in the radiance and / or brightness and / or homogeneity of the complexion of pathological skin and / or pathological mucous membranes, and / or an increase in the appearance of pigment spots of pathological skin and / or pathological mucous membranes, and / or an increase in the activity of melanogenesis, in particular through an increase in melanin and / or tyrosinase activity, and / or an increase in oxidative stress of pathological skin and / or pathological mucous membranes,

[0135] In particular, the subject of the invention is the use of the strain of Lactobacillus crispatus according to the invention or a pharmaceutical composition, preferably dermatological, comprising it, for the preparation of a medicament, advantageously for topical administration, for the treatment and / or prevention of pathologies of the skin and / or mucous membranes associated with an increase in pigmentation of the skin and / or mucous membranes, in particular for depigmenting and / or preventing hyperpigmentation of pathological skin and / or pathological mucous membranes, and / or for the treatment and / or prevention of pathologies of the skin and / or mucous membranes associated with oxidative stress.

[0136] For the purposes of the present invention, from a dermatological point of view, the term "treatment and / or prevention of pathologies of the skin and / or mucous membranes to depigment and / or prevent hyperpigmentation" is understood to mean, in particular, "treatment and / or prevention of pathologies of the skin and / or mucous membranes associated with an increase in the pigmentation of the skin and / or mucous membranes, in particular to depigment and / or prevent hyperpigmentation", a pigmentary disorder under the effect of pathologies of the skin and / or mucous membranes, such as, for example, Addison's disease, liver failure, purpura, melanoma, chloasma, dermatosis papulosa nigra, which is referred to as pathological hyperpigmentation, or else acne, eczema, atopic dermatitis, rosacea, couperose, redness, preferably chosen from Addison's disease, liver failure, purpura, melanoma, chloasma, dermatosis papulosa nigra, acne, atopic dermatitis,rosacea, redness.,

[0137] For the purposes of the present invention, from a dermatological point of view, the term "treatment and / or prevention of pathologies of the skin and / or mucous membranes associated with oxidative stress" means the treatment and / or prevention of numerous skin diseases linked to excessive oxidative stress, including psoriasis, certain eczemas, acne, vitiligo, or even certain skin cancers, preferably chosen from vitiligo.

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

[0139] Advantageously, it is present in the pharmaceutical composition, preferably dermatological, at a concentration of 1x10' 4 % to 10% (w / w) by weight, preferably between 1x10' 4 % and 5% (w / w) by weight, still advantageously between 1x10' 3 % and 0.5% (w / w) by weight relative to the total weight of the composition, said composition further comprising at least one pharmaceutically, preferably dermatologically acceptable excipient.

[0140] The invention will be better understood by reading the description of the figures and examples which follow.

[0141] Examples referring to the description of the invention are presented below. These examples are given for illustration purposes and should not in any way limit the scope of the invention. Each of the examples has a general scope.

[0142] The examples are an integral part of the present invention and any feature appearing new over any prior art from the description taken as a whole, including the examples, is an integral part of the invention.

[0143] On the other hand, in the examples, all percentages are given by weight unless otherwise indicated, temperature is expressed in degrees Celsius unless otherwise indicated, and pressure is atmospheric pressure unless otherwise indicated.

[0144] Examples

[0145] Example 1. Preparation of different forms of Lactobacillus crispatus Example 1.a: Production of viable whole form of Lactobacillus crispatus.

[0146] The isolated Lactobacillus crispatus (CNCM I-5579) bacteria were inoculated into an MRS culture medium (Man, Rogosa, Sharpe agar), incubated at 37°C anaerobically. The pH was maintained at pH = 6, and the medium was incubated for 24 hours. At the end of incubation, the medium was centrifuged, the cell concentrate was lyophilized in the presence of maltodextrin, for a final quantity of maltodextrin of 50% (w / w) by weight relative to the total weight of the ingredient.

[0147] Example 1 .b: production of the inactivated whole form of Lactobacillus crispatus

[0148] The isolated Lactobacillus crispatus (CNCM I-5579) bacteria were inoculated into an MRS culture medium and incubated anaerobically at 37°C. The pH was maintained at 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 it at 80°C for 30 minutes. The cell concentrate was then re-suspended in glycerin.

[0149] Example 1 .c: Production of the lysate of the bacterium Lactobacillus crispatus

[0150] The isolated Lactobacillus crispatus (CNCM I-5579) bacteria were inoculated into an MRS culture medium and incubated anaerobically at 37°C. The pH was maintained at pH 6, and the medium was incubated for 24 hours. At the end of the incubation, the mixture was passed through a high-pressure homogenizer. The lysate was then re-suspended in glycerin.

[0151] Example 1 .d: Production of the secretome of the bacterium Lactobacillus crispatus The isolated Lactobacillus crispatus (CNCM I-5579) bacteria were inoculated into an MRS-type culture medium and incubated anaerobically at 37°C. The pH was maintained at pH 6, and the medium was incubated for 24 hours. At the end of the incubation, the medium was filtered to recover the bacterial secretome. The secretome was then diluted in glycerin.

[0152] Example 1.e: Production of the secretome of the bacterium Lactobacillus crispatus in powder form

[0153] The isolated Lactobacillus crispatus (CNCM I-5579) bacteria were inoculated into an MRS culture medium, incubated at 37°C anaerobically. The pH was maintained at pH = 6, and the medium was incubated for 24 hours. At the end of incubation, the medium was filtered to recover the bacterial secretome. The secretome was then atomized with maltodextrin, for a final maltodextrin quantity of 90% (w / w) by weight relative to the total weight of the ingredient.

[0154] Example 2: Inhibition of melanin synthesis by melanocytes in the presence of the Lactobacillus crispatus strain according to the invention.

[0155] Melanocytes from a murine B-16 line were seeded in 96-well plates at a rate of 8000 cells / cm 2 , then cultured in MEM (Minimum Essential Medium) medium supplemented with 10% FCS (Fetal Calf Serum) at 37°C, 5% CO2 and 95% Relative Humidity for 3 days.

[0156] The medium was then removed and replaced with 200 μL of the previously described medium with the addition of 1 μM of NDP-a-MSH (analogue of melanotropic hormone) used as a melanin synthesis stimulator and in the presence of the Lactobacillus crispatus strain according to Example 1.a at a concentration of 0.1% (w / w), for 5 days.

[0157] The same culture medium without addition of the strain according to the invention but with the addition of 1 μM NDP-a-MSH or with the addition of 0.01% (w / w) kojic acid were used as manipulation controls, respectively as melanin stimulator control and melanin inhibitor control.

[0158] The same culture medium without addition of the strain according to the invention but with ethanol was used as a control (Control).

[0159] After 5 days of incubation, the cells were lysed with sodium hydroxide (NaOH) or potassium hydroxide (KOH) solution. Absorbance was measured at 475 nm. The amount of melanin was deduced from a standard range curve consisting of different concentrations of commercial synthetic melanin.

[0160] The absorbance results were normalized to the absorbance obtained with the same cell medium in the absence of the Lactobacillus crispatus strain according to the invention (Control). The results presented correspond to the average of 6 replicates (n=6).

[0161] Results

[0162] The Lactobacillus crispatus strain according to Example 1.a produced by the invention showed a significant 52% inhibition of melanin synthesis by murine B-16 line melanocytes cultured in vitro. It can therefore be used to reduce pigmentation of the skin and / or mucous membranes and / or prevent its increase.

[0163] Example 3: Inhibition of tyrosinase activity in the presence of the Lactobacillus crispatus strain according to the invention.

[0164] Tyrosinase is a key enzyme in the process of melanin synthesis, which is responsible for skin tone. The tyrosinase used is extracted from normal human melanocytes.

[0165] Lactobacillus crispatus strain according to Example 1.e at a concentration of 0.05% (w / w) or a reference compound (kojic acid) at a concentration ranging from 0.25 mM to 8 mM, were pre-incubated with the enzyme for 10 minutes on ice. Then, the tyrosinase substrate, L-Tyrosine at a final concentration of 1 mM was added to the medium and the plates were incubated at 37°C for 24 hours.

[0166] The same enzymatic extract without addition of the strain according to the invention but with ethanol was used as a control (Control). The enzymatic activity was evaluated by measuring the production of melanin by optical density (OD) at 540 nm using a microplate reader.

[0167] Optical density measurements were also performed without addition of substrate (n=1) in order to detect possible interference from the Lactobacillus crispatus strain according to Example 1.e or from the reference compound for each concentration tested.

[0168] The percentage inhibition of tyrosinase activity was calculated according to the following formula:

[0169] % Inhibition: 100-[OD Value / Mean OD of Control x 100]

[0170] All experimental conditions were performed in n=3. Intergroup comparisons were performed by unpaired Student's t-test.

[0171] The Lactobacillus crispatus strain according to Example 1.e of the invention showed a significant inhibition of the activity of tyrosinase extracted from human melanocytes by 99%. By this action of inhibiting the activity of tyrosinase, the Lactobacillus crispatus strain can therefore be used to reduce the pigmentation of the skin and / or mucous membranes and / or prevent its increase.

[0172] Example 4: Inhibition of melanin synthesis in a co-culture of melanocytes and keratinocytes in the presence of the Lactobacillus crispatus strain according to the invention.

[0173] Normal human keratinocytes and newborn human melanocytes were seeded at 20,000 cells / cm, respectively. 2 and 10,000 cells / cm 2, then cultured for 7 days at 37°C under 5% CO2 and 95% relative humidity in a mixture of 50% / 50% (v / v) culture medium for melanocytes and culture medium for keratinocytes, supplemented with growth factors.

[0174] After one week of culture, the culture medium was replaced with a mixture of DMEM (Dulbecco's Modified Eagle's Minimum Essential Medium) culture medium without antibiotics and melanocyte medium without growth factor, and supplemented with 40pM oleic acid.

[0175] This culture medium without addition of the strain according to the invention was used as an untreated control (Control).

[0176] To this same medium, the secretome of the bacterium Lactobacillus crispatus obtained according to example 1.e was added at a concentration of 0.1% (w / w) relative to the total weight of the culture medium and the secretome.

[0177] The same culture medium without the strain, but in the presence of Rucinol at 0.002% (w / w) was used as a positive control.

[0178] The cultures were continued for 72 hours at 37°C under 5% CO2 and 95% relative humidity, then the culture medium was renewed. After 48 hours of additional culture, the medium was then removed, then the cells were rinsed with PBS (phosphate buffer). Then, a 1 N sodium hydroxide solution containing 10% Dimethylsulfoxide (DMSO) was added to each well of the plate. 200 pL of each well was transferred to a transparent plate. The optical density reflecting the amount of melanin was read at 475 nm.

[0179] The results were the mean of the tests performed ± the standard deviation, expressed as a percentage, relative to the untreated control normalized to 100%. Statistical analysis of the results was done relative to the untreated control using the One-Way AN OVA test.

[0180] Results

[0181] The Lactobacillus crispatus strain according to Example 1.e of the invention showed a significant decrease of 32% in melanin synthesis by melanocytes cocultured in vitro with human skin keratinocytes. Thus, the Lactobacillus crispatus strain, and in particular its secretome, can therefore be used to reduce pigmentation of the skin and / or mucous membranes and / or prevent its increase. Example 5: Reduction of oxidative stress in the presence of the Lactobacillus crispatus strain according to the invention.

[0182] Example 5.a: Reduction in the formation of DPPH° free radicals in tubo in the presence of the Lactobacillus crispatus strain according to the invention.

[0183] The dosage of the antiradical activity of the strain Lactobacillus crispatus was evaluated by a test using DPPH° (2,2-diphenyl-1-picrylhydrazyl).

[0184] The Lactobacillus crispatus strain according to Example 1.a was mixed in PBS (Phosphate Buffered Saline) at a concentration of 0.05% (w / w), and according to Example 1.e at a concentration of 0.7% (w / w).

[0185] A negative control was performed using the strain dissolution solvent, PBS, and a positive control was performed with 1 mM vitamin C.

[0186] Each of the solutions containing the invention, the negative control or the positive control was mixed 50% / 50% (v / v) in a DPPH° solution, in wells of 96-well plates.

[0187] Each of the solutions containing the invention, the negative control or the positive control was also mixed 50% / 50% (v / v) in an ethanol solution which is the solvent of DPPH°, in order to produce a blank, in this same plate.

[0188] The mixture was incubated for 30 minutes with stirring. At the end of incubation, the optical density (OD) was measured at a wavelength of 530 nm to estimate the quantity of free radicals formed under each of the conditions tested.

[0189] The results of the measurement of DPPH° radicals to obtain the anti-radical activity of the invention were the average of 6 tests (n=6) and were expressed as a mean percentage + / - one standard deviation. The product of the invention was compared to its negative control (PBS alone in the absence of the Lactobacillus crispatus strain). The positive control vitamin C was compared to its negative control, water. The statistical analysis was conducted by a One Way ANOVA analysis of variance for the invention and by a student test for vitamin C.

[0190] The Lactobacillus crispatus strain according to Example 1.a of the invention showed a significant in tubo anti-radical activity of 34%. The Lactobacillus crispatus strain according to Example 1.e of the invention showed an in tubo anti-radical activity of 31%. By this action of reducing the production of free radicals, the Lactobacillus crispatus strain can therefore be used to detoxify the skin and / or mucous membranes, to reduce the pigmentation of the skin and / or mucous membranes and / or prevent its increase.

[0191] Example 5. b: Reduction in the release of UV-induced cellular free radicals within fibroblasts in the presence of the Lactobacillus crispatus strain according to the invention.

[0192] So-called "normal" human fibroblasts, i.e. not presenting any pathology, from a healthy donor were seeded in a 24-well plate at a rate of 30,000 cells / cm 2, then cultured in DMEM (Dulbecco's Modified Eagle's Minimum Essential Medium) / Ham's F-12 medium, supplemented with 10% FCS and 0.5% antibiotic, for 96h at 37°C, under 5% CO2 and 95% relative humidity. Then, 0.5mL of the cell suspension was incubated for 72h in the presence of the Lactobacillus crispatus strain according to Example 1.a at a concentration of 0.0125% (w / w), or an antioxidant control (Vitamin E at 0.0003% (v / v)) in EMEM (Eagle's Minimum Essential Medium) medium supplemented with 1% FCS and 0.5% antibiotic.

[0193] This culture medium without addition of the strain according to the invention was used as an untreated control (UVA control alone - Oxidative stress control).

[0194] A DCFH-DA (2',7'-dichlorodihydrofluorescein diacetate) probe solubilized at 10 pM in PBS (phosphate buffer) was incubated with the cells. After the first hour of incubation at 37°C under 5% CO2 and 95% relative humidity, the cells were rinsed with PBS, and irradiated with UVA at 20 J / cm 2 for 3 hours. A non-irradiated control was also performed. After irradiation, the irradiated medium was removed, and the cells were rinsed with PBS. The fluorescence reading on the cell lawn related to the formation of DCF (2', 7-dichlorofluorescein) from the probe (DCFH-DA) was read at 485 nm excitation and 538 nm emission.

[0195] Results were the average of 12 replicates (n=12) and were expressed as Mean + / - one standard deviation. Results were compared to the UVA-only control normalized to 100% and statistically compared using the One Way ANOVA test.

[0196] The strain of Lactobacillus crispatus according to Example 1.a of the invention showed a significant decrease at the cellular level of 56% in the release of intracellular free radicals induced by UVA oxidative stress. By this action of reducing the release of intracellular free radicals induced by UVA, the strain of Lactobacillus crispatus can therefore be used to detoxify the skin and / or mucous membranes and / or preserve and / or improve the radiance and / or brightness and / or homogeneity of the skin and / or mucous membranes, preferentially reduce the presence and / or prevent the appearance of pigment spots on the skin and / or mucous membranes, in particular when these undergo oxidative stress, preferentially by UVA.

[0197] Example 5.c: Reduction of UV-induced membrane lipid peroxidation within fibroblasts in the presence of the Lactobacillus crispatus strain according to the invention.

[0198] So-called "normal" human fibroblasts, i.e. not presenting any pathology, from a healthy donor were seeded in 24-well plates at a rate of 30,000 cells / cm 2 , then cultured in DMEM / Ham's F-12 medium, supplemented with 10% FCS and 0.5% antibiotic, for 96h at 37°C, under 5% CO2 and 95% relative humidity. Then, 0.5mL of the cell suspension was incubated for 72h in the presence of the Lactobacillus crispatus strain according to example 1.a at a concentration of 0.00625% (w / w), or an antioxidant control (Vitamin E at 0.0003% (v / v)) in EMEM medium supplemented with 1% FCS and 0.5% antibiotic.

[0199] This culture medium without addition of the strain according to the invention was used as an untreated control (after irradiation, constitutes the UVA control alone - Oxidative stress control).

[0200] After the first hour of incubation at 37°C under 5% CO2 and 95% relative humidity, the cells were rinsed with PBS, and irradiated with UVA at 20J / cm 2 for 3 hours. A non-irradiated control was also performed. After irradiation, the supernatants were collected and the cells were rinsed with PBS.

[0201] A quantity of 0.5 mL of the supernatants was transferred into a Pyrex glass tube to which a solution of 40% trichloroacetic acid and 2% thiobarbituric acid was added. The resulting mixture was heated for 30 minutes at 100°C. Then, the formation of MDA (MalonDiAldehde), which is an oxidation product released following UVA-induced lipid peroxidation, was measured by fluorescence at 532nm excitation and 560nm emission.

[0202] The results were the average of the tests performed and were expressed as Mean + / - one standard deviation. The results were compared with the UVA-only control normalized to 100% and statistically compared using the One Way ANOVA test.

[0203] The Lactobacillus crispatus strain according to Example 1.a of the invention showed a significant decrease at the cellular level of the formation of MDA induced by UVA of 32%. By this action of reducing lipid peroxidation in the presence of UV stress, the Lactobacillus crispatus strain can therefore be used to detoxify the skin and / or mucous membranes, in particular when these undergo oxidative stress, in particular UVA stress.

[0204] Example 6: Stimulation of cellular antioxidant defenses after UV irradiation within fibroblasts in the presence of the Lactobacillus crispatus strain according to the invention.

[0205] So-called "normal" human fibroblasts, i.e. not presenting any pathology, from a healthy donor were seeded in a 24-well plate at a rate of 30,000 cells / cm 2 , then cultured in DMEM / Ham's F-12 medium, supplemented with 10% FCS and 0.5% antibiotic, for 96 hours at 37°C, under 5% CO2 and 95% relative humidity.

[0206] This culture medium without addition of the strain according to the invention was used as an untreated control (after irradiation, constitutes the UVA control alone - Oxidative stress control).

[0207] Then, 0.5 mL of the cell suspension was incubated for 72 hours with the presence of the Lactobacillus crispatus strain according to example 1.a at a concentration of 0.0125% (w / w), or an antioxidant control (Vitamin E at 0.0003% (v / v)) in an EMEM medium supplemented with 1% FCS and 0.5% antibiotic.

[0208] After the first hour of incubation at 37°C under 5% CO2 and 95% relative humidity, the cells were rinsed with PBS, and irradiated with UVA at 20J / cm 2 for 3 hours. A non-irradiated control was also performed. After irradiation, the irradiated medium was removed, and the cells were rinsed with PBS.

[0209] 1 N sodium hydroxide (NaOH) was added to each well, followed by a solution of o-phthaldialdehyde (OPT) diluted to 1 Z15 eme in GSH buffer (reduced L-glutathione). The whole was incubated for 15 minutes at room temperature. Then the amount of glutathione was measured by fluorescence at 355 nm excitation and 430 nm emission. The results were the average of the tests carried out and were expressed as Mean + / - one standard deviation. The results were compared with the UVA-only control normalized to 100% and statistically compared using the One Way ANOVA analysis of variance test.

[0210] The Lactobacillus crispatus strain according to Example 1.a of the invention showed a significant increase in cellular antioxidant defenses by an increase in glutathione of 54% after UVA irradiation.

[0211] By this action of increasing cellular antioxidant defenses in the presence of oxidative stress by UVAs, the strain of Lactobacillus crispatus can therefore be used to detoxify the skin and / or mucous membranes, particularly when they are subject to oxidative stress, in particular stress by UVAs.

[0212] Example 7: In vivo measurement of the reduction in the quantity of melanin in the skin in the presence of the strain of Lactobacillus crispatus according to the invention.

[0213] A population of 30 Chinese women of phototype II to IV, aged 40 to 50 years, with pigment spots on the face, applied to half of the face a cream in the form of an emulsion comprising a final concentration by weight of the Lactobacillus crispatus strain according to example 1.e of 1% (w / w) relative to the total weight of the cream (Formulation example 8. b), or without said extract replaced by water (placebo), at a rate of 2 applications per day for 2 months.

[0214] The assessment of the melanin content of the skin was carried out using a device called Mexameter which uses the principle of absorption / reflection. From the measurements of the Mexameter, a melanin index is deduced. A decrease in this index corresponds to a decrease in melanin.

[0215] The effectiveness of the composition containing the strain of Lactobacillus crispatus according to Example 1.e was compared with that of the so-called placebo emulsion.

[0216] The melanin index was measured at the beginning of the study (DO) after one month of application (D28) and finally after 2 months of application (D56).

[0217] The efficacy of the formulation containing the Lactobacillus crispatus strain according to Example 1.e and the placebo formulation were expressed as mean values ​​and were compared with baseline values ​​(OD) and compared between the formulation containing the Lactobacillus crispatus strain with that containing the placebo, using the Student test or the Wilcoxon test.

[0218] The formulation of the Lactobacillus crispatus strain according to Example 1.ea significantly decreased the amount of skin melanin measured via the melanin index using a Mexameter.

[0219] These results show the beneficial effect of the Lactobacillus crispatus strain formulated to preserve and / or improve the radiance and / or luminosity and / or homogeneity of healthy skin and / or healthy mucous membranes.

[0220] Example 8: Example of a cosmetic composition comprising the strain of Lactobacillus crispatus according to the invention Example 8. a: Example of a cosmetic composition comprising the strain of Lactobacillus crispatus according to example 1.a

[0221] The strain of Lactobacillus crispatus obtained according to Example 1.a was added just before application to the skin in a formulation as defined below according to Table 8, at a final concentration of 0.05% (w / w) relative to the total weight of the formulation.

[0222] Example 8. b: Example of a cosmetic composition comprising the strain of

[0223] Lactobacillus crispatus according to example 1.e

[0224] The proportions are expressed in % and the names in capital letters correspond to the INCI names of the ingredients.

[0225] The procedure is carried out according to methods known to those skilled in the art to mix together the different phases A, B, C at room temperature, adding B to A while stirring, then C to AB. The pH is adjusted to 4.9 with D. Then phase E is added. Finally, homogenization is carried out by Ultra Turrax to prepare a composition according to the present invention. (original in electronic form)

[0226] (This sheet is not part of the international application nor does it count as a sheet thereof)

[0227] RESERVED FOR THE RECEIVING OFFICE

[0228] FOR INTERNATIONAL OFFICE USE ONLY

Claims

Demands 1. Cosmetic and / or nutraceutical use of a strain of Lactobacillus crispatus, preferably the Lactobacillus crispatus strain being the species registered under the designation CNCM I-5579, to reduce pigmentation of the skin and / or mucous membranes and / or prevent its increase.

2. Use according to claim 1, wherein the strain decreases and / or inhibits melanogenesis, in particular by acting on the decrease and / or inhibition of melanin, preferentially on the decrease and / or inhibition of the synthesis and / or quantity of melanin, and / or by decreasing and / or inhibiting the activity of tyrosinase, and / or by detoxifying healthy skin and / or healthy mucous membranes.

3. Use according to any of the preceding claims to preserve and / or improve the complexion of healthy skin and / or healthy mucous membranes.

4. Use according to any one of the preceding claims, to preserve and / or improve the radiance and / or luminosity and / or evenness of the complexion, of healthy skin and / or healthy mucous membranes.

5. Use according to any one of the preceding claims, to prevent the appearance and / or reduce the presence of pigment spots on healthy skin and / or healthy mucous membranes, preferably brown spots and / or age spots.

6. Use according to any one of the preceding claims, to detoxify healthy skin and / or healthy mucous membranes, in particular by an antioxidant action and / or on lipid peroxidation and / or by an increase and / or stimulation of glutathione synthesis induced by oxidative stress.

7. Use according to any one of the preceding claims, wherein the strain is used in whole form, in particular viable and / or inactivated, in particular dead, and / or in lysate form, and / or in the form of one or more of its fractions, and / or in the form of one or more of its metabolites, preferably its secretome.

8. Use according to any one of the preceding claims, wherein the Lactobacillus crispatus strain is that filed under number CNCM I-5579.

9. Use according to any one of the preceding claims, wherein the Lactobacillus crispatus strain is used in whole viable form and / or in the form of one or more of its metabolites, preferably its secretome.

10. Use according to any one of the preceding claims, wherein the Lactobacillus crispatus strain is associated with its culture medium.

11. Use according to any one of the preceding claims, wherein the Lactobacillus crispatus strain is applied topically to healthy skin and / or healthy mucous membranes.

12. Use according to claim 11 wherein the skin is phototype IV to VI.

13. Use according to any one of the preceding claims, such that the Lactobacillus crispatus strain is present in a cosmetic composition at a concentration of 1x10⁻¹⁰ 4 % to 10% by weight, preferably between 1x10' 4 % and 5% by weight, still advantageously between 1x10' 3 % and 0.5% by weight, relative to the total weight of the composition, said composition further comprising at least one cosmetically acceptable excipient.

14. A cosmetic treatment method characterized in that it comprises the topical application to at least one area of ​​healthy skin and / or healthy mucous membrane of a strain of Lactobacillus crispatus as defined according to any one of claims 7 to 10, or of a cosmetic composition according to claim 13, to reduce pigmentation of the skin and / or mucous membranes and / or prevent its increase, in particular to preserve and / or improve the complexion of healthy skin and / or healthy mucous membranes, preferably to preserve and / or improve the radiance and / or luminosity and / or homogeneity of the complexion of healthy skin and / or healthy mucous membranes, and / or to prevent the appearance and / or reduce the presence of pigment spots on healthy skin and / or healthy mucous membranes and / or detoxify the skin and / or mucous membranes.

15. A cosmetic treatment method according to claim 14, wherein the topical application of a strain of Lactobacillus crispatus as defined according to any one of claims 7 to 10, or of a cosmetic composition according to claim 13, is carried out on healthy skin and / or healthy mucous membranes of all or part of the body and / or face and / or scalp, preferably the legs, thighs, arms, abdomen, décolleté, neck, armpits, lips, again preferably all or part of the face, and preferably the cheeks, forehead, chin, lips, eye contour.

16. A cosmetic treatment method according to any one of claims 14 to 15, wherein the cosmetic composition comprises the Lactobacillus crispatus strain as defined in any one of claims 7 to 10 at a concentration of 1x10⁻¹ 410% to 10% by weight, preferably between 1x10'; 4% to 5% by weight, again advantageously between 1x10' 3 % and 0.5% by weight, relative to the total weight of the composition, said composition further comprising at least one cosmetically acceptable excipient.

17. A strain of Lactobacillus crispatus, or a pharmaceutical composition, preferably dermatological, comprising it, for use, advantageously by topical route, for the treatment and / or prevention of skin and / or mucous membrane pathologies associated with increased pigmentation of the skin and / or mucous membranes, in particular for depigmenting and / or preventing hyperpigmentation of pathological skin and / or pathological mucous membranes, and / or for the treatment and / or prevention of skin and / or mucous membrane pathologies associated with oxidative stress.

18. Strain according to claim 17 wherein the strain is used in whole form, in particular viable and / or inactivated, in particular dead, and / or in lysate form, and / or in the form of one or more of its fractions, and / or in the form of one or more of its metabolites, preferably its secretome, and / or wherein the strain is associated with its culture medium.

19. A strain of Lactobacillus crispatus for use according to any one of claims 17 to 18, characterized in that it is in the form of a pharmaceutical composition, preferably dermatological, comprising at least one dermatologically acceptable excipient. A strain of Lactobacillus crispatus for use according to any one of claims 17 to 19, characterized in that it is present in the pharmaceutical composition, preferably dermatological, at a concentration of 1 x 10⁻¹⁰ 4% to 10% by weight, preferably between 1x10' 4 % and 5% by weight, still advantageously between 1x10' 3 % and 0.5% by weight, relative to the total weight of the composition, said composition further comprising at least one pharmaceutically, preferably dermatologically, acceptable excipient. A strain of Lactobacillus crispatus for use according to any one of claims 17 to 20, characterized in that the strain is as defined according to any one of claims 7 to 10.

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