Cosmetic, nutraceutical or dermatological uses of Lactobacillus crispatus strains and / or compositions containing same

Abstract

The present invention relates to the cosmetic and nutraceutical use of Lactobacillus crispatus strains and / or compositions comprising same for reducing and / or preventing the increase of pigmentation of the skin and / or mucous membranes, in particular for maintaining and / or improving the complexion of the skin and / or mucous membranes, preferably for maintaining and / or improving the radiance and / or brilliance and / or evenness and / or evenness of the complexion of the skin and / or mucous membranes and / or for preventing the appearance and / or reducing the presence of pigmented spots on the skin and / or mucous membranes and / or for detoxifying the skin and / or mucous membranes. The present invention also relates to Lactobacillus crispatus strains or dermatological compositions comprising same for use in the treatment and / or prevention of diseases of the skin and / or mucous membranes associated with increased pigmentation of the skin and / or mucous membranes, in particular for use in depigmenting and / or preventing the pigmentation of diseased skin and / or diseased mucous membranes.

Description

[Technical field]

[0001] The present invention relates to the cosmetic, nutraceutical and / or pharmaceutical, in particular dermatological, use of Lactobacillus crispatus strains and / or compositions comprising same for reducing pigmentation of the skin and / or mucous membranes and / or preventing increased pigmentation of the skin and / or mucous membranes. [Background technology]

[0002] The structure and appearance of the skin change with age and / or under the influence of exogenous and / or endogenous factors. These changes are numerous and include changes in the pigmentation of the skin and / or mucous membranes. The skin becomes dull, less bright, less radiant, its radiance decreases, the complexion becomes less uniform and pigmented spots appear.

[0003] Natural or accelerated aging, especially under the influence of radiation, typically ultraviolet light or pollution, accelerates the appearance of these changes, such as pigmented spots. They are associated with the presence of melanin and lipofuscin, the excess and abnormal distribution of which in the skin causes the appearance of brown spots known as senile or pigmented brown spots, also called "age spots". They may appear as a marker of accelerated melanogenesis and are often considered unattractive. They make the skin uneven and dull.

[0004] Melanogenesis and oxidative stress are the subject of numerous research and innovation efforts in the field of cosmetics for the development and improvement of cosmetic active agents aimed at reducing and / or preventing the increase of pigmentation in healthy skin and / or healthy mucous membranes.

[0005] There is a need in the cosmetics and dermatology fields to develop alternative natural active ingredients capable of reducing the pigmentation of the skin and / or mucous membranes and / or preventing the increase of pigmentation of the skin and / or mucous membranes, in particular in order to maintain and / or improve the complexion, in particular its radiance and / or lightness and / or evenness, in particular of the skin and / or mucous membranes, in particular within populations that are more prone to pigmentation spots, in particular those with skin phototypes IV to VI as defined by the Fitzpatrick classification system.

[0006] There is a continuing need to identify agents that possess one or more of these properties, particularly those with proven efficacy and safety.

[0007] Particularly surprisingly, the Applicant has discovered that Lactobacillus crispatus, a probiotic beneficial for the skin and / or mucous membranes, has properties to reduce and / or prevent an increase in pigmentation of the skin and / or mucous membranes, in particular to maintain and / or improve the complexion of the skin and / or mucous membranes, preferentially to maintain and / or improve the radiance and / or lightness and / or evenness of the complexion of the skin and / or mucous membranes and / or to prevent the appearance and / or reduce the presence of pigmented spots on the skin and / or mucous membranes.

[0008] Applicants have also surprisingly discovered that this bacterium also has detoxifying properties for the skin and / or mucous membranes.

[0009] The Applicant's invention is all the more advantageous since the bacterium Lactobacillus crispatus, and in particular the one deposited under number CNCM I-5579, has the advantage that it is naturally present on young skin, unlike bacteria that have been used up until now in cosmetic and / or dermatological treatments.

[0010] The present invention is even more advantageous since the applicant has demonstrated that with age and / or under the influence of exogenous and / or endogenous factors, the bacterium Lactobacillus crispatus becomes less present and less abundant on the skin.The subject matter of the present invention therefore has the benefit of rebalancing and / or supplementing the microflora of the skin and / or mucous membranes, in particular in conditions where the bacteria of the skin microflora become reduced and rare over time and / or due to exposure to endogenous and / or exogenous factors.

[0011] Thus, the subject matter of the invention is particularly suitable for treating what is called mature skin in certain individuals over the age of 50.

[0012] The strain according to the invention also has the advantage of being an ingredient that is easy to formulate and can be easily produced on an industrial scale. It is an active ingredient that is externally acceptable for the skin and mucous membranes, which does not present any allergy risk.

[0013] This application has already described the use of Lactobacillus crispatus in cosmetics. For example, application WO 9822082 discloses the use of sphingomyelinase extractable from lactic acid bacteria for external application to increase the level of ceramides in the skin and mucous membranes for the preventive or therapeutic treatment of several pathological conditions.

[0014] Application WO2009031099 discloses the use of beneficial bacteria to prevent and / or treat the appearance of discomfort and / or skin symptoms associated with superficial skin treatments for aesthetic purposes or with invasive treatments.

[0015] Application WO 2019 / 111189 describes the use of bacteria, in particular containing Lactobacillus paracasei, for preventing UV-induced inflammatory damage. Summary of the Invention [Problem to be solved by the invention]

[0016] However, none of these applications discloses or suggests the cosmetic, nutraceutical and / or pharmaceutical, especially dermatological, use of Lactobacillus crispatus strains and / or compositions comprising same for reducing pigmentation of the skin and / or mucous membranes and / or preventing its increase, in particular for maintaining and / or improving the complexion of the skin and / or mucous membranes, preferentially for maintaining and / or improving the radiance and / or lightness and / or evenness of the complexion of the skin and / or mucous membranes and / or for preventing the appearance and / or reducing the presence of pigmented spots on the skin and / or mucous membranes.

[0017] Furthermore, none of these applications disclose or suggest the use of Lactobacillus crispatus strains and / or compositions containing same for detoxifying the skin and / or mucous membranes. [Means for solving the problem]

[0018] A first subject of the present invention is the cosmetic and / or nutraceutical use of a Lactobacillus crispatus strain, preferentially the strain of Lactobacillus crispatus deposited under number CNCM I-5579, for reducing and / or preventing its increase in pigmentation of the skin and / or mucous membranes, in particular for maintaining and / or improving the complexion of healthy skin and / or healthy mucous membranes, preferentially for maintaining and / or improving the radiance and / or lightness and / or evenness of the complexion of healthy skin and / or healthy mucous membranes, and / or for preventing the appearance and / or reducing the presence of pigmented spots, preferentially brown spots and / or age spots, on healthy skin and / or healthy mucous membranes and / or for detoxifying healthy skin and / or healthy mucous membranes, in particular skin of phototypes IV to VI as defined by the Fitzpatrick classification system. In the context of the present invention, the Lactobacillus crispatus strain according to the invention is used in particular as a cosmetic and / or nutraceutical active ingredient.

[0019] According to a particular embodiment of this use, the strain according to the invention reduces 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 amount of melanin, and / or by reducing and / or inhibiting tyrosinase activity and / or by detoxifying healthy skin and / or healthy mucous membranes.

[0020] According to one aspect of the invention, the strains according to the invention can be used to maintain and / or improve the complexion of healthy skin and / or healthy mucous membranes. The strains according to the invention can also be used to maintain and / or improve the radiance and / or lightness and / or evenness of the complexion of healthy skin and / or healthy mucous membranes. The strains according to the invention can also be used to prevent the appearance and / or reduce the presence of pigmented spots, preferentially brown spots and / or age spots, on healthy skin and / or healthy mucous membranes. The strains according to the invention can also be used to detoxify healthy skin and / or healthy mucous membranes, in particular by virtue of their antioxidant effect and / or their effect on lipid peroxidation and / or by increasing and / or stimulating the synthesis of glutathione induced by oxidative stress.

[0021] A subject of the present invention is also a cosmetic care process, characterized in that it comprises the external application to at least one area of ​​healthy skin and / or healthy mucous membranes of a Lactobacillus crispatus strain according to the invention, preferentially a strain of Lactobacillus crispatus CNCM I-5579, or a cosmetic composition comprising it, for reducing and / or preventing an increase in pigmentation of the skin and / or mucous membranes, in particular for maintaining and / or improving the complexion of healthy skin and / or healthy mucous membranes, preferably for maintaining and / or improving the radiance and / or lightness and / or evenness of the complexion of healthy skin and / or healthy mucous membranes, and / or for preventing the appearance and / or reducing the presence of pigmented spots on healthy skin and / or healthy mucous membranes, and / or for detoxifying the skin and / or mucous membranes. In the context of the present invention, the Lactobacillus crispatus strain according to the present invention is in particular used as a cosmetic and / or nutraceutical active ingredient.

[0022] A subject of the present invention is also a Lactobacillus crispatus strain according to the invention, preferentially the Lactobacillus crispatus strain CNCM I-5579, for the prevention and / or treatment of pathological conditions of the skin and / or mucosa associated with increased pigmentation of the skin and / or mucosa, in particular for depigmenting and / or preventing pigmentation of pathological skin and / or mucosa, in order to reduce and / or prevent its increase, in particular for maintaining and / or improving the complexion of diseased skin and / or pathological mucosa, preferentially for maintaining and / or improving the radiance and / or lightness and / or evenness of the complexion of diseased skin and / or pathological mucosa, and / or for preventing the appearance and / or reducing the presence of pigmented spots of pathological skin and / or pathological mucosa and / or for detoxifying pathological skin and / or pathological mucosa, or a pharmaceutical composition, in particular a dermatological composition, comprising it. In the context of the present invention, a Lactobacillus crispatus strain according to the invention is used in particular as active ingredient. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] For the purposes of the present invention, the term "cosmetic use" means non-medicinal use, i.e. not intended for therapeutic use and applied to "healthy" parts of the body, in particular to "healthy" areas of the skin and / or mucous membranes.

[0024] For the purposes of the present invention, the expression "nutraceutical use" refers to a use intended to be used for non-medicinal oral administration, i.e., not requiring or including therapeutic treatment.

[0025] The term "skin" means any area of ​​skin on any part of the body and / or face, including the scalp.

[0026] For the purposes of the present invention, the expression "skin of phototype IV to VI" is understood to mean a specific skin type classified according to the Fitzpatrick scale that allows to classify individuals according to the reaction of their skin and hair during exposure to UV rays, and / or classified itself by dermatologists as a function of the skin tone, which depends in particular on the level of melanocyte activity, the creation of these cells and their classification. Preferentially, these dark skins of phototype IV to VI originate from a characteristic ethnic origin, respectively from India, Asia and / or Africa. In particular, - Phototype IV (Mediterranean type) corresponds to light, matte skin and brown or black hair and brown or dark eyes. Tanning is rare, the skin tans well and is brownish-brown; - Phototype V (Latin type) corresponds to light, matte skin, black hair and dark eyes. Tanning is rare, the skin tans well and is brownish-brown; - Phototype VI (African type) corresponds to very dark skin, black hair and dark eyes. Tanning is very rare.

[0027] According to the invention, "mucous membrane" means the ocular, nasal, auricular, vaginal, urogenital and / or oral mucosa, in particular the oral, labial and / or gingival mucosa, preferentially the ocular and / or oral mucosa, more preferentially the labial and / or ocular mucosa. Advantageously, said term does not include the vaginal mucosa.

[0028] For the purposes of the present invention, the term "healthy skin" or "healthy mucosa" refers to an area of ​​skin or mucosa to which a strain according to the invention is applied, which is said by a dermatologist to be "non-pathological", i.e. free of infection, scarring, disease, inflammation or skin diseases such as candidiasis, impetigo, psoriasis, eczema, acne or dermatitis, or injuries or wounds.

[0029] For the purposes of the present invention, the term "Lactobacillus crispatus strain" is understood to mean the bacterium Lactobacillus crispatus in complete or partial form, in particular in the form of a lysate, in the form of a fraction or in the form of a metabolite. Advantageously, the Lactobacillus crispatus strain is not genetically modified.

[0030] The Lactobacillus crispatus strain according to the invention may be derived from any known species of Lactobacillus crispatus. Preferentially, the Lactobacillus crispatus strain is the species deposited under the Budapest Treaty at the Institut Pasteur (28 rue du Docteur Roux, F-75024 Paris cedex 15) on September 9, 2020, under designation CNCM I-5579.

[0031] The advantage of this particular Lactobacillus crispatus strain is that it is the first Lactobacillus crispatus species found naturally on the skin.

[0032] For the purposes of the present invention, the expression "reducing and / or preventing the increase in pigmentation of the skin and / or mucous membranes" is understood to mean depigmenting, whitening, lightening, preventing the increase in, preserving and / or maintaining and / or improving the complexion and / or shade of the skin and / or mucous membranes, preferentially reducing and / or preventing the increase in melanogenesis thereof, and more preferentially reducing and / or inhibiting, totally or partially, the expression of genes and / or proteins and / or the activity of tyrosinase, and / or reducing and / or inhibiting the synthesis and / or amount of melanin.

[0033] The skin pigmentation process, also called "melanogenesis", involves many steps ranging from the synthesis of melanin in melanocytes to its transport by melanosomes to the keratinocytes of the epidermis. Several proteins are involved in this process. Tyrosinase is an enzyme involved in the main steps of melanin synthesis. It cooperates with other proteins to convert melanin into eumelanin and phaeomelanin, the melanins responsible for the pigmentation process.

[0034] The expression "reducing and / or inhibiting melanogenesis" is understood to mean reducing and / or inhibiting, totally or partially, the expression of genes and / or proteins and / or the activity of tyrosinase, and / or reducing and / or inhibiting, totally or partially, the synthesis and / or amount of melanin.

[0035] According to the present invention, the expression "totally or partially reducing and / or inhibiting tyrosinase activity" is understood to mean resulting in a reduction of at least 25%, preferentially at least 50% and more preferentially at least 75% of tyrosinase activity in the presence of a Lactobacillus crispatus strain according to the invention compared to the activity level measured in the absence of the extract.

[0036] Preferentially, the tyrosinase activity is measured against tyrosinase extracted from "normal" human melanocytes, more preferentially in the presence of a Lactobacillus crispatus strain prepared according to example 1.e, advantageously under the conditions described in example 3, more preferentially by measuring the production of melanin by photometric measurement of absorbance at 540 nm against tyrosinase extracted from "normal" human melanocytes.

[0037] According to the present invention, the expression "to totally or partially reduce and / or inhibit the synthesis and / or amount of melanin" means a reduction of at least 10%, preferentially at least 20%, more preferentially at least 30% of the synthesis and / or amount of melanin in the presence of a Lactobacillus crispatus strain according to the invention, relative to the synthesis and / or amount of melanin detected in the absence of said strain. In a preferred embodiment of the invention, it is a question of a reduction in the amount of melanin in the presence of a Lactobacillus crispatus strain according to the invention prepared according to example 1.a or according to example 1.e. More preferentially, the amount of melanin is measured by optical density at 475 nm using B16 melanocytes cultured in vitro, advantageously under the conditions described in example 2, in a medium containing fetal bovine serum. In another preferred embodiment, the amount of melanin is measured by optical density at 475 nm using a co-culture of "normal" human melanocytes and keratinocytes cultured in vitro in a medium containing growth factors, advantageously under the conditions described in Example 4.

[0038] For the purposes of the present invention, the expression "maintaining and / or improving complexion" is understood to mean maintaining and / or preventing a decrease and / or increasing the radiance and / or lightness and / or lustre and / or brightness of the complexion and / or the evenness of the complexion and / or reducing and / or diminishing a dull and / or earthy complexion of the skin and / or mucous membranes.

[0039] The measurement of complexion radiance, the lightness and uniformity of the complexion of the skin and / or mucous membranes can be examined by measurement techniques known to the person skilled in the art, and in particular can be carried out in vivo, for example by chromametry, or by image analysis. This latter in vivo measurement method consists in taking high-resolution photographs of the face of a volunteer at 45°, in a crossed polarized configuration, before and after the application of the test product. On the basis of these digital photographs, by image analysis, certain parameters related to the skin color, radiance and uniformity can be determined, for example: * , a * , b * , C, h°) can be extracted and quantified.

[0040] For the purposes of the present invention, the expression "maintaining and / or improving the radiance of the complexion" is understood to mean maintaining and / or preventing the decrease and / or increasing the brightness and / or lustre and / or brilliance and / or transparency and / or reducing and / or diminishing the dull and / or earthy complexion of the skin and / or mucous membranes.

[0041] For the purposes of the present invention, the expression "maintaining and / or improving the evenness of skin tone" is understood to mean maintaining and / or preventing the increase thereof and / or reducing the amount and / or degree of defects and / or irregularities in skin tone, in particular selected from redness, pigmented spots, comedones, freckles and / or visible pores.

[0042] The measurement of the evenness of skin complexion and the amount of melanin in the skin can be carried out by mexametry, an in vivo measurement method that uses the absorption / reflection principle and consists in deducing therefrom a melanin index that reflects the amount of melanin present in the skin.

[0043] Advantageously, measurements of improvement in skin tone are carried out on a population of women with pigmented spots on the face, preferentially by application of a cream containing a Lactobacillus crispatus strain prepared according to example 1.e, under the conditions described in example 7.

[0044] The expression "maintaining and / or preventing the increase and / or reducing defects and / or irregularities in skin color" is understood to mean preventing the increase in the amount of melanin in the skin and / or reducing the amount of melanin in the skin by at least 2%, advantageously at least 3%, in the presence of the strain according to the invention during 28 days of application to the skin relative to the amount of melanin detected in the absence of the strain, and / or by at least 2%, advantageously at least 5%, in the presence of the strain according to the invention during 56 days of application to the skin relative to the amount of melanin detected in the absence of the strain.

[0045] The expression "preventing the appearance and / or reducing the presence of pigmentation spots" is understood to mean maintaining and / or preventing the increase and / or reducing the number and / or intensity of localized pigmentation spots, in particular pigmentation spots typically in the areas of the face, nape, neck, back, shoulders and / or hands, in particular age spots on the backs of the hands.

[0046] Pigmented spots are "age spots" in particular when they are brown and / or dark and / or white spots and / or localized on parts of the body exposed to UV radiation and / or are associated with natural aging or UV-induced aging, for example during exposure to sunlight. Pigmented spots also include freckles and / or moles and / or post-inflammatory spots and / or spots appearing in response to an insult and / or hormonal spots, otherwise known as pregnancy masks, in particular in the context of chloasma or melasma, and / or drug-induced spots, and / or the prevention and / or elimination of unattractive pigmented signs of the skin and / or mucous membranes associated with non-cutaneous pathological conditions.

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

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

[0049] Detoxification makes it possible to protect the skin from both external and internal insults that cause oxidative stress that tend to impair the skin's natural functions and to adversely alter its appearance, and is in particular a means capable of stimulating the natural systems of protection and repair of skin cells and limiting the induction of cell damage.

[0050] The term "oxidative stress" refers to the formation of hyper-reactive species, including reactive oxygen species and reactive nitrogen species, which form when the skin is exposed to oxidative stress caused by a variety of factors, such as solar ultraviolet, infrared and visible light, environmental pollution, including ozone and particles, and psychological stress.

[0051] In addition to affecting the effectiveness of the skin-detoxification system, oxidative stress can also exacerbate pigmentation, particularly by inducing skin tone irregularities or pigmentation disorders.

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

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

[0054] More preferentially, the formation of the DPPH° free radical 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 rays, in the presence of a Lactobacillus crispatus strain according to the invention prepared according to example 1.a.

[0056] More preferentially, the release of intracellular free radicals induced by oxidative stress, preferentially by UVA rays, is measured by the fluorescence emitted by the formation of DCF (2',7-dichlorofluorescein) from the DCFH-DA (2',7'-dichlorodihydrofluorescein diacetate) probe 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 membrane lipid peroxidation", i.e. of totally or partially preventing and / or inhibiting, in the presence of a Lactobacillus crispatus strain according to the invention, the lipid oxidation induced by oxidative stress, preferentially by UVA rays, by at least 10%, preferentially by at least 30%, 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 peroxidation of membrane lipids induced by oxidative stress, preferentially by UVA rays, in the presence of a Lactobacillus crispatus strain according to the invention prepared according to example 1.a.

[0059] More preferentially, the peroxidation of membrane lipids induced by oxidative stress, preferentially by UVA rays, is measured by the 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 glutathione synthesis, i.e. of preventing the decrease of the expression and / or amount of glutathione in the presence of a Lactobacillus crispatus strain according to the invention, preferentially in the presence of oxidative stress induced by UVA rays, and / or increasing it by at least 20%, preferentially at least 50%, 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 rays, in the presence of a Lactobacillus crispatus strain according to the invention prepared according to example 1.a.

[0062] More preferentially, the synthesis of glutathione 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 invention, the strain of Lactobacillus crispatus may be used in its intact form, in particular live and / or inactivated, in particular killed, and / or in the form of a lysate and / or in the form of one or more of its fragments and / or in the form of one or more of its metabolites, preferentially its secretome.

[0064] In each of its forms, the strain according to the invention may be isolated or combined with its culture medium.

[0065] Preferentially, the strain according to the invention is associated with the medium, i.e. is included in the medium.

[0066] For the purposes of the present invention, the term "culture medium" is understood to mean a carrier containing nutrients allowing the cultivation and / or growth of a Lactobacillus crispatus strain according to the invention. Preferentially, it is the MRS (Mann, Rogosa and Sharpe agar) medium.

[0067] For purposes of the present invention, the term "isolated" means not mixed with one or more compounds that may be associated with it in the medium.

[0068] For the purposes of the present invention, the term "intact" refers to its native form, the form in which the bacterial envelope is intact, as opposed to a dissolved form. When it is used in intact form, the Lactobacillus crispatus strain is viable and / or inactivated and / or killed.

[0069] For the purposes of the present invention, the term "viable" refers to a Lactobacillus crispatus strain according to the invention that is capable of forming colonies in culture.

[0070] The preparation of the Lactobacillus crispatus strain in a viable intact form can be carried out by any method conventionally known to those skilled in the art. Advantageously, it can be carried out, for example, according to the protocol described in Example 1.a.

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

[0072] The inactivation of the Lactobacillus crispatus strain can be carried out by any method conventionally known to the person skilled in the art. It can be advantageously inactivated, inter alia, by irradiation, heat treatment or, under certain conditions, by high pressure treatment or by extrusion, which will more advantageously be carried out by heat treatment, for example according to the protocol described in Example 1.b.

[0073] Heat inactivation may be carried out by incubating the Lactobacillus crispatus strain for a given period of time, advantageously from about 10 seconds to 90 minutes, preferentially from about 15 minutes to 1 hour, advantageously at a temperature of about 60° C. to 150° C. Preferentially, it will be incubated at about 80° C. for about 30 minutes.

[0074] For the purposes of the present invention, the term "killed" refers to a Lactobacillus crispatus strain according to the invention that is definitively no longer capable of forming colonies in culture.

[0075] For the purposes of the present invention, the term "lysate" refers to the material obtained after disruption or lysis of biological cells by a phenomenon known as cell lysis, which causes the release of fragments of intracellular biological components and cell membrane components naturally contained in the cells of the microorganism under consideration. The lysate used is therefore formed from all the intracellular biological components as well as cell wall and membrane components of the Lactobacillus crispatus strain according to the invention.

[0076] The lysate may be obtained by any method conventionally known to those skilled in the art: it may be obtained, inter alia, by osmotic shock, by heat shock, by ultrasound, by enzymatic lysis, by intermittent sterilization, or under mechanical stress of centrifugation type, or by increasing the pressure, or by a combination of these various techniques.

[0077] Preferentially, the lysate will be obtained by a combination of mechanical action and increased pressure. More preferentially, the lysate will be obtained, for example, according to the protocol described in Example 1.c.

[0078] In a preferred embodiment of the invention, the lysate will be combined with a bacterial culture medium.

[0079] Preferentially, the resulting lysate does not contain active enzymes, in particular active sphingomyelinase.

[0080] For the purposes of the present invention, the term "fragment" is understood to mean a fragment of a Lactobacillus crispatus strain according to the invention that is effective in reducing and / or preventing the increase of pigmentation of the skin and / or mucous membranes, in particular for maintaining and / or improving the complexion of the skin and / or mucous membranes, preferentially for maintaining and / or improving the radiance and / or lightness and / or uniformity of the complexion of the skin and / or mucous membranes, and / or for preventing the appearance and / or reducing the presence of pigmented spots on the skin and / or mucous membranes, and / or for detoxifying the skin and / or mucous membranes. This fragment corresponds to a non-total part of the intracellular biological components and cell wall and membrane components obtained by lysis of a Lactobacillus crispatus strain according to the invention.

[0081] According to a particular embodiment of the invention, the fragment may be a bacterial cytoplasm. According to a particular embodiment of the invention, the fragment represents all intracellular biological components, but does not include cell wall and membrane components of the Lactobacillus crispatus strain.

[0082] According to a particular embodiment of the invention, the resulting fragment does not contain an active enzyme, in particular an active sphingomyelinase.

[0083] In a preferred embodiment of the invention, the fragments will be combined with a bacterial culture medium.

[0084] For the purposes of the present invention, the term "metabolites" is understood to mean one or more organic and / or inorganic molecules derived by the metabolism of a strain of Lactobacillus crispatus according to the invention and which are effective in reducing and / or preventing the increase of pigmentation of the skin and / or mucous membranes, in particular for maintaining and / or improving the complexion of the skin and / or mucous membranes, preferentially for maintaining and / or improving the radiance and / or lightness and / or evenness of the complexion of the skin and / or mucous membranes, and / or for preventing the appearance and / or reducing the presence of pigmented spots on the skin and / or mucous membranes, and / or for detoxifying the skin and / or mucous membranes. Preferentially, this is the secretome of a strain of Lactobacillus crispatus according to the invention.

[0085] In certain embodiments of the invention, the metabolites will be combined with the bacterial medium.

[0086] For the purposes of the present invention, the term "secretome" is understood to mean all the organic and / or inorganic molecules secreted by a Lactobacillus crispatus strain according to the invention that are effective in reducing and / or preventing the increase of pigmentation of the skin and / or mucous membranes, in particular for maintaining and / or improving the complexion of the skin and / or mucous membranes, preferentially for maintaining and / or improving the radiance and / or lightness and / or evenness of the complexion of the skin and / or mucous membranes and / or for preventing the appearance and / or reducing the presence of pigmented spots on the skin and / or mucous membranes and / or for detoxifying the skin and / or mucous membranes.

[0087] The secretome may be obtained by any method conventionally known to those skilled in the art. Conveniently, it may be obtained, for example, according to the protocol described in Example 1.e.

[0088] In a preferred embodiment of the invention, the secretome will be combined with a bacterial medium.

[0089] According to a particular embodiment of the invention, the metabolites and / or the secretome do not contain active enzymes, in particular active sphingomyelinase.

[0090] According to a preferential embodiment of the invention, the Lactobacillus crispatus strain according to the invention will be used in viable intact form and / or in the form of one or more of its metabolites, preferentially in the form of its secretome.

[0091] Preferentially, the Lactobacillus crispatus strain according to the invention will be used in an effective amount for reducing pigmentation of the skin and / or mucous membranes and / or preventing an increase in pigmentation of the skin and / or mucous membranes.

[0092] According to an advantageous embodiment, the Lactobacillus crispatus strain according to the invention does not contain sphingomyelinase.

[0093] According to an advantageous embodiment of the invention, the Lactobacillus crispatus strain 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, as defined by the Fitzpatrick classification system.

[0094] For the purposes of the present invention, the term "external route" means direct topical application and / or spraying of a Lactobacillus crispatus strain according to the invention or a composition comprising it according to the invention onto the surface of the involved skin and / or mucous membrane area.

[0095] According to the invention, the Lactobacillus crispatus strain according to the invention may be used alone, in the form of an active ingredient and / or in cosmetic and / or nutraceutical and / or pharmaceutical compositions, especially dermatological compositions, preferentially for external and / or oral application, more preferentially for external application.

[0096] When it is used in a viable intact form according to the invention and when it is used alone in the form of an active ingredient, the strain according to the invention may be in dry form, i.e. in powder form, advantageously in maltodextrin, with a strain content preferentially between 10% and 80% by weight (w / w) of the strain, preferentially between 30% and 70% by weight (w / w) and more advantageously between 40% and 60% by weight (w / w) of the strain, relative to the total weight of the powder.

[0097] Alternatively, the viable intact form according to the invention can be used dispersed and / or diluted in a solvent. Preferentially, this solvent contains less than 20% by volume (v / v) of water, more preferentially less than 5% by volume (v / v) of water. More preferentially, the solvent does not contain any water.

[0098] According to an alternative embodiment, the solvent contains less than 20% by weight (w / w) of water, more preferentially less than 5% by weight (w / w) of water, and even more preferentially the solvent does not contain water. Very preferentially, the solvent is a cosmetically acceptable oil.

[0099] When it is used in its entire inactivated form, in particular killed and / or in the form of a lysate and / or in the form of one or more of its fragments and / or in the form of one or more of its metabolites, in particular in the form of its secretome, and when it is used alone in the form of an active ingredient, the strain according to the invention may be in dry form, i.e. in the form of a powder, advantageously in maltodextrin, in a strain content of preferentially between 1% and 80% (w / w) of the strain by weight, preferentially between 3% and 50% (w / w) of the strain by weight, very preferentially about 5% or 20% (w / w) of the strain by weight, more preferentially about 10% (w / w) of the strain by weight, more preferentially 10% (w / w) of the strain by weight, relative to the total weight of the powder.

[0100] Alternatively, the inactivated intact forms, especially killed forms, and / or lysates, and / or fragments and / or metabolites, especially secretomes according to the invention, may be used soluble and / or diluted and / or dispersed in a solvent, especially a polar solvent, such as water, alcohol, polyol, glycols such as pentylene glycol and / or hexylene glycol and / or caprylyl glycol and / or butylene glycol, or mixtures thereof, preferentially water-glycol or aqueous-alcoholic mixtures, more preferentially comprising glycols selected from hexylene glycol, caprylyl glycol and mixtures thereof.

[0101] In another embodiment, the strain according to the invention may be incorporated into a cosmetic and / or nutraceutical composition comprising at least one cosmetically acceptable and / or nutraceutically acceptable excipient. Preferentially, it may be incorporated into a cosmetic composition comprising at least one cosmetically acceptable excipient.

[0102] For the purposes of the present invention, the term "cosmetically acceptable" excipient means a compound and / or solvent that is acceptable for topical use, i.e., one that does not induce an allergic reaction in contact with the skin, including the human scalp and / or mucous membranes, and is non-toxic and not chemically unstable.

[0103] In a preferential embodiment, the strain according to the invention is incorporated only into a cosmetic composition, i.e. it will not be used to ferment a plant extract, in other words a cosmetic composition comprising a strain according to the invention does not comprise the fermentation of a plant extract by the strain.

[0104] In a preferred embodiment of the invention, the strain according to the invention is present in the cosmetic composition in an effective amount, i.e. in a concentration of 1×10 by weight relative to the total weight of the composition. -4 % to 10% (w / w), preferentially 1×10 by weight -4 % to 5% (w / w), more conveniently 1×10 by weight -3 % to 0.5% (w / w), and said composition also comprises at least one cosmetically acceptable excipient.

[0105] In a particularly advantageous manner, when the strain according to the invention is used in the form of one or more of its metabolites, in particular its secretome, it is present in a cosmetic composition in an amount effective to reduce the pigmentation of the skin and / or mucous membranes and / or prevent an increase in the pigmentation of the skin and / or mucous membranes, i.e. at a concentration of between 0.05% and 0.5% (w / w) by weight, more preferentially at a concentration of about 0.1% (w / w) by weight, relative to the total weight of the composition, said composition also comprising at least one cosmetically acceptable excipient.

[0106] Particularly advantageously, when the strain according to the invention is used in its viable intact 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 an increase in the pigmentation of the skin and / or mucous membranes, i.e. at a concentration of between 0.01% and 0.1% (w / w) by weight, more preferentially at a concentration of about 0.025% (w / w) by weight, relative to the total weight of the composition, said composition also comprising at least one cosmetically acceptable excipient.

[0107] The cosmetic composition of the present invention may be selected from aqueous or oily suspensions or solutions, creams or aqueous or oily gels, especially shower gels, shampoos; milks; emulsions, microemulsions or nanoemulsions, especially oil-in-water or water-in-oil or multiple or silicon-based emulsions; masks; serums; lotions; liquid soaps; dermatological bars; ointments; balms; butters; mousses; patches; anhydrous products, preferably in the form of a liquid, a paste or a solid, such as a make-up powder, a wand or a stick, especially in the form of a lipstick.

[0108] It can also be a make-up product or a make-up removal product.

[0109] Advantageously, the Lactobacillus crispatus strain or the composition of the invention is intended to be applied to the healthy skin and / or healthy mucous membranes of all or part of the body and / or face and / or scalp, preferentially to the legs, thighs, arms, abdomen, nape, neck, armpits or lips, more preferentially to all or part of the face and preferentially to the cheeks, forehead, chin, lips or eye contour.

[0110] Advantageously, the strain and / or the composition comprising it is intended to be applied to areas of healthy skin exhibiting a dull complexion and / or areas of uneven healthy skin and / or areas of healthy skin in which pigmented spots have appeared.

[0111] Preferentially, the Lactobacillus crispatus strain according to the invention is particularly suitable for the formulation of neutral and mild compositions for the respect of the skin, including the sebaceous glands, and especially the scalp and / or mucous membranes.

[0112] Alternatively, the Lactobacillus crispatus strain according to the invention may be in any presentation form conventionally used for oral application, especially in the form of a nutraceutical active ingredient and / or a nutraceutical composition.

[0113] The compositions according to the invention may comprise any suitable solvent and / or any suitable vehicle and / or any suitable excipient, optionally in combination with other compounds of interest.

[0114] As a result, in these compositions, the excipient contains at least one compound selected from the group including, for example, preservatives, emollients, emulsifiers, surfactants, moisturizers, thickeners, conditioners, mattifying agents, stabilizers, antioxidants, texturing agents, gloss agents, film formers, solubilizers, pigments, dyes, fragrances and sunscreens.These excipients are preferably selected from the group consisting of amino acids and derivatives thereof, polyglycerols, esters, cellulose polymers and derivatives, lanolin derivatives, phospholipids, lactoferrin, lactoperoxidase, sucrose-based stabilizers, vitamin E and derivatives thereof, natural and synthetic waxes, vegetable oils, triglycerides, unsaponifiable matter, phytosterols, vegetable esters, silicones and derivatives thereof, protein hydrolysates, jojoba oil and derivatives thereof, fat-soluble / water-soluble esters, betaines, amine oxides, plant extracts, sucrose esters, titanium dioxide, glycine, and parabens, more preferably butylene glycol, steareth-2, steareth-21, glycol-15 stearyl ether, cetearyl alcohol, phenoxyethanol, methylparaben, ethylparaben, propylparaben, butyl Parabens, Butylene Glycol, Natural Tocopherols, Glycerin, Sodium Dihydroxycetyl Phosphate, Isopropyl Hydroxycetyl Ether, Glycol Stearate, Triisononanoin, Octyl Cocoate, Polyacrylamide, Isoparaffin, Laureth-7, Carbomer, Propylene Glycol, Glycerin, Bisabolol, Dimethicone, Sodium Hydroxide, PEG-30 Dipolyhydroxystearate, Capric / Caprylic Triglyceride, 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, glycerides derived from hydrogenated palm kernel oil, glycerides derived from hydrogenated palm oil, lanolin oil, sesame oil, cetyl lactate, lanolin alcohol, castor oil, titanium dioxide, lactose, sucrose, low density polyethylene, isotonic saline solution, and mixtures thereof.

[0115] 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 obtain the best effect. Furthermore, these compounds may have synergistic effects 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 cosmetics and pharmaceutical industries, especially those 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 fragrances, pigments, dyes, essential oils, astringents, etc. (e.g., clove oil, menthol, camphor, eucalyptus oil, eugenol, menthyl lactate, witch hazel distillate), anti-acne agents, anti-flocculants, anti-foaming agents, anti-microbial agents (e.g., iodopropyl butylcarbamate), antioxidants, binders, biological additives, buffers, bulking agents, chelating agents, additives, biocides, denaturants, thickeners, and vitamins and derivatives or equivalents thereof, film-forming materials, polymers, opacifiers, pH adjusters, reducing agents, pigment removers or whitening agents (e.g., hydroquinone, kojic acid, ascorbic acid, magnesium ascorbyl phosphate, ascorbyl glucosamine), conditioning agents (e.g., humectants).

[0116] The cosmetic composition may further comprise other cosmetic and / or nutraceutical agents having the same properties and inducing a synergistic or non-synergistic effect with the Lactobacillus crispatus strain according to the invention, or cosmetic agents having a complementary effect.

[0117] They contain, for example, pigment-removing and / or whitening ingredients and / or ingredients intended to reduce pigment spots on the skin, such as niacinamide or vitamin B3, arbutin, azelaic acid, ascorbic acid or derivatives thereof, a combination of extracts of the plants Saxifraga sarmentosa, Psidium guajava and Carica papaya sold under the name Dermawhite™ WF by BASF Beauty Care Solutions France, a combination of sulfites and extracts of Camellia sinensis, Scutellaria baicalensis, Cucumis sativus, Pyrus malus sold under the name Phytolight™ BG, a combination of Lansium domesticum sold under the name DN-Aura™, a combination of sulphites and extracts of Camellia sinensis, Scutellaria baicalensis, Cucumis sativus, Pyrus malus sold under the name DN-Aura™, a combination of sulphites and extracts of Camellia sinensis, Scutellaria baicalensis, Cucumis sativus, Pyrus malus sold under the name DN-Aura™, a combination of sulphites and extracts of Lansium domesticum ... the standardized extract of Cassia domesticum leaves sold under the name Actiwhite™, a combination of pea extract and sucrose dilaurate sold under the name Actiwhite™, a derivative of 4-hydroxyphenoxyacetic acid, in particular 2-(4-hydroxyphenoxy)propionic acid sold under the name Radianskin™ by BASF Beauty Care Solutions France, or a purified polysaccharide extract from the seeds of Cassia angustifolia sold under the name Hyalurosmooth™, a standardized protease extract isolated from Carica papaya latex sold under the name X-Pressin™, cationic liposome-encapsulated ferulic acid sold under the name Cytovector ferulic™, or a complex of glycolic acid and L-arginine sold under the name AH-Care™ by BASF Beauty Care Solutions France.

[0118] Among other active ingredients that may be combined with the strains according to the invention, it may be a matter of plant extracts with similar properties preventing the appearance of pigmentation and / or increasing the radiance of the complexion, in particular the extract of the fungus Inonotus obliquus sold by the Applicant under the name Inolixir™, the extract of Argania spinosa oil sold by the Applicant under the name Arganyl™, the extract of Moringa oleifera seeds sold by the Applicant under the name Purisoft™, the combination of an extract of Salvia miltiorrhiza and niacinamide sold by the Applicant under the name CollRepair™, the extract of Achillea millefolium sold by the Applicant under the name Neurobiox™, the extract of Cassia alata sold under the name DN-age™, alata leaf extract and / or litchi extract sold under the name Litchiderm™, as antioxidant active agents, chicory extract sold under the name Lox-Age™, yeast extract sold under the name Vitacell™, extract of Polygonum bistorta sold under the name Perlaura™, galangal extract sold under the name Hyalufix™, maize extract sold under the name Deliner™ or extract of Voandzeia subterranea sold by the Applicant under the name Epigenist™, or active agents that promote skin firming, such as synthetic tetrapeptides sold under the name Dermican™, Hibiscus abelmoschus sold under the name Linefactor™. abelmoschus extract, a purified extract of pea sold under the name Proteasyl™, and an extract of Manilkara multinervis sold by the applicant under the name Elestan™.Extracts of the plants Origanum majorana sold under the name Dermagenist™ and / or extracts of Khaya senegalensis sold under the name Collalift™ 18 and / or extracts of Eperua falcata sold under the name Eperulin™ or phytosterols, in particular extracts containing β-sitosterol and / or campesterol and / or brassicasterol originating from rapeseed oil and sold by the Applicant under the name Phytosoothe™, may also be added to the cosmetic composition.

[0119] They may furthermore be anti-aging active ingredients and / or tensioning agents for synergistic effects with Lactobacillus crispatus strains. Advantageously, they are active ingredients that increase the gene and / or protein expression of collagen or that prevent collagen degradation, such as retinol, vitamin C, the extract of Davilla rugosa sold by BASF Beauty Care Solutions under the name Collguard™, the extract of Hibiscus abelmoschus seeds sold under the name Linefactor™, an extract of soy protein hydrolysate sold under the name Phytokine™, peptides sold by the Applicant under the name Dermican™, or the products sold by Sederma under the names Matrixyl™, Matrixyl 3000™ and Regestril™, or the products sold by Codif under Neuroguard, or the products sold by Silab under Eternaline™.

[0120] Tensioning agents that may be used in the present invention may be selected from synthetic polymers such as polyurethane latexes or acrylic latexes, naturally occurring polymers, especially polyholosides in the form of starch or carrageenan, alginate, agar, gellan, cellulosic polymers and pectins; vegetable proteins and protein hydrolysates; mixed silicates; wax microparticles; colloidal particles of inorganic fillers, for example selected from silica, silica-alumina composites; and mixtures thereof.

[0121] Advantageously, the Lactobacillus crispatus strain according to the invention may be combined with a cosmetic agent selected 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 mixtures thereof.

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

[0123] Among the antimicrobial agents that may be combined with the non-viable Lactobacillus crispatus strains in a preferred embodiment of the present invention are 2,4,4'-trichloro-2'-hydroxydiphenyl 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, butadi ... Mention may be made of conazole, oxiconazole, sulfaconazole, sulconazole, terbinafine, undecylenic acid and its salts, benzoyl peroxide, 3-hydroxybenzoic acid, 4-hydroxybenzoic acid, phytic acid, N-acetyl-L-cysteic acid, lipoic acid, azelaic acid and its salts, arachidonic acid, resorcinol, octoxyglycerin, octanoylglycine, caprylyl glycol, 10-hydroxy-2-decanoic acid, farnesol, phytosphingosine and mixtures thereof.

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

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

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

[0127] Slimming active agents may be selected in particular from lipoprotein lipase inhibitors, such as those described in patent US 2003086949 (Coletica) and in particular extracts of Peruvian liana (Uncaria tomentosa); draining active agents, in particular hesperitin laurate (Flavagrum™) or quercitin caprylate (Flavenger™); phosphodiestarase enzyme inhibitors, adenylate cyclase activators, active agents capable of sequestering cAMP and / or spermine and / or spermidine. Examples include Coleus Forskohlii root extract, Cecropia obtusa extract, Uva lactuca extract, caffeine, forskolin, theophylline, theobromine and / or derivatives thereof, a hydrolyzed kappa carrageenan product sold by the applicant called Slimexcess™ and / or mixtures thereof.

[0128] A subject of the present invention is also a cosmetic care process comprising the external application of a Lactobacillus crispatus strain according to the invention, or a cosmetic composition comprising it, to at least one area of ​​healthy skin and / or healthy mucous membranes, in order to reduce and / or prevent an increase in pigmentation of the skin and / or mucous membranes, in particular to maintain and / or improve the complexion of healthy skin and / or healthy mucous membranes, preferentially to maintain and / or improve the radiance and / or lightness and / or evenness of the complexion of healthy skin and / or healthy mucous membranes and / or to prevent the appearance and / or reduce the presence of pigmented spots of healthy skin and / or healthy mucous membranes and / or to detoxify the skin and / or mucous membranes.

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

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

[0131] In an advantageous embodiment of the cosmetic process according to the invention, the cosmetic composition contains a Lactobacillus crispatus strain according to the invention in an amount of 1×10 by weight relative to the total weight of the composition. -4 % to 10% (w / w), preferentially 1×10 by weight -4 % to 5% (w / w), more conveniently 1×10 by weight -3% to 0.5% (w / w), and said composition also contains cosmetically acceptable excipients.

[0132] The aim of the cosmetic care process according to the invention is to reduce and / or prevent the increase of pigmentation of the skin and / or mucous membranes, in particular to maintain and / or improve the complexion of healthy skin and / or healthy mucous membranes, preferentially to maintain and / or improve the radiance and / or lightness 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 pigmented spots on healthy skin and / or healthy mucous membranes, which advantageously comprises the following steps: - reducing and / or preventing the increase of pigmentation of the skin and / or mucous membranes, in particular maintaining and / or improving the complexion of healthy skin and / or healthy mucous membranes, preferentially maintaining and / or improving the radiance and / or lightness and / or uniformity of the complexion of healthy skin and / or healthy mucous membranes, and / or identifying on an individual the areas of healthy skin and / or healthy mucous membranes in which it is desirable to prevent the appearance and / or reduce the presence of pigmented spots on healthy skin and / or healthy mucous membranes, and in an amount effective to reduce and / or prevent the increase of pigmentation of the skin and / or mucous membranes, in particular to maintain and / or improve the complexion of healthy skin and / or healthy mucous membranes, preferentially to maintain and / or improve the radiance and / or lightness and / or evenness of the complexion of healthy skin and / or healthy mucous membranes, and / or to prevent the appearance and / or reduce the presence of pigmented spots on healthy skin and / or healthy mucous membranes, i.e. advantageously in an amount of 1×10 by weight relative to the total weight of the composition -4 % to 10% (w / w), preferentially 1×10 by weight -4 % to 5% (w / w), more conveniently 1×10 by weight -3topical application to said areas of healthy skin and / or healthy mucous membranes of a cosmetic composition comprising a Lactobacillus crispatus strain according to the invention, with a content of the Lactobacillus crispatus strain of 0.5% (w / w).

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

[0134] A subject of the present invention is also the Lactobacillus crispatus strain according to the invention in a pharmaceutical composition, preferentially a dermatological composition, alone or comprising it, for its use, advantageously by external application, for the treatment and / or prevention of pathological conditions of the skin and / or mucous membranes, including pigmentation and / or increased pigmentation, in particular a decrease in the radiance and / or lightness and / or evenness of the complexion of diseased skin and / or pathological mucous membranes and / or an increased appearance of pigmented spots on diseased skin and / or pathological mucous membranes and / or an increased melanogenic activity, in particular an increased melanin and / or tyrosinase activity, and / or an increased oxidative stress of diseased skin and / or pathological mucous membranes.

[0135] In particular, a subject of the present invention is a Lactobacillus crispatus strain according to the invention, or a pharmaceutical composition, preferably a dermatological composition, comprising it, for its use, advantageously by external application, for the treatment and / or prevention of pathological conditions of the skin and / or mucous membranes associated with increased pigmentation of the skin and / or mucous membranes, in particular for the depigmentation and / or prevention of pigmentation of pathological skin and / or pathological mucous membranes and / or for the treatment and / or prevention of pathological conditions of the skin and / or mucous membranes associated with oxidative stress.

[0136] A subject of the present invention is also a method for the treatment and / or prevention of pathological conditions of the skin and / or mucous membranes, including pigmentation and / or increased pigmentation, in particular a decrease in the radiance and / or lightness and / or uniformity of the complexion of diseased skin and / or pathological mucous membranes and / or an increased appearance of pigmented spots in diseased skin and / or pathological mucous membranes and / or an increased melanogenic activity, in particular an increased melanin and / or tyrosinase activity, and / or an increased oxidative stress in diseased skin and / or pathological mucous membranes, which method comprises the administration, advantageously by external application, of a Lactobacillus crispatus strain according to the invention, alone or in a pharmaceutical composition, preferentially a dermatological composition, comprising it in a pharmacologic amount.

[0137] In particular, a subject of the present invention is a method for the treatment and / or prevention of pathological conditions of the skin and / or mucous membranes associated with increased pigmentation of the skin and / or mucous membranes, in particular for the depigmentation and / or prevention of pigmentation of pathological skin and / or pathological mucous membranes and / or for the treatment and / or prevention of pathological conditions of the skin and / or mucous membranes associated with oxidative stress, which method comprises the administration, advantageously by topical application, of a Lactobacillus crispatus strain according to the invention or a pharmaceutical composition comprising it in a pharma- ceutical effective amount, preferentially a dermatological composition, to a subject in need thereof.

[0138] A subject of the present invention is also the use of a Lactobacillus crispatus strain according to the invention, alone or in a pharmaceutical composition comprising it, preferentially a dermatological composition, for the preparation of a medicinal product, advantageously for external administration, for the treatment and / or prevention of pathological conditions of the skin and / or mucous membranes, including pigmentation and / or increased pigmentation, in particular a decrease in the radiance and / or lightness and / or evenness of the complexion of diseased skin and / or pathological mucous membranes and / or an increased appearance of pigmented spots on pathological skin and / or pathological mucous membranes and / or an increased melanogenic activity, in particular an increased melanin and / or tyrosinase activity, and / or an increased oxidative stress of pathological skin and / or pathological mucous membranes.

[0139] In particular, a subject of the present invention is the use of a Lactobacillus crispatus strain according to the invention, or of a pharmaceutical composition comprising it, preferentially a dermatological composition, for the preparation of a medicinal product, advantageously for topical administration, for the treatment and / or prevention of pathological conditions of the skin and / or mucous membranes associated with increased pigmentation of the skin and / or mucous membranes, in particular for depigmenting and / or preventing pigmentation of pathological skin and / or pathological mucous membranes and / or for the treatment and / or prevention of pathological conditions of the skin and / or mucous membranes associated with oxidative stress.

[0140] For the purposes of the present invention, the expression "treatment and / or prevention of pathological conditions of the skin and / or mucous membranes, in order to remove pigmentation and / or prevent pigmentation", in particular "treatment and / or prevention of pathological conditions of the skin and / or mucous membranes associated with increased pigmentation of the skin and / or mucous membranes, in particular to remove pigmentation and / or prevent pigmentation", is understood to mean pigment regulation disorders caused by pathological conditions of the skin and / or mucous membranes, such as Addison's disease, liver failure, purpura, melanoma, melasma, tan papular dermatosis, described as pathological pigmentation, or acne, eczema, atopic dermatitis, rosacea, couperose, redness, from the dermatological point of view, preferentially selected from Addison's disease, liver failure, purpura, melanoma, melasma, tan papular dermatosis, acne, atopic dermatitis, rosacea, and redness.

[0141] For the purposes of the present invention, the expression "treatment and / or prevention of pathological conditions of the skin and / or mucous membranes associated with oxidative stress" is understood to mean, from a dermatological point of view, the treatment and / or prevention of a number of skin diseases associated with excessive oxidative stress, including psoriasis, certain eczemas, acne, vitiligo or alternatively certain skin cancers, preferentially selected from vitiligo.

[0142] The Lactobacillus crispatus strain according to the invention may be in the form of a pharmaceutical composition, preferentially a dermatological composition, comprising at least one pharma- ceutically and / or dermatologically acceptable excipient. In one embodiment of the invention, said composition is applied externally and / or orally, preferentially externally.

[0143] Advantageously, it is present in the pharmaceutical composition, preferentially in the dermatological composition, in an amount of 1×10 by weight relative to the total weight of the composition. -4 % to 10% (w / w), preferentially 1×10 by weight -4 % to 5% (w / w), more conveniently 1×10 by weight -3 % to 0.5% (w / w), said composition also comprising at least one pharma- ceutically acceptable, preferentially dermatologically acceptable excipient.

[0144] The invention will be better understood on reading the following description of the figures and examples.

[0145] Examples are presented below which refer to the description of the present invention. These examples are given for illustrative purposes and are not intended to limit the scope of the present invention in any way. Each of the examples has a general scope.

[0146] The examples form an integral part of the present invention, and any features which are believed to be novel over any prior art from the specification taken as a whole including the examples form an integral part of the present invention.

[0147] Further, in the examples, all percentages are given by weight unless otherwise specified, temperatures are expressed in degrees Celsius unless otherwise specified, and pressures are atmospheric unless otherwise specified. EXAMPLES

[0148] Example 1. Preparation of different forms of Lactobacillus crispatus Example 1.a: Production of viable, intact Lactobacillus crispatus Isolated Lactobacillus crispatus bacteria (CNCM I-5579) were inoculated on MRS type medium (Mann, Rogosa and Sharp agar) and incubated anaerobically at 37 °C. The pH was kept at a value of pH = 6 and the medium was incubated for 24 h. At the end of the incubation, the medium was centrifuged and the cell concentrate was freeze-dried in the presence of maltodextrin with a final maltodextrin amount of 50% by weight (w / w) relative to the total weight of the components.

[0149] Example 1.b: Preparation of inactivated intact Lactobacillus crispatus Isolated Lactobacillus crispatus bacteria (CNCM I-5579) were inoculated into MRS type medium and incubated anaerobically at 37°C. The pH was kept at a value of pH=6 and the medium was incubated for 24 hours. At the end of the incubation, the medium was centrifuged and the cell concentrate was inactivated by heating at 80°C for 30 minutes. The cell concentrate was then resuspended in glycerol.

[0150] Example 1.c: Preparation of Lactobacillus crispatus bacterial lysate The isolated Lactobacillus crispatus bacteria (CNCM I-5579) were inoculated into a medium of MRS type and incubated anaerobically at 37 °C. The pH was kept at a value of pH = 6 and the medium was incubated for 24 h. At the end of the incubation, the whole was treated in a high-pressure homogenizer. The lysate was then resuspended in glycerol.

[0151] Example 1.d: Preparation of Lactobacillus crispatus bacterial secretome Isolated Lactobacillus crispatus bacteria (CNCM I-5579) were inoculated into MRS type medium and incubated anaerobically at 37 °C. The pH was kept at a value of pH = 6 and the medium was incubated for 24 h. At the end of the incubation, the medium was filtered to recover the bacterial secretome. The secretome was then diluted in glycerol.

[0152] Example 1.e: Preparation of Lactobacillus crispatus bacterial secretome in powder form Isolated Lactobacillus crispatus bacteria (CNCM I-5579) were inoculated into a medium of MRS type and incubated anaerobically at 37 °C. The pH was kept at a value of pH = 6 and the medium was incubated for 24 h. At the end of the incubation, the medium was filtered to recover the bacterial secretome. The secretome was then sprayed with maltodextrin with a final maltodextrin amount of 90% (w / w) relative to the total weight of the components.

[0153] Example 2: Inhibition of melanin synthesis by melanocytes in the presence of a Lactobacillus crispatus strain according to the invention. B-16 mouse melanocytes were cultured at 8000 cells / cm 2 into a 96-well plate at a ratio of 1:1 and then cultured in MEM medium (minimum essential medium) supplemented with 10% FCS (fetal calf serum) at 37° C., 5% CO 2 and 95% relative humidity for 3 days.

[0154] The medium was then removed and replaced with 200 μl of the above medium supplemented with 1 μM NDP-α-MSH (melanotropin hormone analogue) used as a melanin synthesis stimulant in the presence of the Lactobacillus crispatus strain according to Example 1.a at a concentration of 0.1% (w / w), for 5 days.

[0155] The same medium without the strain according to the invention but with 1 μM NDP-α-MSH or with 0.01% (w / w) kojic acid was used as procedural controls and as melanin stimulator and melanin inhibitor controls, respectively.

[0156] The same medium containing ethanol but without the strain according to the invention was used as control (control).

[0157] After 5 days of incubation, the cells were lysed with a solution of sodium hydroxide (NaOH) or potassium hydroxide (KOH) and the absorbance was measured at 475 nm.

[0158] The amount of melanin was estimated from a standard range curve consisting of various concentrations of commercially available synthetic melanin.

[0159] The absorbance results were normalized with respect to the absorbance obtained with the same cell culture medium in the absence of the Lactobacillus crispatus strain according to the invention (control). The results presented correspond to the average of six replicate experiments (n=6).

[0160] result

[0161] [Table 1]

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

[0163] Example 3: Inhibition of tyrosinase activity in the presence of a Lactobacillus crispatus strain according to the invention. Tyrosinase is a key enzyme in the process of synthesizing melanin, which is responsible for skin color. The tyrosinase used is extracted from normal human melanocytes.

[0164] Lactobacillus crispatus strain according to Example 1.e at a concentration of 0.05% (w / w) or a reference compound (kojic acid) at concentrations ranging from 0.25 mM to 8 mM were pre-incubated with the enzyme for 10 min on ice. Then, the tyrosinase substrate, i.e. L-tyrosine at a final concentration of 1 mM, was added to the medium and the plates were incubated at 37 °C for 24 h.

[0165] The same enzyme extract without the strain according to the invention but with ethanol was used as control (control).

[0166] Enzyme activity was assessed by measuring melanin production by optical density (OD) at 540 nm using a microplate reader.

[0167] To detect interference of the Lactobacillus crispatus strain according to example 1.e or the reference compounds at each concentration tested, optical density measurements were also performed without the addition of substrate (n=1).

[0168] The percentage of inhibition of tyrosinase activity was calculated by the following formula: Inhibition %: 100-[OD value / mean OD of control x 100]

[0169] All experimental conditions were performed with n = 3. Comparisons between groups were performed by unpaired Student's t-test.

[0170] [Table 2]

[0171] The Lactobacillus crispatus strain according to example 1.e of the present invention showed a significant inhibition of 99% of the activity of tyrosinase extracted from human melanocytes. Therefore, due to its effect of inhibiting tyrosinase activity, the Lactobacillus crispatus strain can be used to reduce and / or prevent the increase of pigmentation of the skin and / or mucous membranes.

[0172] Example 4: Inhibition of melanin synthesis in co-cultures of melanocytes and keratinocytes in the presence of a Lactobacillus crispatus strain according to the invention. Normal human keratinocytes and neonatal human melanocytes were cultured at 20,000 cells / cm 2 and 10,000 cells / cm 2 The cells were seeded at 4° C. and then cultured for 7 days at 37° C., 5% CO2 and 95% relative humidity in a 50% / 50% (v / v) mixture of melanocyte medium and keratinocyte medium supplemented with growth factors.

[0173] After 1 week of culture, the medium was replaced with a mixture of antibiotic-free DMEM medium (Dulbecco's modified minimal essential Eagle's medium) and growth factor-free medium for melanocytes, supplemented with 40 μM oleic acid.

[0174] This medium without the addition of the strain according to the invention was used as an untreated control (Control).

[0175] The secretome of Lactobacillus crispatus bacteria obtained according to Example 1.e was added to this same medium at a concentration of 0.1% (w / w) relative to the total weight of medium and secretome.

[0176] The same medium without the strain but with 0.002% (w / w) rucinol was used as a positive control.

[0177] The culture was continued for 72 hours at 37°C, 5% CO2 and 95% relative humidity, and then the medium was renewed. Then, after an additional culture of 48 hours, the medium was removed and the cells were rinsed with PBS (phosphate buffer). Then, 1N sodium hydroxide solution containing 10% dimethyl sulfoxide (DMSO) was added to each cup of the plate. 200 μl of each cup was transferred to a transparent plate. The optical density, which reflects the amount of melanin, was read at 475 nm.

[0178] Results are the mean ± standard deviation of the assays performed and expressed as a percentage relative to the untreated control normalized to 100%. Statistical analysis of the results was performed using a one-way ANOVA test relative to the untreated control.

[0179] result

[0180] [Table 3]

[0181] The Lactobacillus crispatus strain according to example 1.e of the present invention showed a significant 32% reduction in melanin synthesis by melanocytes co-cultured in vitro with human skin keratinocytes. Therefore, the Lactobacillus crispatus strain, and in particular its secretome, can be used to reduce and / or prevent the increase in pigmentation of the skin and / or mucous membranes.

[0182] Example 5: Reduction of oxidative stress in the presence of Lactobacillus crispatus strains according to the invention Example 5.a: Reduction of the formation of DPPH° free radical in vitro in the presence of a Lactobacillus crispatus strain according to the invention. The free radical scavenging activity assay of Lactobacillus crispatus strains was evaluated by a test using DPPH° (2,2-diphenyl-1-picrylhydrazyl).

[0183] Lactobacillus crispatus strains were mixed in PBS (phosphate buffered saline) at a concentration of 0.05% (w / w) according to example 1.a and at a concentration of 0.7% (w / w) according to example 1.e.

[0184] A negative control was performed using the solvent for lysing the strains, PBS, and a positive control with 1 mM vitamin C.

[0185] Each of the solutions containing the invention, the negative control or the positive control were mixed in a solution of DPPH° at 50% / 50% (v / v) in the wells of a 96-well plate.

[0186] Each of the solutions containing the invention, the negative control or the positive control was also mixed in this same plate at 50% / 50% (v / v) in an ethanol solution, which is the solvent for DPPH°, to create a blank.

[0187] The whole was incubated for 30 minutes under stirring. At the end of the incubation, the optical density (OD) was measured at a wavelength of 530 nm to estimate the amount of free radicals formed under each of the conditions tested.

[0188] The results of the measurement of DPPH° radicals to obtain the free radical scavenging activity of the invention were the average of six tests (n=6) and expressed as the mean percentage ± 1 standard deviation. The product of the invention was compared to its negative control (PBS only without the presence of Lactobacillus crispatus strains). The vitamin C positive control was compared to its negative control, water. Statistical analysis was performed by one-way ANOVA analysis of variance for the invention and by Student's test for vitamin C.

[0189] [Table 4]

[0190] The Lactobacillus crispatus strain according to example 1.a of the invention showed a significant free radical scavenging activity of 34% in vitro. The Lactobacillus crispatus strain according to example 1.e of the invention showed a free radical scavenging activity of 31% in vivo. Thus, due to this effect of reducing the production of free radicals, the Lactobacillus crispatus strain can be used to detoxify the skin and / or mucous membranes and to reduce and / or prevent the increase of pigmentation of the skin and / or mucous membranes.

[0191] Example 5.b: Reduction of UV-induced release of cellular free radicals in fibroblast cells in the presence of a Lactobacillus crispatus strain according to the invention. "Normal" human fibroblasts, i.e. fibroblasts derived from healthy donors and showing no pathology, were cultured at 30,000 cells / cm in 24-well plates. 2 and then cultured in DMEM (Dulbecco's modified minimal essential Eagle's medium) / Ham's F-12 medium supplemented with 10% FCS and 0.5% antibiotics for 96 hours at 37° C., 5% CO 2 and 95% relative humidity.

[0192] Then, 0.5 ml of the cell suspension was incubated for 72 h in EMEM medium (Eagle's Minimum Essential Medium) supplemented with 1% FCS and 0.5% antibiotics in the presence of a Lactobacillus crispatus strain according to Example 1.a at a concentration of 0.0125% (w / w) or an antioxidant control (0.0003% (v / v) vitamin E).

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

[0194] DCFH-DA (2',7'-dichlorodihydrofluorescein diacetate) probe solubilized at 10 μM in PBS (phosphate buffered saline) was incubated with the cells. After an initial 1 h incubation at 37 °C, 5% CO2 and 95% relative humidity, the cells were rinsed with PBS and irradiated with 20 J / cm 2 The cells were irradiated with UVA for 3 h at 4 °C. A non-irradiated control was also performed. After irradiation, the irradiated medium was removed and the cells were rinsed with PBS.

[0195] The fluorescence reading on the cell layer associated with the formation of DCF (2',7-dichlorofluorescein) using the probe (DCFH-DA) was read at 485 nm excitation and 538 nm emission.

[0196] Results are the mean of 12 replicates (n=12) and expressed as ±1 standard deviation. Results were compared to the UVA control alone, normalized to 100%, and statistically compared using a one-way ANOVA analysis of variance test.

[0197] [Table 5]

[0198] The Lactobacillus crispatus strain according to example 1.a of the present invention showed a significant reduction of 56% at the cellular level of the release of intracellular free radicals induced by UVA oxidative stress. Thus, due to this effect of reducing the release of intracellular free radicals induced by UVA, the Lactobacillus crispatus strain can be used to detoxify the skin and / or mucous membranes and / or to protect and / or improve the radiance and / or brightness and / or evenness of the skin and / or mucous membranes, especially when the latter are exposed to oxidative stress preferentially caused by UVA, and preferentially to reduce the presence and / or prevent the appearance of pigmented spots on the skin and / or mucous membranes.

[0199] Example 5.c: Reduction of UV-induced membrane lipid peroxidation in fibroblast cells in the presence of a Lactobacillus crispatus strain according to the invention. "Normal" human fibroblasts, i.e. fibroblasts derived from healthy donors and showing no pathology, were cultured in 24-well plates at 30,000 cells / cm. 2 and then cultured in DMEM / Ham's F-12 medium supplemented with 10% FCS and 0.5% antibiotics for 96 hours at 37° C., 5% CO 2 and 95% relative humidity.

[0200] 0.5 ml of the cell suspension was then incubated for 72 h in EMEM medium supplemented with 1% FCS and 0.5% antibiotics 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 (0.0003% (v / v) vitamin E).

[0201] This medium without the addition of the strain according to the invention was used as an untreated control (after irradiation, constituting only a UVA control - oxidative stress control).

[0202] After an initial 1 h incubation at 37 °C, 5% CO2 and 95% relative humidity, cells were rinsed with PBS and incubated with 20 J / cm 2 The cells were irradiated with UVA for 3 h at 1000 rpm. A non-irradiated control was also performed. After irradiation, the supernatant was collected and the cells were rinsed with PBS.

[0203] A volume of 0.5 ml of the supernatant was transferred to a Pyrex glass tube, to which a solution of 40% trichloroacetic acid and 2% thiobarbituric acid was added. The resulting mixture was heated at 100° C. for 30 min. The formation of MDA (malondialdehyde), an oxidation product released after UVA-induced peroxidation of lipids, was then measured by fluorescence at 532 nm excitation and 560 nm emission.

[0204] Results are the average of the assays performed and are expressed as the mean ± 1 standard deviation. Results were compared to the UVA control only, normalized to 100%, and statistically compared using a one-way ANOVA analysis of variance test.

[0205] [Table 6]

[0206] The Lactobacillus crispatus strain according to example 1.a of the present invention showed a significant reduction of the formation of MDA induced by UVA at the cellular level by 32%. Thus, due to its effect of reducing lipid peroxidation in the presence of UV-induced stress, the Lactobacillus crispatus strain can be used to detoxify, especially the skin and / or mucous membranes, when they are exposed to oxidative stress, especially UVA-induced stress.

[0207] Example 6: Stimulation of the cellular antioxidant defenses after UV irradiation in fibroblast cells in the presence of a Lactobacillus crispatus strain according to the invention. "Normal" human fibroblasts, i.e. fibroblasts derived from healthy donors and showing no pathology, were cultured in 24-well plates at 30,000 cells / cm. 2 and then cultured in DMEM / Ham's F-12 medium supplemented with 10% FCS and 0.5% antibiotics for 96 hours at 37° C., 5% CO 2 and 95% relative humidity.

[0208] This medium without the addition of the strain according to the invention was used as an untreated control (after irradiation, constituting only a UVA control - oxidative stress control).

[0209] 0.5 ml of the cell suspension was then incubated for 72 h in EMEM medium supplemented with 1% FCS and 0.5% antibiotics in the presence of a Lactobacillus crispatus strain according to example 1.a at a concentration of 0.0125% (w / w) or an antioxidant control (0.0003% (v / v) vitamin E).

[0210] After an initial 1 h incubation at 37 °C, 5% CO2 and 95% relative humidity, cells were rinsed with PBS and incubated with 20 J / cm 2 The cells were irradiated with UVA for 3 h at 400 rpm. A non-irradiated control was also performed. After irradiation, the irradiated medium was removed and the cells were rinsed with PBS.

[0211] 1N sodium hydroxide (NaOH) was added to each cup, followed by a solution of o-phthaldialdehyde (OPT) diluted 1 / 15 in GSH buffer (reduced L-glutathione). The whole was incubated for 15 minutes at ambient temperature. The amount of glutathione was then measured by fluorescence at 355 nm excitation and 430 nm emission.

[0212] Results are the average of the assays performed and are expressed as the mean ± 1 standard deviation. Results were statistically compared using a one-way ANOVA analysis of variance test compared to the UVA control alone, normalized to 100%.

[0213] [Table 7]

[0214] The Lactobacillus crispatus strain according to example 1.a of the present invention showed a significant increase in cellular antioxidant defense with a 54% increase in glutathione after UVA irradiation.

[0215] Therefore, due to this action of increasing cellular antioxidant defenses in the presence of UV-induced oxidative stress, Lactobacillus crispatus strains can be used to detoxify the skin and / or mucous membranes, especially when they are exposed to oxidative stress, especially UVA-induced stress.

[0216] Example 7: In vivo measurement of the reduction in the amount of melanin in the skin in the presence of a Lactobacillus crispatus strain according to the invention. A group of 30 Chinese women, aged 40-50 years, with pigmented spots on the face, with phototypes II-IV, applied to half of their face a cream in the form of an emulsion containing a final weight concentration of 1% (w / w) of the Lactobacillus crispatus strain according to Example 1.e with respect to the total weight of the cream (formulation example 8.b) or a cream not containing said extract, in which said extract was replaced by water (placebo), said emulsion being applied at a rate of two applications per day for two months.

[0217] Assessment of the melanin content of the skin was performed using a device called a Megzameter, which utilizes the principle of absorption / reflection. The Melanin Index is estimated from the Megzameter measurements. A decrease in this index corresponds to a decrease in melanin.

[0218] The efficacy of a composition containing a Lactobacillus crispatus strain according to Example 1.e was compared to that of a "placebo" emulsion.

[0219] The melanin index was measured at the start of the study (D0), after one month of application (D28) and finally after two months of application (D56).

[0220] The efficacy of the formulations containing Lactobacillus crispatus strains according to Example 1.e and the placebo formulation were expressed as means and compared with the values ​​at the start of the study (D0) and compared between the formulations containing Lactobacillus crispatus strains and the formulations containing placebo using Student's test or Wilcoxon test.

[0221] [Table 8]

[0222] The preparation of Lactobacillus crispatus strain according to Example 1.e significantly reduced the amount of melanin in the skin as measured by the melanin index using a Megzameter.

[0223] These results show a beneficial effect of Lactobacillus crispatus strains formulated to maintain and / or improve the radiance and / or brightness and / or evenness of healthy skin and / or healthy mucous membranes.

[0224] Example 8: Example of a cosmetic composition containing a Lactobacillus crispatus strain according to the invention Example 8.a: Example of a cosmetic composition containing a Lactobacillus crispatus strain according to example 1.a The Lactobacillus crispatus strain obtained according to Example 1.a was added to the formulation defined below according to Table 8 at a final concentration of 0.05% (w / w) relative to the total weight of the formulation immediately before application to the skin.

[0225] [Table 9]

[0226] Example 8.b: Example of a cosmetic composition containing a Lactobacillus crispatus strain according to example 1.e Ratios are expressed as percentages and the capitalized names correspond to the INCI names of the ingredients.

[0227] [Table 10]

[0228] Using methods known to those skilled in the art, the various phases A, B and C are mixed together at ambient temperature by adding B to A with stirring, followed by adding C to AB. The pH is adjusted to 4.9 with D. Phase E is then added. Finally, homogenization is carried out with an Ultra-Turrax blender to prepare the composition according to the invention.

Claims

1. Cosmetic and / or nutritional use of Lactobacillus crispatus strain, preferredly the Lactobacillus crispatus strain deposited under designation CNCM I-5579, for the purpose of reducing and / or preventing the increase of pigmentation of the skin and / or mucous membranes.

2. The use according to claim 1, wherein the strain reduces and / or inhibits melanin formation, particularly by acting to reduce and / or inhibit melanin, preferentially by acting to reduce and / or inhibit the synthesis and / or amount of melanin, and / or by reducing and / or inhibiting tyrosinase activity, and / or by detoxifying healthy skin and / or healthy mucous membranes.

3. The use according to claim 1 for maintaining and / or improving the skin color of healthy skin and / or healthy mucous membranes.

4. The use according to claim 1 for maintaining and / or improving the luster, brightness, and / or uniformity of the skin tone of healthy skin and / or healthy mucous membranes.

5. The use according to claim 1 for preventing and / or reducing the appearance of pigment spots, preferably brown spots and / or age spots, on healthy skin and / or healthy mucous membranes.

6. In particular, the use according to claim 1 for detoxifying healthy skin and / or healthy mucous membranes by its antioxidant effect and / or effect against lipid peroxidation, and / or by increasing and / or stimulating glutathione synthesis induced by oxidative stress.

7. The use according to claim 1, wherein the strain is used preferentially in its secretome, in its complete form, in particular in a living and / or inactivated form, in particular in a dead form, and / or in the form of a lysate, and / or in the form of one or more fragments thereof, and / or in the form of one or more metabolites thereof.

8. The use according to claim 1, wherein the Lactobacillus crispatus strain is deposited under the number CNCM I-5579.

9. The use according to claim 1, wherein the Lactobacillus crispatus strain is used in a living, complete form and / or in one or more forms of its metabolites, preferably in its secretome.

10. The use according to claim 1, wherein the Lactobacillus crispatus strain is combined with the culture medium.

11. The use according to claim 1, wherein the Lactobacillus crispatus strain is applied topically to healthy skin and / or healthy mucous membranes.

12. The use according to claim 11, wherein the skin is of phototype IV to VI.

13. The Lactobacillus crispatus strain present in the cosmetic composition in an amount of 1 × 10⁻¹⁶ of the total weight of the composition. －４ Weight % to 10% weight, preferably 1 x 10 －４ % by weight to 5% by weight, or more conveniently, 1 × 10 －３ The use according to claim 1, further comprising an excipient present at a concentration of % to 0.5% by weight, wherein the composition is acceptable as at least one cosmetic.

14. A cosmetic care process characterized by the application of a Lactobacillus crispatus strain as defined in any one of claims 7 to 10 or a cosmetic composition as defined in claim 13 to at least one area of ​​healthy skin and / or mucous membranes for the purpose of reducing and / or preventing the increase of pigmentation of the skin and / or mucous membranes, particularly for maintaining and / or improving the skin tone of healthy skin and / or mucous membranes, preferentially for maintaining and / or improving the luster and / or brightness and / or uniformity of the skin tone of healthy skin and / or mucous membranes, and / or for preventing and / or reducing the appearance of pigment spots on healthy skin and / or mucous membranes, and / or for detoxifying the skin and / or mucous membranes.

15. The cosmetic care process according to claim 14, wherein the topical application of the Lactobacillus crispatus strain or the cosmetic composition is performed on healthy skin and / or mucous membranes of the whole or part of the body and / or face and / or scalp, preferably on the legs, thighs, arms, abdomen, nape of the neck, neck, armpits, lips, more preferably on the whole or part of the face, and preferably on the cheeks, forehead, chin, lips, and contours of the eyes.

16. The cosmetic composition contains the Lactobacillus crispatus strain in an amount of 1 × 10¹⁶ of the total weight of the composition. －４ Weight % to 10% weight, preferably 1 x 10 －４ % by weight to 5% by weight, or more conveniently, 1 × 10 －３ The cosmetic care process according to claim 14, further comprising an excipient present in a concentration of % to 0.5% by weight, wherein the composition is acceptable as at least one cosmetic.

17. A pharmaceutical composition, preferably a dermatological composition, for use, conveniently by topical application, comprising Lactobacillus crispatus strain, in the treatment and / or prevention of pathological conditions of the skin and / or mucous membranes associated with increased pigmentation of the skin and / or mucous membranes, particularly in the removal of pigment from pathological skin and / or mucous membranes and / or the prevention of pigmentation, and / or in the treatment and / or prevention of pathological conditions of the skin and / or mucous membranes associated with oxidative stress.

18. The pharmaceutical composition according to claim 17, wherein the strain is used in its secretome, preferably in its complete form, particularly alive and / or inactive, particularly dead, and / or in the form of a lysate, and / or in the form of one or more fragments thereof, and / or in the form of one or more metabolites thereof, and / or the strain is combined with its culture medium.

19. The pharmaceutical composition according to claim 17, characterized in that it comprises at least one dermatologically acceptable excipient, preferably in the form of a dermatological composition.

20. The strain is present in the pharmaceutical composition, preferably in a dermatological composition, at a concentration of 1×10 －４ % by weight to 10% by weight, preferably 1×10 －４ % by weight to 5% by weight, more preferably 1×10 －３ % by weight to 0.5% by weight based on the total weight of the composition, and the composition also contains at least one pharmaceutically, preferably dermatologically acceptable excipient. The pharmaceutical composition according to claim 17.

21. The pharmaceutical composition according to claim 17, characterized in that the strain is as described in any one of claims 7 to 10.