Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K8/00—Cosmetics or similar toiletry preparations
  • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
  • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
  • A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
  • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/74—Bacteria
  • A61K35/741—Probiotics
  • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
  • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/18—Antioxidants, e.g. antiradicals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
  • A61Q19/00—Preparations for care of the skin
  • A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
  • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
  • A23V2200/00—Function of food ingredients
  • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
  • A23V2200/318—Foods, ingredients or supplements having a functional effect on health having an effect on skin health and hair or coat
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
  • A23V2400/00—Lactic or propionic acid bacteria
  • A23V2400/11—Lactobacillus
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
  • A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
  • A61K2800/52—Stabilizers
  • A61K2800/522—Antioxidants; Radical scavengers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
  • A61K2800/74—Biological properties of particular ingredients
  • A61K2800/78—Enzyme modulators, e.g. Enzyme agonists
  • A61K2800/782—Enzyme inhibitors; Enzyme antagonists

Definitions

• the present invention relates to the cosmetic, nutraceutical and/or pharmaceutical, in particular dermatological, use of a strain of Lactobacillus crispatus and/or of a composition comprising it to reduce pigmentation of the skin and/or mucous membranes and/or prevent increased pigmentation of the skin and/or mucous membranes.
• the structure and appearance of the skin are modified with age and/or under the influence of extrinsic and/or intrinsic factors. These changes are numerous and include changes in the pigmentation of the skin and/or mucous membranes.
• the skin becomes dull, less luminous, less radiant, its radiance diminishes and the complexion is less homogeneous with the appearance of pigment spots.
• Melanogenesis and oxidative stress are the subject of numerous studies and innovations in the field of cosmetics for the development and improvement of cosmetic active ingredients aimed at reducing and/or preventing the increase in skin pigmentation. healthy and/or healthy mucous membranes.
• skin and/or mucous membranes in particular to preserve and/or improve the complexion of the skin and/or mucous membranes, in particular its radiance and/or its luminosity and/or its homogeneity, particularly within a population more prone to tasks pigmentary, in particular phototype IV to VI skin as defined by the Fitzpatrick classification method.
• Lactobacillus crispatus a beneficial commensal bacteria of the skin and/or mucous membranes
• the Applicant's invention is all the more interesting since the bacteria Lactobacillus crispatus, and in particular that deposited under the number CNCM I-5579, has the advantage of being naturally present on young skin, unlike bacteria used until now in cosmetic and/or dermatological treatments.
• the object of the present invention has the advantage of rebalancing and/or supplementing the microbiota of the skin and/or mucous membranes, in particular with a bacteria of the skin flora which diminishes and becomes rarer over time and/or exposure to intrinsic and/or extrinsic factors.
• the object of the present invention is thus particularly suitable for the treatment of so-called mature skin, in particular of people over 50 years old.
• the strain according to the invention also has the advantage of being an easily formulated ingredient, and can be easily manufactured on an industrial scale. It is an active ingredient topically acceptable for the skin and mucous membranes, which does not present any risk of allergy.
• Application WO9822082 thus discloses the use of sphingomyelinase which can be extracted from lactic acid bacteria to be applied topically in order to increase the levels of ceramides in the skin and mucous membranes, for the prevention or therapeutic treatment of several pathologies.
• Application W02009031099 discloses the use of a probiotic to prevent the appearance and/or to treat the manifestation of sensations of discomfort and/or skin signs associated with a surface skin treatment or an invasive treatment, for aesthetic purposes .
• Application WO2019111189 describes the use comprising a probiotic in particular Lactobacillus paracasei for the prevention of inflammatory damage induced by UV radiation.
• the first subject of the present invention is the cosmetic and/or nutraceutical use of a strain of Lactobacillus crispatus, preferably the strain of Lactobacillus crispatus deposited under the number CNCM I-5579, to reduce the pigmentation of the skin and/or mucous membranes.
• the strain of Lactobacillus crispatus according to the invention is in particular used as a cosmetic and/or nutraceutical active ingredient.
• the strain according to the invention reduces and/or inhibits melanogenesis, in particular by acting on the reduction and/or inhibition of melanin, preferably on the reduction and/or inhibition the synthesis and/or quantity of melanin, and/or by reducing and/or inhibiting the activity of tyrosinase, and/or by detoxifying healthy skin and/or healthy mucous membranes.
• the strain according to the invention can be used to preserve and/or improve the complexion of healthy skin and/or healthy mucous membranes.
• the strain according to the invention can also be used to preserve and/or improve the radiance and/or luminosity and/or homogeneity of the complexion, healthy skin and/or healthy mucous membranes.
• the strain according to the invention can also be used to prevent the appearance and/or reduce the presence of pigment spots on healthy skin and/or healthy mucous membranes, preferably brown spots and/or age spots.
• the strain according to the invention can also be used to detoxify healthy skin and/or healthy mucous membranes, in particular by an antioxidant action and/or on lipid peroxidation and/or by an increase and/or stimulation of glutathione synthesis. induced by oxidative stress.
• a cosmetic treatment process characterized in that it comprises the topical application to at least one area of healthy skin and/or healthy mucous membrane of a strain of Lactobacillus crispatus according to the invention, preferably the Lactobacillus crispatus strain CNCM I-5579, or a cosmetic composition comprising it, to reduce skin pigmentation and/or mucous membranes and/or prevent its increase, in particular to preserve and/or improve the complexion of healthy skin and/or healthy mucous membranes, preferably to preserve and/or improve radiance and/or luminosity and/or homogeneity of the complexion of healthy skin and/or healthy mucous membranes, and/or to prevent the appearance and/or reduce the presence of pigment spots in healthy skin and/or healthy mucous membranes, and/or detoxify the skin and /or mucous membranes.
• the strain of Lactobacillus crispatus according to the invention is in particular used as a cosmetic and/or nutraceutical active ingredient.
• the invention also relates to a strain of Lactobacillus crispatus according to the invention, preferably the strain of Lactobacillus crispatus CNCM I-5579, or to a pharmaceutical composition, in particular dermatological composition comprising it, for the prevention and/or treatment of pathologies of the skin and/or mucous membranes associated with an increase in pigmentation of the skin and/or mucous membranes, in particular to depigment and/or prevent hyperpigmentation of pathological skin and/or pathological mucous membranes, to reduce pigmentation of the skin and/or mucous membranes and/or to prevent its increase, in particular to preserve and/or improve the complexion of the pathological skin and/or the pathological mucous membranes, preferably to preserve and/or improve the radiance and/or the brightness and/or homogeneity of the complexion of the pathological skin and/or pathological mucous membranes, and/or to prevent the appearance and/or reduce the presence of pigment spots of the pathological skin and/or pathological mucous membranes and
• the term “cosmetic use” means a non-pharmaceutical use, that is to say which is not intended for therapeutic use and is applied to a so-called healthy part of the body, in particular on an area of so-called healthy skin and/or mucous membranes.
• “nutraceutical use” means a use intended for non-pharmaceutical oral use, that is to say which does not require or involve therapeutic treatment.
• “Skin” means any area of skin on any part of the body and/or face, including the scalp.
• phototype IV to VI skin means particular skin types classified according to the Fitzpatrick scale which makes it possible to classify individuals according to the reaction of their skin and hair during exposure to UV rays, and/or classified as such by a dermatologist according to the tone of the skin, which depends in particular on the level of activity of the melanocytes, the creation of these cells and their groupings.
• these dark skin types of phototype IV to VI come from characteristic ethnic origins, respectively from India, Asia and/or Africa.
• Phototype IV (Mediterranean type) corresponds to light matte skin and brown or black hair and eyes. Sunburns are rare, the skin tans well, a dark brown;
• Phototype V corresponds to light matte skin, black hair and eyes. Sunburns are rare, the skin tans well, a dark brown;
• the term "mucosa(s)" means the ocular mucosa, the nasal mucosa, the ear mucosa, the vaginal mucosa, the urogenital mucosa and/or the oral mucosa, in particular buccal, labial and/or gingival, preferably, the ocular and/or oral mucosa, and even more preferably, the labial and/or ocular mucosa.
• it does not include the vaginal mucosa.
• the term “healthy skin” or “healthy mucosa” means an area of skin or mucosa to which the strain according to the invention is applied and called “non-pathological” by a dermatologist, i.e. -meaning no infection, scar, disease, inflammation or skin condition such as candidiasis, impetigo, psoriasis, eczema, acne or dermatitis, or sores or injuries.
• the term “Lactobacillus crispatus strain” means the bacterium Lactobacillus crispatus in whole form or in partial form, in particular in the form of lysate, in the form of a fraction or in the form of metabolites.
• the Lactobacillus crispatus strain is not genetically modified.
• the strain of Lactobacillus crispatus according to the invention may be derived from any known species of Lactobacillus crispatus.
• the strain of Lactobacillus crispatus is the species deposited according to the Budapest Treaty at the Pasteur Institute (28 rue du Dondel Roux, F -75024 Paris cedex 15) on 09/03/2020 under the designation CNCM I-5579 .
• the term "reduce the pigmentation of the skin and/or mucous membranes and/or prevent its increase” means depigmenting, whitening, lightening, preventing the increase, maintaining and/or preserving and/or improving the complexion and/or complexion of the skin and/or mucous membranes, and preferably reduce and/or prevent the increase in melanogenesis, and still preferentially reduce and/or totally or partially inhibit gene and/or protein expression and /or tyrosinase activity and/or reduce and/or inhibit the synthesis and/or quantity of melanin.
• the process of skin pigmentation is also called “melanogenesis” and includes numerous stages ranging from the synthesis of melanin in melanocytes to its transport into the keratinocytes of the epidermis via melanosomes.
• Tyrosinase is the enzyme which intervenes at key stages of melanin synthesis; it cooperates with other proteins to transform melanin into eumelanin and phaeomelanin, the melanins responsible for the pigmentation process.
• reduce and/or inhibit melanogenesis means reducing and/or totally or partially inhibiting gene and/or protein expression and/or the activity of the tyrosinase and/or reduce and/or totally or partially inhibit the synthesis and/or quantity of melanin.
• the term “reduce and/or totally or partially inhibit the activity of tyrosinase” is understood to cause a reduction of at least 25%, preferably of at least 50%, more preferably of at least 75% of the activity of tyrosinase in the presence of the strain of Lactobacillus crispatus according to the invention, compared to the level of activity measured in the absence of the extract.
• the measurement of tyrosinase activity is carried out on the tyrosinase extracted from so-called normal human melanocytes, still preferentially by measuring the production of melanin by photometrically measuring the absorbance at 540 nm from the tyrosinase extracted from melanocytes so-called normal humans, more preferably in the presence of the strain of Lactobacillus crispatus prepared according to Example 1.e, advantageously under the conditions described in Example 3.
• the term "reduce and/or totally or partially inhibit the synthesis and/or the quantity of melanin” means a reduction in melanin of at least 10%, preferably at least 20%, more preferably by at least 30% of the synthesis and/or the quantity of melanin in the presence of the strain of Lactobacillus crispatus according to the invention compared to the synthesis and/or the quantity of melanin detected in the absence of the strain.
• this involves a reduction in the quantity of melanin in the presence of the strain of Lactobacillus crispatus according to the invention prepared according to Example 1.a or according to Example 1 .e.
• the quantity of melanin is measured by optical density at 475 nm from B16 melanocytes cultured in vitro in a culture medium comprising fetal calf serum, advantageously under the conditions described in Example 2.
• the quantity of melanin is measured by optical density at 475 nm from a coculture of so-called normal human melanocytes and keratinocytes, cultured in vitro in a culture medium comprising growth factors, advantageously under the conditions described in Example 4.
• the term "preserve and/or improve the complexion” means maintaining and/or preventing the reduction and/or increasing the radiance and/or luminosity and/or the radiance and/or shine of the complexion and/or the homogeneity of the complexion and/or diminish and/or reduce the dull and/or earthy complexion of the skin and/or mucous membranes.
• the measurement of the radiance of the complexion, the luminosity and the homogeneity of the complexion of the skin and/or the mucous membranes can be studied using measurement techniques known to those skilled in the art, in particular can for example be carried out in vivo, by chromametry or by image analysis.
• This last in vivo measurement method consists of taking high-resolution photographs in a crossed polarized configuration of the faces of volunteers taken at 45° before and after application of the product tested. On the basis of these digital photographs, an image analysis makes it possible to extract and quantify specific parameters (for example: L*, a*, b*, C, h°) related to color, brightness and the homogeneity of the skin.
• the term “preserve and/or improve the radiance of the complexion” means maintaining and/or preventing the reduction and/or increasing luminosity and/or radiance and/or shine and/or clarity. , and/or diminish and/or reduce the dull and/or earthy complexion of the skin and/or mucous membranes.
• the term "preserving and/or improving the homogeneity of the complexion” means maintaining and/or preventing the increase and/or reducing the quantity and/or extent of imperfections and/or irregularities of the complexion.
• complexion color in particular chosen from redness, pigment spots, blackheads, freckles and/or visible pores.
• Measuring the evenness of skin tone and the amount of melanin in the skin can be measured by Mexametrie.
• This in vivo measurement method consists of using the absorption/reflection principle to deduce a melanin index which reflects the quantity of melanin present in the skin.
• the measurement of the improvement in complexion is carried out on a population of women having pigment spots on the face, preferably by application of a cream containing the strain of Lactobacillus crispatus prepared according to example 1.e, in the conditions described in Example 7.
• the term "maintain and/or prevent the increase and/or reduce imperfections and/or irregularities in the color of the complexion” means to prevent the increase and/or decrease the quantity of melanin in the skin, by at least 2%, advantageously at least 3% in the presence of the strain according to the invention during a 28-day application to the skin relative to the quantity of melanin detected in the absence of the strain and/or at least 2%, advantageously at least 5% in the presence of the strain according to the invention during a 56-day application to the skin compared to the quantity of melanin detected in the absence of the strain.
• preventing the appearance and/or reducing the presence of pigment spots means maintaining and/or preventing the increase and/or reducing the pigmentation of localized pigment spots, in particular the number and/or intensity of the pigment spots. typically on areas of the face, beaulleté, neck, back, shoulders and/or hands, particularly spots on the tops of the hands.
• Pigment spots include brown and/or dark and/or white spots and/or “aged spots” when they are located on a part of the body exposed to UV radiation and/or linked to chronological or UV-induced aging, for example during exposure to the sun. Pigment spots also include freckles and/or moles and/or postinflammatory spots and/or which appear in response to attacks, and/or of hormonal origin, in particular in the context of chloasma or melasma, otherwise called mask of pregnancy, and/or of medicinal origin, and/or to prevent and/or combat unsightly pigmentary manifestations of the skin and/or mucous membranes accompanying a non-cutaneous pathology.
• strain of Lactobacillus crispatus in particular that deposited under the designation CNCM I-5579, can be used to detoxify the skin and/or mucous membranes, in particular healthy skin and/or mucous membranes.
• detoxifying the skin and/or mucous membranes is meant the prevention and/or total and/or partial inhibition of oxidative stress, in particular by the prevention and/or total and/or partial inhibition of the formation of free radicals, and/or lipid peroxidation and/or by increasing and/or stimulating the synthesis of glutathione which has antioxidant and detoxifying properties.
• Detoxifying helps protect the skin against attacks, both external and internal, which cause oxidative stress likely to alter its proper functioning and appearance, in particular a means capable of stimulating the natural protection and repair systems of skin cells. and limit the induction of cellular damage.
• Oxidative stress means the formation of excessive reactive species, including reactive oxygen species and reactive nitrogen species, which form when the skin is subjected to oxidative stress caused by various factors. such as solar ultraviolet, infrared and visible light, environmental pollution including ozone and particulate matter, and psychological stress.
• oxidative stress can also exacerbate pigmentation, in particular by inducing uneven skin tone or even a pigmentary disorder.
• this involves reducing and/or inhibiting the formation of DPPH° (2,2-diphenyl-1-picrylhydrazyl) free radicals by at least 10%, preferably by at least 10%. less 30%, in the presence of the Lactobacillus crispatus strain according to the invention compared to the formation of DPPH° free radicals detected in the absence of the strain, and/or reduce and/or inhibit the release of induced intracellular free radicals by oxidative stress, preferably by UVA, of at least 20%, preferably of at least 50%, in the presence of the strain of Lactobacillus crispatus according to the invention in relation to the release of intracellular free radicals detected in absence of the strain.
• oxidative stress preferably by UVA
• this involves reducing and/or inhibiting the spontaneous formation of DPPH° free radicals, in the presence of the strain of Lactobacillus crispatus according to the invention prepared according to example 1.a and/or 1.e.
• the formation of DPPH° free radicals is measured in tubo by optical density at 530 nm, under the conditions described in Example 5.a.
• the invention involves reducing and/or inhibiting the release of intracellular free radicals induced by oxidative stress, preferably by UVA, in the presence of the strain of Lactobacillus crispatus according to the invention prepared according to example 1.a.
• the release of intracellular free radicals induced by oxidative stress is measured by fluorescence emitted by the formation of DCF (2',7-dichlorofluorescein) from the DCFH-DA probe (2', 7'-dichlorodihydrofluorescein diacetate) at wavelengths of 485 nm excitation and 538 nm emission from normal human fibroblasts, under the conditions described in Example 5.
• DCF 2',7-dichlorofluorescein
• DCFH-DA probe 2', 7'-dichlorodihydrofluorescein diacetate
• it involves “preventing and/or inhibiting the peroxidation of membrane lipids”, that is to say preventing and/or totally or partially inhibiting the oxidation of lipids induced by a oxidative stress, preferably by UVA of at least 10%, preferably at least 30% in the presence of the strain of Lactobacillus crispatus according to the invention compared to the lipid peroxidation detected in the absence of the strain.
• this involves preventing and/or inhibiting the peroxidation of membrane lipids induced by oxidative stress, preferably by UVA, in the presence of the strain of Lactobacillus crispatus according to the invention prepared according to example 1.a.
• the peroxidation of membrane lipids induced by oxidative stress is measured by fluorescence of the formation of MalonDiAldehde (MDA) at wavelengths of 532 nm excitation and 560 nm emission from of normal human fibroblasts, under the conditions described in Example 5.c.
• MDA MalonDiAldehde
• it involves maintaining and/or stimulating the synthesis of glutathione, that is to say preventing the decrease and/or increasing the expression and/or quantity of glutathione. in the presence of oxidative stress, preferably induced by UVA, of at least 20%, preferably at least 50%, in the presence of the strain of Lactobacillus crispatus according to the invention in relation to the synthesis of glutathione detected in the absence of the strain.
• oxidative stress preferably induced by UVA
• the invention involves maintaining and/or stimulating the synthesis of glutathione induced by oxidative stress, preferably by UVA, in the presence of the Lactobacillus crispatus strain according to the invention prepared according to Example 1.a.
• 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.
• the strain of Lactobacillus crispatus can be used in whole form, in particular viable and/or inactivated, in particular dead, and/or in the form of lysate, and/or in the form of one or more of its fractions and /or in the form of one or more of its metabolites, preferably its secretome.
• the strain according to the invention can be isolated or associated with its culture medium.
• the strain according to the invention is used associated with its culture medium, that is to say contained in its culture medium.
• the term “culture medium” means a support containing the nutrients allowing the cultivation and/or multiplication of the strain of Lactobacillus crispatus according to the invention.
• it is an MRS medium (Man, Rogosa and Sharp agar).
• isolated means not mixed with one or more compounds likely to be associated with it in its culture medium.
• whole form means its native form, a form in which the bacterial envelope is intact, as opposed to a lysed form.
• the Lactobacillus crispatus strain may be viable and/or inactivated and/or dead.
• the term “viable” means a strain of Lactobacillus crispatus according to the present invention capable of forming colonies in culture.
• the production of the strain of Lactobacillus crispatus in whole viable form can be carried out according to any method conventionally known to those skilled in the art. Advantageously, it will be carried out according to the protocol as described for example in example 1.a.
• the term “inactivated” means a strain of Lactobacillus crispatus according to the present invention which is no longer capable, temporarily or permanently, of forming colonies in culture.
• the inactivation of the Lactobacillus crispatus strain can be carried out using any method conventionally known to those skilled in the art. It can in particular be inactivated by irradiation, heat treatment or, under certain conditions, by high pressure treatment or by extrusion advantageously, it will be carried out by heat treatment, again advantageously according to the protocol as described for example in example 1.b.
• Thermal inactivation can be carried out by incubating the Lactobacillus crispatus strain for a given period of time, advantageously from approximately 10 s to 90 min, preferably from approximately 15 minutes to one hour, and at a temperature advantageously of approximately 60 °C to 150°C. Preferably, it will be incubated for around 30 minutes at around 80°C.
• “dead” means a strain of Lactobacillus crispatus according to the present invention which is no longer capable, definitively, of forming colonies in culture.
• the term "lysate” means a material obtained following the destruction or dissolution of biological cells by a phenomenon called cell lysis thus causing the release of intracellular biological constituents naturally contained in the cells of the considered microorganism and fragments of cell membrane components.
• the lysate used is therefore formed of all the intracellular biological constituents and the constituents of the cell walls and membranes of the Lactobacillus crispatus strain according to the invention.
• the lysate can be obtained by any method conventionally known to those skilled in the art. It may in particular be obtained by an osmotic shock, a thermal shock, by ultrasound, by enzymatic lysis, by tyndallization, or even under mechanical stress such as centrifugation, or by increasing the pressure or combinations of these different technologies.
• the lysate will be obtained by a combination of mechanical action and increased pressure. Still preferably, the lysate will be obtained according to the protocol as described for example in example 1.c.
• the lysate will be associated with the culture medium of the bacteria.
• the lysate obtained does not contain active enzyme, in particular active sphingomyelinase.
• the term "fractions" means a fragment of the strain of Lactobacillus crispatus according to the present invention having an effectiveness in reducing the pigmentation of the skin and/or mucous membranes and/or preventing its increase, in particular to preserve and/or improve the complexion of the skin and/or mucous membranes, preferably to preserve and/or improve the radiance and/or luminosity and/or homogeneity of the complexion of the skin and/or mucous membranes, and/or to prevent the appearance and/or reduce the presence of pigment spots on the skin and/or mucous membranes and/or detoxify the skin and/or mucous membranes.
• This fraction corresponds to a non-total part of the intracellular biological constituents and the constituents of the cell walls and membranes obtained by lysis of the Lactobacillus crispatus strain according to the invention.
• the fractions can be the protoplasm of the bacteria.
• the fraction corresponds to all of the intracellular biological constituents, but does not contain constituents of the cell walls and membranes of the Lactobacillus crispatus strain.
• the fraction obtained does not contain any active enzyme, in particular active sphingomyelinase.
• the fractions will be associated with the culture medium of the bacteria.
• the term “metabolites” means one or more organic and/or inorganic molecules resulting from the metabolism of the strain of Lactobacillus crispatus according to the present invention and also having an effectiveness in reducing the pigmentation of the skin and /or mucous membranes and/or prevent its increase, in particular to preserve and/or improve the complexion of the skin and/or mucous membranes, preferably to preserve and/or improve radiance and/or luminosity and/or homogeneity of the complexion of the skin and/or mucous membranes, and/or to prevent the appearance and/or reduce the presence of pigment spots on the skin and/or mucous membranes and/or detoxify the skin and/or mucous membranes.
• it is the secretome of the Lactobacillus crispatus strain according to the invention.
• the metabolites will be associated with the culture medium of the bacteria.
• secretome means all of the secreted organic and/or inorganic molecules secreted by the strain of Lactobacillus crispatus according to the present invention having an effectiveness in reducing the pigmentation of the skin and/or or mucous membranes and/or to prevent its increase, in particular to preserve and/or improve the complexion of the skin and/or mucous membranes, preferably to preserve and/or improve radiance and/or luminosity and/or homogeneity of the complexion of the skin and/or mucous membranes, and/or to prevent the appearance and/or reduce the presence of pigment spots on the skin and/or mucous membranes and/or detoxify the skin and/or mucous membranes.
• the secretome can be obtained by any method conventionally known to those skilled in the art. Advantageously, it will be obtained according to the protocol as described for example in example 1.e.
• the secretome will be associated with the culture medium of the bacteria.
• the metabolites and/or the secretome do not contain any active enzyme, in particular active sphingomyelinase.
• the strain of Lactobacillus crispatus according to the invention will be used in whole viable form and/or in the form of one or more of its metabolites, preferably its secretome.
• the strain of Lactobacillus crispatus according to the invention will be used in an effective quantity to reduce the pigmentation of the skin and/or mucous membranes and/or prevent the increase in pigmentation of the skin and/or mucous membranes.
• the Lactobacillus crispatus strain according to the invention does not contain sphingomyelinase.
• the strain of Lactobacillus crispatus according to the invention is applied topically to healthy skin and/or healthy mucous membranes.
• the skin and/or mucous membranes are phototype IV to VI.
• Phototypes IV to VI are as defined by the Fitzpatrick classification method.
• the term “topical route” means the direct local application and/or vaporization of the strain of Lactobacillus crispatus according to the invention or of the composition comprising it according to the invention on the surface of the affected area of skin and/or mucous membrane.
• the strain of Lactobacillus crispatus according to the invention can be used alone, in the form of active ingredient and/or in a cosmetic and/or nutraceutical, and/or pharmaceutical composition, in particular dermatological, preferably intended for topical and/or oral application, more preferably for topical application.
• the strain according to the invention can be in dry form, that is to say in of powder, advantageously in maltodextrin, preferably in a strain content of 10% to 80% (w/w) by weight of the strain, preferably from 30% to 70% (w/w), more advantageously from 40 to 60% (w/w) in weight of the strain relative to the total weight of powder.
• the whole living form according to the invention can be used dispersed and/or diluted in a solvent.
• this solvent contains less than 20% (v/v) by volume of water, still preferably less than 5% (v/v) by volume of water, still preferably the solvent does not contain water.
• this solvent contains less than 20% (w/w) by weight of water, still preferably less than 5% (w/w) by weight of water, still preferably the solvent does not contain water .
• this solvent is a cosmetically acceptable oil.
• the strain according to the invention can be in dry form, that is to say in powder form, advantageously in maltodextrin, preferably in a strain content of 1% to 80% (w/w) by weight of the strain, preferably from 3% to 50% (w/w) by weight of the strain, very preferably around 5% or 20% ( p/w) by weight of the strain relative to the total weight of powder, still preferably around 10% (w/w), still preferably 10% (w/w) by weight of the strain relative to the total weight of powder.
• the whole inactivated forms, in particular dead, and/or the lysates, and/or the fractions, and/or the metabolites, in particular the secretome according to the invention can be used soluble and/or diluted and or dispersed in a solvent , in particular polar, such as water, an alcohol, a polyol, a glycol such as pentylene glycol and/or hexylene glycol and/or caprylyl glycol and/or butylene glycol, or one of their mixtures, preferably a hydroglycolic or hydroalcoholic mixture, more preferably comprising a glycol chosen from hexylene glycol, caprylyl glycol and their mixtures.
• a solvent in particular polar, such as water, an alcohol, a polyol, a glycol such as pentylene glycol and/or hexylene glycol and/or caprylyl glycol and/or butylene glycol, or one of their mixtures, preferably a hydroglycolic or
• the strain according to the invention can be incorporated into a cosmetic and/or nutraceutical composition comprising at least one cosmetically acceptable and/or nutraceutically acceptable excipient.
• a cosmetic composition comprising at least one cosmetically acceptable excipient.
• the term “cosmetically acceptable” excipient means a topically acceptable compound and/or solvent, that is to say one which does not induce an allergic response upon contact with the skin, including the scalp. human, and/or mucous membranes, non-toxic, not unstable, chemically.
• the strain according to the invention will be incorporated into a cosmetic composition alone, that is to say it will not be used to ferment plant extracts.
• the cosmetic composition comprising the strain according to the invention does not comprise fermented plant extracts by the strain.
• the strain according to the invention is present in the cosmetic composition in an effective quantity, namely a concentration of 1x10' 4 % to 10% (w/w) by weight, preferably between 1x10' 4 % and 5% (w/w) by weight, still advantageously between 1x10' 3 % and 0.5% (w/w) by weight, relative to the total weight of the composition, said composition further comprising at least one cosmetically acceptable excipient.
• the strain according to the invention when used in the form of one or more of its metabolites, in particular its secretome, it is present in the cosmetic composition in an amount effective for reducing the pigmentation of the skin and/or or mucous membranes and/or prevent the increase in pigmentation of the skin and/or mucous membranes, namely a concentration of 0.05 to 0.5% (w/w) by weight, more preferably at a concentration of approximately 0.1% (w/w) by weight, relative to the total weight of the composition, said composition further comprising at least one cosmetically acceptable excipient.
• the strain according to the invention when used in whole viable form, it is present in the cosmetic composition, in an amount effective for reducing the pigmentation of the skin and/or mucous membranes and/or preventing the increase pigmentation of the skin and/or mucous membranes, namely a concentration of 0.01 to 0.1% (w/w) by weight, more preferably at a concentration of approximately 0.025% (w/w) by weight , relative to the total weight of the composition, said composition further comprising at least one cosmetically acceptable excipient.
• the cosmetic composition of the invention can be chosen from a suspension or a solution, aqueous or oily, an aqueous cream or gel or an oily gel, in particular a shower gel, a shampoo; a milk ; an emulsion, a microemulsion or a nanoemulsion, in particular oil-in-water or water-in-oil or multiple or siliconized; a mask; a serum; lotion; liquid soap; a dermatological bar; an ointment ; a balm; a butter; a foam; a patch; an anhydrous product, preferably liquid, pasty or solid, for example in the form of makeup powders, rods or sticks, in particular in the form of lipstick.
• It can also be a makeup product or a makeup remover product.
• the strain of Lactobacillus crispatus or the composition of the invention is intended to be applied to healthy skin and/or healthy mucous membranes of all or part of the body and/or the face and/or the scalp, preferably the legs, thighs, arms, stomach, neckline, neck, armpits, lips, preferably all or part of the face, and preferably the cheeks, forehead, chin, lips, eye contour .
• the strain and/or the composition comprising it is intended to be applied to an area of healthy skin presenting a dull complexion and/or an area of non-homogeneous healthy skin and/or an area of healthy skin with the appearance pigment spots.
• the strain of Lactobacillus crispatus according to the invention is particularly suitable for the formulation of so-called neutral and gentle composition for respect for the sebaceous gland, particularly the skin including the scalp and/or mucous membranes.
• strain of Lactobacillus crispatus according to the invention can be presented in all the galenic forms conventionally used for oral application, in particular in the form of nutraceutical active ingredient and/or nutraceutical composition.
• compositions according to the invention may contain any suitable solvent and/or any suitable vehicle and/or any suitable excipient, optionally in combination with other compounds of interest.
• the excipient contains for example at least one compound chosen from the group comprising preservatives, emollients, emulsifiers, surfactants, moisturizers, thickeners, conditioners, mattifying agents, stabilizers , antioxidants, texturing agents, shine agents, film-forming agents, solubilizers, pigments, dyes, perfumes and sun filters.
• excipients are preferably chosen from the group consisting of amino acids and their derivatives, polyglycerols, esters, polymers and cellulose derivatives, lanolin derivatives, phospholipids, lactoferrins, lactoperoxidases, stabilizers based on sucrose, vitamins E and its derivatives, natural and synthetic waxes, vegetable oils, triglycerides, unsaponifiables, phytosterols, plant esters, silicones and its derivatives, protein hydrolysates, Jojoba oil and its derivatives derivatives, lipo/water-soluble esters, betaines, am inoxides, plant extracts, sucrose esters, titanium dioxides, glycines, and parabens, and more preferably from the group consisting of butylene glycol, steareth-2, steareth-21, glycol-15 stearyl ether, cetearyl alcohol, phenoxyethanol, methylparaben, ethylparaben, propylparaben, but
• anti-acne agents for example: clove oil, menthol, camphor, eucalyptus oil, eugenol, menthyl lactate, witch hazel distillate
• anti-acne agents for example: clove oil, menthol, camphor, eucalyptus oil, eugenol, menthyl lactate, witch hazel distillate
• antimicrobial agents for example: iodopropyl butylcarbamate
• antioxidants for example: iodopropyl butylcarbamate
• binders biological additives, buffering agents, bulking agents, chelating agents, additives, biocidal agents, denaturants, thickeners, and vitamins, and derivatives or equivalents thereof, film-forming materials, polymers, opacifying agents, pH adjusters, reducing agents, depigmenting or lightening agents (for example: hydroquinone, kojic acid, ascorbic acid, magnesium ascorbyl phosphate, ascorby
• the cosmetic composition may also include other cosmetic and/or nutraceutical agents having the same properties and inducing a synergistic effect or not with the strain of Lactobacillus crispatus according to the invention, or cosmetic agents with complementary effects.
• These may for example be depigmenting and/or lightening ingredients and/or intended to reduce pigment spots on the skin, such as niacinamide or vitamin B3, arbutin, azelaic acid, ascorbic acid or its derivatives, a combination of Saxifraga sarmentosa, Psidium guajava and Carica papaya plant extracts, marketed by BASF Beauty Care Solutions France under the name DermawhiteTM WF, a combination of sulphites and extracts of Camellia sinensis, Scutellaria baicalensis, Cucumis sativus , Pyrus malus, marketed under the name PhytolightTM BG, a standardized extract of Lansium domesticum leaves marketed under the name DN-AuraTM, a combination of a pea extract and sucrose dilaurate marketed under the name ActiwhiteTM, derivatives of 4-hydroxyphenoxy acetic acid, in particular 2-(4-hydroxyphenoxy)-propionic acid marketed under the name Radians
• active ingredients which may be combined with the strain according to the invention, it may be a plant extract having similar properties of preventing the appearance of pigmentation and/or increasing the radiance of the complexion, in particular an extract of the mushroom Inonotus obliquus marketed under the name InolixirTM by the applicant, an extract of Argania spinosa oil marketed under the name ArganylTM by the applicant, an extract of Moringa oleifera seeds marketed under the name of PurisoftTM by the applicant, a combination of an extract of Salvia miltiorrhiza and niacinamide marketed under the name of ColIRepairTM by the applicant, an extract of Achillea millefolium marketed under the name of NeurobioxTM by the applicant, an extract of Cassia alata leaves sold under the name DN-AgeTM and/or an extract of lychee sold under the name LitezermTM as anti-oxidant active ingredients, a chicory extract sold under the name Lox-AgeTM
• these may also include anti-aging active ingredients and/or tightening agents for a synergistic effect with the Lactobacillus crispatus strain.
• active ingredients increasing the gene and/or protein expression of collagen or active ingredients preventing the degradation of collagen such as retinol, vitamin C, an extract of Davilla rugosa marketed under the name CollguardTM by BASF Beauty Care Solutions, a Hibiscus abelmoschus seed extract marketed under the name LinefactorTM, a soy protein hydrolyzate extract marketed under the name PhytokineTM, a peptide marketed under the name DermicanTM by the applicant or the products marketed under the names MatrixylTM, Matrixyl 3000TM and RegestrilTM by the company Sederma or Neuroguard by the company Codif, or even EternalineTM by the company Silab.
• the tensing agents which can be used in the invention can be chosen from synthetic polymers, such as polyurethane latexes or acrylic latexes, polymers of natural origin, in particular polyholosides in the form of starch or in the form of carrageenans, alginates, agars, gellans, cellulose polymers and pectins; plant proteins and protein hydrolysates; mixed silicates; wax microparticles; colloidal particles of inorganic filler chosen for example from silica, silica-alumina composites; as well as their mixtures.
• the Lactobacillus crispatus strain according to the invention may be combined with a cosmetic agent chosen from benzoic acid and/or its salts, levulinic acid and/or its salts such as sodium levulinate, acid sorbic and/or its salts, anisic acid and/or its salts, and their mixture.
• a cosmetic agent chosen from benzoic acid and/or its salts, levulinic acid and/or its salts such as sodium levulinate, acid sorbic and/or its salts, anisic acid and/or its salts, and their mixture.
• the cosmetic and/or dermatological composition of the present invention also contains one or more tensing agents and/or one or more antimicrobial agents and/or one or more anti-radical agents and/or one or more soothing agents and/or one or more agents. slimming agents and/or one or more agents active on the microcirculation.
• antimicrobial agents associated with the Lactobacillus crispatus strain in non-viable form in a preferred embodiment of the present invention mention may be made of 2,4,4'-trichloro-2'-hydroxy diphenyl ether (or triclosan), 3 ,4,4'-trichlorobanilide, phenoxyethanol, phenoxypropanol, phenoxyisopropanol, hexamidine isethionate, metronidazole and its salts, miconazole and its salts, itraconazole, terconazole, econazole, ketoconazole, saperconazole , fluconazole, clotrimazole, butoconazole, oxiconazole, sulfaconazole, sulconazole, terbinafine, undecylenic acid and its salts, benzoyl peroxide, 3-hydroxy benzoic acid, 4-hydroxy acid benzoic acid, phytic acid, N-acetyl
• Anti-free radical agents can be vitamin C and its derivatives including ascorbyl glucoside, phenols and polyphenols, in particular tannins, ellagic acid and tannic acid; epigallocatechin and natural extracts containing it, in particular green tea extracts; anthocyanins; phenolic acids, stilbenes; actives scavengers of mono- or polycyclic aromatic compounds, tannins such as ellagic acid and indole derivatives and/or active scavengers of heavy metals such as EDTA, anti-radical actives such as vitamin E and its derivatives such as tocopheryl acetate; bioflavonoids; coenzyme Q10 or ubiquinone.
• vitamin C and its derivatives including ascorbyl glucoside, phenols and polyphenols, in particular tannins, ellagic acid and tannic acid; epigallocatechin and natural extracts containing it, in particular green tea extracts; anthocyanins; phenolic
• pentacyclic triterpenes ursolic acid and its salts, oleanolic acid and its salts, betulinic acid and its salts, salts of salicylic acid
• pentacyclic triterpenes ursolic acid and its salts
• oleanolic acid and its salts oleanolic acid and its salts
• betulinic acid and its salts salts of salicylic acid
• salicylic acid can be used as soothing agents included in the composition of the invention.
• zinc salicylate bisabolol, Allantoin, unsaturated omega 3 oils, cortisone, hydrocortisone, indomethacin and beta methasone
• anti-inflammatory active ingredients and in particular those described in application FR2847267, in particular the root extract of Pueraria lobata marketed under the name InhipaseTM by the applicant, the extracts of Theobroma cacao.
• the active ingredients acting on microcirculation, vasoprotectors or vasodilators can be chosen from flavonoids, ruscogenins, nicotinates and essential oils.
• the slimming active ingredients can be chosen in particular from lipoprotein lipase inhibitor agents such as those described in patent US2003086949 (Coletica) and in particular an extract of Peruvian liana (Uncaria tomentosa), draining active ingredients, in particular hesperitin laurate (FlavagrumTM), or quercitin caprylate (FlavengerTM); agents that inhibit the enzyme phosphodiestarase, agents that activate adenylate cyclase, cAMP and/or active ingredients capable of trapping spermine and/or spermidine.
• lipoprotein lipase inhibitor agents such as those described in patent US2003086949 (Coletica) and in particular an extract of Peruvian liana (Uncaria tomentosa), draining active ingredients, in particular hesperitin laurate (FlavagrumTM), or quercitin caprylate (FlavengerTM); agents that inhibit the enzyme phosphodiestarase, agents that activate adenylate
• the present invention also relates to a cosmetic treatment process comprising the topical application to at least one area of healthy skin and/or healthy mucous membrane of a strain of Lactobacillus crispatus according to the invention, or of a composition cosmetic comprising it, to reduce the pigmentation of the skin and/or mucous membranes and/or prevent its increase, in particular to preserve and/or improve the complexion of healthy skin and/or healthy mucous membranes, preferably to preserve and/or improve the radiance and/or luminosity and/or homogeneity of the complexion of healthy skin and/or healthy mucous membranes, and/or to prevent the appearance and/or reduce the presence of pigment spots healthy skin and/or healthy mucous membranes and/or detoxify the skin and/or mucous membranes.
• the Lactobacillus crispatus strain according to the invention preferably in the form of a cosmetic composition according to the invention, is used in regular topical application and preferably at least once a day, advantageously twice a day, for at least 10 days, preferably for 20 days, and even more preferably for at least 28 days.
• the topical application of a strain of Lactobacillus crispatus according to the invention or of a cosmetic composition comprising it is carried out on healthy skin and/or healthy mucous membranes of all or part of the body and/or or the face and/or scalp, preferably the legs, thighs, arms, stomach, neckline, neck, armpits, lips, even preferably all or part of the face, and preferably the cheeks, forehead , chin, lips, eye contour.
• the cosmetic composition comprises the strain of Lactobacillus crispatus according to the invention at a concentration of between 1x10' 4 % and 10% (w/w) by weight, preferably between 1x10 ' 4 % and 5% (w/w) by weight, still advantageously between 1x10' 3 % and 0.5% (w/w) by weight, relative to the total weight of the composition, said composition further comprising an excipient cosmetically acceptable.
• the cosmetic treatment process according to the invention aims to reduce the pigmentation of the skin and/or mucous membranes and/or to prevent its increase, in particular to preserve and/or improve the complexion of healthy skin and/or healthy mucous membranes, preferably to preserve and/or improve the radiance and/or luminosity and/or homogeneity of the complexion of healthy skin and/or healthy mucous membranes, and/or to prevent the appearance and/or reduce the presence of pigment spots on healthy skin and/or healthy mucous membranes, advantageously includes the following steps: - The identification on the individual of an area of healthy skin and/or healthy mucous membrane, for which it is desired to reduce the pigmentation of the skin and/or mucous membranes and/or prevent its increase, in particular to preserve and/or improve the complexion of healthy skin and/or healthy mucous membranes, preferably to preserve and/or improve the radiance and/or luminosity and/or homogeneity of the complexion of healthy skin and/or healthy mucous membranes, and/ or to prevent the
• a cosmetic composition comprising the strain of Lactobacillus crispatus according to the invention, in an effective quantity to reduce the pigmentation of the skin and/or mucous membranes and/or prevent its increase, in particular to preserve and/or improve the complexion of healthy skin and/or healthy mucous membranes, preferably to preserve and/or improve the radiance and/or luminosity and/or homogeneity of the complexion healthy skin and/or healthy mucous membranes, and/or to prevent the appearance and/or reduce the presence of pigment spots in healthy skin and/or healthy mucous membranes, namely in a content of Lactobacillus crispatus strain included advantageously between 1x10' 4 % and 10% (w/w) by weight, preferably between 1x10' 4 % and 5% (w/w) by weight, still advantageously between 1x10' 3 % and 0.5% (w/w ) by weight, relative to the total weight of the composition.
• topically and/or orally acceptable means an ingredient suitable for application respectively topically and/or orally, non-toxic, non-irritating to the skin including the scalp and does not does not induce an allergic response, and which is not chemically unstable.
• the subject of the invention is also the strain of Lactobacillus crispatus according to the invention, alone or in a pharmaceutical composition, preferably dermatological, comprising it, for its use, advantageously topically for the treatment and/or prevention of skin pathologies. and/or mucous membranes involving hyperpigmentation and/or an increase in pigmentation, in particular a reduction in the radiance and/or luminosity and/or homogeneity of the complexion of the pathological skin and/or the pathological mucous membranes, and/ or an increase in the appearance of pigment spots in pathological skin and/or pathological mucous membranes, and/or an increase in the activity of melanogenesis, in particular through an increase in melanin and/or tyrosinase activity, and/or an increase in oxidative stress of pathological skin and/or pathological mucous membranes.
• the subject of the invention is the strain of Lactobacillus crispatus according to the invention or a pharmaceutical composition, preferably dermatological, comprising it, for its use, advantageously topically, for the treatment and/or prevention of skin pathologies. and/or mucous membranes associated with an increase in pigmentation of the skin and/or mucous membranes, in particular to depigment and/or prevent hyperpigmentation of pathological skin and/or pathological mucous membranes, and/or for the treatment and/or mucous membranes. or the prevention of skin and/or mucous membrane pathologies associated with oxidative stress.
• the invention also relates to a method for the treatment and/or prevention of pathologies of the skin and/or mucous membranes involving hyperpigmentation and/or an increase in pigmentation, in particular a reduction in radiance and/or brightness and/or homogeneity of the complexion of the pathological skin and/or pathological mucous membranes, and/or an increase in the appearance of pigment spots of the pathological skin and/or pathological mucous membranes, and/or an increase in the activity of melanogenesis, in particular through an increase in melanin and/or tyrosinase activity, and/or an increase in oxidative stress of pathological skin and/or pathological mucous membranes, said method comprising: administration, advantageously topically, of the strain of Lactobacillus crispatus according to the invention, alone or in a pharmaceutical composition, preferably dermatological comprising it, in a pharmaceutically effective quantity in a subject in need.
• the subject of the invention is a method for the treatment and/or prevention of pathologies of the skin and/or mucous membranes associated with an increase in pigmentation of the skin and/or mucous membranes, in particular for depigmentation and/or or prevent hyperpigmentation of pathological skin and/or pathological mucous membranes, and/or for the treatment and/or prevention of pathologies of the skin and/or mucous membranes associated with oxidative stress, said method comprising the administration, advantageously topically, from the strain of Lactobacillus crispatus according to the invention or a pharmaceutical composition, preferably dermatological, comprising it, in a pharmaceutically effective quantity in a subject in need.
• the invention also relates to the use of the strain of Lactobacillus crispatus according to the invention, alone or in a pharmaceutical composition, preferably dermatological comprising it, for the preparation of a medication, advantageously for topical administration, for the treatment and /or the prevention of pathologies of the skin and/or mucous membranes involving hyperpigmentation and/or an increase in pigmentation, in particular a reduction in the radiance and/or luminosity and/or homogeneity of the complexion of the pathological skin and/or pathological mucous membranes, and/or an increase in the appearance of pigment spots in the pathological skin and/or pathological mucous membranes, and/or an increase in the activity of melanogenesis, in particular through an increase in melanin and/or tyrosinase activity, and/or an increase in oxidative stress of pathological skin and/or pathological mucous membranes,
• the subject of the invention is the use of the strain of Lactobacillus crispatus according to the invention or a pharmaceutical composition, preferably dermatological comprising it, for the preparation of a medication, advantageously for topical administration, for the treatment and/or the prevention of pathologies of the skin and/or mucous membranes associated with increased pigmentation of the skin and/or mucous membranes, in particular to depigment and/or prevent hyperpigmentation of pathological skin and/or mucous membranes pathological, and/or for the treatment and/or prevention of pathologies of the skin and/or mucous membranes associated with oxidative stress.
• treatment and/or prevention of pathologies of the skin and/or mucous membranes to depigment and/or prevent hyperpigmentation is understood, in particular by “treatment and/or prevention of pathologies of the skin and/or mucous membranes associated with an increase in pigmentation of the skin and/or mucous membranes, in particular to depigment and/or prevent hyperpigmentation”, a pigmentary disturbance caused by skin pathologies and/or mucous membranes, such as disease Addison's disease, liver failure, purpura, melanoma, chloasma, dermatosis papulosa nigra, which is called pathological hyperpigmentation, or even acne, eczema, atopic dermatitis, rosacea, rosacea, redness, preferably chosen from Addison's disease, liver failure, purpura, melanoma, chloasma,
• treatment and/or prevention of pathologies of the skin and/or mucous membranes associated with oxidative stress means the treatment and/or prevention of numerous skin diseases. linked to excessive oxidative stress, including psoriasis, certain eczemas, acne, vitiligo, or even certain skin cancers, preferably chosen from vitiligo.
• the strain of Lactobacillus crispatus according to the invention can be found in the form of a pharmaceutical composition, preferably dermatological, comprising at least one pharmaceutically and/or dermatologically acceptable excipient.
• said composition is applied topically and/or orally, preferably topically.
• the pharmaceutical composition preferably dermatological, at a concentration of 1x10' 4 % to 10% (w/w) by weight, preferably between 1x10' 4 % and 5% (w/w) by weight, again advantageously between 1x10' 3 % and 0.5% (w/w) by weight relative to the total weight of the composition, said composition further comprising at least one pharmaceutically, preferably dermatologically acceptable, excipient.
• Example 1 Preparation of different forms of Lactobacillus crispatus
• Example 1.a production of whole viable form of Lactobacillus crispatus.
• MRS type culture medium Man's Agar, Rogosa, Sharpe
• Example 1.b production of the entire inactivated form of Lactobacillus crispatus
• Example 1.c production of the lysate of the bacteria Lactobacillus crispatus
• Example 1.d production of the secretome of the bacterium Lactobacillus crispatus
• Example 1.e production of the secretome of the bacteria Lactobacillus crispatus in powder form
• Example 2 Inhibition of melanin synthesis by melanocytes in the presence of the Lactobacillus crispatus strain according to the invention.
• Melanocytes from a B-16 murine line were seeded in a 96-well plate at a rate of 8000 cells/cm 2 , then cultured in MEM medium (Minimum Essential Medium) supplemented with 10% FBS (Fetal Bovine Serum) at 37°C, 5% CO2 and 95% Relative Humidity for 3 days.
• MEM medium Minimum Essential Medium
• FBS Fetal Bovine Serum
• the medium was then removed and replaced with 200pL of the medium previously described with the addition of 1 pM of NDP-a-MSH (analog of melanotropic hormone) used as a stimulator of melanin synthesis and in the presence of the Lactobacillus crispatus strain. according to example 1.a at a concentration of 0.1% (w/w), for 5 days.
• NDP-a-MSH analog of melanotropic hormone
• the strain of Lactobacillus crispatus according to Example 1.a product of the invention showed a significant inhibition of 52% of melanin synthesis by melanocytes of the B-16 murine line cultured in vitro. It can therefore be used to reduce pigmentation of the skin and/or mucous membranes and/or prevent its increase.
• Example 3 Inhibition of tyrosinase activity in the presence of the Lactobacillus crispatus strain according to the invention.
• Tyrosinase is a key enzyme in the melanin synthesis process responsible for skin tone.
• the tyrosinase used is extracted from normal human melanocytes.
• the strain of Lactobacillus crispatus according to Example 1.e of the invention showed a significant inhibition of the activity of tyrosinase extracted from human melanocytes by 99%. Through this action of inhibiting tyrosinase activity, the Lactobacillus crispatus strain can therefore be used to reduce the pigmentation of the skin and/or mucous membranes and/or prevent its increase.
• Example 4 Inhibition of melanin synthesis in a co-culture of melanocytes and keratinocytes in the presence of the Lactobacillus crispatus strain according to the invention.
• Normal human keratinocytes and human melanocytes from newborns were seeded at 20,000 cells/cm 2 and 10,000 cells/cm 2 respectively, then cultured for 7 days at 37°C under 5% CO2 and at 95% relative humidity. in a mixture of 50%/50% (v/v) of culture medium for melanocytes and culture medium for keratinocytes, supplemented with growth factors.
• the culture medium was replaced with a mixture of DMEM culture medium (Dulbecco's modified Eagle's minimum essential medium) without antibiotic and melanocyte medium without growth factor, and supplemented with 40pM oleic acid.
• DMEM culture medium Dulbecco's modified Eagle's minimum essential medium
• This culture medium without addition of the strain according to the invention was used as an untreated control (Control).
• the secretome of the bacteria Lactobacillus crispatus obtained according to Example 1.e was added at a concentration of 0.1% (w/w) relative to the total weight of the culture medium and the secretome.
• the cultures were continued for 72 hours at 37°C under 5% CO2 and at 95% relative humidity, then the culture medium was renewed. After 48 hours of additional culture, the medium was then eliminated, then the cells rinsed with PBS (phosphate buffer). Then, a 1 N sodium hydroxide solution containing 10% Dimethylsulfoxide (DMSO) was added to each well of the plate. 200pL from each well were transferred to a transparent plate. The optical density reflecting the quantity of melanin was read at 475nm.
• PBS phosphate buffer
• DMSO Dimethylsulfoxide
• results were the average of the tests performed ⁇ the standard deviation, expressed as a percentage, relative to the untreated control normalized to 100%.
• Statistical analysis of the results was carried out in relation to the untreated control using the One-Way AN OVA test.
• the strain of Lactobacillus crispatus according to Example 1.e of the invention showed a significant reduction of 32% in the synthesis of melanin by melanocytes cocultured in vitro with human skin keratinocytes.
• the strain of Lactobacillus crispatus, and in particular its secretome can therefore be used to reduce the pigmentation of the skin and/or mucous membranes and/or prevent its increase.
• Example 5 Reduction of oxidative stress in the presence of the Lactobacillus crispatus strain according to the invention.
• Example 5.a Reduction in the formation of DPPH° free radicals in tubo in the presence of the Lactobacillus crispatus strain according to the invention.
• the dosage of the anti-radical activity of the Lactobacillus crispatus strain was evaluated by a test using DPPH° (2,2-diphenyl-1-picrylhydrazyl).
• the strain of Lactobacillus crispatus according to Example 1.a was mixed in PBS (phosphate buffer saline) at a concentration of 0.05% (w/w), and according to Example 1.e at a concentration of 0.7% (w/w).
• PBS phosphate buffer saline
• a negative control was carried out using the strain dissolution solvent, PBS, and a positive control was carried out with vitamin C at 1 mM.
• Each of the solutions containing the invention, the negative control or the positive control was mixed at 50%/50% (v/v) in a DPPH° solution, in wells of 96-well plates.
• Each of the solutions containing the invention, the negative control or the positive control was also mixed at 50%/50% (v/v) in an ethanol solution which is the solvent for DPPH°, in order to produce a blank, in this same plate.
• the product of the invention was compared to its negative control (PBS alone in the absence of the Lactobacillus crispatus strain).
• the positive vitamin C control was compared to its negative control, water.
• the statistical analysis was carried out by a One Way ANOVA analysis of variance for the invention and by a Student test for vitamin C.
• the strain of Lactobacillus crispatus according to example 1.a of the invention showed a significant in tubo anti-radical activity of 34%.
• the Lactobacillus crispatus strain according to Example 1.e of the invention showed an in tubo anti-radical activity of 31%.
• the Lactobacillus crispatus strain can therefore be used to detoxify the skin and/or mucous membranes, to reduce the pigmentation of the skin and/or mucous membranes and/or prevent its increase.
• Example 5 Decrease in the release of cellular free radicals induced by UVs within fibroblasts in the presence of the Lactobacillus crispatus strain according to the invention.
• So-called “normal” human fibroblasts that is to say not presenting pathology, from a healthy donor were seeded in a 24-well plate at a rate of 30,000 cells/cm 2 , then cultured in a DMEM medium ( modified Eagle's minimum essential medium Dulbecco) / Ham's F-12, supplemented with 10% FCS and 0.5% antibiotic, for 96 hours at 37°C, under 5% CO2 and 95% relative humidity.
• DMEM medium modified Eagle's minimum essential medium Dulbecco
• Ham's F-12 supplemented with 10% FCS and 0.5% antibiotic
• Example 1a 0.5 mL of the cell suspension was incubated for 72 hours in the presence of the Lactobacillus crispatus strain according to Example 1.a at a concentration of 0.0125% (w/w), or an antioxidant control (Vitamin E at 0.0003% (v/v)) in EMEM medium (Eagle's minimum essential medium) supplemented with 1% FCS and 0.5% antibiotic.
• EMEM medium Eagle's minimum essential medium
• This culture medium without addition of the strain according to the invention was used as an untreated control (UVA control alone - Oxidative stress control).
• a DCFH-DA (2',7'-dichlorodihydrofluorescein diacetate) probe solubilized at 10 pM in PBS (phosphate buffer) was incubated with the cells. After the first hour of incubation at 37°C under 5% CO2 and 95% relative humidity, the cells were rinsed with PBS, and irradiated with UVA at 20J/cm 2 for 3 hours. A non-irradiated control was also produced. After irradiation, the irradiated medium was removed, and the cells were rinsed with PBS. The reading of the fluorescence on the cell layer linked to the formation of DCF (2', 7-dichlorofluorescein) from the probe (DCFH-DA) was read at 485 nm excitation and 538 nm emission.
• the results were compared against the UVA alone control normalized to 100% and statistically compared using the One Way ANOVA analysis of variance test.
• the Lactobacillus crispatus strain according to example 1.a of the invention showed a significant reduction at the cellular level of 56% in the release of intracellular free radicals induced by UVA oxidative stress.
• the Lactobacillus crispatus strain can therefore be used to detoxify the skin and/or mucous membranes and/or preserve and/or improve radiance and/or luminosity and/or homogeneity of the skin and/or mucous membranes, preferably reducing the presence and/or preventing the appearance of pigment spots on the skin and/or mucous membranes, particularly when these undergo oxidative stress, preferably by UVA.
• Example 5.c Decrease in the peroxidation of membrane lipids induced by UVs within fibroblasts in the presence of the Lactobacillus crispatus strain according to the invention.
• So-called “normal” human fibroblasts that is to say not presenting pathology, from a healthy donor were seeded in a 24-well plate at a rate of 30,000 cells/cm 2 , then cultured in a medium DMEM/Ham's F-12, supplemented with 10% FCS and 0.5% antibiotic, for 96 hours at 37°C, under 5% CO2 and 95% relative humidity. Then, 0.5 mL of the cell suspension was incubated for 72 hours in the presence of the Lactobacillus crispatus strain according to Example 1.a at a concentration of 0.00625% (w/w), or an antioxidant control (Vitamin E at 0.0003% (v/v)) in EMEM medium supplemented with 1% FCS and 0.5% antibiotic.
• This culture medium without addition of the strain according to the invention was used as an untreated control (after irradiation, constitutes the UVA control alone - Oxidative stress control).
• the cells were rinsed with PBS, and irradiated with UVA at 20J/cm 2 for 3 hours. A non-irradiated control was also produced. After irradiation, the supernatants were collected and the cells were rinsed with PBS.
• results were the average of the tests carried out and were expressed as Mean +/- one standard deviation.
• the results were compared against the UVA alone control normalized to 100% and statistically compared using the One Way ANOVA analysis of variance test.
• the Lactobacillus crispatus strain according to example 1.a of the invention showed a significant reduction at the cellular level in the formation of MDA induced by UVA of 32%.
• the Lactobacillus crispatus strain can therefore be used to detoxify the skin and/or mucous membranes, particularly when they undergo oxidative stress, notably UVA stress.
• Example 6 Stimulation of cellular antioxidant defenses after UV irradiation within fibroblasts in the presence of the Lactobacillus crispatus strain according to the invention.
• So-called “normal” human fibroblasts that is to say not presenting any pathology, from a healthy donor were seeded in a 24-well plate at a rate of 30,000 cells/cm 2 , then cultured in a DMEM/ Ham's F-12, supplemented with 10% FCS and 0.5% antibiotic, for 96 hours at 37°C, under 5% CO2 and 95% relative humidity.
• This culture medium without addition of the strain according to the invention was used as an untreated control (after irradiation, constitutes the UVA control alone - Oxidative stress control).
• Example 1a 0.5 mL of the cell suspension was incubated for 72 hours with the Lactobacillus crispatus strain according to Example 1.a at a concentration of 0.0125% (w/w), or an antioxidant control (Vitamin E at 0.0003% (v/v)) in EMEM medium supplemented with 1% FCS and 0.5% antibiotic.
• the cells were rinsed with PBS, and irradiated with UVA at 20J/cm 2 for 3 hours. A non-irradiated control was also produced. After irradiation, the irradiated medium was removed, and the cells were rinsed with PBS.
• Lactobacillus crispatus strain according to example 1.a of the invention showed a significant increase in cellular antioxidant defenses by an increase in glutathione of 54% after UVA irradiation.
• the strain of Lactobacillus crispatus can therefore be used to detoxify the skin and/or mucous membranes, particularly when they undergo oxidative stress, notably a UVA stress.
• Example 7 In vivo measurement of the reduction in the quantity of melanin in the skin in the presence of the Lactobacillus crispatus strain according to the invention.
• the assessment of the skin's melanin content was carried out using a device called Mexameter which uses the absorption/reflection principle. From the Mexameter measurements, a melanin index is deduced. A decrease in this index corresponds to a decrease in melanin.
• the melanin index was measured at the start of the study (DO) after one month of application (D28) and finally after 2 months of application (D56).
• Example 8 Example of a cosmetic composition comprising the strain of Lactobacillus crispatus according to the invention
• Example 8. a Example of a cosmetic composition comprising the strain of Lactobacillus crispatus according to Example 1.a
• Lactobacillus crispatus strain obtained according to Example 1 was added just before application to the skin in a formulation as defined below according to Table 8, at a final concentration of 0.05% (w/w ) relative to the total weight of the formulation.
• Example 8 Example of cosmetic composition comprising the strain of

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