Combination of lactobacillus strain and grape extract for use as anti-aging agent

Abstract

The present invention relates to a composition comprising: an inactivated Lactobacillus strain; and aqueous extracts obtained from grape seeds and / or grape skins, and their use as anti-aging agents, for example in cosmetics.

Description

Technical Field

[0001] The present invention relates to an active ingredient for use, for example, in cosmetics, which comprises a Lactobacillus strain and grape extract.

[0002] It has been shown that such an ingredient exhibits a protective function on the skin barrier, antioxidant activity, and anti-inflammatory activity, and thus can be used as an anti-aging agent. Background Art

[0003] Skin, whose main component is collagen, is the first interface between the human body and its environment, i.e., the interface between external and internal assaults.

[0004] Aging is not a disease but a normal biological phenomenon that affects each of us in different ways. Besides genetic factors, other factors amplify or even exacerbate this natural phenomenon.

[0005] Today, all strategies for attempting to slow down aging, from balanced nutrition to surgery, are possible challenges, but living and aging reasonably remain areas of research.

[0006] To fully understand the impact of time on skin physiology, it is important to understand two types of aging: intrinsic aging and extrinsic aging. Intrinsic aging is related to genetics, while extrinsic aging is of interest to many players in the cosmetics and pharmaceutical fields.

[0007] Among external factors, various environmental factors, such as solar ultraviolet (UV) radiation, visible light, environmental pollution (including ozone and particles), increase the production of reactive oxygen species (ROS) in the skin. Besides the active substances induced exogenously in the skin itself, some ROS are produced as by-products of oxidative metabolism in mitochondria. In addition, the components of the skin microbiota (or microbiome or flora) are affected by environmental factors such as ultraviolet light and pollutants and are also related to skin health. Finally, chronic psychological stress can also induce skin oxidative stress by reducing the anti-toxic defense ability. Oxidative stress and the resulting oxidative damage exacerbate pigmentation and skin aging, leading to skin changes such as skin color uniformity, wrinkles, sagging, dryness, and roughness.

[0008] More generally, aging is the result of an imbalance between degradation and repair systems (e.g., related to collagen metabolism), the result of chronic skin inflammation, and the result of the loss of skin tissue homeostasis (especially at the level of the skin microbiota).

[0009] Document FR 3 110 414 discloses a cosmetic composition for skin care, which comprises at least one ceramide, at least one tyndallized microorganism, such as Lactobacillus acidophilus and / or Lactobacillus casei, and at least one bisabolol.

[0010] Document CN 108 670 943 discloses an antiseptic cosmetic for topical use, which comprises an antioxidant in its oil phase and a lysate of Bifidobacterium longum (

[0055] ; CAS 96507-89-0) and a grape seed extract mainly containing procyanidins (

[0054] ; CAS 84929-27-1) in its aqueous phase.

[0011] Document CN 112 825 990 discloses a cosmetic for topical use, which comprises a grape extract obtained as follows: fermenting red grape juice with yeast and / or lactic acid bacteria at 30 °C for 72 hours, and then concentrating and filtering.

[0012] Document CN 112 244 299 discloses a probiotic drinkable composition, which comprises a freeze-dried Lactobacillus strain with antioxidant activity and a grape powder (i.e., a non-aqueous extract) containing resveratrol.

[0013] Document WO2011 / 070508 discloses the use of probiotics comprising inactivated (e.g., at high temperature) Lactobacillus (e.g., Lactobacillus paracasei or Lactobacillus gasseri) in a topical composition for skin brightening. Various antioxidants, such as grape extract rich in procyanidins, can be added.

[0014] 《Microbiota P3 Balancing Mist》(MINTEL) discloses a topical product, which comprises a mixture of lysates of 3 Lactobacillus species and a grape extract (Vitis Vinifera fruit extract).

[0015] However, there is still a need to provide further skin anti-aging strategies. Summary of the Invention

[0016] As defined in the claims, the present invention relates to a composition which combines a Lactobacillus strain and a specific grape extract rich in polyphenols, the polyphenols particularly including tannins such as crown tannin.

[0017] To the best knowledge of the applicant, such a combination having many advantageous properties reported in the present application and useful, for example, in cosmetics has never been disclosed.

[0018] Definition

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The terms used in the specification are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0020] The articles "a" and "an" are used herein to refer to one or more than one (i.e., at least one) of the grammatical objects of the article. For example, "an element" means one element or more than one element.

[0021] When referring to measurable values such as amounts, durations, etc., the terms "about" or "approximate" as used herein mean a variation of ±20% or ±10% of the specific value, more preferably ±5%, even more preferably ±1%, still more preferably ±0.1%, because such variations are suitable for implementing the disclosed methods.

[0022] Ranges: Throughout the disclosure, various aspects of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an immutable limitation on the scope of the invention. Thus, the description of a range should be considered to have specifically disclosed all possible sub-ranges as well as the individual numerical values within that range. For example, the description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 2, from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as the individual numerical values within that range, e.g., 1, 2, 3, 4, 5, and 6. This applies to any width of the range.

[0023] The term "isolated" refers to being altered or removed from its natural state. For example, a nucleic acid or peptide that naturally exists in a living animal is not "isolated", but the same nucleic acid or peptide that is partially or completely separated from the coexisting substances in its natural state is "isolated". An isolated nucleic acid or protein may exist in a substantially purified form or may exist in, for example, a non-natural environment such as a host cell.

[0024] As used herein, the terms "compound", "composition", "component" or "reagent" may be used interchangeably and refer to a chemical or biological molecule, which may be pure or mixed with other molecules. The compound or composition or molecule may have an activity of interest, such as an activity of interest in beauty or treatment, and may therefore also be referred to as an "active" or "active ingredient". In the case where the compound or composition or molecule is only used for formulating a compound of interest, it may be designated as an "excipient" or "adjuvant". With respect to the compositions of the present invention, cosmetically or pharmaceutically acceptable excipients or adjuvants are preferably used. Detailed Description of the Invention

[0026] According to a first aspect, the present invention relates to a composition comprising:

[0027] - an inactivated Lactobacillus strain; and

[0028] - an aqueous extract extracted from grapes.

[0029] According to a preferred embodiment, the Lactobacillus strain present in the composition belongs to Lactobacillus gasseri or Lactobacillus paragasseri, advantageously Lactobacillus paragasseri.

[0030] According to a specific embodiment, the Lactobacillus strain is the strain deposited with the CNCM (National Collection of Microorganisms, Pasteur Institute, 25 rue du Docteur Roux, 75724 Paris Cedex 15) under number I-5892 by LARENA (1, ZI du Taillis, Champtoceaux, F-49270 Orée d’Anjou) on September 13, 2022.

[0031] According to one embodiment, the Lactobacillus strain is inactivated by sterilization, advantageously by flash pasteurization or high temperature short time (HTST) treatment, more advantageously by flash pasteurization. In practice, the inactivation step is carried out at a high temperature but for a short time, for example under the following conditions:

[0032] - at a temperature above (greater than or equal to) 100 °C, advantageously 105 °C, 110 °C, 115 °C, 120 °C, 125 °C, 130 °C, 135 °C or even 140 °C. This temperature may include from 115 °C to 140 °C, preferably from 115 °C to 130 °C, for example 124 °C. Alternatively, a temperature of 105 to 120 °C may be applied; and / or

[0033] - a duration below (less than or equal to) 1 minute, advantageously 50, 40, 30, 20, 10 or even 5 seconds. This duration may include from 15 to 60 seconds, preferably from 15 to 40 seconds, for example 30 seconds. Alternatively, a time as short as 5 seconds may be used.

[0034] According to a specific embodiment, the inactivation is achieved by the bacteria being subjected to the following conditions:

[0035] - a temperature of 115 - 130 °C for 5 seconds; or

[0036] - a temperature of 105 - 120 °C for 5 seconds.

[0037] According to a specific embodiment, the inactivation of the bacteria is not carried out by tyndallization. As is known in the art, the tyndallization process typically uses an intermittent sterilization method, i.e., cycles of heating (at a temperature below 60 °C and for up to 24 hours) and cooling of the lactic acid bacteria. The purpose of this process is to remove spores and bacteria in the tyndallization product.

[0038] Preferably, the strain is inactivated after being mixed with the grape extract and possibly other components present in the composition according to the invention.

[0039] Thus, the live bacteria are stabilized by the grape extract, and due to a suitable sterilization method, the composition according to the invention contains a high percentage of intact bacteria. The bacterial fraction of the composition according to the invention is different from bacterial biomass lysates or fermentation supernatants.

[0040] According to a preferred embodiment of the invention, the bacterial fraction accounts for 0.1 to 2% w / w (weight / weight) of the composition, advantageously 0.5 to 2% w / w or 1 to 2% w / w.

[0041] Before inactivation, the composition advantageously contains at least 10 6 CFU (colony forming units) of bacteria / ml, more advantageously at least 10 7 or 10 8 or 10 9 or even 10 10 or 10 11 CFU / ml, for example 10 9 CFU / ml.

[0042] After inactivation, the composition advantageously contains at least 50% or 60%, more advantageously at least 70%, 80% or even 90% of intact bacteria. The percentage of intact bacteria can be measured by any technique known to the person skilled in the art, for example by flow cytometry to evaluate the membrane integrity of the bacteria.

[0043] According to a specific embodiment, the composition according to the invention contains an inactivated Lactobacillus strain, advantageously Lactobacillus paracasei, more advantageously strain I-5892, wherein most of the bacterial cells, i.e., at least 50% of the cells, are intact.

[0044] In the context of the present application, "intact" means that the cells are alive, or have a completely intact or slightly damaged membrane. In contrast, non-intact cells represent cells with a highly damaged membrane or even lysates and cell debris. As is known in the art, viable (living) lactic acid bacteria can be counted according to the French standard NF ISO 7889, while intact lactic acid bacteria can be counted according to the French standard NF ISO 19344.

[0045] After inactivation, the bacteria in the composition cannot be evaluated in terms of colony forming units (CFU) because most or all of them are inactivated and cannot reproduce anymore. However, intact cells can be evaluated as described above. In practice and after inactivation, the composition of the present invention advantageously comprises at least 10 5 intact cells / ml, more advantageously at least 10 6 or 10 7 or 10 8 or 10 9 or even 10 10 or 10 11 intact cells / ml.

[0046] According to the present invention, the second component of the present invention is grape extract. According to one embodiment, the grape used in the framework of the present invention is fermented red grape. According to one embodiment, the grape extract is obtained from grape seeds and / or grape skins, advantageously from grape seeds and grape skins. The grape seeds and / or grape skins can be obtained from the pomace and mechanically separated.

[0047] The original plant material, i.e., advantageously the seeds and / or grape skins from fermented red wine grapes present in the pomace, is in the form of fresh matter and can be stored under cold conditions, advantageously under cold negative conditions, or dried by any technique known to the person skilled in the art, such as vacuum drying at 40 - 50°C.

[0048] According to another embodiment, the grape extract is an aqueous extract. In practice, an aqueous extraction method is carried out on the raw material, which allows the dissolution of a group of non-polar substances of interest, especially polyphenols including tannins.

[0049] Aqueous extraction means incubating the grape material with an aqueous extraction solvent. This solvent can be pure water or a mixture of water and a small amount of alcohol, such as 20% volume / volume (V / V) ethanol.

[0050] Generally, this extraction is carried out under the following conditions:

[0051] - Temperature between 50°C and 80°C;

[0052] - For a period of 1 to 2 hours, preferably 1 hour 30 minutes to 2 hours;

[0053] - The volume ratio (V / V) between the solvent and the substance to be extracted is 1 to 10, preferably 1 to 5, such as 4.2.

[0054] After incubation, the extract product (i.e., the liquid crude extract) is preferably clarified by centrifugation (e.g., at 6000 - 11000 g) or filtration through a membrane with a cut-off value of, for example, 15 to 60 μm.

[0055] Advantageously, after extraction and possible clarification or filtration, the dry matter (DM) including the molecules of interest, especially tannins, accounts for at least 0.5% or even 1% of the extract.

[0056] The next stage is to purify the extract by a membrane purification method, advantageously by ultrafiltration, to obtain tannin enrichment.

[0057] Generally, this ultrafiltration is carried out under the following conditions:

[0058] - Using a membrane with a cut-off value ranging from 10 to 200 kDa, preferably 20 to 100 kDa, more preferably 20 to 50 kDa;

[0059] - For a period that can reach several hours.

[0060] Both the aqueous extraction and ultrafiltration aim to concentrate especially proanthocyanidin oligomers and condensed trimeric, tetrameric, and pentameric tannins (including crown tannins).

[0061] The extract of interest corresponds to the retentate after ultrafiltration and generally contains 40 - 50% dry matter, including the molecules of interest, especially tannins.

[0062] The retentate can be diafiltered with water.

[0063] The retentate is usually in liquid form, but can be dried by any technique known in the art, such as by vacuum concentration until a dry extract is obtained.

[0064] According to one aspect, the present invention also relates to a method for obtaining an aqueous extract to be incorporated into the claimed composition, which comprises:

[0065] - Collecting seeds and / or skins, advantageously collecting skins and seeds from grapes, more advantageously skins and seeds from fermented red grapes;

[0066] - Extracting them with water or an aqueous solvent;

[0067] - Ultrafiltering the extract;

[0068] - Collecting the retentate corresponding to the aqueous extract of interest;

[0069] - Optionally drying the retentate.

[0070] According to a preferred embodiment, the aqueous extract to be incorporated into the composition of the invention has at least one of all the following characteristics, and advantageously has all the following characteristics:

[0071] - The total amount of polyphenols advantageously represents at least 2, 3, 4, 5, 6 or even 7, 8, 9 or 10% w / w of the dry matter, such as 2 to 5% or 2 to 6%, such as 4%;

[0072] - The total amount of tannins advantageously represents at least 2, 3, 4, 5, 6, 7 or even 8, 9 or 10% w / w of the dry matter, such as 2 to 7% or 3 to 6%, such as 5%;

[0073] - The amount of molecular tannins advantageously represents at least 0.05, 0.1, 0.15 or even 0.2% w / w of the dry matter, such as 0.05 to 0.2% or 0.05 to 0.15%, such as 0.12%;

[0074] - The amount of crown tannins advantageously represents at least 0.005, 0.01, 0.015 or even 0.02% w / w of the dry matter, such as 0.005 to 0.02% or 0.05 to 0.015%, such as 0.01%.

[0075] As is known in the art, tannins are a specific type of polyphenol, i.e., molecules containing flavan-3-ol units:

[0076]

[0077] wherein

[0078] 1. (+)-Catechin ((+)-Catéchine) R1 = H, R2 = H, R3 = OH

[0079] 2. (-)-Epicatechin ((-)- ) R1 = H, R2 = OH, R3 = H

[0080] 3. (+)-Gallocatechin ((+)-Gallocatéchine) R1 = OH, R2 = H, R3 = OH

[0081] 4. (-)-Epigallocatechin R1 = OH, R2 = OH, R3 = H

[0082] ((-)- )

[0083] 5. (-)-Epicatechin-3-O-gallate R1 = H, R2 = O-gallate, R3 = H

[0084] ((-)- )

[0085] Tannins can exist in monomeric or polymeric form. In the framework of the present invention, molecular tannins (or condensed tannins) correspond to polymers of flavan-3-ols, including dimers, trimers, tetramers, ….

[0086] The so-called crown tannins include tetramers having the following structure:

[0087]

[0088] and pentamers having the following structure:

[0089]

[0090] Methods for determining the nature and quantity of these different types of molecules are known in the art, such as those disclosed by Drinkine, J. et al. (J. Agric. Food Chem. 2007, 55(4), 1109-1116) and Lorrain, B et al. (Food Chem. 2011, 126, 1991–1999), and are illustrated in the examples.

[0091] According to another preferred embodiment, the aqueous extract to be incorporated into the composition of the present invention has the following composition:

[0092] - The total amount of tannins advantageously accounts for at least 2, 3, 4, 5, 6, 7 or even 8, 9 or 10% w / w of the dry matter, for example 2 to 7% or 3 to 6%, such as 5%;

[0093] - The amount of molecular tannins advantageously accounts for at least 0.05, 0.1, 0.15 or even 0.2% w / w of the dry matter, for example 0.05 to 0.2% or 0.05 to 0.15%, such as 0.12%;

[0094] - The amount of crown tannins advantageously accounts for at least 0.005, 0.01, 0.015 or even or 0.02% w / w of the dry matter, for example 0.005 to 0.02% or 0.05 to 0.015%, such as 0.01%.

[0095] After adding the other components of the composition and inactivating the bacteria, the amounts in the extract remain stable.

[0096] According to another preferred embodiment, the aqueous extract accounts for 4-8% w / w of the composition according to the present invention.

[0097] Thus, the composition according to the present invention can advantageously comprise:

[0098] - The total amount of polyphenols accounts for 0.08 to 0.4% w / w of the dry matter; and / or

[0099] - The total amount of tannins accounts for 0.12 to 0.48% w / w of the dry matter; and / or

[0100] - The amount of molecular tannins accounts for 0.002 to 0.012% w / w of the dry matter; and / or

[0101] - The amount of crown tannins accounts for 0.0002 to 0.0012% w / w of the dry matter.

[0102] More preferably, the composition according to the invention may comprise:

[0103] - The total amount of tannins accounts for 0.12 to 0.48% w / w of the dry matter;

[0104] - The amount of molecular tannins accounts for 0.002 to 0.012% w / w of the dry matter; and

[0105] - The amount of crown tannins accounts for 0.0002 to 0.0012% w / w of the dry matter.

[0106] According to another aspect, the present invention relates to a method for preparing the claimed composition, which comprises:

[0107] - Mixing an aqueous extract with Lactobacillus; and

[0108] - Sterilizing the mixture, preferably by flash pasteurization.

[0109] According to a preferred embodiment, the Lactobacillus strain is cultured under fermentation conditions and the bacteria are collected after centrifuging the growth medium. This fraction, i.e., the pellet, is generally referred to as bacterial biomass. It can be stored at -20 - 80°C.

[0110] Preferably, the bacterial fraction generally contains at least 10 8 CFU (colony forming units) of bacteria / ml, more preferably at least 10 9 or 10 10 or 10 11 or even 10 12 CFU / ml, for example 10 9 CFU / ml to 10 11 CFU / ml.

[0111] According to another embodiment, the other components of the composition are added before sterilization.

[0112] According to a specific embodiment, the composition further comprises:

[0113] - Water, preferably accounting for 45 to 70% w / w of the composition, more preferably 50 to 65% w / w;

[0114] - Glycerol, advantageously present in the composition in an amount of 15 to 40% w / w, more advantageously 20 to 40% w / w;

[0115] - Xanthan gum, advantageously present in the composition in an amount of 0.05 to 0.3% w / w, more advantageously 0.1 to 0.25% w / w;

[0116] According to another aspect, the present invention relates to the use of the composition according to the invention in cosmetics or pharmaceuticals.

[0117] Examples of the cosmetic composition according to the invention are as follows:

[0118]

[0119]

[0120] According to a preferred embodiment, the composition according to the invention is used as an anti-aging agent, an antioxidant and / or an anti-inflammatory agent.

[0121] According to a specific embodiment, the composition according to the invention is used for preventing or treating skin irritation and / or skin redness and / or skin hydration and / or skin barrier protection.

[0122] In terms of its ability to protect the skin barrier, the composition according to the invention can be used for preventing or even treating diseases associated with skin dysbiosis, such as: atopic dermatitis (AD), psoriasis and hidradenitis suppurativa (HS).

[0123] In terms of its antioxidant activity, the composition according to the invention can be used for preventing or even treating cancer, especially skin cancer.

[0124] According to its application, the composition of the present invention can be formulated for topical administration or oral administration, and then used topically or orally.

[0125] In the case of oral administration, for example when the composition of the present invention is a nutritional product, it can be in the form of a solid, liquid, solution, suspension, paste or gel.

[0126] According to one embodiment, it is in solid form prepared in a conventional manner using acceptable additives such as binders, fillers, lubricants, disintegrants or wetting agents, such as powders, tablets (including effervescent tablets), pills, capsules or lozenges. The tablets can optionally be coated with sugar, film or enteric coatings in a manner known to those skilled in the art.

[0127] Alternatively, other delivery systems can be used, such as formulations in soft gelatin or gelatin-based capsules, or formulations prepared using nanotechnology, such as nanodispersions, nanoemulsions or nanocapsules.

[0128] This composition may also contain one or more additives, such as colorants, pigments or flavorings...

[0129] When the composition according to the invention is used for cosmetic purposes, especially for topical application, its form can be cream, ointment, mask, serum, lotion, wash, paste, foam, aerosol, stick, deodorant, shampoo, conditioner, patch, water-alcohol or oily solution, oil-in-water (O / W) or water-in-oil (W / O) or multiple emulsion, aqueous or oily gel, liquid, pasty or solid anhydrous product, and / or dispersion of oil in an aqueous phase using spheres, which may be polymeric nanoparticles such as nanospheres and nanocapsules or ionic and / or non-ionic lipid vesicles.

[0130] The composition according to the invention may also contain at least one other cosmetically acceptable compound, preferably selected from soothing agents, restructuring agents, regenerating agents, conditioning agents, sunscreen agents, anti-wrinkle agents, moisturizing agents, anti-aging agents, surfactants, fatty substances, organic solvents, solubilizers, thickeners and gelling agents, smoothing agents, firmness enhancers, elasticity and / or skin barrier effects, antioxidants, opacifiers, stabilizers, foaming agents, fragrances or perfumes, ionic or non-ionic emulsifiers, fillers, polyvalent chelating agents and chelating agents, filters, essential oils, colorants, pigments, hydrophilic or lipophilic active agents, lipid vesicles encapsulating one or more active agents and / or preservatives.

[0131] By way of example, the composition according to the invention may contain an extract of Ganoderma lucidum and / or apricot oil and / or jojoba seed oil and / or squalane and / or niacinamide and / or glycerol and / or fruit extract of pomegranate and / or glyceril stearate and / or glyceryl stearate and / or alkane of coconut and / or hemp oil and / or sunflower seed oil, and / or jojoba (Simmondsia chinensis) seed oil, and / or pentylene glycol and / or fermented root filtrate and / or sclerotic gum and / or lysophosphatidylcholine and / or caprylic / capric triglyceride and / or maltodextrin, and / or xanthan gum and / or pullulan and / or butylene glycol and / or disodium cocoyl glutamate and / or lactic acid and / or sodium cocoyl glutamate and / or sodium hyaluronate and / or tocopherol and / or squalene and / or β-sitosterol and / or pine bark extract and / or red ginseng and / or white hemlock (Conium maculatum) and / or extract of Cornus officinalis and / or extract of Boesenbergia rotunda and / or N-acetylglucosamine and / or hyaluronic acid and / or extract of Amorphophallus konjac and / or collagenase-digested AP peptide and / or extract of Taraxacum mongolicum.

[0132] The skin area to be treated can be the face or the body, advantageously the face, neck and neckline.

[0133] According to a specific embodiment, the composition of the present invention:

[0134] - For skin moisturization or hydration;

[0135] - For protecting or enhancing the protection of the skin barrier of the skin;

[0136] - For alleviating skin irritation;

[0137] - A skin soothing composition; and / or

[0138] - An anti-redness composition for the skin.

[0139] According to another aspect, the present invention relates to a method of beauty care, comprising the step of applying the composition according to the present invention to a skin area.

[0140] Preferably, 0.1 to 50 mg / cm 2 , preferably 0.25 to 2.5 mg / cm 2 of the cosmetic composition according to the present invention is applied to the skin area.

[0141] Preferably, the application step is carried out on the face, neck and neckline.

[0142] The composition according to the present invention can be applied one to two times a day, preferably two times a day, for at least 7 days, preferably at least 15 days, more preferably at least one month, particularly preferably at least two months, and even more particularly preferably at least three months.

[0143] According to a particular embodiment, in the method according to the present invention, the cosmetic composition is applied 2 times a day for at least 3 months.

[0144] Unless otherwise indicated, the practice of the present invention employs conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the knowledge of those of ordinary skill in the art. These techniques are well explained in the literature, such as "Molecular Cloning: A Laboratory Manual", 4th Edition (Sambrook, 2012); "Oligonucleotide Synthesis" (Gait, 1984); "Culture of Animal Cells" (Freshney, 2010); "Methods in Enzymology", "Handbook of Experimental Immunology" (Weir, 1997); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Short Protocols in Molecular Biology" (Ausubel, 2002); "Polymerase Chain Reaction: Principles, Applications and Troubleshooting" (Babar, 2011); "Current Protocols in Immunology" (Coligan, 2002). These techniques are applicable to the production of the polynucleotides and polypeptides of the present invention and can, therefore, be considered in the manufacture and practice of the present invention.

[0145] Techniques that are particularly useful for specific embodiments will be discussed in the following sections.

[0146] The disclosures of each and all patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety.

[0147] Without further description, it is believed that one of ordinary skill in the art can make and utilize the compounds of the present invention and practice the claimed methods using the foregoing description and the following illustrative examples.

[0148] Experimental Examples

[0149] The present invention is further described in detail with reference to the following experimental examples and drawings. These examples are provided for illustrative purposes only and are not intended to be limiting. Brief Description of the Drawings

[0150] Figure 1 ： Enhanced effect of the composition according to the present invention in protecting skin barrier integrity

[0151] Growth of the commensal bacterium Staphylococcus epidermidis on a reconstructed epidermal model colonized with bacteria after the individual application of 0.29% (w / w) of the extract (E), the individual application of 0.079% (w / w) of inactivated bacteria (B), and then the application of 0.3% (w / w) of a combination of both (A = E + B).

[0152] The histograms represent values obtained from 3 replicates for each condition and are expressed as a percentage (%) of the untreated control. Statistical analysis (Student's t-test) was performed, comparing all cases to the untreated control.

[0153] Figure 2 ： Compared to the reference molecule (300 μg / ml of vitamin C), the effect of 0.29% (w / w) of grape extract (E) alone and 0.3% (w / w) of the composition (A) according to the invention on ROS production in keratinocytes subjected to UVA 5 J / cm 2

[0154] The histograms represent values obtained from 3 replicates for each condition and are expressed as a percentage (%) of the untreated control. Statistical analysis (Student's t-test) was performed, comparing all cases to the untreated control. Values of 0.01 < p < 0.05 were considered significant (*), 0.001 < p < 0.01 were considered highly significant (**), and < 0.001 was considered very highly significant (***).

[0155] Figure 3 ： Abundance of pro-MMP1 in NHDF treated for 72 hours with 0.029% (w / w) of the extract (E) alone, 0.0079% (w / w) of inactivated bacteria (B) alone, and 0.03% (w / w) of a combination of both (A = E + B).

[0156] The histograms represent values obtained from 3 replicates for each condition. Statistical analysis (Student's t-test) was performed, comparing all cases to the untreated control. Values of 0.01 < p < 0.05 were considered significant (*), 0.001 < p < 0.01 were considered highly significant (**), and < 0.001 was considered very highly significant (***).

[0157] I / Preparation of the composition according to the invention

[0158] I-1 / Preparation of bacteria (B)

[0159] Strain:

[0160] ​The bacterial strain used in the examples is a strain of the species Lactobacillus paracasei, which was deposited on September 13, 2022, under the number I-5892 at the CNCM (National Collection of Microorganisms, Pasteur Institute, 25 rue du Docteur Roux, 75724 Paris Cedex 15) by LARENA (1, ZI du Taillis, Champtoceaux, F-49270 Orée d’Anjou).

[0161] Growth conditions

[0162] Culture medium:

[0163] A suitable medium for culturing the strain under fermentation conditions has, for example, the following composition (Table 1):

[0164]

[0165]

[0166] Condition

[0167] The strain was cultured under traditional fermentation conditions.

[0168] The bacteria were collected by centrifugation (5000 g, 10 minutes), and the pellet fraction generally contains 10 8 to 10 13 and usually 10 10 to 10 12 CFU (or bacteria) / ml.

[0169] Preparation of I-2 / grape extract (E)

[0170] Extracts containing the molecules of interest, in particular tannins, were obtained from different varieties and different parts of the grape, namely the skins and seeds of fermented red grapes separated from the pomace.

[0171] The raw material was then subjected to aqueous extraction by mixing it with 4.2 (w / w) parts of water at a temperature of 50 °C to 80 °C for 1 hour 30 minutes to 2 hours.

[0172] The extract was then clarified by filtration using a membrane with a cut-off value of 50 μm. At this stage, the dry matter (DM) of the extract, expressed as weight / weight (w / w), was estimated to be 0.9%, which was measured for a few minutes with a 3 - 5 g probe in a dryer (Mettler Toledo) at a temperature of 110 - 115 °C.

[0173] In the next stage, the extract is ultrafiltered to concentrate the molecules of interest. In practice, ultrafiltration is carried out using a membrane with a cut-off value of 10 - 50 kDa.

[0174] The extract of interest corresponds to the retentate after ultrafiltration and usually contains 40 to 50% dry matter, while the permeate containing the remaining dry matter is discarded.

[0175] Percolation is carried out with water, and then the retentate is concentrated in vacuo until an extract (E) is obtained.

[0176] The following protocol is used to determine the composition of this extract:

[0177] Reagents and standards

[0178] Deionized water was purified using a Milli-Q water system (Millipore, Bedford, MA, USA). HPLC-grade acetonitrile, ethyl acetate, chloroform, methanol, ethanol, and acetone were purchased from Scharlau (Sentmenat, Barcelona, Spain). The following chemicals were from Sigma Aldrich (St. Louis, MO, USA): (+)-catechin, (-)-epicatechin, B1 [(-)-epicatechin-(4β-8)-(+)-catechin], procyanidin dimer B2 [(-)-epicatechin-(4β-8)-(-)-epicatechin], gallic acid.

[0179] Procyanidin dimer B3 [(+)-catechin-(4α-8)-(+)-catechin], B4 [(+)-catechin-(4α-8)-(-)-epicatechin], and trimer (C1) [(+)-catechin-(4β-8)-(+)-catechin-(4β-8)-(-)-epicatechin]

[55] were synthesized in the Organic Chemistry and Organometallic Laboratory (Université Bordeaux 1).

[0180] Analysis of grape extracts

[0181] Tannin extraction

[0182] Separate grape extract (E) (1 g of dry matter) was dissolved in water / ethanol (250 mL, 95:5, v / v) and partitioned three times with chloroform (250 mL) to remove lipophilic substances. The aqueous phase was then extracted three times with ethyl acetate (250 mL) to obtain two different fractions: a low molecular weight procyanidin fraction (monomers / oligomeric tannins) in the organic phase and a high molecular weight procyanidin fraction (polymeric tannins) in the aqueous phase. These two fractions were concentrated and lyophilized.

[0183] Polyphenols and total tannins

[0184] The crude extract was dissolved at an appropriate concentration in water / ethanol (90:10, v / v; pH 3.5, using tartaric acid). The polyphenol content was measured using the Folin-Ciocalteu reagent as described by Singleton and Rossi (Am J Enol Vitic. 1965, 16(3), 144 - 158). The total tannin content was determined by acid hydrolysis using the method of Ribereau-Gayon and Stonestreet (Chim. Anal. 1966, 48, 188 - 196).

[0185] Determination of individual tannins by HPLC analysis

[0186] The extract was dissolved at an appropriate concentration in a methanol / water solution (50:50, v / v), and monomeric / oligomeric tannins (catechin, epicatechin, dimers B1, B2, B3, B4; trimer C2) were analyzed according to the method of Silva et al. (Eur. Food Res. Technol. 2012, 234, 361–365). The data were expressed as catechin equivalents / g dry weight GPE.

[0187] Determination of the mean degree of polymerization (mDP)

[0188] The mean degree of polymerization (mDP) of proanthocyanidins in the monomeric / oligomeric and polymeric tannin fractions of GPE was determined by phloroglucinolysis (Drinkine, J. et al., J. Agric. Food Chem. 2007, 55(4), 1109 - 1116). Analysis was carried out using the same method described by Lorrain et al. (Food Chem. 2011, 126, 1991–1999).

[0189] In an acidic medium, in the presence of the nucleophile phloroglucinol, oligomeric and polymeric proanthocyanidins are depolymerized. Reverse-phase HPLC analysis of the products formed allows the determination of the structural composition of the proanthocyanidins, characterized by the nature of their constitutive extension units (released as flavan-3-ol phloroglucinol adducts) and terminal units (released as flavan-3-ols). To calculate the apparent mDP, the sum of all subunits (flavan-3-ol monomers and phloroglucinol adducts, in moles) is divided by the sum of all flavan-3-ol monomers (in moles). GPE samples were analyzed using a Surveyor series instrument (Thermo-Finnigan, Les Ullis, France) equipped with a 100x 4.6 mm i.d., 3.5 μm X-Terra reverse-phase C18 column (Waters), thermostated at 25 °C. Detection was carried out at 280 nm using a Finnigan Surveyor PDA Plus detector. Mass detection was performed using a Finnigan LCQ DECAXP MAX mass spectrometer with an ESI interface, operating in positive mode with the following parameters: capillary temperature 325 °C, capillary voltage 4 V, nebulizer gas flow rate 1.75 L / min, desolvation gas flow rate 1 L / min, and spray voltage 5 kV. The solvents used were solvent A: H2O / AcOH (99:1 v / v), and solvent B: MeOH. The gradient consisted of 5% B for 25 minutes, a linear gradient of 5%–20% B in 20 minutes, then 20%–32% B in 15 minutes, and finally 32%–100% B in 2 minutes. The column was washed with 100% B for 5 minutes and then equilibrated with the initial conditions for 10 minutes. The injection volume was 20 μL. The flow rate was 1 mL / min.

[0190] Determination of the procyanidins in E

[0191] The UPLC-MS system used was an Agilent 1290 Infinity equipped with an ESI-Q-TOF-MS (Agilent 6530 Accurate Mass). Chromatographic separation was carried out on an Eclipse Plus C18 column (2.1 x 100 mm, 1.8 μm). The solvents used were: water containing 0.1% formic acid as solvent A, and methanol containing 0.1% formic acid as solvent B, with a flow rate of 0.3 ml / min. The gradient of solvent B for analysis was as follows: 6% B for 80.5 minutes; 6 - 95% B in 13.5 minutes; 95% B held for 4 minutes, and the UPLC column was equilibrated with the starting conditions for 3 minutes. The ESI conditions were as follows: the gas temperature and flow rate were set at 300 °C and 9 L / min, respectively; the jacket gas temperature and flow rate were set at 350 °C and 11 L / min, respectively; the capillary voltage was set at 4000 V. The fragmenter was always set at 200 V. The data obtained were processed using Mass Hunter qualitative analysis software. A calibration curve was established using previously purified procyanidin coronatetramer for the quantification of procyanidin coronatetramer and procyanidin coronapentamer. The results were expressed as mg coronatetramer equivalent / g dry matter.

[0192] The results are shown in Table 2 below:

[0193]

[0194] I-3 / Preparation of the composition according to the invention (A = B + E)

[0195] The active substance (A) according to the present invention can have the following composition (Table 3):

[0196]

[0197] In practice, the different components are mixed in the weight ratios shown in Table 3 above. The resulting liquid-form composition is sterilized (pasteurization) at 124 °C for 30 seconds. This sterilization technique can inactivate bacteria while minimizing their damage and maintaining their integrity. As a result, the composition contains more than 80% intact cells and less than 20% damaged cells.

[0198] This composition is stored under aseptic conditions and is further formulated into a cosmetic composition as described, for example, below.

[0199] II / Cosmetic solution according to the invention

[0200] The composition according to the present invention can be in the form of a solution containing the composition (A) of the present invention and other cosmetically acceptable excipients. Such a formulation can have the composition disclosed in Table 4 below:

[0201]

[0202]

[0203] III / Beneficial effects of the composition according to the invention

[0204] Materials and methods:

[0205] 1 / Cell models

[0206] 1.1 / Normal human epidermal keratinocytes (NHEK)

[0207] Normal human foreskin-derived epidermal keratinocytes isolated from 3 Caucasian donors (NHEK, Lonza, 00192906) were grown in Epilife medium (Fisher Scientific, M-EPI-500-A) supplemented with human keratinocyte growth supplement (HKG, Fisher Scientific, S-001-5). The cells were maintained in a humidified incubator at 37 °C and 5% CO2 atmosphere.

[0208] 1.2 / Recombinant human epidermis (RHE)

[0209] Epidermis was reconstructed in vitro with NHEK keratinocytes (RHE, StratiCELL). Tissues were cultured at the air-liquid interface in Epilife medium (Fisher Scientific, M-EPI-500-A) containing supplements. They were maintained in a humidified gas environment at 37 °C and 5% CO2 atmosphere.

[0210] 1.3 / Normal human dermal fibroblasts (NHDF)

[0211] Normal human foreskin-derived dermal fibroblasts (NHDF; ATCC, CRL-2522) cells were grown in Dulbecco's Modified Eagle Medium (DMEM, Gibco / Life Technologies, 31885) supplemented with 10% fetal bovine serum (FBS, Gibco / Life Technologies, 10270). The cells were maintained in a humidified incubator at 37 °C and 5% CO2 atmosphere.

[0212] 2 / Growth of Staphylococcus epidermidis on 3D RHE

[0213] 2.1 / Inoculation of RHE with Staphylococcus epidermidis

[0214] After 16 days (D16) at the air-liquid interface, reconstructed human epidermis (RHE) was inoculated with Staphylococcus epidermidis (ATCC 12228, Fisher Scientific, 12999068, lot: 465973). Staphylococcus epidermidis stored at -80 °C was initially plated on tryptic soy brain agar (TSB + agar = TSA, VWR, 20767.232) and incubated at 37 °C for one day. Colonies were transferred to liquid medium TSB and grown at 37 °C until mid-log phase. Bacteria were harvested to prepare an inoculum of 10 3 CFU (colony-forming units) / RHE for monitoring bacterial growth in Epilife. The bacterial suspension was added topically to the RHE for 24 hours. The determined concentrations of the test items prepared in Epilife were: 0.079% for B, 0.29% for E, and 0.3% for A.

[0215] 2.2 / Control of inoculum and sterility

[0216] The inoculum was serially diluted and spotted onto TSA agar plates. After incubation at 37 °C for one day, colonies were counted and the number of CFU / mL (N) was determined using the following formula: N = n x (1 / v) x (1 / d), where:

[0217] n: average number of colonies

[0218] v: deposition volume

[0219] d: dilution factor

[0220] Once the RHE was inoculated with Staphylococcus epidermidis and treated with B, E, and A (at T0 h), 20 μL of medium was collected from each RHE and placed on TSA-agar plates. After incubation at 37 °C, sterility control was checked. The same verification was performed at the end of the experiment (T24 h). In addition, the sterility of all used products was controlled.

[0221] 2.3 / Bacterial growth assessment

[0222] At the end of the RHE contact period with Staphylococcus epidermidis and the test compounds (B, E, and A), bacteria were collected with a swab in PBS 0.1%-Tween 80 and inoculated onto TSA agar plates (according to the decimal dilution method). After incubation at 37 °C for one day, colonies were counted to determine the number of CFU.

[0223] Measurement of 3 / ROS production

[0224] The amount of intracellular ROS in 2D NHEK keratinocytes or 3D RHE was detected and quantified using the H2DCFDA (2’,7’-dichlorodihydrofluorescein diacetate; Fisher Scientific; D399) probe. After the probe diffuses into live cells, it is deacetylated by intracellular esterases into a non-fluorescent compound. This compound can be oxidized by ROS and converted into highly fluorescent DCF (2’,7’-dichlorofluorescein). Therefore, the ROS produced by cells can be measured by the fluorescence level emitted by the oxidized dye.

[0225] 3.1 / RHE treatment and ROS analysis

[0226] After 14 days of differentiation (D14), the test compound was topically applied to RHE (3 mg / cm 2 ). Meanwhile, the effect of the antioxidant reference molecule (ascorbic acid 300 μg / mL) was analyzed by applying it in the medium for 24 hours. On the 15th day (D15), the RHE was rinsed, placed in fresh PBS, the reference and compound were renewed, and the RHE was incubated with the H2DCFDA probe for 40 minutes (at a concentration of 14.5 μM in PBS). Then, the RHE was irradiated with UVA rays at 30 J / cm 2 for ~1 hour 20 minutes to induce the production of ROS. After irradiation, the RHE was transferred to a new culture plate, and the fluorescence directly related to the ROS produced by the cells was measured using a Glomax Explorer microplate reader (Promega) (excitation: 475 nm; emission: 500 - 550 nm). As described above, the data were normalized to the cell metabolic activity measured by the MTS assay (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium).

[0227] 3.2 / NHEK keratinocyte treatment and ROS analysis

[0228] NHEK keratinocytes in 24-well plates were pretreated with 1 concentration of the compound or 300 μg / mL of the antioxidant reference ascorbic acid in the medium for 24 hours. Then the NHEK keratinocytes were placed in HBSS and subjected to the same reference treatment test for 30 minutes (15 minutes at a concentration of 10 μM and 15 minutes at a concentration of 5 μM) in the presence of the H2DCFDA probe under a 37 °C and 5% CO2 atmosphere. Then the cultures were rinsed and placed in a fresh solution containing the test or reference compound, and irradiated with 5 J / cm 2UVA irradiation (Biosun, Vilber Lourmat, ~20 min) was used for challenge. After challenge, fluorescence was measured using a fluorescence microplate reader (Glomax Explorer, Promega) (excitation: 475 nm; emission: 500 - 550 nm). The data of ROS production were normalized to cell metabolic activity (MTS assay).

[0229] 4 / Quantification of pro-MMP1 and MMP1 activities was performed by ELISA

[0230] 4.1 / NHDF treatment

[0231] NHDF fibroblasts were cultured on 24-well plates in complete medium (containing 10% FBS) for 24 hours and then treated with the compounds. Then, the cells were treated with the test compounds in medium without FBS for 72 hours. In parallel, a reference treatment (a combination of 20 ng / ml TGF-β; 20 ng / mL TNFα and 2.5 ng / mL IL1β) was applied according to the same method. All treatments were performed in triplicate cultures (n = 3).

[0232] 4.2 / ELISA assay

[0233] At the end of the 72-hour treatment, all the supernatants were collected, aliquoted, and stored at -20 °C until further use in specific ELISA assays. By these assays, the extracellular release of pro-MMP1 produced by NHDF fibroblasts was evaluated according to the supplier's instructions (R&D systems, DMP100).

[0234] Results:

[0235] III-1 / Growth of Staphylococcus epidermidis

[0236] The purpose of this test was to study the growth of Staphylococcus epidermidis, the most abundant commensal bacterium on the skin, which participates in skin homeostasis by combating pathogens and producing beneficial bacterial metabolites.

[0237] As Figure 1As shown, the individual extract (E) slightly reduced the growth of Staphylococcus epidermidis. This can be explained by the antimicrobial properties of polyphenols previously reported in relation to Staphylococcus aureus. It should be noted that this same extract (E) did not show any harmful effects on Lactobacillus paracasei CNCM I-5892 (B). It was observed that the grape extract (E) and the Lactobacillus paracasei CNCM I-5892 strain (B) forming the active composition (A) according to the invention increased the growth of Staphylococcus epidermidis, and this growth was significant for the combination, which confirmed this potential stimulating effect. Thus, the Lactobacillus paracasei CNCM I-5892 strain of the present invention appears to be a prebiotic that stimulates the growth of the commensal skin bacterium Staphylococcus epidermidis.

[0238] III-2 / Antioxidant activity

[0239] The aim of the tests was to evaluate the antioxidant activity of the grape extract (E) and the active ingredient (A).

[0240] As Figure 2 shown, the extract (E) and the active substance (A) had an antioxidant effect on the target molecule ascorbic acid in the population of monolayer keratinocytes (NHEK) irradiated with UVA. The potential antioxidant effects of 0.29% extract and 0.3% active ingredient according to the invention were tested.

[0241] Exposure of NHEK to UVA irradiation induced a very significant increase in ROS production, which was partially inhibited by treatment with ascorbic acid. These results validated the assays and analyses. Treatment with the extract very significantly reduced the production of ROS, to a level similar to that of vitamin C. In the presence of the composition according to the invention, the effect was less important but significant.

[0242] III-3 / Inhibitory activity against pro-MMP1

[0243] Figure 3 The summary results of NHDF fibroblasts treated with the test item and the combination of TGF-β1 20 ng / mL (negative reference) or TNFα 20 ng / mL – IL1β 2.5 ng / mL (positive reference) are shown, with the untreated condition set at 100%.

[0244] As expected, the negative reference (TGF-β1) and the positive reference (TNFα + IL1β) highly significantly reduced and increased the release of pro-MMP1 in the supernatant of NHDF fibroblast cultures, respectively, and validated the experiment.

[0245] Compared to the untreated control, treatment with only the strain of Lactobacillus paracasei CNCM I-5892 (B) at 0.0079% had no effect on pro-MMP1 abundance. However, compared to the untreated control, grape extract (E) at 0.029% and the composition according to the invention (A) at 0.03% significantly reduced the release of pro-MMP1.

[0246] Thus, the active ingredients have an impact on the remodeling of the extracellular matrix, as shown by measuring the abundance of the biomarker pro-MMP1. The results show that the presence of the active ingredients significantly reduces the amount of pro-MMP1, an interstitial collagenase involved in the degradation of fibrillar collagen.

[0247] Conclusion

[0248] As shown in the above examples, a combination of a specific bacterium and a specific grape extract makes it possible to obtain a powerful cosmetic combining the positive effects of the two ingredients, the specific bacterium being inactivated in a specific way to preserve and maintain its biological activity, in particular its membrane integrity and adhesiveness, and the specific grape extract being rich in specific tannins compatible with or even protective of the bacterium.

Claims

1. A composition, comprising: - an inactivated Lactobacillus strain; and - an aqueous extract obtained from grape seeds and / or grape skins, advantageously an aqueous extract obtained from fermented red grapes.

2. The composition according to claim 1, wherein the Lactobacillus strain belongs to the species Lactobacillus paracasei.

3. The composition according to claim 2, wherein the strain is the strain deposited at the CNCM (National Collection of Microorganisms, Pasteur Institute, 25 rue du Docteur Roux, 75724 Paris Cedex 15) under the number I-5892 on September 13, 2022.

4. The composition according to any one of claims 1 to 3, wherein the bacterial cells are inactivated by a sterilization method, advantageously by flash pasteurization.

5. The composition according to any one of claims 1 to 4, wherein at least 50% of the bacterial cells are intact.

6. The composition according to any one of claims 1 to 5, wherein the bacteria account for 0.1% to 2% w / w of the composition, advantageously 0.5% to 2% w / w of the composition.

7. The composition according to any one of claims 1 to 6, wherein the extract is ultrafiltered after aqueous extraction.

8. The composition according to any one of claims 1 to 7, wherein the extract contains: - tannins, the total amount of which advantageously accounts for 2 to 7 or 3 to 6% w / w of the dry matter; - molecular tannins, the amount of which advantageously accounts for 0.05 to 0.2 or 0.15 to 0.15% w / w of the dry matter; - crown tannins, the amount of which advantageously accounts for 0.005 to 0.02 or 0.005 to 0.015% w / w of the dry matter.

9. The composition according to any one of claims 1 to 8, wherein the aqueous extract accounts for 4 to 8% w / w of the composition.

10. A method for preparing the composition according to any one of claims 1 to 9, comprising: - Mix the aqueous extract with a Lactobacillus strain, advantageously at least 10 8 CFU (colony forming units) of bacteria per ml of extract; and - sterilizing the mixture, advantageously by flash pasteurization.

11. The composition according to any one of claims 1 to 9, wherein it further comprises excipients, advantageously glycerol and xanthan gum.

12. The composition according to any one of claims 1 to 9, wherein it is in the form of a cream, ointment, mask, serum, lotion, wash, paste, foam, aerosol, stick, deodorant, shampoo, conditioner, patch, water-alcohol or oily solution, water-in-oil or oil-in-water emulsion or multiple emulsion, aqueous or oily gel, liquid, pasty or solid anhydrous product, and / or a dispersion of oil in an aqueous phase by means of spheres, which spheres may be polymeric nanoparticles such as nanospheres and nanocapsules or ionic and / or non-ionic lipid vesicles.

13. The cosmetic use of the composition according to any one of claims 1 to 9 and 11 to 12, advantageously the topical cosmetic use of the composition.

14. The use of the composition according to any one of claims 1 to 9 and 11 to 12 as an anti-aging agent.

15. A composition according to any one of claims 1 to 9 and 10 to 11, for treating skin irritation and / or skin redness and / or skin hydration and / or skin barrier protection.

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