Lacto-fermented extract of at least one red algae of the order Palmariales, preparation process and cosmetic use of this extract

Abstract

The present invention relates to a lacto-fermented extract of at least one red alga of the order Palmariales, this red alga having been lacto-fermented by lactic acid bacteria. The invention also relates to a process for producing such a lacto-fermented extract. The invention further relates to the cosmetic use of such a lacto-fermented extract and of a composition comprising it. Figure: none

Description

Title of the invention: Lacto-fermented extract of at least one red alga of the order Palmanales, process for preparing and cosmetic use of this extract. Technical field

[0001] The present invention belongs to the technical field of cosmetics, and in particular to fermented and seaweed-based natural active extracts for use in cosmetics, notably in a cosmetic composition to combat skin aging and / or improve skin firmness and / or improve skin elasticity and / or in a cosmetic composition for whitening and / or brightening the skin. Previous art

[0002] It is known to those skilled in the art to extract active compounds, such as seaweed extracts, from algal biomass for cosmetic use. The use of algal biomass, which is a natural biomass, offers numerous advantages. In particular, it reduces the use of synthetic chemicals, which has a positive impact on the health of users and on the environment.

[0003] By way of example, we can cite document FR 2 911 278 in the name of the company SILAB, which describes a process for obtaining a cosmetic active ingredient from the enzymatic hydrolysis of Palmaria palmata. The demonstrated action is strictly limited to demonstrating an effect on melanin synthesis by melanocytes and its transfer into keratinocytes, suggesting the potential benefit of using them as a depigmenting agent.

[0004] Furthermore, it is also known to those skilled in the art to produce, for cosmetic use, extracts of certain lacto-fermented algae, particularly with lactic acid bacteria. Indeed, certain benefits that lactic acid bacteria can provide to the skin, by acting in particular on the regulation of the skin microbiota, have made it possible, for example, to improve the skin's barrier function.

[0005] Thus, Chinese application CN 113 383 917 describes the lactic acid fermentation of the red alga Porphyra haitanensis by Lactobacillus plantarum for antimicrobial effects. Application CN 113 383 917 cites a fermented extract of the red alga Porphyra for anti-lipase effects.

[0006] Korean patent KR101409764B1, for its part, teaches the production of a lacto-fermented extract of the red algae Gelidium amansii, used for an anti-wrinkle cosmetic action. In this Korean patent, this lacto-fermented extract of Gelidium amansii It is notably used to produce, among other things, cosmetic compositions such as a softening lotion, a nourishing lotion, or a nourishing cream. KR101409764B1 specifically teaches that the lacto-fermented extract of Gelidium amansii is used to improve collagen production in fibroblasts. This application can, for example, be implemented to combat skin aging caused by ultraviolet (UV) radiation, as this radiation can inhibit the production of the procollagen protein.

[0007] Furthermore, the publication by Perez-Alva A. et al. in Algal Research vol. 64, May 1, 2022, page 102884, XP093242185 describes the fermentation of brown, red, and green algae, and cites 14 different species of red algae (Gracilaria, Grateloupia, Porphyra, etc.). This publication mentions the production of acids by bacteria during fermentation, notably lactic acid, as well as glucose derivatives such as glucose 6P. This publication cites Lactic Acid Bacteria (LAB) type bacteria, but for applications in the field of nutrition. Various effects are associated with lacto-fermented extracts, which vary depending on the extract: antioxidant, antimicrobial, anticoagulant, and anti-inflammatory effects, and other effects on human health (anti-glycation, anti-diabetic, liver protection, etc.) of lacto-fermented extracts used in oral administration.

[0008] Thus, it appears that lacto-fermented algae extracts do not have any recognized common activity, and even less so any recognized common activity in cosmetics. Furthermore, lacto-fermentation modifies the composition of the extract obtained, particularly compared to an extract obtained by enzymatic hydrolysis, which can negatively affect the activity of the extract obtained.

[0009] Furthermore, there is no known in the current state of the art of broad-spectrum lacto-fermented algae extracts which make it possible to act not only on pigmentary mechanisms related to skin whitening / lightening, in particular by acting on melanocytes, but also having an advantageous effect to combat skin aging and / or improve skin elasticity, in particular in an anti-wrinkle or anti-aging cosmetic composition, or even having an anti-inflammatory activity.

[0010] Nowadays, the skin, and especially facial skin, is significantly impacted by numerous daily environmental stressors such as pollution in urban areas, or ultraviolet (UV) or infrared (IR) radiation. These stressors accelerate skin cellular aging, resulting in the appearance of pigment spots, wrinkles, and fine lines.

[0011] Therefore, new active ingredients that have a broad spectrum of action and allow for multiple effects are always being sought in cosmetics. Summary of the invention

[0012] The aim of the invention is therefore to meet this constant demand in the field of cosmetics by providing cosmetic extracts that offer multiple effects, in particular those that reduce pigment spots by whitening / lightening the skin, but also those that have an effect on skin aging, and in particular on the loss of elasticity and the appearance of wrinkles. To this end, the inventors focused on lacto-fermented extracts of a specific algae.

[0013] In particular, the present invention relates to a lacto-fermented extract of at least one red alga of the order Palmariales, this red alga having been lacto-fermented by lactic acid bacteria.

[0014] The inventors of the present invention have demonstrated in a completely surprising and innovative way that a lacto-fermented extract of a specific type of red algae, of the order Palmariales, acts on skin pigmentation, by acting, in particular, on different skin pigmentation mechanisms, and also had an effect on the production of elastin and collagen and therefore had a beneficial effect in combating skin aging, and in particular the loss of firmness and elasticity.

[0015] A first pigmentary mechanism impacted by the extract according to the invention is the mechanism related to melanocytes, linked to the production of melanin (a phenomenon called melanogenesis). Melanin is a pigment, generally black or brown, which develops in reaction to UV radiation (UVA and UVB), and which can cause skin pigmentation in the form of spots, called, for example, age spots or sun spots. The lacto-fermented extract according to the invention slows down, or even stops, melanogenesis, thus reducing these pigment spots on the skin, which are generally considered undesirable.

[0016] A second pigmentary mechanism on which the extract according to the invention acts is the mechanism related to lipofuscin production. Lipofuscin is a non-melanin brown pigment that develops in the skin during the aging of lysosomes, which are present in all cells. The accumulation of lipofuscin in skin cells results in the appearance of brown spots on the skin. This phenomenon is caused in particular by exposure to UV radiation, and especially UVA radiation. The lacto-fermented extract according to the invention reduces the accumulation of lipofuscin, thus reducing these brown spots on the skin. The inventors have demonstrated both a protective and repairing effect after exposure to UVA or infrared (IR) radiation.

[0017] A third mechanism related to aging and modified by the extract according to the invention is the mechanism related to protein carbonylation. During their lifespan in the human body, proteins can undergo modifications that alter their structural and functional properties. One example of protein modification is carbonylation. Carbonylation corresponds to the irreversible and cumulative attachment of a carbonyl group (CO) to the amino acids of proteins. It can be caused by environmental stresses, and in particular oxidative stresses, such as those caused by air pollution or exposure to UV radiation (especially UVA) or IR radiation.Protein carbonylation in the skin is one of the causes of cellular and tissue aging, and this phenomenon can be illustrated by a modification of the skin's intrinsic structure, particularly of keratin in the epidermis and collagen in the dermis. The lacto-fermented extract according to the invention reduces this protein carbonylation, which can be harmful to the skin. The inventors have demonstrated both a protective and repairing effect after exposure to UVA or infrared radiation.

[0018] A fourth mechanism related to aging modified by the extract according to the invention is the mechanism linked to protein carbamylation. Carbamylation corresponds to the irreversible and cumulative attachment of isocyanic acid (of formula HN=C=O) to the amino groups of proteins, in particular to lysine residues, to form homocitrulline. During aging, there is also an accumulation of carbamylated proteins.

[0019] The inventors have discovered and demonstrated that the lacto-fermented red algae extract according to the invention can act on each of these phenomena: firstly, phenomena related to skin pigmentation (action on melanin and lipofuscin), making it possible to reduce skin discoloration, and in particular to reduce pigment spots, which are harmful to health and often considered unsightly; and secondly, phenomena related to skin aging (action on carbonylation, carbamylation of proteins, and on the degradation of elastin and collagen), making it possible to reduce the loss of firmness, and in particular to reduce the appearance of wrinkles. The invention therefore provides the skin with a whitening / brightening effect, but also a more general effect on skin aging.A reduction in pigment spots also has an impact on the skin's luminosity and radiance, which are also improved, as well as on the skin's texture, which is also improved.

[0020] Furthermore, the inventors also demonstrated that the lacto-fermented red algae extract according to the invention reduced skin inflammation. Inflammation, particularly chronic inflammation, is well known as a factor that aggravates skin aging, especially the appearance of wrinkles. It is even referred to as " "inflammaging." This anti-inflammatory effect supports the anti-stain, anti-aging and soothing effect of the extract according to the invention.

[0021] Finally, the inventors have also demonstrated that the lacto-fermented extract has a beneficial effect on the protection and / or repair of collagen and elastin. Therefore, the lacto-fermented extract according to the invention is of interest for combating skin aging and / or improving skin elasticity, particularly in an anti-wrinkle or anti-aging cosmetic composition.

[0022] Advantageously, the red algae of the order Palmariales is of the genus Palmaria.

[0023] More advantageously, the red algae of the genus Palmaria is the species Palmaria palmata.

[0024] The inventors have demonstrated that these specific red algae act on the pigmentary phenomena mentioned above, with very effective results on skin whitening / lightening for each of these mechanisms, on inflammation and on the production of collagen and elastin.

[0025] Advantageously, the red algae was lacto-fermented with lactic acid bacteria of the species Lactiplantibacillus plantarum. It should be noted that Lactiplantibacillus plantarum is the new name for the species formerly known as Lactobacillus plantarum until the 2020s, due to the change in the genus name Lactobacillus to Lactiplantibacillus (Zheng et al. Int. J. Syst. Evol. Microbiol. 2020, 70, 2782-2858). For example, the strain filed by POLYMARIS BIOTECHNOLOGY with the CNCM under number 1-6105 can be used.

[0026] Lactic acid bacteria, and in particular those of the species Lactiplantibacillus plantarum, advantageously strengthen the populations of microorganisms naturally present in the skin microbiota, thereby preventing, or at least reducing, the risk of disruption and imbalance of the skin microbiota. They also allow the production of an extract that offers other beneficial effects on the skin, particularly mature skin, by providing various advantageous and desirable properties, such as moisturizing, antioxidant, and anti-aging properties.

[0027] The inventors have demonstrated the presence of lactic acid and four cyclodipeptides, namely cyclo Pro-Ala, cyclo Pro-Leu, cyclo Pro-Ile, and cyclo Pro-Val (where Pro denotes proline, Ala alanine, Leu leucine, Ile isoleucine, and Val valine), in the lacto-fermented red algae extract according to the invention. Without being linked by any specific mechanism of action, the cyclodipeptides present in the cosmetic extracts and ingredients according to the invention could be responsible for the observed effect on collagen and elastin production and on the Soothing effects, notably a decrease in PGE2 in fibroblasts and IL8 in keratinocytes, are demonstrated in the examples. Such cyclodipeptides have never been associated with lacto-fermented extracts and are not present in non-lacto-fermented extracts of red algae of the order Palmariales, as shown by the analyses presented in the examples. Thus, the extract according to the invention comprises metabolites and constituents from the red algae of the order Palmariales specifically linked to lacto-fermentation. The specific composition of the extract confers upon it a very broad spectrum of activity that goes well beyond the effect on melanin production known for extracts obtained by enzymatic hydrolysis of Palmaria palmata, which was not easy to preserve, and the effects on the microbiota that lacto-fermentation can produce.

[0028] Preferably, the extract according to the invention is an aqueous extract comprising preferably 0.1% to 10% by weight of dry extract, and preferably 1% to 5% by weight of dry extract, and typically 2.5% to 3.5% by weight of dry extract. In general, the dry extract consists of at least 70% by weight of constituents derived from the red algae used, of the order Palmariales, in particular of the genus Palmaria, and preferably Palmaria palmata.

[0029] In particular, the extract according to the invention is obtained by lacto-fermentation in a medium consisting solely of water and sea salt.

[0030] Advantageously, the extract according to the invention has a pH which belongs to the range of 2 to 5, and preferably of 3.5 to 4.5.

[0031] According to an advantageous embodiment of the invention, the lactic acid bacteria are present in the lacto-fermented extract in the form of fragments.

[0032] Indeed, according to a technique for preparing the extracts of the invention, the lactic acid bacteria are not separated from the reaction medium, but are inactivated and fragmented. They therefore remain in the final algal extract in the form of fragments. These fragments include, in particular, pieces of the cell walls of the lactic acid bacteria. Such a lacto-fermented extract containing bacteria is therefore an aqueous extract, comprising water-soluble molecules, which also includes lipid compounds from the fragments of the bacterial membranes present in the extract.

[0033] According to another embodiment of the invention, the lacto-fermented extract no longer includes lactic acid bacteria, nor even fragments of lactic acid bacteria.

[0034] Advantageously, the dry extract of the extract according to the invention comprises: - 10 to 50% by weight of sugars, including, in particular, floridoside, galactose, and possibly other polysaccharides. Floridoside represents, in particular, 15 to 35% of the dry extract; and / or - 0.0005% to 0.005% of vitamins, and in particular B vitamins, including, in particular, vitamin B2, vitamin B3, vitamin B5, vitamin B6 and vitamin B7, preferably vitamin B3, vitamin B6 and vitamin B7; and / or - 2 to 10% by weight of protein comprising amino acids selected from glutamic acid, alanine, threonine and arginine; and / or - 3 to 15% by weight of lactic acid; and / or - from 0.001% to 0.25% by weight, typically from 0.01 to 0.05% by weight of cyclodipeptides, and in particular from 0.001% to 0.25% by weight, typically from 0.01 to 0.05% by weight of cyclodipeptides cyclo Pro-Ala, cyclo Pro-Leu, cyclo Pro-Ile and cyclo Pro-Val, notably present at a rate of 0.0003% to 0.06% by weight each.

[0035] The present invention also relates to a process for producing a lacto-fermented extract as defined above, comprising the following steps: - a step A) of lacto-fermentation of a red algae by lactic acid bacteria in an aqueous medium comprising sea salt, the red algae being of the order Palmariales, without aeration; - a step B) of filtration by clarification, to separate the residues of the red algae from the reaction medium; - a step C) of treating the reaction medium to form the lacto-fermented extract.

[0036] To implement this process for preparing the extract according to the invention, the red algae used may have been cultivated or directly harvested. It may be dried and / or cut before being introduced into the reaction medium.

[0037] Step A) of lactofermentation is generally carried out in the presence, in the lactofermentation medium used, of 1 to 10% by weight, preferably 5 to 8% by weight of the selected red algae of the order Palmariales, and an inoculum enabling the achievement of 0.5 to 5% by weight, preferably 1 to 3% by weight, of lactic acid bacteria at the end of lactofermentation, in particular lactic acid bacteria of the species Lactiplantibacillus plantarum, of 0.1 to 3% by weight, preferably 0.5 to 1.5% by weight of sea salts (NaCl), these % being given in relation to the total weight of the lactofermentation medium (bacteria + red algae + aqueous medium).

[0038] The duration of the lacto-fermentation stage is generally between 1 and 7 days.

[0039] According to one embodiment, step C) of treatment constitutes an additional filtration step, to remove lactic acid bacteria from the lactofermented extract, this second filtration step being a sterilizing filtration.

[0040] According to this first embodiment, the process according to the invention results in the production of a lacto-fermented extract of a red algae that is free of bacteria lactic acid bacteria. The sterilizing filtration step, which is a finer filtration than clarification filtration, which is coarser than membrane filtration, makes it possible to eliminate the lactic acid bacteria that cause the lacto-fermentation of the red algae used.

[0041] According to another embodiment, step C) of treatment constitutes an inactivation step of lactic acid bacteria within the lactofermented extract leading to their fragmentation.

[0042] According to this second embodiment of the process of the invention, unlike the first embodiment detailed above, the final extract comprises fragments of lactic acid bacteria, which are, among other things, pieces of bacterial cell walls. This is due to the fact that this implementation of the process does not involve a second filtration step.

[0043] Preferably, the lactic acid bacteria in the final extract are inactivated by a heat treatment step. This heat treatment also fragments the bacteria, so that they are present in fragment form in the final extract. Furthermore, this heat treatment sterilizes the final extract. This heat treatment is, in particular, carried out in an autoclaving chamber at a temperature of 120°C for a duration of between 10 and 30 minutes, and preferably 20 minutes. Bacterial lysis can also be achieved by other heat treatment methods (pasteurization, etc.) or mechanical lysis methods (high-pressure homogenizer, etc.).

[0044] It is also possible to inactivate the bacteria, preferably as previously described, and then to carry out a finer filtration, in particular with a membrane having in particular a cut-off threshold of 0.2pm to eliminate bacterial fragments and thus obtain an extract without bacterial fragments.

[0045] Thus, the two types of extracts according to the invention, i.e. respectively with and without fragments of lactic acid bacteria, comprise cosmetic actives which have been extracted directly from the algae, as well as compounds which are derived from the transformation of algal biomass by lactic acid bacteria.

[0046] The invention also relates to extracts of algae of the order Palmariales obtained by the processes described above. The particular characteristics described in this description for the extracts according to the invention apply mutatis mutandis to the processes for manufacturing extracts and to the extracts obtained by such processes.

[0047] The present invention also relates to a cosmetic ingredient comprising a lacto-fermented extract of red algae according to the invention, with at least one cosmetic additive selected from stabilizing and antimicrobial agents. Such an ingredient can be stored and used for the manufacture of cosmetic compositions.

[0048] Examples of stabilizing and / or antimicrobial agents include glycerin, pentylene glycol, propylene glycol, butylene glycol, propanediol, phenoxyethanol, phenethyl alcohol, hexanediol, or mixtures of potassium sorbate and sodium benzoate.

[0049] In particular, the cosmetic ingredient according to the invention comprises glycerin and pentylene glycol.

[0050] A cosmetic ingredient according to the invention may comprise (in % by weight relative to the total weight of the cosmetic ingredient) in particular 0.05 to 5% of lacto-fermented dry extract according to the invention, preferably 0.5% to 2.5%, 10 to 80% of water, preferably 40% to 50%, 20 to 60% of glycerin, preferably 30 to 50%, and 2 to 20% of pentylene glycol, preferably 5 to 15%.

[0051] The present invention also relates to the use of a lacto-fermented extract of red algae according to the invention or of a cosmetic ingredient according to the invention in a cosmetic composition and the cosmetic use of a lacto-fermented extract of red algae according to the invention or of a cosmetic ingredient according to the invention.

[0052] A cosmetic composition is defined as a composition compatible with keratinous materials, and in particular the skin, that has a pleasant color, odor, and feel, and that does not generate unacceptable discomfort (tingling, tightness, redness) for the consumer. A lacto-fermented extract of red algae according to the invention or a cosmetic ingredient according to the invention can be incorporated into a lotion, cream, stick, or any other formulation suitable for application to keratinous materials, in particular the skin.

[0053] The cosmetic compositions and cosmetic ingredients according to the invention may contain one or more other cosmetic additives or active ingredients, in particular chosen from among anti-wrinkle actives, anti-aging or antioxidant actives, anti-inflammatory and anti-redness actives, anti-blemish or peeling actives, anti-swelling or anti-puffiness actives, moisturizing agents, skin barrier restoring actives, slimming actives.

[0054] By way of example, a cosmetic composition according to the invention shall comprise a lacto-fermented extract of red algae according to the invention, the dry extract of which represents from 0.001 to 1% by weight, and preferably from 0.01 to 0.1% by weight of the cosmetic composition, and one or more cosmetic additives listed in Table 1 below. Table 1 shows the advantageous weight percentages of certain cosmetic additives in cosmetic compositions, it being understood that the total quantity of cosmetic additives and the extract or cosmetic ingredient according to the invention represents 100% or less.

[0055] [Tables] Cosmetic Additives Example % Benefits Anti-Wrinkle Retinol 0.1 to 3.5% Retinyl Palmitate 0.1 to 0.5% Polyethoxylated Retinamide 0.05 to 0.2% Adenosine 0.01 to 0.04% Vitamin C and its stabilized derivatives 0.1 to 99.9% Resveratrol 0.05 to 3% Babuchiol 0.1 to 1% Hyaluronic Acid Bpm 0.1 to 0.5% Coenzyme Q10 0.1 to 1% Matrixyl Peptide: Palmitoyl Tripeptide-1 & Palmitoyl Tetrapeptide-7 3 to 10% Ectoin 0.3 to 3% Silanol Derivatives: Silanetriol... 3 to 6% Anti-Aging - Antioxidant Ferulic Acid 0.5% to 3% Vitamin E 0.1% to 3.9% Astaxanthin 0.01% to 0.3% Brightening - Radiance Niacinamide - Vitamin B3 0.5% to 99.9% Ethyl Ascorbyl Ether 0.5% to 2% Broussonetia Extract (powder) 0.1% to 2% Arbutin 0.1% to 2% Soluble Licorice Oil (Glycyrrhiza extract) 0.1% to 0.5% Alpha Bisabolol 0.1% to 1% Ascorbyl Glucoside 0.1% to 12% Magnesium Ascorbyl Phosphate 0.1% to 10% Ascorbyl Tetraisopalmitate 0.1% to 20% Stabilized Vitamin C and its derivatives 0.1% to 99.9% Glabridin 0.01% to 0.04% Cysteamine 3 to 5% Anti-inflammatory - Soothing - Anti-redness Allantoin 0.1 to 2% Alpha Bisabolol 0.1 to 1% Enoxolone 0.1 to 2% Alpha Glucan Oligosaccharide 0.25 to 3% Anti-blemish / Peeling Azelaic Acid 0.1 to 10% Glycolic Acid 0.1 to 20% Salicylic Acid 0.1 to 2% Malic Acid 0.1 to 5% Lactic Acid 0.1 to 10% Citric Acid 0.1 to 20% Gluconolactone 3 to 15% Zinc Gluconate 0.1 to 3% Papain 0.1 to 0.5% Anti-dark circles, Anti-puffiness or slimming Caffeine 0.1 to 5% Hydration Hyaluronic Acid HPM 0.01 to 3% PCA 0.015 to 0.15% Betaine, 0.1 to 3% Aloe Vera, 0.1 to 0.5% Skin Barrier Restoration Ceramides, 0.05 to 1% Pro-Vitamin B5, 0.1 to 2% Squalane, 1 to 100%

[0056] The invention also relates to cosmetic uses of a lacto-fermented extract, a cosmetic ingredient, or a cosmetic composition according to the invention. Advantageously, the use of a lacto-fermented red algae extract according to the invention, or of a cosmetic ingredient according to the invention, or of a cosmetic composition comprising it, is for topical cosmetic application to the skin.

[0057] The extract according to the invention does not exhibit cytotoxic effects on skin cells. It can therefore be applied safely to the skin, without risk, and in particular without side effects.

[0058] More advantageously, the use according to the invention is for an application of whitening, and / or skin lightening, and / or improvement of skin brightness, and / or improvement of skin radiance.

[0059] More advantageously, the use according to the invention is for an application to combat skin aging and / or improve skin elasticity and / or improve skin firmness and / or texture, particularly in an anti-wrinkle or anti-aging cosmetic composition.

[0060] These different uses can be combined, given the broad spectrum of activity of the extracts according to the invention. In particular, the cosmetic use according to the invention is for an application of whitening, and / or skin lightening, and / or improvement of skin luminosity, and / or improvement of skin radiance, and / or combating skin aging, and / or improvement of skin elasticity, and / or improvement of skin firmness and / or texture, particularly in an anti-wrinkle or anti-aging cosmetic composition, and / or soothing and / or anti-inflammatory effect.

[0061] Indeed, the lacto-fermented red algae extracts according to the invention are capable of reducing protein carbonylation, which has the effect of reducing the denaturation of keratin fibers in the epidermis and collagen fibers in the dermis, and thus reducing loss of firmness and the appearance of wrinkles in the skin. This also has the effect of improving skin texture and firmness, for example, through an action on keratinocytes. Furthermore, the lacto-fermented red algae extracts according to the invention have a beneficial effect on the protection and / or repair of collagen and elastin. Brief description of the figures

[0062] Other features and advantages of the invention will become apparent upon reading the detailed description that follows, for the understanding of which reference should be made to the accompanying figures (as well as to the examples referring to these figures), in which:

[0063] [Fig.1] - [Fig.1] is a graph illustrating the evolution of the cell viability of fibroblasts as a function of the concentration of extracts of the red algae Palmaria palmata, respectively lacto-fermented and non-lacto-fermented;

[0064] [Fig.2] - [Fig.2] is a graph illustrating the evolution of cell viability of keratinocytes as a function of the concentration of extracts of the red algae Palmaria palmata, respectively lacto-fermented and non-lacto-fermented;

[0065] [Fig.3] - [Fig.3] is a set of photos comparing the accumulation of lipofuscin in skin expiants, treated or not with the extracts according to the invention;

[0066] [Fig.4] - [Fig.4] is a set of photos comparing the carbonylation rate of proteins in skin expiants, treated or not with the extracts according to the invention. Detailed description of the invention

[0067] Other advantages and features of the present invention will result from the following description, given by way of non-limiting example and made with reference to the examples. EXAMPLES

[0068] Example 1: preparation of lacto-fermented extracts of Palmaria palmata

[0069] Example a: Lacto-fermented extract of Palmaria palmata with fragments of lactic acid bacteria

[0070] A 7 g / L NaCl solution in water with 5% dried, ground Palmaria palmata was placed in an autoclave at 121°C for 20 minutes. The pre-culture of Lactiplantibacillus plantarum (strain registered by POLYMARIS BIOTECHNOLOGY with the CNCM under number 1-6105) was then inoculated at 10% by weight. Fermentation was maintained for 3 days at 20°C without aeration. The algae underwent coarse filtration, followed by sterilization at 121°C.

[0071] The extract obtained had a dry extract representing 3+ / -1% of the total weight of the extract and a pH of 4+ / -1, depending on the production batches. The portion representing bacterial fragments corresponded to approximately 0.05% by weight.

[0072] Example 1b: Lacto-fermented extract of Palmaria palmata without fragments of lactic acid bacteria

[0073] In this case, the procedure was followed as in example 1a, except that sterilization was replaced by sterile filtration with a cutoff threshold of 0.2 µm. The resulting extract had a dry extract representing 3 + / - 1% of the total weight of the extract and a pH of 4 + / - 1, depending on the production batch.

[0074] The extracts obtained were characterized by HPLC with an RI, MS, or UV detector. Table 2 summarizes the data concerning the composition of the extracts obtained (data as % by weight relative to the dry extract), and, by comparison, the composition of a Palmaria palmata extract obtained without fermentation:

[0075] [Tables2] Example 1a (with fragments) Example 1b (without fragments) Extract without lacto-fermentation (comparative) Mineral matter 50 + / - 3% 50 + / - 3% 50 + / - 3% Floridoside 27 + / - 5% 27 + / - 5% 27 + / - 5% Vitamin B3 0.001-0.002% 0.001-0.002% 0.001-0.002% Vitamin B6 present present present Vitamin B7 0.0005-0.001% 0.0005-0.001% 0.0005-0.001% Protein with amino acids: 6.5 + / - 3% 6.5 + / - 3% 1.6 + / - 1% Glutamic acid 2.7 + / - 1.5% 2.7 + / - 1.5% Not determined (ND) Alanine 0.7 + / - 0.5% 0.7 + / - 0.5% ND Threonine 0.3 + / - 0.2% 0.3 + / - 0.2% ND Arginine 0.2 + / - 0.1% 0.2 + / - 0.1% ND Cyclodipeptides (Pro-Ala, Pro-Leu, Pro-Ile, Pro-Val) 0.015 + / - 0.010% 0.015 + / - 0.010% absent Lactic acid 6.7 + / - 3% 6.7 + / - 3% absent Succinic acid present present absence Bacterial fragments 2.0 + / - 0.5% 0 0

[0076] The mineral matter originates from both sea salts (NaCl) and constituents of the algae. The presence of cyclodipeptides has notably been demonstrated by LC / MS.

[0077] Also, it appears from the data in this table that apart from the presence or absence of bacterial fragments, the composition of the two lacto-fermented extracts is identical.

[0078] Furthermore, comparison with the non-lactofermented extract shows the appearance of lactic acid and succinic acid in the lactofermented extracts, proving that the fermentation process was successful. An increase in protein content and the appearance of cyclodipeptides are also noted after fermentation. For floridoside (the main sugar), the variations observed between batches do not allow us to conclude that it was consumed during lactofermentation. Similarly, the observed variations in vitamins do not allow us to draw any conclusions.

[0079] Example 2: preparation of a cosmetic ingredient comprising a lacto-fermented extract of Palmaria palmata

[0080] Glycerin and pentylene glycol were added to the previously prepared lacto-fermented extracts of Palmaria palmata, in quantities such that glycerin constituted 40% by weight of the total weight of the resulting cosmetic ingredient and pentylene glycol constituted 10% by weight of the total weight of the resulting cosmetic ingredient. The resulting cosmetic ingredients (Example 2a, in the case of the one with lactic acid bacteria fragments, and Example 2b, in the case of the one without lactic acid bacteria fragments) proved stable after storage for 6 months at 4°C, 23°C, and 40°C.

[0081] Example 3: Analysis of the cytotoxicity of lacto-fermented extracts of the red algae Palmaria palmata according to the invention, with and without fragments of lactic acid bacteria, respectively on fibroblasts and on keratinocytes of the skin.

[0082] The graph in [Fig. 1] illustrates the cell viability (in percent) of fibroblasts as a function of the concentration (in percent by weight) of a lacto-fermented extract of Palmaria palmata according to Example 1b without bacterial fragments according to the invention (curve with triangles). The cosmetic ingredient is added to the cell culture medium at a variable concentration.

[0083] By comparison, this graph also illustrates the cellular viability of fibroblasts as a function of the concentration of the cosmetic ingredient comprising an extract of Palmaria palmata outside the invention, i.e. an extract of Palmaria palmata which is not fermented (curve with the points).

[0084] This study was carried out on normal human dermal fibroblasts (NHDF), which were treated respectively with the extract according to the invention (lacto-fermented) and with the extract outside the invention (non-fermented) for 48 hours.

[0085] This graph demonstrates that a lacto-fermented extract according to the invention does not have a toxic effect on fibroblasts, and that it even stimulates the cell viability of these skin cells, compared with the behavior of the unfermented extract.

[0086] Similarly, the graph in [Fig.2] illustrates the cell viability (in percent) of keratinocytes as a function of the concentration (in percent) of a lacto-fermented extract of Palmaria palmata according to the invention (curve with triangles).

[0087] By comparison, this graph also illustrates the cellular viability of keratinocytes as a function of the concentration of an extract of Palmaria palmata outside the invention, i.e. an extract of Palmaria palmata which is not fermented (curve with the points).

[0088] This study was carried out on normal human epidermal keratinocytes (NHEK), which were treated respectively with the extract according to the invention (lacto-fermented) and with the extract outside the invention (non-fermented) for 48 hours.

[0089] This graph demonstrates that a lacto-fermented extract according to the invention does not have a toxic effect on skin keratinocytes.

[0090] Similar results were obtained with a lacto-fermented extract of Palmaria palmata with bacterial fragments according to example 1a, which shows that the presence of bacterial fragments does not have a negative influence on cytotoxicity.

[0091] Example 4: analysis of the effect of lacto-fermented extracts of the red algae Palmaria palmata according to the invention, with and without fragments of lactic acid bacteria, on skin pigmentation.

[0092] Example 4a: In vitro analysis of the effect of the lacto-fermented extract of the red alga Palmaria palmata according to the invention, without fragments of lactic acid bacteria, on the pigment mechanism related to melanogenesis Test 1

[0093] Test conditions:

[0094] Control: 0.5mM L-tyrosine solution. L-tyrosine is known to stimulate the expression of many genes involved in melanogenesis.

[0095] Control: 0.02% by weight alpha-arbutin solution. Alpha-arbutin is known to be used to combat pigment spots. It is also a control used to reduce melanin production.

[0096] Sample (invention): cosmetic ingredient comprising a fermented extract of the red algae Palmaria palmata not containing fragments of lactic acid bacteria (example 2b). The cosmetic ingredient is used at a concentration of 0.1% by weight in the melanocyte culture medium.

[0097] These three solutions were respectively applied to a biological model of normal human epidermal melanocytes (NHEM), for a period of 12 days.

[0098] A quantitative PCR (qPCR) analysis was performed on these NHEMs to determine the expression of certain genes involved in the mechanism of melanogenesis.

[0099] The results are presented in Table 3 below.

[0100] [Tables3] Genes Reduction in the expression of these genes (in %): application of the control, compared to the control Reduction in the expression of these genes (in %): application of the sample (invention), compared to the control CRTC1 -51 -63 MITF -29 -36 PMEL -46 -58 TYR -33 -52 TYRP1 -30 -52 SLC45A2 -37 -50 CD63 -43 -44 MILANA -42 -45 RAB38 -28 -40 FZD1 -42 -54 WLS -36 -45 MAPK3 -40 -55 RASA4 -34 -60 EDNRB -41 -47 PRKCA -43 -54

[0101] The CRTC1, MITF, PMEL, TYR and TYRP1 genes encode enzymes and structural proteins involved in melanogenesis.

[0102] The SLC45A2 gene codes for channels and support proteins involved in melanogenesis.

[0103] The CD63 and MILANA genes code for melanosome proteins.

[0104] The RAB38 gene codes for proteins involved in the transport of melanosomes.

[0105] The FZD1 and WLS genes are involved in the WNT pathway.

[0106] The MAPK3 and RASA4 genes are involved in the SCF / KIT pathway.

[0107] The EDNRB and PRKCA genes are involved in the endothelin (ET1) pathway.

[0108] Thus, the extract according to the invention reduces the expression of genes involved in melanin production. In particular, the reduction in the expression of these genes by the extract according to the invention is greater than for the comparative alpha-arbutin control solution.

[0109] The extract according to the invention therefore has a strong impact on melanocytes, effectively reducing melanin synthesis, which allows for an improved whitening / lightening effect on the skin compared to the active ingredients usually used for this cosmetic application. Test 2

[0110] Test conditions:

[0111] Control: 0.5mM L-tyrosine solution.

[0112] Control 1: 5 pg / mL lipoic acid solution mixed with 0.5 mM L-tyrosine solution. Lipoic acid is known to be used to combat pigment spots. It is also a control used to reduce melanin production.

[0113] Control 2: 0.02% alpha-arbutin solution, mixed with 0.5mM L-tyrosine solution.

[0114] Sample (invention): cosmetic ingredient comprising a fermented extract of the red algae Palmaria palmata not containing fragments of lactic acid bacteria (example 1b), mixed with a 0.5 mM L-tyrosine solution. The cosmetic ingredient is used at a concentration of 0.1% by weight in the melanocyte culture medium.

[0115] These four solutions were respectively applied to a biological model of normal human epidermal melanocytes (NHEM), for a period of 10 days.

[0116] An analysis was performed on these NHEM cells to determine melanin production. This analysis is based on spectrophotometric measurement by reading the absorbance at a wavelength of 405 nm, and total protein measurement by a colorimetric protein assay method (Lowry (Pierce) method), to relate the amount of melanin to the protein content.

[0117] The results are presented in Table 4 below.

[0118] [Tables4] Reduction in melanin production (in %), compared to control: Control 1 -45, Control 2 -57, Invention sample -51

[0119] The lacto-fermented extract of Palmaria palmata according to the invention reduces melanin production by 51%. Furthermore, the extract according to the invention reduces melanin production more significantly compared to the first control solution, which is based on lipoic acid, and almost equivalently compared to the second control solution, which is based on alpha-arbutin.

[0120] The results of this second test relating to the pigmentary mechanism in connection with melanogenesis are consistent with the results of the first test, and demonstrate that the extract according to the invention makes it possible to effectively reduce melanin synthesis, which allows a whitening / lightening effect on the skin at least equal to, or even improved upon, compared to the active ingredients usually used for this cosmetic application.

[0121] Example 4b: In vitro analysis of the effect of the lacto-fermented extract of the red alga Palmaria palmata according to the invention, with fragments of lactic acid bacteria, on the pigmentary mechanism related to melanogenesis using the same human melanocyte model

[0122] Test conditions:

[0123] Control: 0.5mM L-tyrosine solution.

[0124] Control: 0.02% alpha-arbutin solution mixed with 0.5 mM L-tyrosine solution. Alpha-arbutin is known to be used to combat pigment spots. It is a control used to reduce melanin production.

[0125] Sample (invention): cosmetic ingredient comprising a fermented extract of the red algae Palmaria palmata including fragments of lactic acid bacteria (example 2a), mixed with a 0.5 mM L-tyrosine solution. The cosmetic ingredient is used at a concentration of 0.1% by weight in the cell culture medium.

[0126] These three solutions were respectively applied to the same biological model as in Experiment 4b, namely normal human epidermal melanocytes (NHEM), for a period of 10 days. Melanin analysis was performed as in Test 2 of Experiment 4b, i.e., a spectrophotometric assay by reading the absorbance at a wavelength of 405 nm, and a total protein assay by a colorimetric protein assay method.

[0127] The results are presented in Table 5 below.

[0128] [Tables5] Reduction in melanin production (in %), compared to the control. Control -41 Invention sample -52

[0129] The lacto-fermented extract of Palmaria palmata according to the invention, with bacterial fragments, reduces melanin production by 52%. Furthermore, the extract according to the invention reduces melanin production more significantly compared to the control solution, which is based on alpha-arbutin.

[0130] The results of this test relating to the pigmentary mechanism in connection with melanogenesis are consistent with the results of the first tests, and demonstrate that the extract according to the invention with fragments of bacteria makes it possible to effectively reduce melanin synthesis, which allows a whitening / lightening effect on the skin at least equal to, or even improved upon, compared to the active ingredients usually used for this cosmetic application.

[0131] Example 4c: In vitro analysis of the effect of the lacto-fermented extract of the red alga Palmaria palmata according to the invention, with and without fragments of lactic acid bacteria, on the pigmentary mechanism related to melanogenesis in another melanocyte model

[0132] Test conditions:

[0133] In this case, the cell model used was human melanocytes derived from human pluripotent stem cells (hiPSCs) of the PCi-MEL_AFR cell line (from Phenocell). The cells were seeded at a density of 5,000 cells per cm² onto culture plates pre-coated with fibronectin and cultured in PhenoCULT-MEL culture medium (prepared by Phenocell) for 5 days. After 5 days, extracts 1a and 1b, with and without fragments respectively, were added after predilution in the complete PhenoCell culture medium. The final percentages of extracts 1a and 1b were 0.25% and 0.5%. A positive control was performed in parallel with a cell depigmentation agent (the nature of which was not disclosed). After 48 hours, the treatments were repeated. Analyses were performed after 96 hours of treatment.On day 9 of culture, image analysis was performed using a CX5 CellInsight platform (ThermoFischer). The cells were then rinsed with PBS buffer and treated with a TrypLE Express solution (ThermoFischer) supplemented with 0.4 pM Calcein-AM (Life Technology) and 0.1 pM SytoxRed (Life Technology) to label live and dead cells, respectively. Data acquisition was performed using an Accuri C6+ analyzer (BD). Melanin was extracted from the cells by adding sodium hydroxide (NaOH) and heating to 80°C. The extracted melanin was quantified by optical density measurement using a plate reader at 405 nm.

[0134] The results obtained are presented in Table 6 below.

[0135] [Tableauxô] Reduction in melanin production (in %), compared to the control Positive control -48% Sample invention without fragments at 0.25% -19% Sample invention without fragments at 0.5% -21% Sample invention with fragments at 0.25% -5% Sample invention with fragments at 0.5% -16%

[0136] In this melanogenesis model, the lacto-fermented extract of Palmaria palmata according to the invention, without bacterial fragments, tested at 0.25% and 0.5%, reduces melanin production by 19% and 21%, respectively. Furthermore, the extract according to the invention, with lactic acid bacteria fragments, tested at 0.25% and 0.5%, also reduces melanin production by 5% and 16%, respectively.

[0137] Example 4d: effect of lacto-fermented extract with bacterial fragments (cosmetic ingredient tested at 1% and 2% by weight) on melanin production (protection against UVA)

[0138] Test conditions:

[0139] Two samples comprising a cosmetic ingredient according to the invention (example 2a), comprising an extract comprising fragments of lactic acid bacteria, either at 1% in water or at 2% in water, were prepared.

[0140] These two samples according to the invention were respectively applied to fragments of human skin (expiants) belonging to a 35-year-old donor with phototype III skin. Twenty-four hours after the application of the samples comprising an extract according to the invention, these expiants were exposed to UVA radiation (6 J / cm²). Two hours after this UVA exposure, the expiants were fixed in an OCT solution and then frozen at a temperature of -80°C.

[0141] A skin expiant that has not been subjected to the application of an extract according to the invention was also treated with UVA in the same way as the skin expiants with extracts according to the invention. A control sample was also prepared, which corresponds to a skin expiant that has not been subjected to the application of an extract according to the invention, and which has not been treated with UVA.

[0142] Sections were then made using a microtome on these different expiants, and melanin was detected by Fontana Masson staining.

[0143] The results of the protection test are presented in Table 7 below.

[0144] [Tables?] Mean versus control (in %) Standard deviation Efficacy of melanin production reduction (in %) p-value (versus UVA stress) Control 100 1 100 ***; <0.001 Skin expiry without extract and exposed to UVA 119 3 0 / Skin expiry with cosmetic ingredient 2a (extract with bacteria) at 1% for 24h and exposed to UVA 116 4 14 ns, 0.67 Skin expiry with cosmetic ingredient 2b (extract with bacteria) at 2% for 24h and exposed to UVA 101 3 93 ***; <0.001

[0145] Thus, the lacto-fermented extracts of Palmaria palmata according to the invention, with fragments of lactic acid bacteria, significantly reduce melanin production when the skin is exposed to UVA radiation. A decrease in melanin production of 14% and 93% was observed for the extract with bacterial fragments tested at 1% and 2% before UVA irradiation.

[0146] Example 4e: Ex vitro analysis of the effect of lacto-fermented extracts of the red alga Palmaria palmata according to the invention, respectively with and without fragments of lactic acid bacteria, on the pigmentary mechanism related to lipofuscin production

[0147] Test conditions:

[0148] Two samples comprising a cosmetic ingredient according to the invention (examples 2a or 2b, respectively, comprising an extract including fragments of lactic acid bacteria, and an extract not including fragments of lactic acid bacteria) at 2% in water were prepared.

[0149] These two samples according to the invention were respectively applied to fragments of human skin (expiants) belonging to a 44-year-old donor with phototype III skin. Twenty-four hours after the application of the extracts according to the invention, these expiants were exposed to UVA radiation (6 J / cm²). Two hours after this UVA exposure, the expiry samples were fixed in an OCT solution and then frozen at a temperature of -80°C.

[0150] A skin expiant that has not been subjected to the application of an extract according to the invention was also treated with UVA in the same way as the skin expiants with extracts according to the invention. A control sample was also prepared, which corresponds to a skin expiant that has not been subjected to the application of an extract according to the invention, and which has not been treated with UVA.

[0151] Sections were then made using a microtome on these different expiants, and lipofuscin was detected by staining with Sudan Black, a dye known to detect lipids and to allow visualization of lipofuscin in particular.

[0152] The results are presented in Table 8 below.

[0153] [Tables8] Mean vs. control (in %) Standard deviation Lipofuscin production reduction efficacy (in %) p-value (vs. UVA stress) Control 100 1 100 ***; <0.001 Skin expiry without extract and exposed to UVA 115 1 0 / Skin expiry with cosmetic ingredient 2b (bacteria-free extract) and exposed to UVA 102 2 89 ***; <0.001 Skin expiry with cosmetic ingredient 2a (bacteria-containing extract) and exposed to UVA 105 1 68 ***; 0.001

[0154] Thus, the lacto-fermented extracts of Palmaria palmata according to the invention, with and without fragments of lactic acid bacteria, significantly reduce lipofuscin production when the skin is exposed to UVA radiation. A decrease in lipofuscin production of 89% is observed for the extract without bacterial fragments, and a still significant decrease of 68% for the extract with bacterial fragments.

[0155] These results are confirmed with [Fig.3], which illustrates the presence of lipofuscin at the level of these different skin expiants tested.

[0156] The first image, top left, illustrates the control skin expiant which has not been treated with UVA, and therefore does not contain lipofuscin, or contains only a very minimal amount.

[0157] The second image, top right, illustrates the skin expiry which does not include any extract according to the invention, and which has been treated with UVA: there are areas in dark black visible, which correspond to areas of the skin expiry where lipofuscin has accumulated, due to UVA radiation.

[0158] The third and fourth images, on the bottom line, respectively on the left and right, illustrate UVA-treated skin expiants comprising an extract according to the invention, respectively with and without a fragment of lactic acid bacteria. These images are visually similar to the first image: they do not include areas of dark black, but only areas of gray, or even light gray, which illustrates that these skin expiants do not include areas in which lipofuscin has accumulated significantly.

[0159] Thus, the results of these tests demonstrate that the extracts according to the invention, with and without lactic acid bacteria fragments, effectively reduce lipofuscin production. This demonstrates that both types of extracts according to the invention act on skin pigmentation and produce a whitening and / or lightening effect on the skin.

[0160] Example 4f: effect of lacto-fermented extract with bacterial fragments (cosmetic ingredient 2a tested at 1% and 2% by weight) on lipofuscin production (protection against UVA)

[0161] Test conditions:

[0162] The test conditions are equivalent to those of the test in Example 4e, except that the ingredient according to the invention, containing bacterial fragments, will be applied at two concentrations (1% and 2%), either before UVA irradiation to evaluate protection, or after UVA irradiation to evaluate repair. Lipofuscin was detected as before.

[0163] The results of the protection test are presented in Table 9 below.

[0164] [Tables9] Mean versus control (in %) Standard deviation Efficacy of reducing lipofuscin production (in %) p-value (versus UVA stress) Control 100 1 100 ***; <0.001 Skin expiratory without extract and exposed to UVA 119 1 0 / Skin expiratory with cosmetic ingredient 2a (extract with bacteria) at 1% for 24h and exposed to UVA 113 1 32 **; 0.001 Skin expiratory with cosmetic ingredient 2a (extract with bacteria) at 2% for 24h and exposed to UVA 104 1 78 ***; <0.001

[0165] The results of the repair test are presented in Table 10 below.

[0166] [Tables 10] Mean versus control (in %) Standard deviation Efficacy of reducing lipofuscin production (in %) p-value (versus UVA stress) Control 100 1 100 ***; <0.001 Skin expiry without extract and exposed to UVA 119 1 0 / Skin expiry exposed to UVA then treated with cosmetic ingredient 2a (extract with bacteria) at 1% for 24h 107 1 62 ***; <0.001 Skin expiry exposed to UVA then treated with cosmetic ingredient 2a (extract with bacteria) at 2% for 24h 107 2 62 ***; <0.001

[0167] Thus, the lacto-fermented extracts of Palmaria palmata according to the invention, with fragments of lactic acid bacteria, significantly reduce lipofuscin production when the skin is exposed to UVA radiation after treatment (protection test), but also before treatment (repair test). A decrease in lipofuscin production of 32% and 78% is observed for the extract with bacterial fragments tested in the form of cosmetic ingredient 2a used at 1% and 2% before UVA irradiation (protection test), and a decrease of 62% for the extract with bacterial fragments tested in the form of cosmetic ingredient 2a used at 1% and 2% after UVA irradiation (repair test).

[0168] Example 4g: effect of lacto-fermented extract with bacterial fragments (cosmetic ingredient 2a tested at 1% and 2% by weight) on lipofuscin production (protection against IR infrared).

[0169] Test conditions:

[0170] The test conditions are equivalent to those of the test in Example 4d, except that the ingredient according to the invention, containing bacterial fragments, will be applied at two concentrations (1% and 2%) before IR irradiation (at a wavelength of 850 nm for 1.5 hours) to evaluate protection. Lipofuscin was detected as before.

[0171] The results of the IR protection test are presented in Table 11 below.

[0172] [Tableauxll] Mean versus control (in %) Standard deviation Efficacy of reducing ipofuscin production (in %) p-value (versus stress to IR) Control 100 2 100 ***; <0.001 Skin expiratory without extract and exposed to IR 120 3 0 / Skin expiratory with cosmetic ingredient 2a (extract with bacteria) at 1% for 24h and exposed to IR 100 4 99 ***; <0.001 Skin expiratory with cosmetic ingredient 2a (extract with bacteria) at 2% for 24h and exposed to IR 105 1 74 ***; <0.001

[0173] Thus, the lacto-fermented extracts of Palmaria palmata according to the invention, with fragments of lactic acid bacteria, significantly reduce lipofuscin production when the skin is exposed to IR after treatment (protection test). A decrease in lipofuscin production of 99% and 74% was observed for the extract with bacterial fragments tested in the form of cosmetic ingredient 2a used at 1% and 2% before IR irradiation.

[0174] Example 5: Effect of lacto-fermented extracts of the red alga Palmaria palmata according to the invention, respectively with and without fragments of lactic acid bacteria, on the aging mechanism related to protein carbonylation

[0175] Example 5a: Ex vitro analysis of the effect of lacto-fermented extracts of the red alga Palmaria palmata according to the invention, respectively with and without fragments of lactic acid bacteria, on the aging mechanism related to protein carbonylation

[0176] Test conditions:

[0177] The cell model was a fragment of human skin from a 44-year-old donor, phototype III.

[0178] Two samples comprising a cosmetic ingredient according to the invention (examples 2a or 2b, respectively, comprising an extract including fragments of lactic acid bacteria, and an extract not including fragments of lactic acid bacteria) at 2% in water were prepared.

[0179] These two samples according to the invention were respectively applied to fragments of human skin (expiants), which were exposed to UVA radiation (6J / cm2).

[0180] A skin expiant that has not been subjected to the application of an extract according to the invention has also been treated with UVA, in the same way as skin expiants with extracts according to the invention.

[0181] A control sample was also prepared, which corresponds to an expiant that has not been subjected to the application of an extract according to the invention, and which has not been treated with UVA.

[0182] Carbonylated proteins in all of these expiants were then detected and analyzed by specific labeling and fluorescence imaging (Oxi-Proteome), at the level of the different layers of the skin.

[0183] The results are presented in Tables 12 to 15 below.

[0184] [Tables 12] Mean vs. control (in %) Standard deviation Carbonylation production reduction efficacy (in %) p-value (vs. UVA stress) Whole skin Control 100 10 100 ***; <0.001 Skin expiratory without extract and exposed to UVA 201 8 0 / Skin expiratory with cosmetic ingredient 2b (bacteria-free extract) and exposed to UVA 120 15 80 ***; <0.001 Skin expiratory with cosmetic ingredient 2a (bacteria-containing extract) and exposed to UVA 112 2 88 ***; <0.001

[0185] [Tables 13] Mean vs. control (in %) Standard deviation Carbonylation production reduction efficacy (in %) p-value (vs. UVA stress) Stratum Coma Control 100 13 100 ***; <0.001 Skin expiratory without extract and exposed to UVA 302 34 0 / Skin expiratory with cosmetic ingredient 2b (extract without bacteria) and exposed to UVA 160 21 71 ***; <0.001 Skin expiratory with cosmetic ingredient 2a (extract with bacteria) and exposed to UVA 134 19 83 ***; <0.001

[0186] [Tables 14] Mean vs. control (in %) Standard deviation Efficacy of reducing carbonylation production (in %) p-value (vs. UVA stress) Epidermis Control 100 5 100 ***; <0.001 Skin expiratory without extract and exposed to UVA 167 5 0 / Skin expiratory with cosmetic ingredient 2b (extract without bacteria) and exposed to UVA 114 8 79 ***; <0.001 Skin expiratory with cosmetic ingredient 2a (extract with bacteria) and exposed to UVA 120 13 70 ***; <0.001

[0187] [Tables 15] Mean vs. control (in %) Standard deviation Efficacy of reducing carbonylation production (in %) p-value (vs. UVA stress) Dermis Control 100 2 100 ***; <0.001 Skin expiratory without extract and exposed to UVA 271 40 0 ​​ / Skin expiratory with cosmetic ingredient 2b (extract without bacteria) and exposed to UVA 135 14 79 ***; <0.001 Skin expiratory with cosmetic ingredient 2a (extract with bacteria) and exposed to UVA 119 20 89 ***; <0.001

[0188] The lacto-fermented extracts of Palmaria palmata according to the invention, with and without fragments of lactic acid bacteria, make it possible to reduce protein carbonylation significantly, when the skin is exposed to UVA. This reduction in protein carbonylation is observed throughout the skin, as well as at the individual level, i.e. at the level of each layer of the skin (the Stratum Comeum, the epidermis and the dermis).

[0189] In particular, the lacto-fermented extract according to the invention, which includes fragments of lactic acid bacteria, has a greater impact on reducing protein carbonylation than the lacto-fermented extract according to the invention that does not include bacteria, and this result is observed in virtually all layers of the skin, with the exception of the epidermis. However, in the latter case (epidermis), the reduction of protein carbonylation by the lacto-fermented extract containing protein fragments remains very effective.

[0190] The results of reducing protein carbonylation by each of the extracts of the invention (with and without bacteria) are always advantageously very close to the results of the control expiant.

[0191] These results are confirmed with [Fig.4], which illustrates the presence of carbonylated proteins at the level of these different skin expiants tested.

[0192] The first image, top left, illustrates the control skin expiant which has not been treated with UVA, and therefore includes a very small amount of carbonylated proteins (bleached area of ​​the image).

[0193] The second image, top right, illustrates the skin expiant which does not include any extract according to the invention, and which has been treated with UVA: it is visible that almost the entire surface of the expiant, which is white in color, comprises carbonylated proteins, which is due to UVA radiation.

[0194] The third and fourth images, on the bottom line, respectively on the left and right, illustrate UVA-treated skin expiants comprising an extract according to the invention, respectively with and without fragments of lactic acid bacteria. These two images are visually almost identical and visually resemble the first control image: a very small proportion of the surface of these expiants is white, which corresponds to the fact that a very small proportion of the surface of these expiants comprises carbonylated proteins.

[0195] Thus, the results of these tests demonstrate that the extracts according to the invention, with and without fragments of lactic acid bacteria, very effectively reduce protein carbonylation at all levels of the skin. This demonstrates that these two types of extracts according to the invention act on skin pigmentation and provide a whitening and / or lightening effect on the skin. In particular, these tests demonstrated the ability of the lacto-fermented extracts according to the invention to protect skin cells.

[0196] Example 5b: effect of lacto-fermented extract with bacterial fragments (cosmetic ingredient 2a tested at 1% and 2% by weight) on protein carbonylation (protection against UVA)

[0197] Test condition:

[0198] The procedure was carried out as in example 5a, but the sample was applied before or after the application of UVA.

[0199] The cell model was a fragment of human skin from a 35-year-old donor, phototype III.

[0200] The sample tested was a cosmetic ingredient 2a according to the invention (extract containing fragments of lactic acid bacteria), tested at 1% and 2%, and applied either for 24 hours before UVA irradiation (6J / cm2) to evaluate the protective effect, or just after UVA irradiation to evaluate the repairing effect.

[0201] The results are presented in Tables 16 (for the protection effect) and 17 (for the repair effect) below.

[0202] [Tables 16] Mean versus control (in %) Standard deviation Efficacy of reducing carbonylation production (in %) p-value (versus stress to UVA) Protection effect / Whole skin Control 100 7 100 ***; <0.01 Skin expiratory without extract and exposed to UVA 255 23 0 / Skin expiratory with cosmetic ingredient 2a (extract with bacteria), tested at 1% for 24h, and exposed to UVA 206 15 32 *; <0.05 Skin expiratory with cosmetic ingredient 2a (extract with bacteria), tested at 2% for 24h and exposed to UVA 202 4 34 *; <0.05

[0203] [Tables 17] Mean vs. control (in %) Standard deviation Carbonylation production reduction efficacy (in %) p-value (vs. UVA stress) Repair effect / Whole skin Control 100 7 100 ***; <0.001 Skin expiry without extract and exposed to UVA 255 23 0 / Skin expiry exposed to UVA then treated with cosmetic ingredient 2a (extract with bacteria) tested at 1% for 24h 121 9 86 ***; <0.001 Skin expiry exposed to UVA then treated with cosmetic ingredient 2a (extract with bacteria) tested at 2% for 24h 122 13 86 ***; <0.001

[0204] UVA stress increases protein carbonylation by +155%, significantly.

[0205] The lacto-fermented extracts of Palmaria palmata according to the invention, with fragments of lactic acid bacteria, significantly reduce protein carbonylation when the skin is exposed to UVA radiation after treatment (protective effect). This reduction in protein carbonylation is observed throughout the skin (as shown in Table 16), as well as at the individual level, i.e., at the level of each skin layer: the stratum corneum, the epidermis, and the dermis (not shown).

[0206] Furthermore, the lacto-fermented extracts of Palmaria palmata according to the invention, with fragments of lactic acid bacteria, significantly reduce protein carbonylation when the skin has been exposed to UVA radiation prior to treatment (repairing effect). Again, the reduction of protein carbonylation can be observed on the whole of the skin (as shown in Table 17), as well as at the individual level, that is to say at the level of each layer of the skin, the Stratum Comeum, the epidermis and the dermis (not shown).

[0207] Example 5c: effect of lacto-fermented extract with bacterial fragments (cosmetic ingredient 2a tested at 1% and 2% by weight) on protein carbonylation (protection against infrared IR)

[0208] Test condition:

[0209] The procedure was carried out as in example 5b.

[0210] The cell model was a fragment of human skin from a 35-year-old donor, phototype III. The sample was applied before the application of IR.

[0211] The sample tested was a cosmetic ingredient 2a (extract according to the invention containing fragments of lactic acid bacteria), tested at 1% and 2%, and applied either for 24 hours before IR irradiation (at a wavelength of 850nm for 1h30) to evaluate the protective effect.

[0212] The results are presented in Table 18 below.

[0213] [Tables 18] Mean vs. control (in %) Standard deviation Carbonylation production reduction efficiency (in %) p-value (vs. IR stress) Whole skin Control 100 11 100 ***; <0.001 Skin expiratory without extract and exposed to IR 160 4 0 / Skin expiratory with cosmetic ingredient 2a (extract with bacteria), tested at 1% for 24h, and exposed to IR 117 1 72 ***; <0.001 Skin expiratory with cosmetic ingredient 2a (extract with bacteria), tested at 2% for 24h and exposed to IR 119 1 69 ***; <0.001

[0214] UVA stress increases protein carbonylation by +60%, significantly.

[0215] The lacto-fermented extracts of Palmaria palmata according to the invention, with fragments of lactic acid bacteria, make it possible to significantly reduce protein carbonylation when the skin is exposed to IR. This reduction in protein carbonylation is observed over the whole skin (as shown in Table 18), as well as at the individual level, i.e. at the level of each layer of the skin, the Stratum Corneum, the epidermis and the dermis (not shown).

[0216] Example 6: effect of lacto-fermented extract with bacterial fragments (cosmetic ingredient 2a tested at 1% and 2% by weight) on protein carbamylation (protection against UVA)

[0217] Test condition:

[0218] The procedure was carried out as in example 5a, but the sample was applied before or after the application of UVA.

[0219] The cell model was a fragment of human skin from a 35-year-old donor, phototype III.

[0220] The sample tested was a cosmetic ingredient 2a (extract according to the invention containing fragments of lactic acid bacteria), tested at 1% and 2%, and applied either for 24 hours before UVA irradiation (6J / cm2) to evaluate the protective effect, or just after UVA irradiation to evaluate the repairing effect.

[0221] Carbamylation was measured by specific labeling with a carbamyl-lysine specific antibody (Euromedex) diluted in a PBS solution containing 3% BSA. The labeling was then visualized with a fluorophore-labeled secondary antibody (Alexa fluor 647 from Invitrogen). Images were acquired using an epifluorescence microscope (EVOS M5000 Imaging System).

[0222] The results are presented in Tables 19 (for the protection effect) and 20 (for the repair effect) below.

[0223] [Tables 19] Mean vs. control (in %) Standard deviation Efficacy of reducing carbamylation production (in %) p-value (vs. UVA stress) Protective effect / Whole skin Control 100 2 100 ***; <0.001 Skin expiratory without extract and exposed to UVA 112 2 0 / Skin expiratory with cosmetic ingredient 2a (extract with bacteria), tested at 1% for 24h, and exposed to UVA 95 1 100 ***; <0.001 Skin expiratory with cosmetic ingredient 2a (extract with bacteria), tested at 2% for 24h and exposed to UVA 92 1 100 ***; <0.001

[0224] [Tables20] Mean vs. control (in %) Standard deviation Efficacy of reducing carbamylation production (in %) p-value (vs. UVA stress) Repair effect / Whole skin Control 100 2 100 ***; <0.001 Skin expiry without extract and exposed to UVA 112 2 0 / Skin expiry exposed to UVA then treated with cosmetic ingredient 2a (extract with bacteria) tested at 1% for 24h 91 1 100 ***; <0.001 Skin expiry exposed to UVA then treated with cosmetic ingredient 2a (extract with bacteria) tested at 2% for 24h 90 1 100 ***; <0.001

[0225] UVA stress significantly increases protein carbamylation by +12%.

[0226] The lacto-fermented extracts of Palmaria palmata according to the invention, with fragments of lactic acid bacteria, significantly reduce protein carbamylation when the skin is exposed to UVA radiation after treatment (protective effect). This marked reduction in protein carbamylation is observed throughout the skin (as shown in Table 19).

[0227] Furthermore, the lacto-fermented extracts of Palmaria palmata according to the invention, with fragments of lactic acid bacteria, significantly reduce protein carbamylation, even when the skin has been exposed to UVA prior to treatment (repairing effect). Here again, the marked reduction in protein carbamylation is observed throughout the skin (as shown in Table 20).

[0228]

[0229] Example 7: Effect of lacto-fermented extract with bacterial fragments (cosmetic ingredient 2a tested at 0.1% and 1% by weight) on inflammation

[0230] Example 7a: In vitro analysis of the effect of the lacto-fermented extract of the red algae Palmaria palmata according to the invention, with fragments of lactic acid bacteria, on the measurement of prostaglandins of type E2 (PGE2).

[0231] Test condition:

[0232] The cell model was a primary culture of normal human dermis fibroblasts (NHDF) from a 38-year-old donor, used in the 5th pass (R5).

[0233] The products tested were as follows: - a positive control with the reference molecule Dexamethasone (Dex) tested at 0.2 pg / ml, - the lacto-fermented extract of the red algae Palmaria palmata according to the invention with fragments of lactic acid bacteria, tested at 0.1% and 1% by weight in the cell culture medium.

[0234] The products were diluted in the appropriate culture medium and incubated for 24 hours at 37 °C, 5% CO2.

[0235] Pro-inflammatory stress was induced by the addition of phorbol myristate acetate (PMA) at 0.1 pg / ml.

[0236] The PGE2 assay was performed using a commercial PGE2 assay kit (Enzo Life Science).

[0237] The results are presented in Table 21 below.

[0238] [Tables21] Reduction in PGE2 production (in %), compared to the control Positive control (Dex) - 100% Sample invention with 0.1% fragments -22% Sample invention with 1% fragments -41%

[0239] PMA stress significantly increases PGE2 production by fibroblasts.

[0240] The lacto-fermented extracts of Palmaria palmata according to the invention, with fragments of lactic acid bacteria, make it possible to reduce the release of PGE2 induced by the inflammatory agent.

[0241] Example 7b: In vitro analysis of the effect of the lacto-fermented extract of the algae Palmaria palmata red according to the invention, with fragments of lactic acid bacteria, on the measurement of interleukin (IL8)

[0242] Test condition:

[0243] The cell model was a primary culture of normal human epidermal keratinocytes (NHEK), from a 27-year-old donor, used in the 3rd pass (R3).

[0244] The products tested were as follows: - a positive control with the reference molecule Epigallocatechin gallate (EGCG) tested at 1 pg / ml, - the lacto-fermented extract of the red algae Palmaria palmata according to the invention with fragments of lactic acid bacteria, tested at 0.1% and 1% by weight in the cell culture medium.

[0245] The products were diluted in the appropriate culture medium and incubated for 24 hours at 37 °C, 5% CO2.

[0246] Pro-inflammatory stress was induced by the addition of poly IC at 2 pg / ml.

[0247] IL8 was measured using a commercial IL8 assay kit (R&D System).

[0248] The results are presented in Table 22 below.

[0249] [Tables22] Reduction in IL8 production (in %), compared to the control Positive control (EGCG) -79% Sample invention with 0.1% fragments -44% Sample invention with 1% fragments -27%

[0250] Poly IC stress significantly increases IL8 production by keratinocytes.

[0251] The lacto-fermented extracts of Palmaria palmata according to the invention, with fragments of lactic acid bacteria, make it possible to reduce the release of IL8 induced by the inflammatory agent.

[0252] Example 8: effect of lacto-fermented extract with bacterial fragments (cosmetic ingredient 2a tested at 1% and 2% by weight) on collagen (protection against UVA)

[0253] Test condition:

[0254] The cell model was a fragment of human skin from a 35-year-old donor, phototype III.

[0255] The sample tested was cosmetic ingredient 2a (extract according to the invention containing fragments of lactic acid bacteria), tested at 1% and 2% in water, and applied either for 24 hours before UVA irradiation (6J / cm2) to evaluate the protective effect, or just after UVA irradiation to evaluate the repairing effect.

[0256] Collagen measurement was performed by specific labeling using a type 1 collagen-specific antibody (Abcam) diluted in a PBS solution containing 3% BSA. The labeling was then visualized using a fluorophore-labeled secondary antibody (Invitrogen's Alexa fluor 647). Images were acquired using an epifluorescence microscope (EVOS M5000 Imaging System).

[0257] The results are presented in Tables 23 (for the protection effect) and 24 (for the repair effect) below.

[0258] [Tables23] Mean vs. control (in %) Standard deviation Collagen 1 protection efficacy (in %) p-value (vs. UVA stress) Protection effect / Dermis Control 100 1 100 ***; <0.001 Skin expiratory without extract and exposed to UVA 79 1 0 / Skin expiratory with cosmetic ingredient 2a (extract with bacteria), tested at 1% for 24h, and exposed to UVA 92 1 61 ***; <0.001 Skin expiratory with cosmetic ingredient 2a (extract with bacteria), tested at 2% for 24h and exposed to UVA 116 1 100 ***; <0.001

[0259] [Tables24] Mean vs. control (in %) Standard deviation Collagen 1 repair efficacy (in %) p-value (vs. UVA stress) Repair effect / Dermis Control 100 1 100 ***; <0.001 Skin expiry without extract and exposed to UVA 79 1 0 / Skin expiry exposed to UVA then treated with cosmetic ingredient 2a (extract with bacteria) tested at 1% for 24h 125 3 100 ***; <0.001 Skin expiry exposed to UVA then treated with cosmetic ingredient 2a (extract with bacteria) tested at 2% for 24h 127 1 100 ***; <0.001

[0260] UVA stress significantly reduces the quality and density of type 1 collagen by -21%.

[0261] The lacto-fermented extracts of Palmaria palmata according to the invention, with fragments of lactic acid bacteria, make it possible to reduce the decline of collagen 1 significantly, when the skin is exposed to UVA after treatment (protective effect), as shown in Table 23.

[0262] In addition, the lacto-fermented extracts of Palmaria palmata according to the invention, with fragments of lactic acid bacteria, make it possible to reduce the decline of collagen 1 significantly, even when the skin has been exposed to UVA prior to treatment (repairing effect), as shown in Table 24.

[0263]

[0264] Example 9: effect of lacto-fermented extract with bacterial fragments (cosmetic ingredient 2a tested at 1% and 2% by weight) on elastin (protection against UVA)

[0265] Test condition:

[0266] The cell model was a fragment of human skin from a 35-year-old donor, phototype III.

[0267] The sample tested was a cosmetic ingredient 2a (extract according to the invention containing fragments of lactic acid bacteria), tested at 1% and 2% in water, and applied either for 24 hours before UVA irradiation (6J / cm2) to evaluate the protective effect, or just after UVA irradiation to evaluate the repairing effect.

[0268] Elastin was measured by specific labeling using an elastin-specific antibody (Abcam) diluted in a PBS solution containing 3% BSA. The labeling was then visualized using a fluorophore-labeled secondary antibody (Invitrogen's Alexa fluor 647). Images were acquired using an epifluorescence microscope (EVOS M5000 Imaging System).

[0269] The results are presented in Tables 25 (for the protection effect) and 26 (for the repair effect) below.

[0270] [Tables25] Mean vs. control (in %) Standard deviation Elastin protection efficacy (in %) p-value (vs. UVA stress) Protection effect / Dermis Control 100 2 100 ***; <0.001 Skin expiratory without extract and exposed to UVA 68 5 0 / Skin expiratory with cosmetic ingredient 2a (extract with bacteria), tested at 1% for 24h, and exposed to UVA 88 4 64 ***; <0.001 Skin expiratory with cosmetic ingredient 2a (extract with bacteria), tested at 2% for 24h and exposed to UVA 99 10 97 ***; <0.001

[0271] [Tables26] Mean vs. control (in %) Standard deviation Telastin repair efficacy (in %) p-value (vs. UVA stress) Repair effect / Dermis Control 100 2 100 ***; <0.001 Skin expiry without extract and exposed to UVA 68 5 0 / Skin expiry exposed to UVA then treated with cosmetic ingredient 2a (extract with bacteria) tested at 1% for 24h 91 1 73 ***; <0.001 Skin expiry exposed to UVA then treated with cosmetic ingredient 2a (extract with bacteria) tested at 2% for 24h 90 4 69 ***; <0.001

[0272] UVA stress significantly reduces the quality and density of elastin fibers by -32%.

[0273] The lacto-fermented extracts of Palmaria palmata according to the invention, with fragments of lactic acid bacteria, make it possible to reduce the decrease in elastin significantly when the skin is exposed to UVA after treatment (protective effect), as shown in Table 25.

[0274] In addition, the lacto-fermented extracts of Palmaria palmata according to the invention, with fragments of lactic acid bacteria, make it possible to reduce the loss of elastin significantly, when the skin has been exposed to UVA prior to treatment (repairing effect), as shown in Table 26.

Claims

Demands [Clause 1] Lacto-fermented extract of at least one red alga of the order Palmariales, said red alga having been lacto-fermented by lactic acid bacteria.

2. Extract according to claim 1, wherein said red algae of the order Palmariales is of the genus Palmaria.

3. Extract according to claim 2, wherein said red algae of the genus Palmaria is the species Palmaria palmata.

4. Extract according to any one of claims 1 to 3, wherein said red algae has been lacto-fermented by lactic acid bacteria of the species Lactiplantibacillus plantarum.

5. Extract according to any one of claims 1 to 4, said extract being an aqueous extract comprising preferably from 0.1% to 10% by weight of dry extract, and preferably from 1% to 5% by weight of dry extract.

6. Extract according to any one of claims 1 to 5, characterized in that it comprises from 3 to 15% by weight of lactic acid, relative to the total weight of dry extract.

7. Extract according to any one of claims 1 to 6, characterized in that it comprises cyclodipeptides, and in particular cyclodipeptide Pro-Ala, cyclodipeptide Pro-Leu, cyclodipeptide Pro-Ile, and cyclodipeptide Pro-Val, said cyclodipeptides being preferably present in an amount of 0.001% to 0.25% by weight, relative to the total weight of the dry extract.

8. Extract according to any one of claims 1 to 7, wherein said lactic acid bacteria are present in said lacto-fermented extract in the form of fragments.

9. A process for producing a lacto-fermented extract as defined according to any one of claims 1 to 8, comprising the following steps: - a step A) of lacto-fermentation of a red alga by lactic acid bacteria in an aqueous medium comprising sea salt, said red alga being of the order Palmariales, without aeration; - a step B) of filtration by clarification, to separate the residues of said red alga from the reaction medium; - a step C) of treatment of said reaction medium, to form said lacto-fermented extract.

10. A process for producing according to claim 9 a lacto-fermented extract according to any one of claims 1 to 7, wherein said processing step C) constitutes an additional filtration step, for removing said lactic acid bacteria from said lacto-fermented extract, said additional filtration step being a sterilizing filtration.

11. A method of producing according to claim 9 a lacto-fermented extract according to claim 8, wherein said processing step C) constitutes an inactivation step of said lactic acid bacteria within said lacto-fermented extract leading to their fragmentation.

12. Cosmetic ingredient comprising an extract according to any one of claims 1 to 8 with at least one cosmetic additive selected from stabilizing agents and antimicrobial agents.

13. Cosmetic ingredient according to claim 12 characterized in that it comprises glycerin and pentylene glycol.

14. Use of a lacto-fermented extract according to any one of claims 1 to 8 or of a cosmetic ingredient according to claim 12 or 13 in a cosmetic composition.

15. Cosmetic composition comprising a lacto-fermented extract according to any one of claims 1 to 8 or a cosmetic ingredient according to claim 12 or 13.

16. Cosmetic composition according to claim 15 characterized in that the dry extract of the lacto-fermented extract according to any one of claims 1 to 8 represents from 0.001 to 1%, and preferably from 0.01 to 0.1% of the total weight of the cosmetic composition.

17. Cosmetic use of a lacto-fermented extract as defined in any one of claims 1 to 8, of a cosmetic ingredient according to claim 12 or 13, or of a cosmetic composition according to claim 15 or 16.

18. Use according to claim 17 for topical cosmetic application on the skin.

19. Use according to claim 17 or 18 for an application of whitening, and / or skin lightening, and / or skin brightness enhancement, and / or skin radiance enhancement.

20. Use according to claim 17 or 18 to combat skin aging and / or improve skin elasticity and / or to improve skin firmness and / or texture, particularly in an anti-wrinkle or anti-aging cosmetic composition.

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