EP4271208A1

EP4271208A1 - Fermented extract of longoza seeds

Info

Publication number: EP4271208A1
Application number: EP21854927.7A
Authority: EP
Inventors: Lorène GOURGUILLON; Patrick Choisy; Carine Nizard; Olivier JEANNETON
Original assignee: LVMH Recherche GIE
Current assignee: LVMH Recherche GIE
Priority date: 2020-12-30
Filing date: 2021-12-17
Publication date: 2023-11-08
Also published as: JP2024501552A; KR20230128047A; FR3118422B1; FR3118422A1; WO2022144513A1; CN116916763A; US20240099961A1

Abstract

The present invention relates to a fermented extract of Longoza seeds, as well as to the preparation method thereof and to cosmetic compositions containing same. The fermented extract of Longoza seeds can in particular be used as an active ingredient to promote and/or stimulate epidermal renewal, desquamation of the skin, vitality of the skin cells, and/or cohesion of the skin cells, and/or to prevent and/or reduce the signs of skin ageing, in particular loss of firmness, loss of elasticity, loss of density, appearance of wrinkles and/or fine lines, dryness of the skin, alteration of the skin's barrier function, alteration of the skin's suppleness and/or alteration of the homogeneity or radiance of the complexion.

Description

DESCRIPTION

TITLE: Fermented Longoza Seed Extract

FIELD OF THE INVENTION

The present invention relates to the cosmetic field and in particular to a fermented extract of Longoza seeds as well as the fermentation process for obtaining it, a cosmetic composition containing said fermented extract of Longoza seeds, and its use in a cosmetic care process. and/or for making up keratin materials.

STATE OF THE ART

We know that aging is induced by endogenous (eg: stress, hormones, etc.) and exogenous (eg: temperature, climate, UV radiation, atmospheric pollution, cigarette smoke, etc.) factors leading to a slowdown and/or an alteration of various cellular functions. At the level of the skin, aging is manifested by various clinical signs, in particular by a reduction in epidermal renewal, an alteration of the cutaneous barrier, a loss of firmness and elasticity of the skin, a reduction in the thickness of the skin, the appearance of fine lines or wrinkles, the appearance of a less even complexion, and/or drier skin.

There is therefore a constant need to find agents capable of preventing and/or reducing the signs of skin aging, in particular making it possible to combat stress and/or making it possible to stimulate and/or promote skin renewal as well as the vitality of skin cells. .

Unexpectedly, the Applicant demonstrated the 'anti-aging' effects of a fermented extract of Longoza seeds in vitro on a skin model. Indeed, the fermented extract of Longoza seeds advantageously makes it possible to improve the structure of the skin, which notably becomes thicker, with a very clear basal organization and structured dermo-epidermal junctions. Furthermore, the fermented extract of Longoza seeds advantageously makes it possible to increase the expression of various proteins involved in the mechanical solidity of the skin as well as its renewal and its elasticity, such as elafin, desmoglein 1 , loricrine, transglutaminase and ZO-1 , ultimately promoting cellular integrity and skin cell homeostasis. These effects therefore make it possible to consider the use of this fermented extract of Longoza seeds to prevent and/or reduce the signs of skin aging. The fermented extract of Longoza seeds is in particular used according to the invention as a cosmetic active ingredient intended to promote and/or stimulate epidermal renewal, desquamation of the skin, the vitality of the skin cells and/or the cohesion of the skin cells, and /or prevent and/or reduce the signs of skin aging, in particular loss of firmness, loss of elasticity, loss of density, appearance of wrinkles and/or fine lines, dryness of the skin, alteration of the barrier function of the skin, the alteration of the suppleness of the skin and/or the alteration of the homogeneity or the radiance of the complexion.

DISCLOSURE OF THE INVENTION

The first object of the invention therefore relates to a fermented extract of Longoza seeds comprising a quinic acid content of between 0.05 and 2% and an acetic acid content of between 5 and 15%, by weight relative to the weight of dry extract of the fermented extract.

Another object of the invention relates to a process for the fermentation of Longoza seeds comprising the following steps: a) the incubation of a consortium of microorganisms in a solution comprising a carbohydrate at a temperature between 17 and 38° C for 1 to 10 days in order to obtain a culture of microorganisms, said consortium comprising at least one lactic acid bacterium belonging to the genus Lactobacillus or Pediococcus, at least one yeast belonging to the genus Saccharomyces, Schyzosaccharomyces or Torulaspora, and at least one bacterium acetic acid belonging to the genus Acetobacter, Gluconobacter or Komagataeibacter; b) the grinding of Longoza seeds in order to obtain a powder composed of particles having an average size of between 100 μm and 1 mm; c) adding said powder to a fermentation medium comprising the culture of microorganisms in order to obtain a fermentation mixture, said fermentation medium comprising a carbohydrate chosen from sucrose, glucose, fructose, molasses or a mixture of one or more of these, d) incubating the fermentation mixture at a temperature between 25 and 35°C for 7 to 13 days, and e) filtering the fermentation mixture to obtain a fermented extract of Longoza seeds.

Also, another object of the invention relates to a fermented extract of Longoza seeds obtained by the method as described according to the invention.

The invention also relates to a cosmetic composition comprising, in a physiologically acceptable medium, a fermented extract of Longoza seeds.

Another object of the invention relates to a cosmetic process for caring for and/or making up keratin materials, in particular the skin and/or the lips, comprising the application to said keratin materials of at least one layer of the composition cosmetic as described according to the invention.

The invention finally relates to the cosmetic use of a fermented extract of Longoza seeds according to the invention as an active ingredient for promoting and/or stimulating epidermal renewal, desquamation of the skin, the vitality of skin cells, and/or the cohesion of skin cells, and/or preventing and/or reducing the signs of skin aging, in particular loss of firmness, loss of elasticity, loss of density, appearance of wrinkles and/or fine lines, dryness of the skin, the alteration of the barrier function of the skin, the alteration of the suppleness of the skin and/or the alteration of the homogeneity or the radiance of the complexion.

DESCRIPTION OF FIGURES

[Fig. 1] Effect of fermented Longoza seed extract on loricrin expression

[Fig. 2] Effect of Fermented Longoza Seed Extract on Filaggrin Expression

[Fig. 3] Effect of Fermented Longoza Seed Extract on Elafin Expression

[Fig. 4] Effect of Fermented Longoza Seed Extract on Kallikrein Expression

5

[Fig. 5] Effect of Fermented Longoza Seed Extract on Desmoglein 1 Expression

[Fig. 6] Effect of Fermented Longoza Seed Extract on Transglutaminase Expression

[Fig. 7] Effect of Fermented Longoza Seed Extract on ZO-1 Expression [Fig. 8] Histological analysis of the effect of Longoza seed fermented extract on a skin model

[Fig. 9] Effect of Fermented Longoza Seed Extract on Elastic Modulus of Skin

DETAILED DESCRIPTION OF THE INVENTION

Aframomum ansustifolium or Longoza

Originally from Madagascar, the Longoza is a fruit of ['Aframomum an ustifolium, a plant of the ginger family. This plant is characterized by an exceptional regeneration by the root since it grows even after cutting and burning. It is one of the pioneer plants that grow back on land burned during forest fires in Madagascar.

In the context of the present invention, the fermented extract of Aframomum an ustifolium or Longoza is obtained from the seeds of the Aframomum an ustifolium or Longoza plant. Although we use the expression “seeds of Longoza” here for simplification, this obviously means indifferently seeds of Aframomum an ustifolium or Longoza.

Longoza seed fermented extract

Longoza seeds are used in a fermentation process in order to generate a fermented extract of Longoza seeds, object of the present invention.

It is specified that in the present description, the expression "fermented extract" designates the product resulting from the fermentation process by fermentation, with or without filtration, therefore both with and without the microbial biomass of the fermentation medium, as well as any product having undergone additional steps, such as a formulation step (eg by adding a preservative).

According to a particular embodiment, the fermented extract of Longoza seeds according to the invention is in the form of an aqueous solution of fermented extract comprising the fermented dry extract, water and a preservative. The fermented extract in the form of an aqueous solution is also called 'raw material' in the description, as opposed to 'dry matter' or fermented dry extract. According to a particular embodiment, the aqueous solution of fermented extract comprises the fermented extract in an active content (dry matter) ranging from 0.5 to 3% by weight, in particular 1 to 2% by weight and in particular 1.2 to 1.7% by weight of active material relative to the total weight of raw material (aqueous solution of fermented extract), water and a preservative, preferably glycerin. The water content will generally range from 20 to 40% by weight, in particular from 25 to 30% in particular from 27 to 29% by weight relative to the total weight of raw material (aqueous solution of fermented extract) and the preservative content (for example: glycerin) will generally range from 60 to 80% by weight, in particular from 65 to 75% by weight, in particular 70% by weight relative to the total weight of raw material (aqueous solution of fermented extract).

Thus, the invention also relates to a fermented extract of Longoza seeds in the form of a solution comprising 1 to 2% by weight of active ingredient of fermented extract of Longoza seeds, 65 to 75% by weight of glycerin and 25 to 30% by weight of water.

Preferably, the aqueous solution of fermented extract, otherwise called 'raw material' in the characterization which is illustrated below, comprises 1.2 to 1.65% in active matter (dry matter) of fermented extract, 70% of glycerine and 28.35 to 28.8% of water relative to the total weight of raw material (aqueous solution of fermented extract). According to a particular mode, the aqueous solution of fermented extract, otherwise called 'raw material' in the characterization which is illustrated below, comprises 1.2 to 1.35% in active matter (dry matter) of fermented extract, 70% glycerin and 28.65 to 28.8% water relative to the total weight of raw material (aqueous solution of fermented extract). According to another particular mode, the aqueous solution of fermented extract, otherwise called 'raw material' in the characterization which is illustrated below, comprises 1.35 to 1.65% in active matter (dry matter) of fermented extract , 70% glycerin and 28.35 to 28.65% water relative to the total weight of raw material (aqueous solution of fermented extract).

According to a particular mode, the fermented extract of Longoza seeds comprises a quinic acid content greater than 0.005%, preferably between 0.005 and 10%, and an acetic acid content greater than 0.5%, preferably between 0.5 and 25%, by weight relative to the weight of dry extract (dry matter) of the fermented extract. Preferably, the fermented extract of Longoza seeds comprises a quinic acid content of between 0.01 and 5%, more preferably between 0.05 and 2% by weight relative to the dry extract weight of the extract fermented. Preferably, the extract fermented Longoza seeds comprises an acetic acid content of between 1 and 20%, more preferably between 5 and 15% by weight by weight relative to the dry extract weight of the fermented extract.

Longoza seed fermented extract may include other organic acids, such as citric acid, gluconic acid, lactic acid, malic acid, and succinic acid. Also, the fermented extract of Longoza seeds may include:

- a citric acid content of between 0.01 and 5%, preferably between 0.05 and 2%, more preferably between 0.1 and 1%, even more preferably between 0.2 and 0.4% by weight relative the dry extract weight of the fermented extract,

- a gluconic acid content greater than 0.5%, preferably between 0.5 and 25%, more preferably between 1 and 20%, even more preferably between 1 and 15% by weight relative to the weight of dry extract fermented extract,

- a lactic acid content of between 0.001 and 10%, preferably between 0.005 and 5%, more preferably between 0.01 and 2%, more preferably between 0.01 and 1.5% by weight relative to the weight of dry extract of the fermented extract,

- a malic acid content of between 0.001 and 5%, preferably between 0.005 and 1%, more preferably between 0.008 and 0.5% by weight relative to the dry extract weight of the fermented extract, and/or

- a succinic acid content of between 0.001 and 5%, preferably between 0.005 and 1%, more preferably between 0.01 and 0.05% by weight relative to the dry extract weight of the fermented extract.

Preferably, the fermented Longoza seed extract does not include glucuronic acid or propionic acid.

The fermented Longoza seed extract may also include one or more preservatives. By “preservative” is meant a compound whose purpose is to preserve the composition of the extract or the cosmetic containing it from any physico-chemical and/or microbial alteration. Preferably, at least one preservative is chosen from butylene glycol, glycerin, propylene glycol, sodium benzoate benzoate and potassium sorbate, more preferentially glycerin. The fermented extract may in particular comprise one or more preservative(s) at a final concentration of between 60 and 80%, preferably between 65 and 75%, more preferably 70% by weight relative by total weight, preferably at least one of the preservatives described above chosen from butylene glycol, glycerin and propylene glycol.

Fermentation process

This fermented extract of Longoza seeds is obtained by a fermentation process comprising steps of: a) incubation of a consortium of microorganisms in a solution comprising a carbohydrate and optionally in addition an aqueous extract of tea leaves at a temperature between 17 and 38°C in order to obtain a culture of microorganisms; b) grinding seeds of Aframomum angustifolium or Longoza to obtain a fine powder; c) adding said powder to a fermentation medium comprising the culture of microorganisms and a carbohydrate in order to obtain a fermentation mixture; c) incubation of the fermentation mixture at a temperature between 12 and 42° C. for 2 to 20 days; d) filtration of the fermentation mixture, in order to obtain a fermented extract of Longoza seeds; and, optionally, e) adding at least one preservative.

By “fermentation process” is meant a process making it possible to obtain a fermented extract of Longoza seeds by culturing a consortium of microorganisms as defined here in a medium containing a carbohydrate and allowing their growth. The fermentation can take place under conditions of discontinuous culture (in “batch”), of continuous culture, or in continuous-discontinuous (in “Fed batch”). Fermentation can be carried out aerobic, micro-aerobic and/or anaerobic.

By "consortium of microorganisms" is meant an assembly of at least three species of microorganisms that can coexist in a stable and reproducible manner.

Advantageously, the microorganisms make it possible to combine complementary activities. By way of non-limiting example, the use or assimilation of a carbon source by a first species makes it possible to produce metabolites which can then be metabolized by a second species. The consortium of microorganisms according to the present invention comprises: i) at least one microorganism belonging to the group of lactic acid bacteria, ii) at least one microorganism belonging to the group of yeasts, and iii) at least one microorganism belonging to the group of acetic acid bacteria.

Preferably, at least one microorganism belonging to the group of lactic acid bacteria is a microorganism of the order Lactobacillales of the Lactobacillaceae family, in particular those belonging to the genera Lactobacillus and Pediococcus, such as Lactobacillus plantarum and/or Lactobacillus acidophilus , more preferably L. plantarum.

Preferably, at least one microorganism belonging to the yeast group is a microorganism of the order Saccharomycetales of the family Saccharomycetaceae or of the order Schyzosaccharomycetales of the family Schyzosaccharomycetaceae, in particular those belonging to the genera Saccharomyces, Schyzosaccharomyces or Torulaspora.

Preferably, at least one microorganism belonging to the yeast group is Saccharomyces cerevisiae, Torulaspora delbrueckii, Saccharomyces boulardii and/or Schyzosaccharomyces pombe.

Preferably, at least one microorganism belonging to the group of acetic acid bacteria is a microorganism of the order Rhodospirillales of the family Acetobacteraceae, in particular those belonging to the genera Acetobacter, Gluconobacter and Komagataeibacter (also called Gluconacetobacter). Acetic acid bacteria can originate from cider vinegar or unpasteurized wine with an acidity level of 4% to 5%, preferably commercial cider vinegar, labeled "organic farming" according to the definition of the European Union. Preferably, the addition of the acetic acid bacteria to the consortium is done by direct addition of an aliquot of vinegar in the composition comprising the other species of microorganisms. In vinegars, alongside the dominant acetic bacteria, there is a complex natural indigenous flora, part of which will be able to cohabit with the added microorganisms. Another part will collapse during the establishment of the consortium, in the same way as the development in symbiosis of all the micro-organisms forming the consortium prevents the development of undesirable species, including pathogenic species. According to a preferential mode, the consortium includes:

- at least one lactic acid bacterium belonging to the genus Lactobacillus or Pediococcus, more preferably chosen from Lactobacillus plantarum and Lactobacillus acidophilus,

- at least one yeast belonging to the genus Saccharomyces, Schyzosaccharomyces or Torulaspora more preferably chosen from Saccharomyces cerevisiae, Saccharomyces boulardii, Schyzosaccharomyces pombe and Torulaspora delbrueckii, and

- at least one acetic bacterium from cider vinegar or wine vinegar, preferably belonging to the genus Acetobacter, Gluconobacter or Komagataeibacter.

Step a): preparation of a culture of microorganisms

According to a first step, a), of the method, a culture of microorganisms (in other words, a consortium comprising the microorganisms as described above) is prepared. This step advantageously makes it possible to activate and multiply the microorganisms while establishing a stable and balanced consortium. During this step, the different microorganisms that will be present in the consortium are introduced into a solution comprising a carbohydrate (otherwise called a "nutrient medium") and possibly also an aqueous extract of tea leaves in order to generate a microbial suspension. The suspension is then incubated for 1 to 21 days in order to prepare the culture of the microorganisms (in other words, the consortium).

The solution comprises at least one carbohydrate, generally one or more mono- or disaccharides. It can be a pure sugar, such as sucrose, glucose or fructose, or a processed or co-produced product, such as molasses. Also, according to a preferential mode, the solution comprises a carbohydrate chosen from sucrose, glucose or fructose, molasses, or a mixture of one or more of these. Preferably, the carbohydrate is sucrose or a mixture of glucose and fructose. Preferably, at least one carbohydrate is present at a concentration comprised between 20 g/l and 100 g/l, preferably comprised between 50 to 95 g/l of solution. Preferably, the nutrient medium comprises sucrose, or a mixture of glucose and fructose, at a concentration of 50 g/l.

According to a particular embodiment, the solution may also comprise an aqueous extract of tea leaves (Camellia sinensis). Preferably, the aqueous extract is obtained from an infusion of tea leaves, preferably black tea leaves. Preferably, the extract aqueous tea leaves may be a black tea infusion prepared with 2 g/l to 50 g/l of tea leaves, more preferably with 5 g/l to 10 g/l of tea leaves.

The tea is prepared by adding dry leaves infused in water at a temperature of 70° C to 90° C for 10 to 60 min. Preferably, 5 g/l to 10 g/l of tea leaves are infused for 15 min at 80° C. The infusion can be used as is with the leaves, or more conveniently the aqueous phase is filtered. .

The microorganisms can be introduced into the nutrient medium in dry and/or wet form; the inoculation rate may in particular be between 10 ³ CFU/g and 10 ⁵ CFU/g of solution. The microbial suspension can then be incubated between 12°C and 45°C, preferably between 25°C and 35°C, more preferably at 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, or 35°C even more preferably at 26°C. According to a particularly advantageous mode, the incubation takes place in an open reactor, without the need to ensure aseptic conditions.

The suspension can be incubated for 1 to 21 days, preferably for 1 to 10 days, more preferably for 3 to 4 days, even more preferably for 3 days or 4 days. At the end of the incubation period, the total flora in the suspension may be between 10 ⁴ and 10 ⁷ CFU/g.

The consortium thus obtained is capable of reacting with a natural substrate, such as Longoza seeds, in an aqueous medium. Advantageously, it is capable of directing the extractive fermentation of Longoza seeds, to make accessible, and possibly transform, a great diversity of the compounds it contains.

Step b): Preparation of Longoza seeds

According to step b), the Longoza seeds are ground until a fine powder is obtained. The particles of this powder advantageously have an average size of between 0.01 and 1 mm, preferably between 100 μm and 1 mm, more preferentially between 250 and 500 μm, even more preferentially an average size of 500 μm. Preferably, the seeds are raw. Preferably, the seeds are dried before being ground.

Step b) can be carried out either before, at the same time or after step a).

Step c): preparation of a fermentation mixture According to step c) of the process, the Longoza seed powder obtained in step b) is added to a fermentation medium, comprising the culture of the microorganisms obtained according to step a) as well as a carbohydrate, in order to obtain a fermentation mixture.

Preferably, the culture of microorganisms is added to the fermentation medium at a rate of between 10 ² CFU/g and 10 ⁴ CFU/g of fermentation medium. Preferably, the culture of microorganisms is added to the fermentation medium at a concentration of between 0.5% and 15% by weight based on the weight of fermentation medium, preferably between 0.75 and 10%, more preferably at 5% .

Preferably, the Longoza seeds in powder form are added to the fermentation medium at a concentration of between 1 and 75 g of seeds per liter of fermentation medium (g/L), preferably between 10 and 60 g/L, plus preferably between 12 and 50 g/L, even more preferably 12 g/L or 50 g/L. In other words, the Longoza seeds in powder form are added to the fermentation medium at a concentration of between 0.1 and 7.5%, preferably between 1 and 6%, more preferably between 1.2 and 5%, even more preferably at a concentration of 1, 2 or 5% by weight relative to the total volume of the fermentation medium.

The fermentation medium also comprises a carbohydrate, preferably chosen from sucrose, glucose, fructose, molasses or a mixture of one or more of these.

According to a particular mode, the solution can comprise an aqueous extract of tea leaves, as described above. Preferably, the aqueous extract is obtained from an infusion of tea leaves, preferably black tea leaves.

In order to facilitate the dissolution of the carbohydrate in the fermentation medium, the latter can be heated before or after addition of the carbohydrate, for example at a temperature between 25° C. and 90° C., preferably 35° C. Preferably, in the event of heating of the fermentation medium, this is done before adding the culture of microorganisms or else at a temperature less than or equal to 45° C., preferably less than or equal to 35° C., if after adding the culture.

Step d): incubation According to the next step d) of the method, the fermentation mixture obtained in step c) is then incubated in order to ensure the fermentation of the Longoza seeds.

The incubation takes place at a temperature between 12°C and 45°C, preferably between 25°C and 35°C, more preferably at 25°C, 26°C, 27°C, 28°C, 29°C. C, 30°C, 31°C, 32°C, 33°C, 34°C, or 35°C, even more preferably at 26°C. The incubation is carried out for a period of 2 to 20 days, preferably for 7 to 13 days, more preferably for 7, 8, 9, 10, 11, 12, or 13 days, even more preferably 10 days.

According to a particular aspect, the incubation takes place in an open reactor, without the need to ensure aseptic conditions. According to a particular aspect, the incubation takes place at a humidity level of between 20% to 60%, preferably between 30% to 50%, more preferably 40%.

Step e): filtration

The fermentation mixture is then filtered during step e). By “filtration” is meant here a step making it possible to separate the solid and liquid fractions. The solid fraction may in particular comprise the remaining Longoza seeds and/or microorganisms. Advantageously, filtration makes it possible to remove all the microorganisms present in the mixture, in order to obtain a fermented extract of sterile Longoza seeds. Filtration can be achieved by using one or more sieves and/or filters. Also, according to one embodiment, step e) corresponds to one or more filtration steps, preferably comprising at least one sieving step and/or at least one sterilizing filtration step. Preferably, step e) comprises a sieving step and a sterilizing filtration step. Preferably, the sterilizing filtration is carried out on a filter having an average pore size of between 0.1 μm and 0.2 μm, preferably 0.2 μm. Advantageously, the sieving makes it possible to remove the powder debris. Advantageously, sterilizing filtration makes it possible to remove any cellular debris quickly and inexpensively. Preferably, at least one of the filters is a cellulose filter.

Step f): concentration and/or formulation (optional)

The process described above makes it possible to obtain a fermented extract of clarified Longoza seeds. However, in certain cases, it may be advantageous to carry out one or more additional steps, for example concentration or formulation. The process according to the invention can thus also comprise at least one additional step and subsequent to step e) of concentration or formulation.

In a particular embodiment, the method according to the invention further comprises, after step f) of filtration, one or more of the following steps aimed in particular at adapting the composition to a particular formulation:

- a concentration step; and or

- a formulation step.

Each optional step identified above may be present as a single additional process step, or in combination with one or more other optional steps. Preferably, the fermentation process comprises a step f) of adding at least one preservative to the fermented extract of Longoza seeds. Preferably, said preservative is chosen from butylene glycol, glycerin, propylene glycol, sodium benzoate benzoate and potassium sorbate, more preferentially glycerin. In some cases, two or more preservatives may be added to the fermented Longoza seed extract. Preferably, the preservative is added to the fermented Longoza seed extract of step f) at a final concentration of between 50 and 80%, preferably between 65 and 75%, more preferably 70%, by weight per relative to the total weight of the extract in the form of a solution. When the preservative is added to the fermented extract of Longoza seeds from step e) at a final concentration of between 50 and 80%, said preservative is selected from butylene glycol, glycerin and propylene glycol.

The fermented extract of Longoza seeds is preferably stored at around 4°C (4 ± 2°C) and/or in the dark at the end of step e) of filtration or at the end of any other step when an additional step is present after step e). When the method includes the addition of at least one preservative, the fermented extract of Longoza seeds is preferably stored at room temperature.

The fermented extract of Longoza seeds is referenced under the international nomenclature, INCI: “Water, glycerin, Aframomum angustifolium seed extract, lactobacillus ferment lysate, yeast ferment extract”. This product contains in particular 1.2 to 1.65% by weight of dry extract of fermented extract of Longoza seeds in a water/glycerin mixture. Cosmetic composition

Surprisingly, the inventors have demonstrated that the fermented extract of Longoza seeds has beneficial effects on the skin in vitro, making it possible in particular to increase the number and/or density of fibroblasts, as well as the expression of various proteins involved in the mechanical strength of the skin, its renewal and its elasticity, such as elafin, desmoglein 1 and loricrin. Thus, the fermented extract of Longoza seeds makes it possible to fight against the signs of skin aging linked to age and/or stress, in particular the loss of firmness and/or elasticity and the alteration of the barrier function of the skin.

According to the invention, the fermented extract of Longoza seeds is used in an effective amount to obtain the desired effect. It can be used as it is or advantageously incorporated into a cosmetic composition suitable for topical application to keratin materials.

In particular, the fermented extract of Longoza seeds is used as such or is present in a cosmetic composition.

Also, another object of the invention relates to a cosmetic composition comprising, in a physiologically acceptable medium, a fermented extract of Longoza seeds according to the invention.

By “cosmetic composition” is meant any composition for cosmetic purposes, that is to say aesthetic, which can be brought into contact with the superficial parts of the human body and more particularly with keratin materials, in particular the skin and / or the human lips. .

By “physiologically acceptable medium” is meant any excipient suitable for topical use, in contact with keratin materials, without risk of toxicity, incompatibility, instability and/or allergic response.

By “keratin materials” according to the invention, is meant the skin and/or its appendages, and more particularly the human skin and/or lips. In particular, it will be the skin of the face and/or of the neck and/or of the body, and of the lips. The keratin materials according to the invention are in particular healthy keratin materials (“healthy” subjects), that is to say not exhibiting disorders or disorders which would be part of a pathological state (“non-healthy” subjects, affected of a pathology). Reference will be made indiscriminately to healthy skin and/or lips or to healthy skin and/or lips in the rest of the description.

The fermented extract of Longoza seeds is present in the composition of the invention in a content ranging from 0.0001% to 5%, preferably from 0.0005% to 2%, more preferably from 0.001% to 1% by weight of raw material (solution of fermented extract obtained according to the method described above) relative to the total weight of the composition. Thus the fermented extract of Longoza seeds is present in the composition of the invention in a content ranging from 0.0000012% to 0.0825%, preferably from 0.000006% to 0.033%, more preferably from 0.000012 % to 0.0165% by weight of active ingredient (fermented extract of Longoza seeds) relative to the total weight of the composition.

The additional ingredients advantageously used in the composition of the invention may be present in a content ranging from 0.0001% to 10%, preferably from 0.001% to 5% by weight (of raw material) relative to the total weight of the composition.

The physiologically acceptable medium generally represents from 1 to 99% by weight, relative to the total weight of said composition. The physiologically acceptable medium of the composition according to the invention comprises water and optionally the preservative as defined above.

In a particular embodiment, said cosmetic composition comprising a fermented extract of Longoza seeds according to the invention is a composition for caring for and/or making up keratin materials, in particular the skin and/or the lips and in particular the face and/or neck skin.

The cosmetic composition of the invention generally comprises, in addition to the fermented extract of Longoza seeds and the physiologically acceptable medium, one or more cosmetic excipients acceptable from those known to those skilled in the art with a view to obtaining a composition for the topical application, for example in the form of a milk, a cream, an ointment, a water-in-oil or oil-in-water emulsion, a balm, a stick, a gel, a lotion, a serum, or even a powder. The cosmetic composition of the invention may also be in the form of a patch or a mask, in particular a mask in the form of a thick cream, or else in the form of a cellulose mask soaked in fermented extract of seeds of Longoza or a composition containing it.

In a preferred embodiment, said composition is in the form of a cream, emulsion, solution, suspension, gel, milk, lotion, or serum. When the composition is in the form of an emulsion, the emulsion may be an oil-in-water or water-in-oil emulsion or multiple emulsion.

The cosmetic composition of the invention may be in any galenic form suitable for topical application to keratin materials, in particular of the skin and/or of the lips and in particular of the skin of the face and/or of the neck comprising the fermented extract of Longoza seeds and at least one cosmetic ingredient chosen from antioxidants, perfumes, vitamins, thickening agents, emollient agents, moisturizing agents, anti-aging agents, lifting agents, tensing agents, plumping agents , soothing agents, antipollution agents, lightening or depigmenting agents, fillers, nacres and mixtures thereof.

Also, according to a preferred subject of the invention, the cosmetic composition may also comprise at least one cosmetic adjuvant chosen from the group consisting of antioxidants, emollients, moisturizing agents, anti-aging agents, and mixtures thereof. .

Depending on the nature of the composition, one or more cosmetically acceptable excipients will be selected from emulsifiers, polymers, surfactants, rheology agents, electrolytes, pH adjusters, antioxidants, preservatives, colorants, and their mixtures.

By way of specific example, the cosmetic composition may comprise hydrophilic gelling agents, antioxidants, preservatives and mixtures thereof.

Cosmetic process The present invention also relates to a cosmetic process for caring for and/or making up keratin materials, in particular the skin and/or the lips and in particular the skin of the face and/or the neck, comprising the topical application on said keratin materials, of at least one layer of the fermented extract of Longoza seeds or of a cosmetic composition containing it as described according to the invention.

The cosmetic process of the present invention is in particular intended to promote and/or stimulate epidermal renewal, desquamation of the skin, the vitality of the skin cells, and/or the cohesion of the skin cells, and/or prevent and/or reduce the signs of skin aging, in particular loss of firmness, loss of elasticity, loss of density, appearance of wrinkles and/or fine lines, dryness of the skin, alteration of the barrier function of the skin, the alteration of the suppleness of the skin and/or the alteration of the homogeneity or the radiance of the complexion.

By “promoting and/or stimulating epidermal renewal” is meant that the fermented extract of Longoza seeds is intended to promote keratinocyte differentiation.

By "promoting and/or stimulating the desquamation of the skin", we mean in particular that the fermented extract of Longoza seeds is intended to promote the renewal and development of cutaneous cells and tissues, in particular of the skin and/or the lips.

By “promoting and/or stimulating the vitality of skin cells”, we mean in particular that the fermented extract of Longoza seeds is intended to promote the general condition of the skin, its radiance.

By “preventing and/or reducing the signs of skin ageing” is meant in particular that the fermented extract of Longoza seeds is intended to reduce and/or delay the loss of firmness, the loss of elasticity, the loss of density, the dryness of the skin, loss of suppleness, and/or the appearance of wrinkles and/or fine lines.

By "preventing and/or reducing the alteration of the homogeneity or radiance of the complexion" is meant in particular that the fermented extract of Longoza seeds is intended to reduce and/or delay the appearance of imperfections or microreliefs of the skin, dark circles, a hazy, dull and/or uneven complexion. By "preventing and/or reducing the alteration of the barrier function of the skin" is meant in particular that the fermented extract of Longoza seeds is intended to reduce and/or delay changes in the integrity of the skin, which can be associated, for example, with modifications of the interactions between cells and/or between cells and the extracellular matrix, likely to generate an alteration of the various functions of the skin barrier (eg: mechanical, water, antioxidant, etc.).

According to a particular embodiment, said fermented extract of Longoza seeds according to the invention or said cosmetic composition containing it is applied to aged skin or showing signs of aging and/or skin exposed to stress, preferably aged skin or showing signs of aging.

By "skin exposed to stress", we mean that the skin is subjected to stress of endogenous or exogenous origin, in particular mechanical or chemical stress likely to induce unsightly non-pathological skin reactions - e.g. the skin is visibly tired and/or more sensitive to stimuli.

The skins and lips to which the compositions of the invention are applied are healthy, that is to say not showing any disorders or disorders that would come under a pathological condition (“non-healthy” subjects, suffering from pathology).

Advantageously, the compositions comprising the fermented extract of Longoza seeds used in the context of the cosmetic process of the invention are those described above.

Use of Fermented Longoza Seed Extract

Another object of the invention relates to the cosmetic use of the fermented extract of Longoza seeds according to the invention for topical application to keratin materials, in particular the skin and/or the lips and in particular the skin of the face and/or neck, as a cosmetic active ingredient intended to promote and/or stimulate epidermal renewal, desquamation of the skin, the vitality of the skin cells, and/or the cohesion of the skin cells, and/or to prevent and /or reduce the signs of skin aging, in particular loss of firmness, loss of elasticity, loss of density, appearance of wrinkles and/or fine lines, dryness of the skin, alteration of the barrier function of the skin, the alteration of the suppleness of the skin and/or the alteration of the homogeneity or the radiance of the complexion.

Examples

The invention is illustrated by the following non-limiting examples. The % are expressed by weight of raw material relative to the total weight of the composition, unless otherwise indicated.

Example 1: Process for Fermenting Longoza Seeds

Material and methods

Consortium of microorganisms

The microorganisms that are part of the consortium include:

- at least one lactic acid bacterium belonging to the genus Lactobacillus or Pediococcus,

- at least one yeast belonging to the genus Saccharomyces, Schyzosaccharomyces or Torulaspora, and

- at least one acetic bacterium belonging to the genus Acetobacter, Gluconobacter or Komagataeibacter.

Preparation of Longoza seeds

Raw Longoza seeds are crushed to obtain a powder composed of particles having an average size of about 250 to 500 pm.

Preparation of the nutrient medium

The tea is prepared from 5 g/l to 10 g/l of tea leaves infused for 10 to 60 min at 70°C to 90°C. The sucrose is then dissolved in the aqueous phase at a concentration between 20 g/l and 100 g/l, generally 50 g/l. Sucrose can in particular be replaced by a mixture of glucose and fructose.

Fermentation process

Fermentation of Longoza seeds was carried out according to a process comprising the following steps: a) incubation of a consortium of microorganisms in a solution comprising a carbohydrate and an aqueous extract of black tea leaves (i.e. the " nutrient medium”) at 26°C for 3 to 4 days in order to obtain a culture; b) the grinding of Longoza seeds in order to obtain a fine powder, as described below above ; c) the addition, in a fermentation medium comprising dechlorinated water and a carbohydrate, of:

- Longoza seed powder at a concentration of 1.2% or 5% by weight based on the liter of fermentation medium, and

- the culture obtained in step a) at a concentration of 5% by weight relative to the weight of fermentation medium, in order to obtain a fermentation mixture; d) incubation of the fermentation mixture obtained in step c) in a fermenter at 26° C. for 10 days; e) filtering the fermentation mixture by coarse sieving followed by filtration through a cellulose filter having an average pore size of 0.2 µm to obtain a fermented Longoza seed extract, and, f) adding the glycerin with fermented extract of Longoza seeds at a final concentration of 70% by weight relative to the total weight.

Results

A fermented extract of Longoza seeds is obtained in the form of a solution at a concentration of 1.2 to 1.35% by weight of active ingredient of fermented Longoza seeds, 70% glycerin and 28.65 to 28, 8% water. Alternatively, a fermented extract of Longoza seeds is obtained in the form of a solution at a concentration of 1.35 to 1.65% by weight of active ingredient of fermented Longoza seeds, 70% glycerin and 28, 35 to 28.65% water.

The fermented extract of Longoza seeds obtained from a concentration of Longoza seeds of 1.2% by weight relative to the liter of fermentation medium (i.e. 12 g/L) in step c) is named below the “L3” extract.

The fermented extract of Longoza seeds obtained from a concentration of Longoza seeds of 5% by weight relative to the liter of fermentation medium (i.e. 50 g/L) in step c) is named below the extract “L2”.

The effect of these extracts will be illustrated in the following examples.

Example 2: Characterization of a fermented extract of Longoza seeds

Material and methods Quantification of organic acids by HPLC analysis

The assay was carried out on an agilent HPLC-DÀD-MS 1260.

The extract is injected at 0.02%, diluted to 1/ ^50th in 0.1% HCOOH (weighing 20 pL qsp 1 mL) The parameters in the acquisition method cited above are as follows:

Injection volume: 5 L Temperature: 30° C Column: Phenomenex Hydro-RP 150x4.6mm 4pm (n°8) Solvents:

Isocratic 100% À: H2O + 0.1% formic acid

Flow rate: 0.400mL/min

Analysis time 25 min

NMR

Centrifugal partition chromatography (CPC)

Longoza seed fermented extract has been freeze-dried to reduce the amount of water. The mass injected was about 4 g.

After column equilibration, the fermented extract of Longoza seeds was dissolved in 11 ml lower phase + 6 ml upper phase and injected into the CPC column (303 mL column, FCPE300® instrument (Rousselet Robatel Kromaton)) via a 20 ml sample loop. The mobile phase was pumped upwards for 65 min. The column was extruded by switching the mode selector valve for 10 minutes. The flow rate was 20 mL/min and the column rotation speed was 1300 rpm. The two-phase solvent system is ethyl acetate/acetonitrile/water (3/3/4, v/v). The stationary phase corresponds to the lower phase of the two-phase solvent system (bottom-up mode) while the mobile phase corresponds to the upper phase of the two-phase solvent system.

20 ml fractions were collected throughout the experiment (elution and extrusion) and combined according to their thin layer chromatography profiles. 10 fractions were thus obtained.

Identification of major metabolites An aliquot of each fraction from F1 to F10 was dissolved in 700 μL of DMSO-d6 and analyzed by ¹³ C NMR at 298 K on a Bruker Avance AVII I-600 spectrometer (Karlsruhe, Germany) equipped with a cryoprobe. After the spectra were processed, the absolute intensities of all ¹³ C NMR signals were automatically collected and aggregated onto the fraction series spectra using a computer script developed by Natexplore. The resulting table was subjected to a hierarchical clustering analysis (PermutMatrix, 1.9.3, LIRAAM, Montpellier, France). The resulting 13C NMR chemical shift clusters were visualized as dendrograms on a two-dimensional map. For the identification of metabolites, each ¹³ C NMR chemical shift cluster obtained by HCA was manually submitted to the structure search engine of the ACD/NMR Workbook Suite 2012 database management software (ACD/Labs, Ontario, Canada ) understanding the predicted chemical structures and shifts of low molecular weight natural products. 2D NMR experiments (HSQC, HMBC and COZY) were carried out on fractions containing supposedly identified compounds in order to confirm the molecular structures proposed by the database at the end of the dereplication process.

Results

The fermentation process generates a fermented Longoza seed extract having a very different composition from non-fermented Longoza extracts, for example an aqueous non-fermented Longoza extract extracted with 100% water.

In particular, the fermented extract of Longoza seeds includes different organic acids, as shown in Table 1 below.

Table 1: Organic acids present in the fermented extract of Aframomum angustifolium or Longoza seeds compared to an aqueous extract of Longoza (non-fermented) (in mg compared to g of dry extract) [Table 1] nd: not detected

Example 3: Effect of Fermented Longoza Seed Extract on Skin Protein Expression

The effect of fermented Longoza seed extract on the expression of several protein markers of skin barrier function, epidermal cohesion, skin elasticity and radiance was evaluated.

Material and methods

Cultivation and treatment of KHN

Normal Human Keratinocytes (KHN) are seeded in TPP150 flasks in supplemented Epilife medium. The KHNs are trypsinized at pre-confluence (0.05% Trypsin-EDTA, GIBCO-Invitrogen), and neutralized with a medium containing serum. They are then seeded in 96-well imaging plates (96-well p-plate, ibiTreat, Ibidi) at a rate of 10,000 cells per well.

At confluence, the KHNs are treated with the fermented extract of Longoza L2 or L3 seeds in complete Epilife medium containing 500 M of CaC (0.2 mL/well). The L2 and/or L3 extract was tested at a concentration of 0.02% and/or 0.1% v/v. The treatment is carried out for 3 or 5 days with replacement of the culture medium after 48 hours. For the control conditions (untreated), the medium is replaced with new medium containing 500 μM of CaC.

Cell labeling

At the end of the treatments, the cells are fixed with formalin (Formalin Solution 10% Neutral Buffered, Sigma) for 10 minutes then permeabilized with a solution of PBS-Triton 0.1% (Triton X-100, Sigma). They are then saturated with PBS/BSA 1% (PBS: phosphate-buffered saline solution BSA: bovine serum albumin), for 30 minutes and at room temperature. The 1% PBS/BS solution is then replaced with a solution of primary antibody corresponding to each labeled protein (see Table 2) diluted in PBS/1% BS. The plates are incubated overnight at 4°C.

After rinsing with PBS, the cells are covered with a solution of secondary antibody (anti-rabbit or anti-mouse goat antibody, Alexa Fluor 568 from Invitrogen) depending on the primary antibody to be targeted at 1/ ^300th and of DAPI at 1/500th (4′,6′- ^diamidino -2-phenylindole, dihydrochloride, Invitrogen Molecular Probes) diluted in PBS/BSA 1%. The plates are incubated for one hour in the dark and at room temperature.

After rinsing with PBS followed by distilled water, PBS is deposited in each well at the rate of 1 mL. Plates are stored at 4°C in the dark until image acquisition.

Table 2: Summary of primary antibodies used [Table 2]

Image acquisition by HCS and analysis

The plates are scanned with the “ArrayScan XTi” from Thermo Cellomics.

Vesting conditions:

Detection:

DAPI: filter XF53_386_23 Alexa Fluor 568: filter XF53_572_15

Resolution: 1104 x 1104

Objective: 10x dry

Number of images: 25 per well

The images were analyzed using “Spot detector” image analysis software, which detects the red labeling of the targeted protein, corresponding to its expression. The surface of the measurement area is delimited as being the entire surface of the image. The number of cells is determined by counting the nuclei by detecting the blue label.

Depending on the markers and their cellular expression, the results can be expressed by:

- Intensity of the labeling detected (threshold) / number of cells

- Marking intensity in the total field / number of cells

The significance of the differences in the ratios observed between the controls and the treated is measured by the Student test (*p<0.05, **p<0.01).

Results

3.1. Expression of loricrin

Loricrin is a structural protein expressed at the level of the horny envelope which contributes to the barrier function of the skin.

The fermented extract of Longoza seeds significantly increases the expression of loricrin with a dose effect observed when the concentration of extract is increased (+41% for the L2 extract at a concentration of 0.02% and + 57% for the L2 extract at a concentration of 0.1%, p<0.01, see Figure 1).

3.2. Filaggrin expression

Like loricrin, filaggrin is a protein involved in the barrier function of the skin.

The fermented extract of Longoza seeds increases the expression of filaggrin significantly with a dose effect observed when the concentration of extract is increased (+7.5% for the L3 extract at a concentration of 0.1% and +17.8% for the L2 extract at a concentration of 0.1%, p<0.01, see Figure 2). 3.3. Expression of elafin

Elafin is a protein involved in the skin barrier function. It also has an inhibitory effect on elastase, thus preventing the degradation of elastic fibers.

The fermented extract of Longoza seeds significantly increases the expression of elafin with a dose effect observed when the concentration of extract is increased. Indeed, the expression is increased by +38.9% for the L3 extract at a concentration of 0.02% and by +52.6% for the L3 extract at a concentration of 0.1% (p< 0.05, see Figure 3). Similarly, the expression is increased by +37% for the L2 extract at a concentration of 0.02% and by +45.8% for the L2 extract at a concentration of 0.1% (p<0 .05, see Figure 3).

3.4. Expression of kallikrein 5

Kallikrein 5 is a protein involved in skin homeostasis and desquamation. It also contributes to the radiance of the skin.

The fermented extract of Longoza seeds increases the expression of kallikrein 5 significantly (+15.2% for the L3 extract at a concentration of 0.02% and +11% for the L3 extract at a concentration 0.1%, p<0.05, see Figure 4).

3.5. Expression of desmoglein 1

Desmoglein 1 is a protein essential to the integrity of the epidermis, providing its structure through its adhesive function within intracellular junctions (desmosomes). It also promotes the differentiation of keratinocytes.

The fermented extract of Longoza seeds increases the expression of desmoglein 1 significantly (+30.4% for the L3 extract at a concentration of 0.02% and +17% for the L2 extract at a concentration 0.02%, p<0.01, see Figure 5).

3.6. Expression of transglutaminase

Transglutaminase is an enzyme involved in the cross-linking of structural proteins to form the horny envelope in the epidermis. It thus contributes to the barrier function of the skin.

The fermented extract of Longoza seeds increases the expression of transglutaminase significantly (+73.8% for the L2 extract at a concentration of 0.1% and +97.6% for the L3 extract at a concentration of concentration of 0.1%, p<0.01, see Figure 6).

3.7. Expression of ZO-1 ZO-1 is an intercellular protein involved in the formation of tight junctions between skin cells. It thus contributes to the barrier function and the integrity of the skin.

The fermented extract of Longoza seeds increases the expression of ZO-1 significantly (+25.8% for the L2 extract at a concentration of 0.1% and +49.7% for the L3 extract at 0.1% concentration, p<0.01, see Figure 7).

Findings

Two fermented Longoza seed extracts, L2 and L3, were tested on the expression of epidermal differentiation markers on a model of normal human keratinocytes in culture, in particular to determine their activity on the skin barrier.

The extracts have positive effects on the expression of skin barrier markers such as elafin involved in the constitution of the horny envelope or desmoglein 1 constitutive of desmosomal junctions. In addition, the L3 extract increases the expression of kallikrein 5, involved in homeostasis and desquamation of the skin.

Example 4: Effect of Fermented Longoza Seed Extract on a Skin Model

The effect of the fermented Longoza seed extract according to the present invention on the elasticity, firmness, and cell density of the skin was evaluated in a stem cell-depleted skin model.

Material and methods

Establishment of the bio-printed reconstructed skin model depleted in epidermal stem cells (ESC)

Bio-printing of papillary and reticular dermis by extrusion

The powders entering into the composition of the bio-ink were dissolved in calcium-free DMEM and placed under stirring at 37° C. overnight.

The following day, confluent young or old papillary/reticular fibroblasts were trypsinized and counted. The necessary quantity of cells was removed, at the rate of 250,000 fibroblasts per milliliter of ink prepared, then centrifuged. The cell pellet was directly resuspended in the appropriate volume of bio-ink. Then the bio-ink/cell mixture was transferred into a sterile syringe mounted on a 20 gauge cannula. The printed objects were modeled with the software Slic3r (GNU Àffero General Public License) and Repetier (Hot-World GmbH & Co. KG). The papillary dermis and reticular dermis layers were printed in 2 stages, one on top of the other. “Aged” dermis and “young” dermis were printed.

Initially, cubes of dimensions 1.5 cm×1.5 cm×0.2 cm were printed with the inks containing the papillary fibroblasts. The height of these objects corresponded to two superimposed layers. Following this first printing, cubes of dimensions 1.5 cm×1.5 cm×0.1 cm (equivalent to 1 layer) were printed above the first cubes of 2 layers. These constructs were then placed for 1 hour in a solution of calcium and thrombin allowing the polymerization and consolidation of the bio-ink. After three rinses in a saline buffer, the dermal equivalents were cultured in MCF (internal preparation from LabSkin Creations, medium adapted to the culture of fibroblasts) and placed in an incubator (37° C., 5% CO2) then cultured 7 days before the seeding of the sorted keratinocytes on their surface. The culture medium was renewed every 2 days.

Magnetic sorting according to the CD71 marker on primary keratinocytes in culture

First, primary adult keratinocytes were thawed at passage 1 in culture flasks in MCK culture medium (LabSkin Creations internal preparation, medium suitable for keratinocyte culture). After one week of amplification, at confluence, the cells were trypsinized and then counted. All of these cells were sorted with the CD71 magnetic bead kit 130-046-201 (Miltenyi) according to the supplier's instructions.

Briefly, the keratinocytes were incubated for 15 minutes at +4° C. with the magnetic beads according to a defined cell/bead volume ratio. This mixture was then centrifuged and then the supernatant was discarded to get rid of the magnetic beads not bound to the cells of interest. The pellet of keratinocytes was resuspended in culture medium, and was passed over the magnetic columns installed on the magnet provided for this purpose. The negative fraction which contains cells not expressing the CD71 surface marker, corresponding to a population enriched in epidermal stem cells not retained in the column, was harvested first in a sterile tube.

Subsequently, the column was unhooked from the magnetic support and rinsed, making it possible to harvest the cells expressing the surface marker CD71 (fraction depleted in stem cells) which were bound to the magnetic beads. The fractions enriched and depleted in cells were counted in order to calculate the CD71 −/CD71 + ratio. In parallel with this extraction, foreskin keratinocytes previously cultured were also trypsinized and counted to be seeded the same day on the dermis. These constitute the “young control”.

Seeding of CD71+ keratinocytes on bio-printed dermis

Once the magnetic sorting was carried out and the different fractions counted, the culture medium of the “young” and “old” bioprinted dermis was eliminated.

The equivalent of 500,000 CD71+ keratinocytes have been deposited on the surface of aged dermis. The same was done with a suspension of foreskin keratinocytes, seeded on the surface of the “young control” dermis, serving as a reference.

After 30 minutes of adhesion at +37°C, medium was used to submerge the dermo-epidermal assemblies.

The dermo-epidermal assemblies were cultured for 3 days in MCK medium allowing the proliferation of keratinocytes. They were then raised at the air/liquid interface in order to initiate epidermal differentiation. This final culturing step lasted a week in order to obtain pluristratified but not completely mature epidermis.

The L2 or L3 extract was added to the culture medium. The medium was then filtered to avoid any risk of contamination. The extracts were applied to the cell cultures daily for the last 5 days of culture (cultures stopped at day 35).

Samples were fixed in 4% buffered formalin solution (Alphapath, France) then embedded in paraffin.

Histological staining with hematoxylin phloxine saffron

5 µM paraffin sections were prepared. After deparaffinization and rehydration, the samples were stained with Hematoxylin-Phloxine-Saffron (HPS).

Immunofluorescence on sections embedded in paraffin

Alpha 6 anti-integrin labeling was performed on sections 5 and 20 micrometers thick. After heat unmasking, a saturation step in PBS/BSA 4% made it possible to block the aspecific antigenic sites. The cells were then incubated overnight with a primary antibody specific for the alpha 6 integrin antigen at 1/ ^10,000th (rabbit monoclonal antibody, Abeam) then with an anti-rabbit Alexa-Fluor 568 secondary antibody diluted to 1 /1000 ^th (Thermo Fisher Scientific, MA, USA) and the dye nuclear Hoechst (Thermo Fisher Scientific). A negative control without primary antibody was carried out in parallel.

Image acquisition

Histological staining and immunohistological staining were observed with the Axio Observer D1 optical microscope system/Axiocam high-resolution camera (Zeiss, Le Pecq, France). Images were acquired using ZEN pro 2012 software and saved in “tif” (high resolution) format.

Data Acquisition Atomic Force Microscopy (AFM)

The AFM used is a Resolve bioscope (Bruker) on which was mounted an epifluorescence microscope (Leica DMi8) allowing the realization of correlative studies (AFM/Fluorescence image).

PeakForce® QNM/Fast-Force volume mode was used. The AFM probe that was chosen has a theoretical stiffness constant of 0.4N/m and a radius of curvature <10nm. Before each use, the deflection sensitivity of the probe was measured on Saphir and its stiffness constant was also calibrated by the thermal noise method. The lever of the AFM was positioned on the Dermo-Epidermal Junction (DEJ) visible thanks to the anti-integrin alpha 6 immunostaining carried out on frozen sections.

A complete AFM analysis consisted of the acquisition of a force volume of the zone of interest (JDE) in aqueous conditions (PBS 1X).

The parameters used for the acquisitions were:

- Force/volume matrix: 20pm x 20pm - 64px2

- Force curves: Force = 20nN - Ramp size = 10 pM

For AFM imaging of the dermis, the Force Volume mode was also used. Volume forces were acquired on different areas of the cuts with the same parameters as before.

Quantitative analyses: extraction of the elastic modulus (Ea)

Ea quantifications from raw force curves were performed using BioMeca Analysis processing software (BioMeca).

The raw approach curves obtained for each section were processed to extract an accurate quantification of the Ea (stiffness) of the JDE. Here, Sneddon's theoretical model was applied to an indentation between 0 and 1 M. Using the different force matrices, the elasticity variations of the dermal-epidermal junction for each section were extracted. Statistical analyses: Young control versus elderly depleted CSE untreated: t-test, $$$ p<0.001; Depleted elderly CSE untreated versus Depleted elderly CSE treated with L3 at 0.05% or 0.1%: t-test, *** p<0.001.

Results

4.1. Histological analysis

Treatment with the fermented extract of Longoza seeds (L2 or L3, at a concentration of 0.05 or 0.1%) made it possible to obtain cellularized dermis presenting a layer of newly synthesized fibroblasts greater than that observed in the untreated samples. The epidermis also have an improved structure: they appeared thicker, with a very clear basal organization and a structured JDE. The differentiation of this compartment was not disturbed by the administration of the extract - quite the contrary, a stratum corneum is present in all conditions. Finally, the fermented extract of Longoza L3 seeds seems to present a dose effect with a thicker layer of fibroblasts under the dermo-epidermal junction with the 0.1% dose.

4.2. Immunolabelling

An analysis of the expression profile of the integrin apha-6 allowed a more precise study of the basal part of the epidermis containing the populations of epidermal stem cells. The reconstructed skins depleted in CSE and treated with the fermented extract of Longoza L3 seeds, at a concentration of 0.05 or 0.1%, saw their expression of integrin alpha 6 largely increased, revealing cell populations epidermal strains, firmly anchored at the dermo-epidermal junction, more important. The expression levels of this marker reach levels comparable or even higher than that observed in samples of “young” reconstructed skin.

4.3. AFM

An analysis of the elastic modulus of the JDEs obtained under each of the AFM treatment conditions made it possible to confirm the results cited above (see FIG. 9). Treatments with the L3 extract at 0.05 or 0.1% led to an increase in the population of epidermal stem cells and thus to an increase in the elastic modulus (L3 at 0.05%: +48%, p<0.001; L3 at 0.1%: +96%, p<0.001), measured at the JDE, meaning that these junctions are stiffer. The fermented extract of Longoza seeds has also showed a dose effect, the elastic modulus increasing with the concentration of active ingredient applied in the culture medium.

Also, the fermented extract of Longoza L3 seeds, by inducing an increase in the proliferative compartment at the basal level of the epidermis, makes it possible to obtain a stiffer and more strongly anchored JDE as well as thicker and proliferative epidermis which however, showed satisfactory differentiation.

Example 5: Cosmetic formulations

The fermented extract of Longoza seeds as prepared in Example 1 is formulated in the following illustrative and non-limiting compositions, prepared according to conventional cosmetic formulation methods.

5.1. Composition in the form of a lotion

[Table 3]

* prepared according to example 1

Applied to the skin, in particular to the face, this composition provides an anti-aging action with beneficial effects on the firmness and/or elasticity of the skin.

5.2. Composition in the form of a serum

[Table 4]

‘prepared according to example 1

The serum is applied all over the face and especially on areas showing signs of aging. The serum comprising the fermented extract of Longoza seeds according to the invention promotes epidermal renewal, for elastic, firmer skin and a more even complexion.

Claims

34 CLAIMS

1 . Fermented extract of Aframomum angustifolium or Longoza seeds comprising:

- a quinic acid content of between 0.05 and 2%; and

- an acetic acid content of between 5 and 15%, by weight relative to the dry extract weight of the fermented extract.

2. Fermented extract of Aframomum angustifolium or Longoza seeds according to claim 1, characterized in that it is in liquid form and that it comprises at least one preservative, preferably selected from butylene glycol, glycerin, propylene glycol, sodium benzoate benzoate and potassium sorbate.

3. Process for the fermentation of Aframomum angustifolium or Longoza seeds comprising the following steps: a) the incubation of a consortium of microorganisms in a solution comprising a carbohydrate at a temperature of between 17 and 38°C for 1 to 10 days in order to obtain a culture of microorganisms, said consortium comprising at least one lactic bacterium belonging to the genus Lactobacillus or Pediococcus, at least one yeast belonging to the genus Saccharomyces, Schyzosaccharomyces or Torulaspora, and at least one acetic bacterium belonging to the genus Acetobacter, Gluconobacter or Komagataeibacter b) the grinding of seeds of Aframomum angustifolium or of Longoza in order to obtain a powder composed of particles having an average size of between 100 μm and 1 mm; c) adding said powder to a fermentation medium comprising the culture of microorganisms in order to obtain a fermentation mixture, said fermentation medium comprising a carbohydrate chosen from sucrose, glucose, fructose, molasses or a mixture of one or more of these, d) incubating the fermentation mixture at a temperature between 25 and 35°C for 7 to 13 days; e) filtration of the fermentation mixture to obtain a fermented extract of Aframomum angustifolium or Longoza seeds.

4. Process according to claim 3, in which approximately 60 g of powder or less is added per liter of fermentation medium in step a), more preferentially approximately 50 g of powder or less, even more preferentially approximately 12 g/L or 50g/L. 35

5. Method according to claim 3 or A, in which the fermentation medium further comprises an aqueous extract of tea leaves.

6. Fermented extract of Aframomum angustifolium or Longoza seeds obtained by the process according to any one of claims 3 to 5.

7. Cosmetic composition comprising, in a physiologically acceptable medium, a fermented extract of Aframomum an ustifolium or Longoza seeds according to claim 1, 2 or 6.

8. Cosmetic composition according to claim 7, characterized in that the fermented extract of Aframomum an ustifolium or Longoza seeds is present in a content ranging from 0.0000012% to 0.0825%, preferably from 0.000006% to 0.033%, more preferably from 0.000012% to 0.0165% by weight of active ingredient (dry matter of fermented extract of Aframomum an ustifolium or Longoza seeds) relative to the total weight of said composition.

9. Cosmetic composition according to any one of claims 7 to 8, characterized in that it further comprises at least one cosmetic adjuvant chosen from the group consisting of antioxidant agents, emollient agents, moisturizing agents, anti-aging agents. -age, perfumes, and mixtures thereof.

10. Cosmetic composition according to any one of claims 7 to 9, characterized in that it is in the form of a cream, oil-in-water emulsion, or water-in-oil or multiple emulsion, solution, suspension , gel, milk, lotion, or serum.

11. Cosmetic process for caring for and/or making up keratin materials, in particular the skin and/or the lips, comprising the application to said keratin materials of at least one layer of a cosmetic composition as defined in any of claims 7 to 10.

12. Cosmetic process according to claim 11, characterized in that it is intended to promote and / or stimulate epidermal renewal, desquamation of the skin, the vitality of skin cells, and / or the cohesion of skin cells, and / or warn and/or reduce the signs of skin aging, in particular loss of firmness, loss of elasticity, loss of density, appearance of wrinkles and/or fine lines, dryness of the skin, alteration of the barrier function of the skin , the alteration of the suppleness of the skin and/or the alteration of the homogeneity or the radiance of the complexion.

13. Cosmetic use of a fermented extract of Aframomum angustifolium or Longoza seeds as defined according to one of claims 1, 2 or 6 as an active ingredient for promoting and/or stimulating epidermal renewal, desquamation of the skin, the vitality of skin cells, and/or the cohesion of skin cells, and/or preventing and/or reducing the signs of skin aging, in particular loss of firmness, loss of elasticity, loss of density, the appearance of wrinkles and/or fine lines, dryness of the skin, alteration of the barrier function of the skin, alteration of the suppleness of the skin and/or alteration of the homogeneity or radiance of the complexion.