FR3121935A1 - METHOD FOR SELECTING TOPICAL ECOBIOLOGICAL INGREDIENTS AND COMPOSITIONS SUITABLE FOR RESTORING OR STRENGTHEN THE SKIN MICROBIOTA - Google Patents

Abstract

The invention relates to a selection method combining quantification of the biomass of the cutaneous microbiota, and analysis of its biodiversity to select the substances or compositions, ecobiological, which make it possible to increase the biomass of the cutaneous microbiota, in particular without reducing its biodiversity, for their ability to maintain and/or restore a healthy skin microbiota. The invention makes it possible in particular to effectively strengthen the skin in the face of attacks, in particular repeated attacks, on its cutaneous microbiota, in particular by topical virucidal or bactericidal, hygiene or care products.

Description

[Title] METHOD FOR SELECTING TOPICAL ECOBIOLOGICAL INGREDIENTS AND COMPOSITIONS SUITABLE FOR RESTORING OR STRENGTHEN THE CUTANEOUS MICROBIOTA

Technical field of the invention

The present invention relates to the study of the skin microbiota and its use for restoring and/or maintaining a healthy skin microbiota.

State of the art

The skin, the largest human organ, is colonized by billions of microorganisms such as bacteria, yeasts, fungi or viruses collectively called microbiota or microflora. These microorganisms inhabit the epidermis, and are mainly found in the upper layers of the stratum corneum as well as in the ducts of sweat glands and pilosebaceous follicles (Kong et al. , 2012a).

Historically, the study of the human microbiota has focused first on the intestinal microflora, whose crucial role in maintaining human physiological balance is now indisputably recognized. The cutaneous microbiota, which is much less abundant, had to wait for the development and fine-tuning of metagenomic methods allowing its detailed characterization. These techniques, now available, allow the amplification and sequencing of the genetic material of the microbial communities of the skin. To date, the composition of the skin flora is only partially known. Only the bacterial composition of the skin is studied in detail. The number of bacteria present on the skin, whether on its surface, in the glands or on the appendages, can reach nearly 1 billion per cm ² (Kong et al ., 2012a).

The human skin flora can be subdivided into two groups:
- The resident flora is composed of commensal germs, that is to say living at the expense of their host, without causing them any harm. The resident human flora is dominated by Proteobacteria, including about ten types of Gram-positive aerobic bacteria such as Staphylococcus epidermidis and Staphylococcus hominis (Otto, 2010). The resident flora is also composed of Gram-positive anaerobic bacteria belonging to the Actinobacteria division (genera Cutibacterium , Corynebacterium , Dermabacter and Micrococcus ) (Kligman et al ., 1976). Among the microorganisms of the other kingdoms, Malassezia is the fungal species most frequently found on the skin. Finally, the mites Demodex folliculorum and Demodex brevis , or "eyelash mites", are also considered members of the resident human flora.
- Transient flora is composed of mostly harmless fungi, viruses and bacteria, called saprophytes, that is to say, which feed on decomposing organic matter from the environment. This flora can also consist of opportunistic pathogenic bacteria that can cause disease in the host. The transient flora does not establish itself permanently on the surface of the skin, varying during the day, according to the activities carried out and the surrounding conditions. However, it can persist for hours or even days. Its density is low on dry areas and particularly high on hairy areas, folds and areas subject to perspiration. The most common transient species are Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and several species of the genus Bacillus .

Several studies have shown that the skin microbiota has considerable differences between genders, ethnic groups and even individuals and that the distribution of microbial residents in different areas of the body is also highly variable (Grice et al., 2009). Despite these marked interpersonal and biogeographical differences, it is firmly established that all of the resident microflora plays an important role in the health of the skin, particularly in terms of its immune defenses and its resistance to colonization by pathogenic microorganisms, also implying imbalances in the microbiota and the appearance or aggravation of dermatological conditions such as atopic dermatitis or psoriasis (Nakatsuji et al ., 2017; Gao et al., 2008; Kong et al., 2012b).

Indeed, just as in large-scale ecosystems, a loss of diversity of the microbiota is accompanied by a sometimes irreversible deregulation of its balances, in which a limited number of opportunistic species proliferate in an uncontrolled manner at the expense of the others. In the case of the skin microbiota, it is for example possible to cite in particular the species responsible for serious infections, often of nosocomial origin, Staphylococcus aureus and Pseudomonas aeruginosa . It is difficult to treat the skin infections caused by these strains which have sometimes acquired during their evolution, one or more resistances to conventional antibiotics, in particular beta-lactams. For example, methicillin-resistant Staphylococcus aureus (MRSA) represents a concrete public health risk to date, having in 2020 been responsible for more than a third of all community and hospital infections associated with mortality and morbidity high. The management of MRSA requires the use of new antibiotics, which can however further destabilize the entire human microbiota by slowing down the recovery of its diversity.

Several indices making it possible to evaluate the diversity of the microbiota are developed, among which one can quote the indices of Shannon or Simpson, the most used being the index of Shannon which makes it possible at the same time to evaluate the number of species of this environment (specific richness) and the distribution of individuals within these species (specific equitability; Lemos et al., 2011).

It is known that the use of topical products can have a significant impact on the composition and balances of cutaneous microbial communities (Kong, 2011; Kong et al., 2012a). Antiseptic products (i.e. fungicides, bactericides and/or virucides) in particular, including ethanol in particular, impoverish not only the quantity, but also the diversity of the skin microbiota and this potentially more durably (San Miguel et al., 2018).

The application of biocides to the surface of the skin was relatively rare and limited to the hospital setting until 2020. However, since the global pandemic of COVID-19, the use of this category of products has become commonplace and widespread. to levels never before achieved. For alcohol-based products, the indiscriminate inhibition of the growth of all of the microbiota is added to its effect of destruction of the hydrolipidic film, which leads to an alteration of its barrier effect, with increased penetration of allergens and irritants.

The reduction in the quality of the skin microbiota, caused in particular by biocidal products and antibiotic treatments, requires the development of a new class of topical ecobiological care products to maintain and/or quickly restore a healthy and diversified skin microbiota.

There is therefore still an obvious need to develop methods allowing the selection of ingredients, or even topical compositions, capable of maintaining and/or restoring, preferably quickly, a healthy microbiota.

Description of the invention

The present invention is based on the demonstration of the interest, under non-pathological conditions, of promoting the increase in the biomass of the cutaneous microbiota (multiplication of the microbiota), without reducing its overall diversity (conservation or increase in the diversity said microbiota).

The healthy skin microbiota and its balance are then reinforced and/or reconditioned to better resist potential opportunistic dysregulation and colonization.

Surprisingly, the Applicant has thus found a way that is simple, effective, affordable and usable on an industrial scale, in particular in cosmetics or dermatology, for selecting ingredient(s) and/or composition(s) suitable to restore and/or strengthen the healthy skin microbiota, by seeking to increase the biomass of the skin microbiota, in particular without reducing its overall diversity.

For this, in one embodiment of the invention, two methods are combined: a method for measuring the biomass on a skin area and a method for measuring the microbial diversity on this same area.

According to the invention, the “microbiota” concerns all of the microbial flora, including microbes, bacteria, yeasts, fungi and viruses.

According to the invention, by “cutaneous microbiota” or “keratinous material(s)”, is meant the microbiota of the skin, of the semi-mucous membranes (in particular the lips), of the mucous membranes and of the appendages including appendages.

According to the invention, the "microbiome" concerns all the genes or genomes present in the microbiota.

According to the invention, by “biodiversity” or “diversity” or “diversity of the microbiota” or “diversity of the microbiome” or “bacterial diversity”, is meant all of the bacterial populations or species present in the skin microbiota. This biodiversity can be characterized in particular by the number of different bacterial genetic sequences within said microbiome (also denoted OTU, for “Operational Taxonomic Unit”) and/or their relative abundance. Several indices making it possible to evaluate the diversity of the microbiota have been developed and are well known to those skilled in the art, among which the Shannon or Simpson indices can be mentioned.

According to the invention, by “biomass of the skin microbiota” or “biomass” or “bacterial load”, we mean the number or the quantity of bacteria on an area of skin. This biomass can be characterized in particular by the amount of bacterial genetic material, in particular 16S rDNA, from said microbiota.

The biomass and diversity levels of a microbiota can be measured by any appropriate technique well known to those skilled in the art, such as quantitative amplification and 16S rDNA sequencing.

According to the invention, “healthy” means non-pathological.

According to the invention, by “healthy skin area”, is meant a skin area not exhibiting dermatosis.

According to the invention, by “healthy skin microbiota” or “healthy skin microbiota”, is meant a stable and diversified skin microbiota on a given skin area, such as on the same healthy skin area not subjected to stress conditions or particular aggressions (such as, but not limited to, treatments or care or non-ecobiological environments, in particular dermatological, cosmetics, hygiene, pollution, etc.). The microbiota of healthy skin protects in particular against invasive pathogens, but also against drying out of the skin.

According to the invention, by "stress" or "aggression" is meant any event, of any nature whatsoever, single or repeated, in particular of external origin, which may be the cause of destabilization or modifying a system.

At the cutaneous level, such an attack can in particular lead to a reduction in the biomass and/or the diversity of the cutaneous microbiota. In particular, it may be linked to non-ecobiological treatments or care or environments, in particular dermatological, cosmetic, hygiene, pollution, etc., and, more particularly, to the action of bactericides or virucides or fungicides or soaps or hygiene or care products that are aggressive towards the microbiota.

By “ecobiological”, we mean respectful of people, their interactions with the world and the planet.

The present invention relates to a method for selecting or screening ingredients and compositions, preferably ecobiological, cosmetic or dermopharmaceutical or dermatological, advantageously applicable topically, according to their effect on the cutaneous microbiota, in particular their ability to induce the increasing the biomass of the cutaneous microbiota, in particular without reducing its biodiversity.

The present invention also relates to a method for preparing a composition, preferably topical, capable of inducing an increase in the biomass of the cutaneous microbiota, as well as its use for the preparation of care compositions, preferably topical, advantageously ecobiological and ecobiological care methods, preferably topically, based on the use of the aforementioned compositions or ingredients.

The present invention also relates to a composition for its use for maintaining and/or restoring a healthy skin microbiota via an increase in the biomass of the skin microbiota, in particular without reducing its biodiversity.

In other words, the invention aims for the first time to analyze and quantify the biomass of the cutaneous microbiota, as well as, where appropriate, its biodiversity and to select the substances and compositions which make it possible to increase the biomass of the cutaneous microbiota, in particular without diminishing its biodiversity.

The present invention therefore relates to a method for selecting or screening ingredients, substances or topical compositions making it possible to maintain and/or restore, preferably quickly, a healthy microbiota.

The present invention relates in particular to a screening method whose objective is to select ingredients, preferably topically acceptable, individually and/or compositions comprising a mixture of several aforementioned ingredients, optionally combined with excipients, preferably topically acceptable, capable of increase the biomass of the cutaneous microbiota, in particular without reducing its biodiversity.

The present invention also relates to a composition, preferably topical, selected by the screening method.

The present invention also relates to one or more ingredients, optionally in the form of a composition, preferably topical, to be used for the manufacture of a composition, preferably topical, for the care of the skin, mucous membranes or appendages capable of increase the biomass of the cutaneous microbiota, in particular without reducing its biodiversity, to maintain and/or restore the healthy nature of the skin, mucous membranes or appendages.

The present invention finally relates to a care method, preferably by topical route, using one or more ingredients, advantageously topically acceptable, capable of increasing the biomass of the cutaneous microbiota, in particular without reducing its biodiversity, in order to maintain and/or restore the healthy character of the skin, mucous membranes or appendages.

Thus, according to a first aspect, the invention relates to a method, preferably in vitro , for selecting an ingredient or composition, preferably ecobiological, advantageously applicable topically, in particular cosmetic or dermatological, useful in maintaining or/and restoring a healthy skin microbiota, including the steps of:

a) putting an ingredient or composition to be tested in contact with bacteria from a reference microbiota,

b) analyzing the quantity of biomass and, advantageously, the biodiversity of the microbiota after contacting, and

c) selecting as an ingredient or composition useful in maintaining and/or restoring a healthy skin microbiota, an ingredient or a composition capable of increasing the biomass of the skin microbiota, advantageously without reducing its biodiversity, compared the quantification of the biomass and, where appropriate, the biodiversity of the reference microbiota.

According to a particular embodiment, the method according to the invention is personalized, based on the conditions, specificities and reactivity of the skin microbiota of a given individual.

According to a particular embodiment, step a is carried out once or repeatedly and/or step b is carried out from 1 hour to 31 days, preferably 4 days, after step a.

According to a variant embodiment, the invention relates to a method, preferably in vitro , for screening or evaluating or selecting the activity of at least one ingredient or composition, preferably applicable topically, in particular an ingredient or cosmetic and/or dermatological composition, optionally formulated in the form of a topical composition, characterized in that the method comprises the following steps, preferably in vitro :
i) before the application of the ingredient or the composition, optionally formulated in the form of a topical composition, on at least one skin area to be studied presenting a so-called reference microbiota, from a first sample taken from said skin area , analyzes of the amount of biomass of the skin microbiota and, where appropriate, of its biodiversity, thus obtaining a first so-called reference measurement, for quantifying the biomass and, where appropriate, a first so-called reference measurement, of the biodiversity of the bacteria of said reference microbiota; and
ii) after at least one application of the ingredient or of the composition, optionally formulated in the form of a topical composition, on the said skin area, and after a predetermined time of action of the ingredient or of the composition, from a second sample taken from said skin area, the analyzes of the amount of biomass of the skin microbiota and, where appropriate, of its biodiversity, thus obtaining a second so-called biomass quantification activity measurement and, where appropriate, a second so-called biodiversity activity measurement; and
iii) comparing the second activity measurement(s) with the first reference measurement(s), and selecting the ingredient or composition, optionally formulated as a topical composition , increasing the biomass of the cutaneous microbiota, advantageously without reducing its biodiversity.

In other words, the invention relates to a method, preferably in vitro , of screening or selection of at least one ingredient or composition, optionally formulated in the form of a topical composition, to induce an increase in the biomass of a cutaneous microbiota, advantageously without reduce its biodiversity including the following steps:
i) quantifying the biomass of the skin microbiota and possibly measuring the biodiversity of a first sample from a skin area, thus making it possible to obtain a first quantification measurement of the biomass and, where appropriate, a first measurement of the biodiversity of a so-called reference skin microbiota; and
ii) quantifying the biomass of the skin microbiota and optionally measuring the biodiversity of a second sample from said skin area to which the at least one ingredient or composition, optionally formulated in the form of a topical composition, has been applied, thus making it possible to obtain a second biomass quantification measurement and, if necessary, a second biodiversity measurement; and
iii) comparing the second biomass quantification measurement with the first biomass quantification measurement, and where appropriate the second biodiversity measurement with the first biodiversity measurement and selecting the at least one ingredient or composition, optionally formulated in the form of a topical composition, capable of increasing the biomass of the cutaneous microbiota, if necessary without reducing its biodiversity.

According to a preferred embodiment, the predetermined action time of the ingredient can vary between 1 hour and several days, in particular 31 days, advantageously 4 days.

According to the invention, a “reference skin microbiota” or “so-called reference skin microbiota” or “reference microbiota” or “so-called reference microbiota” is for example a microbiota characteristic of a skin zone to be studied.

A reference microbiota can also be a medium comprising different bacteria in various proportions which may or may not correspond to a microbiota existing in nature.

The reference microbiota can be obtained in particular from a sample of a skin zone of an individual or by preparing a medium comprising the different bacteria in the proportions corresponding to said reference microbiota.

By "medium" is meant a medium suitable for the growth of bacteria. It can be a solid or liquid medium.

According to a particular embodiment, the levels of biomass and diversity of a reference microbiota characteristic of a skin area is an average of the levels of biomass and diversity and/or of microbiota diversity profile measured on samples from said area of skin in several individuals.

According to a preferred embodiment of the invention, said reference microbiota is that of a healthy skin area and/or is depleted, advantageously following stress or cutaneous aggression, more advantageously following the action of bactericides or virucides or soaps or hygiene or care products that are aggressive towards the microbiota.

According to a particular embodiment of the invention, a method is provided for screening or selecting or evaluating the activity of at least one ingredient which is preferentially applicable topically, in particular a cosmetic and/or dermatological ingredient, optionally formulated in the form of a topical composition, characterized in that the method comprises:
a) before any operation, the selection of a skin area to be studied of one or more human beings,
b) taking at least a first sample of biological or genetic material from the microbiota present in this selected area, from one or more human beings, constituting a so-called reference microbiota, followed by analysis of the quantity of biomass and the biodiversity of the skin microbiota, thus obtaining the first so-called reference measurements respectively of quantification of the biomass of the skin microbiota and of the biodiversity of the skin microbiota,
c) the application of at least one ingredient or a composition to this area, in a single or repeated manner,
d) after a period of 1 hour to 31 days, advantageously 4 days, after the single application or repeated applications, again taking at least a second sample of biological or genetic material from the microbiota present in this selected area, of one or more human beings,
e) the use of at least one method making it possible to quantify the biomass present in each of the aforementioned first and second samples, thus obtaining respectively a first measurement, called a reference, for quantification of the biomass of the skin microbiota and a second measurement, so-called activity, quantification of the biomass of the skin microbiota,
f) the use of at least one method making it possible to measure the microbial biodiversity present in each of the aforementioned first and second samples, thus obtaining respectively a first measurement, called a reference, of the biodiversity of the skin microbiota and a second measurement, called of activity, the biodiversity of the skin microbiota,
g) evaluation of the activity of the ingredient, or possibly of the composition, applied topically, on the biomass and biodiversity of the microbiota sampled by comparison of the biomass and biodiversity of the microbiota between said first measurements obtained at from the first sample and said second measurements obtained from the second sample, and
h) the selection of the ingredient, optionally formulated in the form of a topical composition, which increases the biomass of the cutaneous microbiota, in particular without reducing its biodiversity.

According to a particular embodiment, said ingredient or said composition to be tested in the selection or screening method according to the invention is chosen from the group comprising:
a) at least one carbon source which is capable of being used by bacteria as a growth and multiplication substrate, and preferably chosen from:
- partially or totally hydrolyzed proteins, from cereals (wheat, corn, barley, rye, oats), legumes (soya, fava beans, broad beans, peas), milk (caseins, albumins), yeast, as well as peptides and amino acids alone or as a mixture;
- short-chain oligosaccharides or polysaccharides, such as fructo-oligosaccharides or FOS; hyaluronic acids, pullulan, chitosan, dextran sulphate, galactoarabinan, carrageenan, alginate, cellulose, advantageously microcrystalline cellulose, amylose, amylopectin and their respective hydrolysates;
b) at least one antioxidant which has an activity on reactive oxygen species (ROS), advantageously free radicals, in particular chosen from the group consisting of octadecyl di-t-butyl-4-hydroxyhydrocinnamate; pentaerythrityl tetra-di-t-butyl hydroxyhydrocinnamate; 2,6-Bis(1,1-dimethylethyl)-4-methylphenol; bis-ethylhexyl hydroxydimethoxy benzylmalonate; tert-butylhydroquinone; tetrabutyl ethylidenebisphenol; thioglycolic acid; thiotaurine; thioctic acid; dilauryl thiodipropionate; sodium erythorbate; sorbityl furfural; erythorbic acid; perillyl alcohol; pyridyloxide t-butylnitrone; ergothioneine; melatonin; acetyl cysteine; cysteine; lysine hydrochloride; carnosic acid; tyrosyl histidine HCl; histidine hydrochloride; pyridoxine serinate; superoxide dismutase; aminopropyl ascorbyl phosphate; ascorbic acid; ascorbyl dipalmitate; ascorbyl glucoside; ascorbyl linoleate; ascorbyl methylsilanol pectinate; ascorbyl palmitate; ascorbyl tetraisopalmitate; ascorbyl tocopheryl maleate; trisodium ascorbyl palmitate phosphate; disodium ascorbyl sulphate; calcium ascorbate; methylsilanol ascorbate; sodium ascorbate; sodium ascorbyl phosphate; sodium ascorbyl/cholesteryl phosphate; tetrahexyldecyl ascorbate; magnesium ascorbyl phosphate; tocopherol; tocopheryl acetate; tocopheryl linoleate; tocopheryl oleate; tocopheryl nicotinate; tocopheryl retinoate; sodium tocopheryl phosphate; dioleyltocopheryl methylsilanol; potassium ascorbyl tocopheryl 5 phosphate; dodecyl gallate; propyl gallate; octyl gallate; epigallocatechin gallate (EGCG); propyl gallate; ethylferulate; ethylhexyl ferulate; chitosan ascorbate; chitosan glycolate; apigenin; tiliroside; alpha-arbutin; arbutin; baicalin; quercetine; quercetin caprylate; isoquercetin; diethylhexyl syringylidenemalonate; dihydroxymethylchromone; dimethoxy di-p-cresol; dimethylmethoxy chromanol; ethylbisiminomethylguaiacol manganese chloride; hesperidin methyl chalcone; kojic acid; kojic dipalmitate; madecassoside; asiaticoside; magnolol (5,5'-diallyl-2,2'-dihydroxybiphenyl); nordihydroguaiaretic acid; phenylethyl resorcinol; resveratrol; troxerutin; glucosylrutin, rutin (4H-1-benzopyran-4-one); disodium rutinyl disulfate; tetrahydrobisdemethoxydiferuloylmethane; tetrahydrodemethoxydiferuloylmethane; tetrahydrodiferuloylmethane; tococysteamide; totarol; hydroxydecyl ubiquinone; ubiquinone; carotenoids; niacinamide or one of its derivatives; creatine; creatinine, carnosine, hypotaurine, inositol, trehalose, betaine, xylitol, mannitol, and rhamnose; more particularly, in the group comprising tocopherol, carnosine, hypotaurine, inositol, trehalose, betaine, xylitol, mannitol, rhamnose; even more particularly carnosine;
c) at least one topically acceptable inorganic or organic salt, advantageously chosen from the group comprising a salt of sodium, magnesium, calcium, potassium, phosphorus, copper, zinc, cobalt and their mixtures, in particular the sodium citrate, sodium lactate, magnesium, copper or zinc sulphate, potassium or sodium phosphate, copper or zinc gluconate, more particularly sodium citrate.

According to another aspect, the invention also relates to a method for preparing a composition, advantageously topical, in particular cosmetic or dermatological, preferably ecobiological, characterized in that it comprises
(i) the implementation of a selection or screening method according to the invention
(ii) the selection of at least one composition, advantageously topical, in particular a cosmetic and/or dermatological composition, comprising at least one ingredient selected by the selection or screening method according to the invention and capable of inducing an increase in the biomass of the cutaneous microbiota, in particular without reducing its biodiversity and
(iii) packaging of said composition.

According to another aspect, the invention relates to a composition, advantageously topical, preferentially ecobiological, in particular cosmetic and/or dermatological, comprising at least one ingredient or a composition, selected by the selection or screening method according to the invention, for its use for its use in maintaining and/or restoring a healthy skin microbiota.

Advantageously, the composition for its use according to the invention comprises at least one carbon source, an antioxidant and an inorganic or organic salt, more advantageously said carbon source, said antioxidant and/or said salt being selected by selection or screening method according to the invention.

According to a preferred embodiment:
a) the at least one carbon source is capable of being used by bacteria as a growth and multiplication substrate, and, preferably, is chosen from:
- partially or totally hydrolyzed proteins, from cereals (wheat, corn, barley, rye, oats), legumes (soya, fava beans, broad beans, peas), milk (caseins, albumins), yeast, as well as peptides and amino acids alone or as a mixture;
- short-chain oligosaccharides or polysaccharides, such as fructo-oligosaccharides or FOS; hyaluronic acids, pullulan, chitosan, dextran sulphate, galactoarabinan, carrageenan, alginate, cellulose, advantageously microcrystalline cellulose, amylose, amylopectin and their respective hydrolysates;
b) The at least one antioxidant exhibits activity on reactive oxygen species (ROS), advantageously free radicals, and, in particular, is chosen from the group consisting of octadecyl di-t-butyl-4-hydroxyhydrocinnamate ; pentaerythrityl tetra-di-t-butyl hydroxyhydrocinnamate; 2,6-Bis(1,1-dimethylethyl)-4-methylphenol; bis-ethylhexyl hydroxydimethoxy benzylmalonate; tert-butylhydroquinone; tetrabutyl ethylidenebisphenol; thioglycolic acid; thiotaurine; thioctic acid; dilauryl thiodipropionate; sodium erythorbate; sorbityl furfural; erythorbic acid; perillyl alcohol; pyridyloxide t-butylnitrone; ergothioneine; melatonin; acetyl cysteine; cysteine; lysine hydrochloride; carnosic acid; tyrosyl histidine HCl; histidine hydrochloride; pyridoxine serinate; superoxide dismutase; aminopropyl ascorbyl phosphate; ascorbic acid; ascorbyl dipalmitate; ascorbyl glucoside; ascorbyl linoleate; ascorbyl methylsilanol pectinate; ascorbyl palmitate; ascorbyl tetraisopalmitate; ascorbyl tocopheryl maleate; trisodium ascorbyl palmitate phosphate; disodium ascorbyl sulphate; calcium ascorbate; methylsilanol ascorbate; sodium ascorbate; sodium ascorbyl phosphate; sodium ascorbyl/cholesteryl phosphate; tetrahexyldecyl ascorbate; magnesium ascorbyl phosphate; tocopherol; tocopheryl acetate; tocopheryl linoleate; tocopheryl oleate; tocopheryl nicotinate; tocopheryl retinoate; sodium tocopheryl phosphate; dioleyltocopheryl methylsilanol; potassium ascorbyl tocopheryl 5 phosphate; dodecyl gallate; propyl gallate; octyl gallate; epigallocatechin gallate (EGCG); propyl gallate; ethylferulate; ethylhexyl ferulate; chitosan ascorbate; chitosan glycolate; apigenin; tiliroside; alpha-arbutin; arbutin; baicalin; quercetine; quercetin caprylate; isoquercetin; diethylhexyl syringylidenemalonate; dihydroxymethylchromone; dimethoxy di-p-cresol; dimethylmethoxy chromanol; ethylbisiminomethylguaiacol manganese chloride; hesperidin methyl chalcone; kojic acid; kojic dipalmitate; madecassoside; asiaticoside; magnolol (5,5'-diallyl-2,2'-dihydroxybiphenyl); nordihydroguaiaretic acid; phenylethyl resorcinol; resveratrol; troxerutin; glucosylrutin, rutin (4H-1-benzopyran-4-one); disodium rutinyl disulfate; tetrahydrobisdemethoxydiferuloylmethane; tetrahydrodemethoxydiferuloylmethane; tetrahydrodiferuloylmethane; tococysteamide; totarol; hydroxydecyl ubiquinone; ubiquinone; carotenoids; niacinamide or one of its derivatives; creatine; creatinine, carnosine, hypotaurine, inositol, trehalose, betaine, xylitol, mannitol, and rhamnose; more particularly, in the group comprising tocopherol, carnosine, hypotaurine, inositol, trehalose, betaine, xylitol, mannitol, rhamnose; even more particularly, carnosine;
c) The at least one inorganic or organic salt is topically acceptable and, advantageously, is chosen from the group comprising a salt of sodium, magnesium, calcium, potassium, phosphorus, copper, zinc, cobalt and their mixtures, in particular sodium citrate, sodium lactate, magnesium, copper or zinc sulphate, potassium or sodium phosphate, copper or zinc gluconate, more particularly sodium citrate.

According to a preferred embodiment, the composition for its use according to the invention comprises sodium citrate and carnosine.

According to a particular embodiment, the composition for its use according to the invention is an aqueous composition, comprising at least 60% by weight of the total composition of water, in particular at least 60%, better still at least 70%, or at least 80% or at least 90% or at least 95%.

Preferably, the aqueous phase represents at least 60% by weight of the total composition, better still at least 70%, or at least 80% or at least 90% or at least 95%.

Advantageously, the aqueous phase is characterized by:
- a resistivity, at ambient temperature, of between 12.5 and 12,500 Ohms.cm, in particular between 80 and 8,000 Ohms.cm; and or
- an osmotic pressure between 70 and 2500 mOsmol/l and in particular between 100 and 800 mOsmol/l as well as a pH between 5.0 and 9.0 and in particular between 6.0 and 8.0.

According to a particular embodiment, the composition for its use according to the invention has a pH comprised between 5.0 and 9.0 and in particular comprised between 6.0 and 8.0.

According to a particular embodiment, the composition for its use according to the invention is applied to a healthy skin area, before or following a skin attack, advantageously the action of bactericides or virucides or soaps or hygiene products or aggressive treatments for the microbiota.

According to a particular embodiment, said cutaneous aggression according to the invention can be single or repeated, preferably repeated.

According to a particular embodiment, the composition for its use according to the invention is for its use for combating cutaneous aggression on healthy skin, by stimulating the biomass of the microbiota present at the cutaneous level, or present on a keratin material, for increasing the quantity of this biomass of said microbiota in order to prevent the arrival and/or installation of new microbial strains linked to the environment.

According to a particular embodiment, the composition according to the invention is for its use in combating cutaneous aggression on healthy skin by stimulating the biomass of the microbiota present at the cutaneous level, or present on a keratin material, to restore the quantity of this biomass of said microbiota, greatly reduced during a skin attack and for example after the action of bactericides or virucides or soaps or hygiene or care products that are aggressive towards the microbiota.

According to another aspect, the invention relates to a method of treatment, advantageously topical, in particular non-therapeutic or dermatological cosmetic, preferably ecobiological, for maintaining and/or restoring a healthy skin microbiota, characterized by the use of a composition such as previously described allowing the increase in the biomass of the cutaneous microbiota, in particular without reducing its biodiversity.,

According to another aspect, the invention relates to the use of at least one ingredient, or at least one substance, or at least one composition, selected by the selection or screening method according to the invention for the preparation of a composition, preferably topical, in particular ecobiological, for maintaining and/or restoring a healthy skin microbiota.

According to another aspect, the invention relates to a method of topical care, in particular ecobiological, advantageously cosmetic or dermatological, for treating the skin requiring maintenance or restoration of its healthy microbiota, by increasing the biomass of the cutaneous microbiota, in particular without reducing its biodiversity, characterized in that at least one area of the skin requiring this care is selected, and an ingredient is applied to at least this area of the skin, advantageously formulated in a composition, or a composition selected at through the selection or screening method described above or in the following description.

According to a particular embodiment, the invention also relates to a topical care method, preferably ecobiological, characterized in that it comprises (i) the implementation of a selection or screening method defined previously or in the description following, and the selection of at least one ingredient or a composition, in particular an ingredient or a cosmetic and/or dermatological composition, capable of inducing the increase in the biomass of the cutaneous microbiota described previously, in particular without reducing its biodiversity ( ii) the formulation of the ingredients or compositions thus selected in a formula for cosmetic and/or dermatological use, capable of inducing an increase in the biomass of the cutaneous microbiota without reducing its diversity (iii) the packaging of said formula (iv) the topical application of said formulation to an area of skin or a surface of the selected appendages.

According to yet another aspect, the invention relates to a treatment method, advantageously topical, preferably cosmetic or dermatological, in particular ecobiological, for combating cutaneous aggression on healthy skin, characterized in that at least one zone is selected of the skin requiring this care and the administration is carried out, preferably by topical application, of at least one ingredient or composition, advantageously applicable topically, in particular a cosmetic and/or dermatological ingredient or composition, in particular formulated in the form of topical composition, selected by the screening or selection method described previously or in the following description, stimulating the biomass of the microbiota present in the skin, or present on a keratin material, to increase the quantity of this biomass of said microbiota in order to avoid the arrival of new microbial strains linked to the environment.

According to another aspect, the invention relates to a treatment method, advantageously topical, preferably cosmetic or dermatological, in particular ecobiological, for combating cutaneous aggression on healthy skin, characterized in that at least one area of the skin requiring this care and the application is carried out, preferably topically, of at least one ingredient which can be applied topically, in particular a cosmetic and/or dermatological ingredient, preferably formulated in the form of a topical composition, selected by the method of selection or screening described previously or in the following description, stimulating the biomass of the microbiota present at the cutaneous level, or present on a keratin material, to restore the quantity of this biomass of said microbiota against its reduction in quantity by a cutaneous aggression and for example after the action of bactericides or virucides or soaps or aggressive hygiene or care products vis-à-vis the mic robot.

Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the field to which the disclosed methods and compositions relate. General definitions can be found in technical books relating to the field of molecular biology, for example, Dictionary of Microbiology and Molecular Biology, 2nd edition. (Singleton et al. , 1994) or The Harper Collins Dictionary of Biology (Hale et al., 1991). As used in the present invention, the following terms and expressions have the meaning attributed to them, unless otherwise indicated.

By “keratin materials”, is meant according to the invention the skin, the semi-mucous membranes (in particular the lips), the mucous membranes and the appendages including the appendages.

By "method for quantifying the biomass of the microbiota", we mean according to the invention, the use of a quantitative or semi-quantitative method which is based, for example:
- on quantitative PCR (or qPCR) techniques, or
- on flow cytometry, or
- on enumeration methods on agar, or
- on any related method.

By "method for measuring the biodiversity of the microbiota", we mean according to the invention, the use:
- a method based on the analysis of part of the hypervariable gene of the 16S ribosome of bacteria after their targeted amplification, or
- a method using the metagenomic shotgun technique (on microbiome DNA), or
- a so-called metatranscriptomic “shotgun” sequencing method (on microbiome RNA), or
- any related method,
followed by a method for calculating the microbial diversity from the data of the previous method used, such as the calculation:
- the Shannon index,
- Simpson's index,
- the Inverse-Simpson index,
- the number of species or OTUs,
- Evenness or the Pielou index,
- the Hurlbert index,
- the Hill index.

These calculation methods and indices are well described in the literature and known to those skilled in the art. They can be found, for example, in the document by Eric Marcon, “Measurement of Biodiversity, Kourou, France, ffcel-01205813v5ff”.

The sampling of biological or genetic material from the microbiota can be done according to methods known to those skilled in the art. Typically, the biological or genetic material is recovered using a swab which allows the bacteria to be collected, always performing the same manipulation to standardize the quantities taken as much as possible. As an example the Sigma Trans Swab liquid Maies swab can be used. In a preferred embodiment, the sample is taken by performing 10 return trips in Z by applying light pressure on the swab. At this stage, it is possible to freeze the swabs, which can be stored at -80°C. From the swabs, the bacterial pellets can be recovered by centrifugation. The extraction of genomic DNA (gDNA) can be carried out according to methods known to those skilled in the art, for example with the Nucleospin Microbial DNA kit (Macherey Nagel, France).

The samples can be taken from several areas of the human body, such as the cheek, forehead, forearm, in order to study the response of the microbiota of the different biogeographical areas to the ingredients or compositions applied topically.

In a preferred embodiment, the sample is taken from the face, which makes it possible to test the ingredients or compositions on a realistic area exposed to the environment, or on the back, which makes it possible to test a set of different conditions. on a relatively homogeneous microbiota (Grice et al., 2009).

According to a preferred embodiment, the area concerned can be marked by a mask, advantageously delimiting a sampling surface comprised between 10 and 20 cm ² , advantageously 15 cm ² .

In a preferred embodiment, this biomass is evaluated starting from the biological or genetic material recovered from the keratin materials, but it can also be quantified directly according to the methods known to those skilled in the art, for example by immunomarking or flow cytometry. on labeled bacteria.

By “standardization” within the meaning of the invention, is meant the standardization of the quantity of biomass sampled.

By "amplification" within the meaning of the invention is meant any in vitro procedure which produces multiple copies of a target nucleic acid sequence, or of its complementary sequence, or of fragments thereof (i.e. i.e. an amplified sequence containing less than the complete target nucleic acid). Advantageously, this amplification is carried out by polymerase chain reaction or polymerase chain reaction (PCR), but other methods known to those skilled in the art, such as isothermal amplification, can also be used.

By “quantitative amplification” is meant within the meaning of the invention any in vitro procedure which produces multiple copies of a target nucleic acid sequence and which at the same time makes it possible to determine the concentration of target sequence initially present in the reaction mixture. . Several types of quantitative amplification reactions are described. We can distinguish quantitative amplifications based on the use of an external standard, competitive amplifications, using an internal standard, and finally kinetic amplifications, which consist in measuring in real time, the increase in the quantity of the target sequence . Advantageously, the quantitative amplification is a kinetic amplification carried out by PCR and corresponds to the technique known to those skilled in the art under the name of quantitative PCR (qPCR) or even real-time PCR. The PCR reactions, whether quantitative or non-quantitative, can be carried out with the “Light Cycler” system (Roche Molecular Systems Inc.) and according to the procedures recommended by the supplier.

The term "target sequence", as used in the present invention, refers to the particular nucleotide sequence of the target nucleic acid which is to be amplified and/or detected. "Target sequence" includes complexing sequences to which oligonucleotides (eg, primer oligonucleotides and/or promoter oligonucleotides) complex during amplification processes (eg, PCR). When the target nucleic acid is originally single-stranded, the term “target sequence” will also designate the sequence complementary to the “target sequence” as present in the target nucleic acid. When the target nucleic acid is originally double-stranded, the term "target sequence" refers to both sense (+) and antisense (-) strands.

The term "region", as used in the present invention, means a portion of a nucleic acid, said part being smaller than the entire nucleic acid. In particular, with “regions V1, V2, V3, V4” is meant hypervariable regions of the bacterial 16S ribosomal DNA (rDNA) defined for example in Gray et al., 1984, which constitutes a portion of the gDNA and which allows the identification of the species to which they belong during sequencing.

By "amplicon" or "amplification product" is meant a nucleic acid molecule generated in an amplification reaction, whether quantitative or not, of nucleic acid and which is derived from a target nucleic acid. An amplicon or amplification product contains a target nucleic acid sequence which may be of the same or opposite sense to the target nucleic acid.

By “sequencing” within the meaning of the invention is meant the decoding of at least one strand of a target sequence, and the reading of the sequence of nucleotides composing it in their order. Several methods for sequencing and assembling the sequences read are known to those skilled in the art.

Advantageously, the sequencing is carried out using the Illumina Miseq™ platform. For example, the documents WO03048387A2, WO2005024010A1 WO2006064199A1 and WO2007123744A1 or also WO2012096703A1 and the references cited in these documents describe sample preparation and sequencing methods suitable for the present invention. Sequencing can be performed directly on amplicons, or alternatively on amplicon libraries.

According to a preferred embodiment, the sequencing is carried out using an amplicon library, advantageously the amplicon libraries obtained according to the Metabiote™ solution described in Guillaume et al., 2014.

A "primer" is an oligomer that hybridizes to nucleic acid and has a 3' end that is extended by polymerization. A primer can optionally be modified, for example by including a 5' region that is not complementary to the target sequence. Such modification may include functional additions, such as tags, promoters or other non-specific target sequences, used or useful to manipulate or amplify the primer or target oligonucleotide. Primers can be "degenerate". Within the meaning of the invention, “degenerate primer” denotes primers allowing incorporation of different nucleotides at determined positions. Degenerate primers are known to those skilled in the art; the following code is universally used to note the degeneracy of primers at a given nucleotide position: I=Inosine (Nichols et al., 1994); R = G or A; K= G or T; S = G or C; W = A or T; M = A or C; Y = T or C; D = G or A or T; V = G or A or C; B = G or T or C; H = A or T or C; N = G or A or T or C.

By “16S universal primers” within the meaning of the invention, is meant oligonucleotides allowing the amplification of highly conserved regions of 16S rDNA. According to a particular embodiment, these are the primers corresponding to SEQ ID. 1 8F (5′-AGAGTTTGATCCTGGCTCAG-3′) and SEQ ID. 2 1391R (5′-GACGGGCGGTGWGTRCA-3′). (Kong et al., 2012b). According to an alternative embodiment, these are the primers corresponding to SEQ ID. 3533F (5′-GTGCCAGCAGCCGCGGTAA-3′) and SEQ ID. 4902R (5′-GTCAATTCITTTGAGTTTYARYC-3′) (San Miguel et al., 2018).

By “16S specific primers” within the meaning of the invention is meant oligonucleotides allowing the amplification of hypervariable regions of 16S rDNA. Advantageously, these are the V1-V4 regions of 16S rDNA, preferably the V1-V3 and/or V3-V4 regions, advantageously the V3-V4 regions. Advantageously, the 16S specific primers make it possible to obtain amplicons of size between 100 and 500 base pairs, advantageously between 200 and 400 base pairs. According to one embodiment, these are the primers for the V1-V3 regions corresponding to SEQ. ID 5 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and SEQ. ID 6534R (5-ATTACCGCGGCTGCTGG-3) (San Miguel et al., 2018). According to a preferred embodiment, these are the primers for the V3-V4 regions corresponding to SEQ ID, 7338F (ACTCCTACGGGAGGCAGCAG) and SEQ ID. 8,806R (GGACTACHVGGGTWTCTAAT) (Li et al., 2020).

The expressions “bank(s)” or “library(s)” will be used interchangeably in the present description, these two expressions having the same meaning. Within the meaning of the invention, the term "bank" means a collection of bacterial nucleotide sequences preferably resulting from the amplification of 16S rDNA and which are capable of being sequenced or detected individually. The constitution of a DNA library can be done according to methods known to those skilled in the art, for example by using kits such as the NEBNext™ Ultra™ DNA Library Prep Kit.

According to a particular embodiment, it is considered within the scope of the invention that the selected ingredient or composition applied topically has a significant effect on the increase in the biomass of the cutaneous microbiota, if the biomass increases statistically significant between two samples, in particular between the first sample and the second sample mentioned above, in particular after 1 hour to 31 days of treatment.

According to the invention, the evaluation of the activity of the ingredient or composition applied topically on the biomass of the sampled microbiota can be carried out by comparing the total bacterial genetic material present on the sampled area before and after the treatment with the ingredient or composition through quantitative amplification with universal 16S primers, which makes it possible to estimate the genetic material present at the time of sampling before and after treatment.

In a particular embodiment, the at least one amplification of the standardized amounts of genetic material is a quantitative amplification carried out using the universal 16S primers. For this, bacterial lysis followed by a genomic DNA extraction step with the PureLink Mini kit (Life Technologies, Grand Island, NY) is carried out. 16S rRNA gene-targeted qPCR is performed using specific primers (Bact-8F: AGAGTTTGATCCT GGCTCAG, Bacte338R: CTGCTGCCTCCCGTAGGAGT) with SYBR Green detection (Applied Biosystems, Foster City, CA) on equipment Mx300P (Agilent Technology). Standard curves are prepared by amplifying known amounts of E. coli DNA. By comparing the number of cycles to the standard curve it is possible to calculate the number of copies of the RNA16S gene per microliter of sample.

In another particular embodiment, the evaluation of the biomass of the cutaneous microbiota is carried out by quantification in flow cytometry. For this, the swab is discharged into 1 mL of Amies liquid then the bacterial suspension is labeled with SYBRGreen, incubated for 15 min at 37°C protected from light and analyzed by a BD Accuri C6 flow cytometer (cytometer BD Accuri, Belgium) equipped with a 50 mW laser emitting at a fixed wavelength of 488 nm. Fluorescence intensity is measured at Fl1 = 533 +/- 30 nm, Fl3 > 670 nm. Data are processed with BD Accuri CFlow software. A number of bacteria is thus obtained per mL of sample.

In another particular embodiment, the evaluation of the biomass of the cutaneous microbiota is carried out by quantification by counting on agar. For this, after unloading the swabs in the Amies liquid, the bacterial suspensions are deposited on nutrient agar in a Petri dish, and are incubated for 48 hours at 35°C. A count of the bacterial colonies makes it possible to obtain the bacterial concentration by multiplying the number of CFU by the dilution factor, and is expressed in CFU/ml.

In yet another particular embodiment, the evaluation of the biomass of the cutaneous microbiota is carried out by quantification by direct counting under a microscope. For this, the bacterial suspensions are inoculated in a nutrient broth such as Mueller-Hinton or Brain Heart Infusion. After incubation, a direct enumeration under the microscope can be carried out on a Thoma cell.

According to a particular embodiment of the invention, it is considered that there has been no reduction in the bacterial diversity of the skin microbiota if the variations are not statistically significant.

According to a particular embodiment of the invention, the evaluation of the activity of the ingredient or composition applied topically on the diversity of the sampled microbiota can be carried out by comparing the bacterial species present in the different experimental conditions by the bias of the amplification with the specific 16S primers and the sequencing of the amplicons obtained. Several indices making it possible to estimate the diversity of a genetic material consisting of mixtures of different species are known to those skilled in the art. According to a particular embodiment, the index is the so-called Shannon index, which is calculated by following the method described in Lemos et al., 2011. The Shannon index integrates two distinct parameters: the number of operational taxonomic units ( Operational Taxonomic Units or OTUs) and the distribution of these OTUs in each sample. This last parameter can be calculated by taking into account the abundance of the 15 most represented bacterial genera, or alternatively, representing the 90% of the amplicons obtained in the amplification with the specific 16S primers.

The bioinformatics processing of the target sequences can be carried out according to the methods known to those skilled in the art, for example by using a PERL script.

According to another particular embodiment, the analysis by amplification with the specific 16S primers then, optionally, sequencing is carried out only for the samples of genetic material from a sample making it possible to obtain, during the quantitative amplification with the 16S universal primers in a reaction volume of 4.38 µl, at least 400 copies of 16S rDNA amplicon/µL after 30 cycles of amplification and/or at least 500 copies of amplicon of 16S/µL rDNA after 35 cycles of amplification.

According to yet another particular embodiment, the analysis of the diversity of the cutaneous microbiota is carried out by metagenomics in the following way: after extraction of the gDNA, the microbial diversity is determined for each sample by targeted amplification of part of the gene ribosomal. A fragment of the 16S rRNA gene comprising the V3 to V4 (or V1-V3) hypervariable regions is amplified with primers 338F (ACTCCTACGGGAGGCAGCAG) and 806R (GGACTACHVGGGTWTCTAAT) (Munyaka et al. , 2015). The amplification is carried out and the products resulting from PCR are purified and normalized using Agencourt AMPure XP magnetic beads (Beckman Coulter). Once purified, amplicons are pooled for Illumina sequencing, performed in 2x300 bp on an Illumina MiSeq platform using MiSeq Reagent Kit v3 (600 cycles). The sequences are grouped into operational taxonomic units (OTUs) with an identity threshold of 97%. Taxonomic assignment is performed using the Ribosomal Database Project (RDP) classification on the Greengenes V13_8 database. The analysis is carried out with the R software. The Shannon-Weaver index, the number of observed OTUs and the Bray-Curtis dissimilarity index are calculated with the Phyloseq and Vegan packages. Statistical significance is determined using the Wilcoxon test. A p-value <0.05 is considered statistically significant.

According to another embodiment, the analysis of the diversity of the cutaneous microbiota is carried out by Shotgun: after extraction of the gDNA, the microbial diversity is determined for each sample by Whole metagenome sequencing/WMS or Shotgun metagenome sequencing, i.e. i.e. complete sequencing of the entire bacterial genome. The libraries are prepared using the NexteraXT preparation kit (Illumina). Sequencing is performed at the Penn Next Generation Sequencing Core on Illumina MiSeq chemistry to obtain final paired reads of 150 bp. Sequencing data is obtained in fastq format. Sequences shorter than 80 nucleotides are removed from the analysis. DeconSeq is used to filter fastq files and one of the paired reads (SE1) is entered into MetaPhlAn version 1.7.7 for taxonomic classification. One of the matched ends (SE1) of the MCC sample is used to perform a BLAST against a personalized database of genomes among the 20 expected bacterial species (blastn, max_target_seqs 1, e <10−10; alignment length > 50 ) to determine the composition of bacterial communities. The alpha diversity index is then calculated using the R software and the Phyloseq and Vegan packages using the Shannon index.

According to another embodiment, the analysis of the diversity of the cutaneous microbiota is carried out by Metatranscriptomic shotgun sequencing (MTS): for this the total RNA is extracted from the bacterial sample. The polyadenylated mRNAs are separated from the non-polyadenylated ones (rRNA, mitochondrial RNA, and tRNA) by affinity on a poly-dT column (Dynabeads Oligo (dT) kit, Dynal). The enriched mRNA is mechanically fragmented to a size range of +/- 200 bp with an ultrasonicator. mRNA fragmentation is assessed using the Agilent RNA 6000 Pico Kit on a 2100 Bioanalyzer instrument (Agilent Technologies, Inc.). Metatranscriptome libraries are prepared using the NEBNext Ultra RNA Library Prep Kit for Illumina (New England BioLabs Inc). The quality and quantity of all final libraries are analyzed with an Agilent DNA 1000 kit on the 2100 Bioanalyzer and Qubit instrument and are quantified and validated by a qPCR assay using the PerfeCTa NGS library quantification kit for Illumina (Quanta Biosciences, Inc.) using the CFX Connect real-time PCR detection system (Rad Laboratories, Inc.). Sequencing of one of the MT libraries is performed on an Illumina HiSeq 2000 using TruSeq SBS v3 reagent for 100 pair read length (BGI Americas) (labeled HS100) and on Illumina MiSeq using the v3-600 cycle kit for 301 base pairs (labeled MS301). Another set of twelve libraries is sequenced on Illumina MiSeq using 151 “paired-end chemistry” (marked MS151). The manufacturer's recommended protocol is used to perform the sequencing reaction on the HiSeq and MiSeq platforms.

For PCR-amplified 16S ribosomal sequences, BLASTN searches (default settings, except word size set to 7) are performed against the GenBank nr nucleotide database (http://www.ncbi.nlm.nih.gov /). The 16S sequences are then assigned to major phyla to perform phylogenetic analyzes (BioNJ and PhyM) using the Best Blast Hit (BBH) algorithm and a set of reference sequences for each of the different phyla. Analyzes (sequence alignments and phylogenetic analyses) are performed using SeaView. Using BLASTX output files, cDNAs are taxonomically assigned using MEGAN V 3.5 (www-ab.informatik.uni-tuebingen.de/software/megan).

According to yet another particular embodiment, the method according to the invention comprises at least two samples of biological or genetic material from the microbiota:
- a sample of biological or genetic material of the microbiota present in the skin, or present on a keratin material of said human being, said sample being taken in an untreated area before the treatment,
- a sample of biological or genetic material of the microbiota present in the skin, or present on a keratin material of said human being, said sample being taken from an area treated with the ingredient or composition, preferably applicable topically, after the treatment.

According to another particular embodiment, the method according to the invention comprises at least four samples of biological or genetic material of the microbiota present in the skin, or present on a keratin material, from one or more human beings:
- A sample taken from an area to be treated with the ingredient or composition, preferably applied topically, before the treatment;
- A sample taken from an untreated area before treatment;
- A sample taken from an area treated with the ingredient or composition, preferably applicable topically, after the treatment;
- A sample taken in an untreated area after the treatment period.

Advantageously, the “untreated area” according to the invention is a skin area equivalent or close, functionally and/or in microbial composition, to said “area to be treated”.

Thus, according to another particular embodiment, the invention relates to a method for selecting or screening or evaluating the activity of at least one ingredient or composition, preferably applicable topically, in particular a cosmetic ingredient and / or dermatological, optionally formulated in the form of a topical composition, characterized in that the method comprises the following steps:
- a sample of biological or genetic material of the microbiota present in the skin, of one or more human beings, said sample being taken from an area to be treated with the ingredient or composition applicable topically before the treatment;
- a sample of biological or genetic material of the microbiota present in the skin, or present on a keratinous material, of one or more human beings, said sample being taken in an untreated area before the treatment;
- a sample of biological or genetic material of the microbiota present on the skin, or present on a keratin material, of one or more human beings, said sample being taken from an area treated with the ingredient or composition applicable topically after the treatment ;
- a sample of biological or genetic material of the microbiota present in the skin, or present on a keratin material, of one or more human beings, said sample being taken in an untreated area after the treatment;
- an analysis of the biomass, for example by quantitative amplification of the standardized quantities of genetic material carried out using the universal primers 16S;
- an analysis of the biodiversity of the sampled microbiota, for example by amplification of the standardized quantities of genetic material sampled, then sequencing of the amplicons thus produced using the specific 16S primers, then calculation of a diversity index allowing this analysis.

The duration of the treatment with the ingredient or the composition is variable. The application of the product can be done daily or several times a day or over the test period. According to one embodiment, several samples can be taken from the treated zone, at, for example, 5 minutes, 1 hour, 24 hours, 2 days, 4 days, 7 days and 31 days from the start of the treatment.

According to a particular embodiment, the sample is taken from the treated zone after 2 to 7 days of daily treatment. According to one embodiment, the application of the product is done due to a volume of between 0.1 and 100 μL/cm ² , advantageously between 1 and 10 μl/cm ² , by standardizing the application as much as possible, for example using a robotic arm.

In a particular embodiment, at least one sample is taken from several human beings. This has the advantage of supporting the statistical significance of the data obtained.

According to a particular embodiment, the particular group of subjects at the origin of the samples can share characteristics (ethnicity, age, phototype, chronic dermatological conditions, type of skin, diet, lifestyle, rate of hydration of the skin, geolocation and other similar parameters) which allow the development of treatment methods, preferably cosmetic and/or dermatological, specifically targeting said groups of particular subjects, also called "populations".

According to an advantageous and preferred embodiment, the population at the origin of the samples is characterized by an impoverished skin microbiota at the time of the samples.

By "depleted skin microbiota" within the meaning of the invention is meant any decline in the biomass of the skin microbiota.

In particular, the depletion can be linked to a skin aggression according to the invention, more particularly to the action of bactericides or virucides or fungicides or soaps or aggressive hygiene or care products vis-à-vis of the microbiota.

In a particular embodiment, the depletion of the cutaneous microbiota is caused by a topical treatment with an antiseptic agent (that is to say fungicide, bactericide and/or virucide), advantageously a composition comprising a C1-C8 alcohol chosen from ethanol, isopropanol, propanol, butanol, pentanol, hexanol, heptanol, octanol, benzyl alcohol and mixtures thereof; and/or an antiseptic agent other than an alcohol, such as for example benzalkonium chloride, chlorhexidine gluconate, sodium hypochlorite, a soap produced by saponification of a fatty acid; and/or also a composition comprising an acid such as lactic, malic, tartaric or glycolic acid; in an alternative embodiment, the depletion of the skin microbiota is caused by treatment with a virucidal and/or antifungal and/or bactericidal and/or antibiotic agent, administered topically or systemically.

According to a particular embodiment, the sampling of biological or genetic material of the microbiota present in the skin, or present on a keratinous material, is carried out on a single human being.

This method makes it possible to develop care methods, advantageously cosmetic and/or dermatological, preferably ecobiological, personalized, based on the conditions, specificities and responsiveness to care, of the skin microbiota of each individual.

Thus, according to another particular embodiment, the invention also relates to a personalized method for selecting or screening or evaluating, preferably in vitro , the activity of at least one ingredient or composition, preferably applicable topically, in particular a cosmetic and/or dermatological ingredient, optionally formulated in the form of a composition, advantageously topical, characterized in that the method comprises the following steps:
- the selection of a skin area or appendages to study,
- at least one sample of biological or genetic material from the microbiota present in this area, from a single human being,
- the application of at least one ingredient or a composition to this area, in a single or repeated manner,
- after a period of 1 hour to 7 days, at least one sample of biological or genetic material from the microbiota present in this area, from a single human being,
- at least one method to quantify the biomass present in the samples,
- at least one method for measuring the microbial diversity present in the samples,
- evaluation of the activity of the ingredient or composition applied topically on the biomass and biodiversity of the sampled microbiota,
- the selection of the ingredient or ecobiological composition applicable topically allowing the increase of the biomass, in particular without reducing the diversity of the cutaneous microbiota.

According to a particular embodiment, the personalized method according to the invention can be accompanied by an analysis of the dermatological profile of the single human being at the origin of the at least one sample on the basis of his ethnicity, age, phototype, chronic dermatological conditions, skin type, diet, lifestyle, skin hydration level, geolocation and other similar parameters. This information can be collected by direct analysis or through a form. For example, the methods for weighting cutaneous, capillary, or nail characteristics and the computer program described in document FR 2 983 328 can be combined with the results of the personalized screening method mentioned above to select an ecobiological dermatological treatment suitable to induce an increase in the biomass of the skin microbiota of an individual, in particular without reducing its diversity, and in accordance with the overall dermatological needs of said individual.

The invention also relates to a method for preparing a composition, preferably topical, characterized in that it comprises (i) the implementation of a screening or selection method defined previously and the selection of at least one ingredient or a composition, in particular an ingredient or a cosmetic and/or dermatological composition, capable of inducing an increase in the biomass of the cutaneous microbiota in particular without reducing its biodiversity (ii) the formulation of the ingredients or compositions thus selected in a formula with cosmetic and/or dermatological use, capable of inducing an increase in the biomass of the cutaneous microbiota, in particular without reducing its biodiversity (iii) the packaging of the said formula.

The invention also relates to a topical care method, preferably cosmetic or dermatological, advantageously ecobiological, characterized in that it comprises (i) the implementation of a screening method defined previously or in the following description and the selection of at least one ingredient or a composition, in particular an ingredient or a cosmetic and/or dermatological composition, capable of inducing the increase in the biomass of the cutaneous microbiota described above, in particular without reducing its biodiversity (ii) the formulation of the ingredients or compositions thus selected in a composition for cosmetic and/or dermatological use, capable of inducing an increase in the biomass of the cutaneous microbiota, in particular without reducing its biodiversity (iii) the packaging of the said formula (iv) the topical application of said composition on a skin area or a surface of the selected appendages.

As mentioned before for the screening or selection method, the advantageously ecobiological topical care method can target specific populations, or be personalized and adapted to the microbiota specific to each individual. According to a particular embodiment, the population to be treated is characterized by an impoverished skin microbiota. According to an alternative embodiment, it is a personalized method capable of treating an individual characterized by an impoverished cutaneous microbiota.

Thus, according to another aspect of the invention, the invention relates to an advantageously cosmetic or dermatological topical care method, preferably ecobiological, capable of inducing an increase in the biomass of the cutaneous microbiota, in particular without impact or reduction on its biodiversity, characterized in that a composition selected through the selection or screening method mentioned above or described below is applied to the skin.

Within the meaning of the invention, by increase in biomass is meant a statistically significant increase. By way of example, it may be an increase of at least 50% in the biomass compared to a reference value, that is to say before application of the ingredient or of the composition of the invention.

In an alternative embodiment, the invention relates to a care method, preferably cosmetic or dermatological, for combating a skin attack (for example against a bactericidal and/or virucidal and/or fungicidal action), and/or maintaining and /or restore a healthy microbiota, characterized in that the method comprises the topical application of at least one topical composition selected in the screening or selection method described previously or in the following description, said composition being capable of inducing the increase of the biomass of the cutaneous microbiota, in particular without reducing its biodiversity, on the skin or on a keratin material.

According to yet another particular embodiment, the composition selected through the screening or selection method mentioned above or in the following description and capable of inducing an increase in the biomass of the cutaneous microbiota, in particular without reducing its biodiversity, is associated with the action of maintaining and/or restoring a healthy skin microbiota.

According to another particular embodiment, the composition to be used in particular in the implementation of the preferably ecobiological topical care method capable of inducing an increase in the biomass of the cutaneous microbiota, in particular without reducing its diversity, according to the The invention is an aqueous composition, comprising in particular at least 60%, better still at least 70%, or at least 80%, or at least 90%, or even at least 95% of water.

Thus, the invention relates to a preferably ecobiological topical care method capable of inducing an increase in the biomass of the cutaneous microbiota in particular without reducing its diversity, according to the invention, with an aqueous composition, advantageously comprising at least 60% of water, preferably 70%, 80%, 90%, or even at least 95% water.

A particular embodiment, the aqueous composition to be used in particular in the care method mentioned above or in the following description, comprises water which is characterized by a resistivity of between 12.5 and 12,500 Ohms.cm, in particular between 80 and 8,000 Ohms.cm, at room temperature

Preferably, the composition to be used in particular in the care method mentioned above or in the following description has an osmotic pressure of between 70 and 2500 mOsmol/l, and in particular between 100 and 800 mOsmol/l.

According to a particular embodiment of the invention, the composition to be used in particular in the implementation of the preferably ecobiological topical care method capable of inducing an increase in the biomass of the cutaneous microbiota, in particular without reducing its biodiversity , comprises at least one antioxidant and at least one inorganic or organic salt, each or at least one of these components or ingredients having, advantageously, been selected beforehand by the screening method described above or in the following description.

By "antioxidant" is meant a compound which exhibits antiradical activity, in particular on reactive oxygen species (ROS), carbonyls (RCS) or nitrogen (RNS).

According to a particular embodiment, the at least one antioxidant is chosen from the group comprising octadecyl di-t-butyl-4-hydroxyhydrocinnamate; pentaerythrityl tetra-di-t-butyl hydroxyhydrocinnamate; 2,6-Bis(1,1-dimethylethyl)-4-methylphenol; bis-ethylhexyl hydroxydimethoxy benzylmalonate; tert-butylhydroquinone; tetrabutyl ethylidenebisphenol; thioglycolic acid; thiotaurine; thioctic acid; dilauryl thiodipropionate; sodium erythorbate; sorbityl furfural; erythorbic acid; perillyl alcohol; pyridyloxide t-butylnitrone; ergothioneine; melatonin; acetyl cysteine; cysteine; lysine hydrochloride; carnosic acid; tyrosyl histidine HCl; histidine hydrochloride; pyridoxine serinate; superoxide dismutase; aminopropyl ascorbyl phosphate; ascorbic acid; ascorbyl dipalmitate; ascorbyl glucoside; ascorbyl linoleate; ascorbyl methylsilanol pectinate; ascorbyl palmitate; ascorbyl tetraisopalmitate; ascorbyl tocopheryl maleate; trisodium ascorbyl palmitate phosphate; disodium ascorbyl sulphate; calcium ascorbate; methylsilanol ascorbate; sodium ascorbate; sodium ascorbyl phosphate; sodium ascorbyl/cholesteryl phosphate; tetrahexyldecyl ascorbate; magnesium ascorbyl phosphate; tocopherol; tocopheryl acetate; tocopheryl linoleate; tocopheryl oleate; tocopheryl nicotinate; tocopheryl retinoate; sodium tocopheryl phosphate; dioleyltocopheryl methylsilanol; potassium ascorbyl tocopheryl 5 phosphate; dodecyl gallate; propyl gallate; octyl gallate; epigallocatechin gallate (EGCG); propyl gallate; ethylferulate; ethylhexyl ferulate; chitosan ascorbate; chitosan glycolate; apigenin; tiliroside; alpha-arbutin; arbutin; baicalin; quercetine; quercetin caprylate; isoquercetin; diethylhexyl syringylidenemalonate; dihydroxymethylchromone; dimethoxy di-p-cresol; dimethylmethoxy chromanol; ethylbisiminomethylguaiacol manganese chloride; hesperidin methyl chalcone; kojic acid; kojic dipalmitate; madecassoside; asiaticoside; magnolol (5,5'-diallyl-2,2'-dihydroxybiphenyl); nordihydroguaiaretic acid; phenylethyl resorcinol; resveratrol; troxerutin; glucosylrutin, rutin (4H-1-benzopyran-4-one); disodium rutinyl disulfate; tetrahydrobisdemethoxydiferuloylmethane; tetrahydrodemethoxydiferuloylmethane; tetrahydrodiferuloylmethane; tococysteamide; totarol; hydroxydecyl ubiquinone; ubiquinone; carotenoids; niacinamide or one of its derivatives; creatine; creatinine, carnosine, hypotaurine, inositol, trehalose, betaine, xylitol, mannitol, rhamnose. More particularly, the at least one antioxidant is chosen from the group comprising tocopherol, carnosine, hypotaurine, inositol, trehalose, betaine, xylitol, mannitol, rhamnose. According to an even more particular embodiment, the at least one antioxidant according to the invention is carnosine.

According to a particular embodiment, the invention relates to a composition comprising:

- at least one inorganic or organic salt chosen from the group comprising a salt of sodium, magnesium, calcium, potassium, phosphorus, copper, zinc, cobalt and mixtures thereof, in particular sodium citrate, sodium lactate, magnesium, copper or zinc sulphate, potassium or sodium phosphate, copper or zinc gluconate, sodium chloride, sodium hydrogen carbonate, sodium hydrogen phosphate, sodium citrate, magnesium gluconate,

- carnosine.

According to another particular embodiment, the invention relates to a composition comprising:

- at least one inorganic or organic salt chosen from the group comprising a salt of sodium, magnesium, calcium, potassium, phosphorus, copper, zinc, cobalt and mixtures thereof, in particular sodium citrate, sodium lactate, magnesium, copper or zinc sulphate, potassium or sodium phosphate, copper or zinc gluconate, sodium chloride, sodium hydrogen carbonate, sodium hydrogen phosphate, sodium citrate, magnesium gluconate,

- carnosine

for its use to maintain and/or restore a healthy skin microbiota.

According to another particular embodiment, the invention relates to a composition comprising:

- sodium citrate,

- carnosine.

According to another particular embodiment, the invention relates to a composition comprising:

- sodium citrate,

- carnosine

for its use to maintain and/or restore a healthy skin microbiota.

According to a particular embodiment, the composition according to the invention, or for its use according to the invention, also comprises at least one carbon source.

By “carbon source”, we mean a compound likely to be used by bacteria as a substrate for growth and multiplication, such as:

- partially or totally hydrolyzed proteins, from cereals (wheat, corn, barley, rye, oats), legumes (soya, fava beans, broad beans, peas), milk (caseins, albumins), yeast, as well as peptides and amino acids and their derivatives alone or as a mixture;

- oligosaccharides or polysaccharides, advantageously short-chain, such as fructooligosaccharides, hyaluronic acid, pullulan, alginate, cellulose, advantageously microcrystalline cellulose, carrageenan, chitosan, dextran and dextran sulphate, galactoarabinan, amylose, amylopectin, maltodextrins, pectins, and their respective hydrolysates, as well as sugars, oses, osides and their derivatives such as glucose, galactose, fructose, xylose, mannose, glucosamine, lactose, ribose , rhamnose and their phosphate and acetylated derivatives in particular.

Fructo oligosaccharides or FOS can be derived from plant materials such as beets, artichokes or corn. The raw material corresponding to the INCI designation fructooligosaccharides can be used in the context of the present invention, such as for example the product marketed by the company Shaanxi Bolin Biotechnology Co under the name FRUCTOOLIGOSACCHARIDES. Advantageously, the FOS represent from 0.001% to 20% by total weight of the composition, advantageously from 0.01% to 10%, even more advantageously from 0.1% to 5%.

According to a particular embodiment, the invention relates to a composition comprising:

- at least one inorganic or organic salt chosen from the group comprising a salt of sodium, magnesium, calcium, potassium, phosphorus, copper, zinc, cobalt and mixtures thereof, in particular sodium citrate, sodium lactate, magnesium, copper or zinc sulphate, potassium or sodium phosphate, copper or zinc gluconate, sodium chloride, sodium hydrogen carbonate, sodium hydrogen phosphate, sodium citrate, magnesium gluconate,

- carnosine

- at least one carbon source chosen from hyaluronic acid, and cellulose, advantageously microcrystalline cellulose.

According to a particular embodiment, the invention relates to a composition comprising:

- at least one inorganic or organic salt chosen from the group comprising a salt of sodium, magnesium, calcium, potassium, phosphorus, copper, zinc, cobalt and mixtures thereof, in particular sodium citrate, sodium lactate, magnesium, copper or zinc sulphate, potassium or sodium phosphate, copper or zinc gluconate, sodium chloride, sodium hydrogen carbonate, sodium hydrogen phosphate, sodium citrate, magnesium gluconate,

- carnosine;

- at least one carbon source chosen from hyaluronic acid, and cellulose, advantageously microcrystalline cellulose

for its use to maintain and/or restore a healthy skin microbiota.

According to another particular embodiment, the invention relates to a composition comprising:

- sodium citrate,

- carnosine.

- at least one carbon source chosen from hyaluronic acid, and cellulose, advantageously microcrystalline cellulose

According to another particular embodiment, the invention relates to a composition comprising:

- sodium citrate,

- carnosine

- at least one carbon source chosen from hyaluronic acid, and cellulose, advantageously microcrystalline cellulose

for its use to maintain and/or restore a healthy skin microbiota.

According to a particular embodiment, a pure or highly purified form of carnosine is used in the composition according to the invention. According to a variant, the carnosine is obtained by chemical synthesis. By way of example, mention may be made of the cosmetic material L-Carnosine corresponding to the INCI name carnosine and marketed by the company MERCK KgaA.

According to another embodiment of the invention, the carnosine represents between 0.0001% and 5% by total weight of the composition, advantageously between 0.001% and 2%.

In practice, in the composition according to the invention, a pure or highly purified form of hypotaurine can be used. Alternatively, hypotaurine is obtained by chemical synthesis. By way of example, mention may be made of the cosmetic raw material hypotaurine corresponding to the INCI name aminoethanesulfinic acid and marketed by the company DKSH.

According to a particular embodiment of the invention, the hypotaurine represents between 0.0001% and 5% by total weight of the composition, advantageously between 0.001% and 2%.

According to another particular embodiment, the tocopherol used is a mixture of purified natural tocopherols, in particular α-tocopherol, β-tocopherol, γ-tocopherol, and δ-tocopherol; this mixture can be used in particular in an oil chosen from vegetable, mineral, silicone, and preferably vegetable oils.

As a mixture of natural tocopherols that can be used according to the invention, mention may be made of the mixture sold by the company Nisshin OilliO Group, Ltd under the name Tocopherol 80 and corresponding to the INCI name “Tocopherol”.

According to another particular embodiment of the invention, the tocopherol represents between 0.0001% and 5% by total weight of the composition, advantageously between 0.001% and 2%.

Betaine is an osmolyte and an antioxidant found in particular in the plant kingdom. Betaine can be of vegetable origin or obtained by biotechnology. Advantageously, a highly purified form of betaine, with a purity of at least 95%, is used in the compositions according to the invention. As a source of betaine that can be used according to the invention, mention may be made of the raw material sold by the company Evonik Nutrition & Care GmbH under the name of Tego Natural Betaine (Evonik Nutrition & Care GmbH) and corresponding to the INCI name “Betaine”.

According to another particular embodiment of the invention, the betaine represents between 0.0001% and 5% by total weight of the composition, advantageously between 0.001% and 2%.

The xylitol can be powdered xylitol, for example of vegetable origin, in particular obtained from wood by hydrogenation of xyloses, having in particular a purity greater than 95%. By way of example, the company Orient Star LCC markets the product OriStar XLT corresponding to the INCI name “Xylitol” which can be implemented within the framework of the invention.

Suitably, the xylitol represents from 0.001% to 5% by total weight of the composition, advantageously from 0.1% to 2%.

The mannitol can for example be powdered mannitol, of plant origin (corn, potato, wheat), preferably having a purity greater than 98%. By way of example, the company American Biochemicals, Inc. markets the product D-mannitol corresponding to the INCI name mannitol which can be used in the context of the invention.

Suitably, the mannitol represents from 0.001% to 5% by total weight of the composition, advantageously from 0.1% to 2%.

Inositol is a cyclic compound derived from cyclohexane and carrying six secondary alcohol functions. In accordance with a particular embodiment of the invention, inositol of plant origin is used, synthesized naturally from glucose-6-phosphate (G6P), resulting from photosynthesis or from the hydrolysis of the carbon reserves of the plant. Preferably, inositol extracted from rice by techniques known to those skilled in the art is used. It is also possible to use inositol prepared by chemical or enzymatic synthesis. By way of example, the company Dupont Industrial Biosciences markets the product Genencare OSMS MI corresponding to the INCI name inositol which can be used in the context of the invention.

Suitably, the inositol represents from 0.001% to 5% by total weight of the composition, advantageously from 0.1% to 2%.

All cosmetically or pharmaceutically acceptable inorganic or organic salts are suitable for use in the composition according to the invention or for its use according to the invention. Advantageously, it is at least one inorganic or organic salt chosen from the group consisting of a salt of sodium, magnesium, calcium, potassium, phosphorus, copper, zinc, cobalt and their mixtures, in particular sodium citrate, sodium lactate, magnesium, copper or zinc sulfate, potassium or sodium phosphate, copper or zinc gluconate, and mixtures thereof, more particularly sodium citrate.

According to a particular embodiment of the invention, the at least one inorganic or organic salt is chosen from the group comprising calcium chloride; potassium chloride; potassium phosphate; magnesium sulfate; sodium chloride; sodium hydrogen carbonate; sodium hydrogen phosphate; sodium citrate; magnesium gluconate.

These mineral salts are available from several suppliers of cosmetic raw materials such as, for example, COOPER INDUSTRIE or BRENNTAG FRANCE.

In a particular embodiment, the composition according to the invention comprises at least one of the following mineral salts, in percentage by weight:
- From 0.000001% to 0.1% by total weight of the calcium chloride composition, in particular from 0.0001% to 0.1%;
- From 0.0001% to 0.1% by total weight of the potassium chloride composition, in particular from 0.001% to 0.1%;
- From 0.00001% to 0.1% by total weight of the potassium phosphate composition, in particular from 0.0001% to 0.1%;
- From 0.0001% to 0.1% by total weight of the magnesium sulphate composition, in particular from 0.001% to 0.1%;
- From 0.001 to 4% by total weight of the composition of sodium chloride, in particular from 0.1 to 4%;
- From 0.0001 to 0.1% by total weight of the sodium hydrogencarbonate composition;
- From 0.0001% to 0.1% by total weight of the sodium hydrogen phosphate composition;
- From 0.0001% to 1% by total weight of the citric acid composition, in particular from 0.001% to 1%;
- From 0.001% to 4% by total weight of the sodium citrate composition;
- From 0.001% to 4% by total weight of the magnesium gluconate composition.

According to another particular embodiment of the invention, the total weight of the mineral and/or organic salts is less than or equal to 1% by weight, in particular is less than or equal to 0.1% by weight of the total weight of the composition. In particular, the inorganic and organic salts are used at a concentration which makes it possible to be within the range of osmotic pressure sought.

According to a particular embodiment, the sodium citrate represents from 0.001% to 4% by total weight of the composition and the carnosine represents from 0.0001% to 5% by total weight of the composition, advantageously from 0.001% to 2%.

Several cosmetic sources of hyaluronic acid are known. According to a particular embodiment of the invention, hyaluronic acid has a molecular weight between between 20 and 50 kDa. By way of example, the raw materials PRIMALHYAL™ 50 or HyaCare 50 marketed respectively by the companies SOLIANCE and EVONIK and corresponding to hyaluronic acid with a molecular weight (Mw) of between 20 and 50 kDa (INCI: hyaluronic acid) can be used in the context of the present invention.

According to a particular embodiment, the hyaluronic acid represents from 0.001 to 4% by total weight of the composition, advantageously from 0.001% to 2%.

Any commercial source of microcrystalline cellulose suitable for topical and cosmetic application can be used in the compositions according to the invention. By way of example, we can cite Avicel PH-101 NF marketed by the company FMC Corporation and corresponding to the INCI designation microcrystalline cellulose.

According to a particular embodiment, the microcrystalline cellulose represents from 0.001 to 5% by total weight of the composition, advantageously from 0.001% to 3%.

According to another particular embodiment, the composition according to the invention or for its use according to the invention or to be used in particular in the care method mentioned above or in the following description is intended for populations characterized by an impoverished cutaneous microbiota.

The way in which the invention can be implemented and the resulting advantages will emerge better from the examples of embodiment which follow, given by way of indication and not limitation, in support of the appended figures.

In the examples the percentages are given by weight, the pressure is atmospheric pressure and the temperature is room temperature usually between about 18° C. and about 30° C., unless otherwise indicated.

and show the effect of pure petroleum jelly ingredients ( ) and silicon ( ) after 4 days of contact versus untreated area (ZNT), on the bacterial biomass of the skin microbiota, measured by the number of 16S gDNA copies in the volunteers taken into account in the analysis. Statistical significance is determined by a Wilcoxon test. (ns, not significant: p>0.05; significant*: p≤0.05; significant**: p≤0.01; significant***: p≤0.001).

and show the effect of formula 1 (fle 1; ) and formula 2 (fle 2 ; ) after 4 days of contact versus untreated area (ZNT), on the bacterial biomass of the skin microbiota, measured by the number of 16S gDNA copies in the volunteers taken into account in the analysis. Statistical significance is determined by a Wilcoxon test. (ns, not significant: p>0.05; significant*: p≤0.05; significant**: p≤0.01; significant***: p≤0.001).

and show the effect of pure Vaseline ingredients and silicon after 4 days of contact versus untreated area (ZNT), on the bacterial diversity of the skin microbiota, measured by the Shannon index, in the volunteers taken into account for the analysis. Statistical significance is determined by a paired Wilcoxon test.

(ns, not significant: p>0.05; significant*: p≤0.05; significant**: p≤0.01; significant***: p≤0.001).

and show the effect of formula 1 (fle 1; ) and formula 2 (fle 2 ; ) after 4 days of contact versus untreated area (ZNT), on the bacterial diversity of the skin microbiota, measured by the Shannon index, in the volunteers taken into account for the analysis. Statistical significance is determined by a paired Wilcoxon test.

(ns, not significant: p>0.05; significant*: p≤0.05; significant**: p≤0.01; significant***: p≤0.001).

Examples 1 of carrying out the Evaluation of the effect of two cosmetic compositions and two pure ingredients on the skin microbiota of healthy volunteers

The objective of the study is to evaluate the effect at 4 days of 2 cosmetic ingredients (vaseline and silicone) and 2 finished products (formula 1 and formula 2) on the skin microbiota by examining their impact on the biomass sampled as well as on the diversity of the skin microbiota.

The following products are tested simultaneously in the clinical study:

[Table 1] – products tested

[Table 2] - Formula 1 (fle 1) – O/W emulsion

[Table 3] - Formula 2 (fle 2) – aqueous lotion

A standardized application (1.5 μL/cm ² ) of each of the products to be tested is carried out on the back of 19 volunteers for 4 days at the rate of 1 application per day. On the day of the sample, the volunteers are instructed not to shower at least 12 hours before the samples.

The sample is taken using a swab (Sigma Trans Swab liquid Amies ref MW176S from Elitech) by carrying out 10 return trips in Z over a surface of 15 cm ² and by exerting slight pressure on the swab. The swab is then kept on ice and analyzed.

In order to measure the effect of the ingredient or the composition (finished product) on the biomass and on the diversity of the skin microbiota, the methodological approach is based on a process consisting of the following steps:
a) Extraction of gDNA (genomic DNA) from swabs
b) Analysis of the quantity of biomass by a qPCR approach
c) Analysis of microbial diversity based on:
- the preparation of amplicon libraries according to the Metabiote® solution, limiting amplification biases between samples and including a positive control (artificial bacterial community "ABC control", as well as a negative control (PCR background noise of the process library prep total).
- the sequencing of amplicon libraries on a single "run" (or single cycle) Illumina MiSeq "paired-end" in 2x300 base chemistry and the sorting of sequences by sample and the reassembly of "paired-end" reads allowing obtain full-length 16S rDNA (16S ribosomal DNA) reads.

In detail, step a) of extracting the genomic DNA from the swabs consists of an optimized bacteria recovery step (consisting of wiping the swab and recovering the bacterial pellets by centrifugation) which is carried out before the extraction step. The genomic DNA extraction was then carried out with the Nucleospin Microbial DNA kit (Macherey Nagel, France). The extracted gDNAs are not “assayable”, a quantification of the biomass and a quality control was therefore carried out by 16S qPCR.

In detail, step b) of biomass analysis and quality control by qPCR16S consists of carrying out a standard range under standardized experimental conditions. On all the dilution points of the range, the use of 4 points makes it possible to obtain an efficiency close to 94%, knowing that it is commonly considered that the efficiency of qPCR is optimal between 90 and 110%. It is considered that this 16S copy number zone of the artificial community had the characteristics necessary to be used in the standard quantification range. Since the extracted DNA samples cannot be quantified because they are too weakly concentrated, the qPCRs are carried out, in triplicate, from a fixed volume of 4.38 μL corresponding to the maximum volume that it is possible to integrate into the qPCR reaction volume.

The negative control named "Tneg" is the negative qPCR control carried out with sterile water.

From the standard curves and their equation on the efficiency zone, it is possible to calculate the number of bacterial 16S rDNA copies contained within each sample.

The quantitative amplifications (qPCR) are carried out with 16S universal primers [SEQ ID. 1 8F (5′-AGAGTTTGATCCTGGCTCAG-3′) and SEQ ID. 2 1391R (5′-GACGGGCGGTGWGTRCA-3′)] (Kong et al. 2012b). The amplification conditions and equipment were as described in SanMiguel et al. 2018.

In detail, and concerning step c) of analysis of the diversity of the cutaneous microbiome, it should be noted that for this type of rare biomass samples, a maximum quantity of material is used for the creation of specific Illumina MiSeq libraries . The amplification step is carried out with the primers for the V1-V3 regions corresponding to SEQ. ID 5 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and SEQ. ID 6534R (5-ATTACCGCGGCTGCTGTGG-3) according to San Miguel et al. 2018, with the following modifications. Two additional controls are integrated during the preparation of the Metabiote® libraries. A positive internal control, named “ABCv2” for “Artificial Bacterial Community”. It is an artificial community made from a mixture of gDNA from different bacterial strains. The second control is a negative control called “BF”, which is also integrated into the preparation process and makes it possible to evaluate the background noise inherent in the reagents used after the extraction step. The quantitative control of the final libraries is carried out by a fluorometric method. Then, the ABC control community whose composition is known (17 bacteria including 2 archaebacteria) is introduced into the sequencing; it serves as a positive control during our MetaBiote® process, but also as a positive control when performing the taxonomic affiliation of the sequences. The negative library creation control (BF.V1V3) is used to identify what profile can be obtained if no initial biomass is introduced and the MetaBiote® preparation process is pushed. This control makes it possible to distinguish the samples to be excluded from the comparisons.

The alpha diversity indices of the samples as well as the measures of rarefaction are defined, thus making it possible to observe the taxonomic diversity (in number of OTUs) of the samples.

The bioinformatics analysis carried out made it possible to eliminate the samples which presented poor quality sequencing.

Results :
The results are presented in figures 1 to 4. The analysis on the biomass, carried out by means of quantitative amplifications with the universal primers 16S, shows that petroleum jelly and silicone induce a slight reduction in the biomass after 4 days of daily application (respectively, an average reduction of 4.5% and 2%; figures 1 and 2).

These two ingredients are therefore not suitable, at least in pure form, for the implementation of a composition for its use according to the invention. They are therefore discarded.

On the other hand, formula 1 (fle 1) induces an average increase in biomass of 134% after 4 days of application ( ) in the volunteers taken into account for the analysis, while formula 2 (fle 2) induces an average increase of 150% ( ). The difference between the untreated zone (ZNT) and the treated zone (ZT) is significant in both cases (Wilcoxon test). The formulas, selected using the screening method described in the present application, are therefore suitable for the implementation of a composition for its use according to the invention, namely to induce an increase in the biomass of the cutaneous microbiota.

Amplification with specific 16S primers followed by sequencing of the amplicons obtained shows that petroleum jelly and silicone have, in practice, no effect on the diversity of the skin microbiota (figures 5 and 6; p>0.05 in both case therefore difference not significant; see tables 4 and 5). This observation is consistent with the results of the biomass analysis and indicates that these ingredients are substantially inert with respect to the skin microbiota.

[Table 4] – Shannon index and OTUs and percentages relating to treatment with petroleum jelly

[Table 5] – Shannon index and OTUs and percentages relating to treatment with silicone

The study of formulas 1 and 2 (fle 1 and fle 2) shows that in both cases the variations observed in the Shannon index by comparing the untreated zone to the zones treated with the compositions under study is non- significant (p = 0.054 and p = 0.76; see also tables 6 and 7 for the variation of the Shannon index and the percentage OTUs).

[Table 6] - Shannon index and OTUs and percentages relating to treatment with fle 1

[Table 7] - Shannon index and OTUs and percentages relating to treatment with fle 2

In both cases, it can be observed that:
- the Shannon index does not vary significantly,
- the number of OTUs does not vary significantly.

The negligible impact of the fle 1 and fle 2 formulas on the balance of the cutaneous microbiota is also confirmed by the detailed analysis of the modification of the relative abundance of 15 more represented bacterial genera between ZNT and ZT - fle 1 and - fle 2 ( tables 8 and 9).

[Table 8] - Comparison of the relative abundance of 15 more represented bacterial genera between ZNT and ZT with fle 1

[Table 9] - Comparison of the relative abundance of 15 more represented bacterial genera between ZNT and ZT with fle 2

It is possible to describe the results of the tests carried out in example 1, in the form of a summary table (see table 10):

[Table 10] – Summary presentation of results

Legend: no significant variation (=),
significant increase (+), significant decrease (-)

It can therefore be concluded that the increase in the biomass of the cutaneous microbiota observed in response to the application of the two formulas fle 1 and fle 2, which contain an organic salt (sodium citrate), an antioxidant (carnosine) and two sources of carbon according to the invention (hyaluronic acid and microcrystalline cellulose) was not accompanied by a reduction in the diversity of the bacterial genera, in particular the most represented. The two formulas, selected using the screening method described above, are suitable for use, particularly in the context of a treatment aimed at increasing the overall biomass of the cutaneous microbiota, while respecting the proportions between its constituents.

Example 2 – Study of ingredients and compositions
The techniques described in Example 1 are reproduced identically. The ingredients listed in Table 11 are placed at a percentage indicated in grams per 100 grams (qsp 100 g of water), the compositions are applied neat. Their pH is adjusted between 5 and 9, and their osmotic pressure is adjusted to obtain a value of between 100 and 800 mOsm.l ^-1 .

The products are applied to the back of series of 10 volunteers. Applications are daily for 4 days and samples of biological material from the skin are taken before the first application and after the ^4th day.
The biomass analysis is carried out by qPCR (16S universal primers) and the analysis of the microbial diversity by amplification with specific 16S primers, then analysis of the results by measuring the Shannon index.
The summary results are grouped in the following table 11:

We note “=” the absence of significant variation, “+” a significant increase and “-” a significant reduction.

[Table 11] – Summary

Legend: no significant variation (=),
significant increase (+), significant decrease (-)

Example 3: Study on a skin area characterized by a reduced skin microbiota biomass after the action of an ethanolic solution

Composition tested

The study is carried out on the forehead of 5 volunteers. A biomass sample is taken at T0 on the forehead of the volunteers. Then, disinfection of the entire forehead with a compress soaked in 5 mL of 70° alcohol (modified Cooper alcohol, batch 013199) is carried out by rubbing for 30 seconds and leaving for an additional 3 minutes. After 5 min of drying, 2 samples are taken: T5 min on the left (G) and T5 min on the right (D). Then a compress soaked in 5 mL of the above composition is placed on the right half of the forehead for 3 min. After 2 hours, a 2 sec mist of the above composition is applied to the right half of the forehead. After 2 additional hours, 2 samples are taken: T4h on the alcohol side and T4h on the composition to be tested side.

The samples are taken using swabs (Sigma Trans Swab liquid Amies ref MW176S from Elitech) at times T0, T5min, T2h and T4h, by performing 10 round trips in Z over an area of 3.75 cm ² and exerting light pressure on the swab in a standardized manner. The swab is then kept on ice.

Effect on biomass:
This suspension is then diluted 1/2, 1/10 and 1/50 in nutrient broth. A seeding of 1 mL of each dilution is then carried out on TSA agar in a Petri dish and incubated at 30-35° C. for 48 hours. After 48 hours of incubation, the colonies (CFU) are counted in their entirety on the dishes. The bacterial biomass is obtained by multiplying the number of CFU counted by its dilution factor and is expressed in CFU/mL.
Disinfection with 70° alcohol induced a sharp decrease in the amount of biomass of cultivable bacteria by 99.8%, 5 min after application of the alcohol. Application of the product to be tested after 4 hours of contact on the depleted microbiota increases this biomass by 160% compared to the 70° alcohol control alone.

Effect on diversity :
The tests carried out according to the protocol described in example 1 show that the diversity of the cutaneous microbiome is not reduced after application of the product evaluated. We note “=” the absence of significant variation, “+” a significant increase and “-” a significant reduction.

The results are collated in Table 13 below.

[Table 13] – Summary

Legend: no significant variation (=),
significant increase (+), significant decrease (-)

Example 4: Study on a skin area characterized by a reduced skin microbiota biomass after the action of a hygiene product based on Marseille soap

Composition tested:

The study is carried out on the forehead of 5 volunteers. A biomass sample is taken at T0 on the forehead of the volunteers. Then, washing the entire forehead with a compress soaked in 5 mL of a 2% by weight Marseille soap solution (Marseille soap, Persavon) is carried out by rubbing for 30 seconds. Two rinses are then performed with compresses soaked in water. After 5 min of drying, a sample is taken: T5 min. Then a compress soaked in 5 mL of the above composition is placed on the right half of the forehead for 3 min. After 2 hours, a 2 sec mist of the above composition is applied to the right half of the forehead. After 2 additional hours, 2 samples are taken: T4h on the Marseille soap side and T4h on the composition to be tested side.

The samples are taken using swabs (Sigma Trans Swab liquid Amies ref MW176S from Elitech) at times T0, T5min, T2h and T4h, by performing 10 round trips in Z over an area of 3.75 cm ² and exerting light pressure on the swab in a standardized manner. The swab is then kept on ice.

Effect on biomass:
This suspension is then diluted 1/2, 1/10 and 1/50 in nutrient broth. A seeding of 1 mL of each dilution is then carried out on TSA agar in a Petri dish and incubated at 30-35° C. for 48 hours. After 48 hours of incubation, the colonies (CFU) are counted in their entirety on the dishes. The bacterial biomass is obtained by multiplying the number of CFU counted by its dilution factor and is expressed in CFU/ml.

Cleaning with the Marseille soap solution induced a strong decrease in the amount of biomass of cultivable bacteria by 78%, 5 min after application of the Marseille soap solution. Application of the product to be tested after 4 hours of contact on the depleted microbiota increases this biomass by 3 times compared to the Marseille soap control.

Effect on diversity :
The tests carried out according to the protocol described in example 1 show that the diversity of the cutaneous microbiome is not reduced after application of the product evaluated. We note “=” the absence of significant variation, “+” a significant increase and “-” a significant reduction.

The results are collated in Table 15 below.

[Table 15] – Summary

Legend: no significant variation (=),
significant increase (+), significant decrease (-)

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Claims (20)

Method for selecting an ingredient or composition, preferably ecobiological, advantageously applicable topically, in particular cosmetic or dermatological, useful in maintaining and/or restoring a healthy skin microbiota, comprising the steps consisting in:
a/ putting an ingredient or composition to be tested in contact with bacteria from a reference microbiota,
b/ analyze the quantity of biomass and, advantageously, the biodiversity of the microbiota after contacting, and
c/ selecting, as an ingredient or composition useful in maintaining and/or restoring a healthy skin microbiota, an ingredient or a composition capable of increasing the biomass of the skin microbiota, advantageously without reducing its biodiversity, compared the quantification of the biomass and, where appropriate, the biodiversity of the reference microbiota. Selection method according to claim 1, comprising the steps of:
a/ putting an ingredient or composition to be tested in contact with bacteria from a reference microbiota,
b/ analyze the quantity of biomass and the biodiversity of the microbiota after contacting, and
c/ select as an ingredient or composition useful in maintaining and/or restoring a healthy skin microbiota, an ingredient or a composition capable of increasing the biomass of the skin microbiota without reducing its biodiversity, compared to the quantification of the biomass and biodiversity of the reference microbiota. Selection method according to Claim 1 or 2, characterized in that the said reference microbiota is that of a healthy skin area. Selection method according to one of the preceding claims, characterized in that the said reference microbiota is depleted, advantageously following a skin attack, more advantageously following the action of bactericides or virucides or soaps or hygiene products or aggressive treatments for the microbiota. Selection method according to one of the preceding claims, characterized in that step b is carried out from 1 hour to 31 days, preferably 4 days, after step a. Selection method according to one of the preceding claims, characterized in that it comprises the following in vitro steps:
i) before the application of said ingredient or composition, optionally formulated in the form of a topical composition, on at least one skin area to be studied, from a first sample taken from said skin area, analyzes of the quantity of biomass of the microbiota cutaneous, as well as its biodiversity, thus obtaining a first so-called reference measurement of biomass quantification and a first so-called reference measurement of the biodiversity of the bacteria of said reference microbiota; and
ii) after at least one application of said ingredient or of said composition, optionally formulated in the form of a topical composition, on said skin area, and after a predetermined time of action of the ingredient or of the composition, from a second sample taken from said cutaneous area, the analyzes of the quantity of biomass of the cutaneous microbiota, as well as of its biodiversity, thus obtaining a second measurement called biomass quantification activity and a second measurement called biodiversity activity ; and
iii) comparing the second activity measurements with the first reference measurements, and selecting the ingredient or the composition, optionally formulated in the form of a topical composition, increasing the biomass of the cutaneous microbiota, in particular without reducing its biodiversity. Selection method according to one of the preceding claims, characterized in that at least one method is used making it possible to quantify the biomass of the microbiota chosen from the following quantitative or semi-quantitative methods:
- Quantitative PCR (or qPCR),
- flow cytometry,
- enumeration methods on agar. Selection method according to one of the preceding claims, characterized in that at least one method is used which makes it possible to measure the biodiversity of the microbiota chosen from the following methods:
- analysis of part of the hypervariable gene of the 16S ribosome of bacteria after their targeted amplification,
- metagenomic shot gun technique,
- metatranscriptomic shotgun sequencing technique,
followed by a method for calculating the microbial diversity from the data of the previous method used, such as the calculation:
- the Shannon index,
- Simpson's index,
- the Inverse-Simpson index,
- the number of species or OTUs,
- Evenness or the Pielou index,
- the Hurlbert index,
- the Hill index. Selection method according to one of the preceding claims, characterized in that it comprises at least two stages of amplification of standardized quantities of genetic material:
- a quantitative amplification carried out using universal 16S primers in order to measure the biomass,
- an amplification carried out using specific 16S primers in order to measure the biodiversity of the microbiota,
- measurement of the Shannon index. Selection method according to one of the preceding claims, characterized in that the said ingredient or the said composition to be tested is chosen from the group comprising:
a) at least one carbon source which is capable of being used by bacteria as a growth and multiplication substrate, and preferably chosen from:
- partially or totally hydrolyzed proteins, from cereals (wheat, corn, barley, rye, oats), legumes (soya, fava beans, broad beans, peas), milk (caseins, albumins), yeast, as well as peptides and amino acids alone or as a mixture;
- short-chain oligosaccharides or polysaccharides, such as fructo-oligosaccharides or FOS; hyaluronic acids, pullulan, chitosan, dextran sulphate, galactoarabinan, carrageenan, alginate, cellulose, advantageously microcrystalline cellulose, amylose, amylopectin and their respective hydrolysates;
b) at least one antioxidant which has an activity on reactive oxygen species (ROS), advantageously free radicals, in particular chosen from the group consisting of octadecyl di-t-butyl-4-hydroxyhydrocinnamate; pentaerythrityl tetra-di-t-butyl hydroxyhydrocinnamate; 2,6-Bis(1,1-dimethylethyl)-4-methylphenol; bis-ethylhexyl hydroxydimethoxy benzylmalonate; tert-butylhydroquinone; tetrabutyl ethylidenebisphenol; thioglycolic acid; thiotaurine; thioctic acid; dilauryl thiodipropionate; sodium erythorbate; sorbityl furfural; erythorbic acid; perillyl alcohol; pyridyloxide t-butylnitrone; ergothioneine; melatonin; acetyl cysteine; cysteine; lysine hydrochloride; carnosic acid; tyrosyl histidine HCl; histidine hydrochloride; pyridoxine serinate; superoxide dismutase; aminopropyl ascorbyl phosphate; ascorbic acid; ascorbyl dipalmitate; ascorbyl glucoside; ascorbyl linoleate; ascorbyl methylsilanol pectinate; ascorbyl palmitate; ascorbyl tetraisopalmitate; ascorbyl tocopheryl maleate; trisodium ascorbyl palmitate phosphate; disodium ascorbyl sulphate; calcium ascorbate; methylsilanol ascorbate; sodium ascorbate; sodium ascorbyl phosphate; sodium ascorbyl/cholesteryl phosphate; tetrahexyldecyl ascorbate; magnesium ascorbyl phosphate; tocopherol; tocopheryl acetate; tocopheryl linoleate; tocopheryl oleate; tocopheryl nicotinate; tocopheryl retinoate; sodium tocopheryl phosphate; dioleyltocopheryl methylsilanol; potassium ascorbyl tocopheryl 5 phosphate; dodecyl gallate; propyl gallate; octyl gallate; epigallocatechin gallate (EGCG); propyl gallate; ethylferulate; ethylhexyl ferulate; chitosan ascorbate; chitosan glycolate; apigenin; tiliroside; alpha-arbutin; arbutin; baicalin; quercetine; quercetin caprylate; isoquercetin; diethylhexyl syringylidenemalonate; dihydroxymethylchromone; dimethoxy di-p-cresol; dimethylmethoxy chromanol; ethylbisiminomethylguaiacol manganese chloride; hesperidin methyl chalcone; kojic acid; kojic dipalmitate; madecassoside; asiaticoside; magnolol (5,5'-diallyl-2,2'-dihydroxybiphenyl); nordihydroguaiaretic acid; phenylethyl resorcinol; resveratrol; troxerutin; glucosylrutin, rutin (4H-1-benzopyran-4-one); disodium rutinyl disulfate; tetrahydrobisdemethoxydiferuloylmethane; tetrahydrodemethoxydiferuloylmethane; tetrahydrodiferuloylmethane; tococysteamide; totarol; hydroxydecyl ubiquinone; ubiquinone; carotenoids; niacinamide or one of its derivatives; creatine; creatinine, carnosine, hypotaurine, inositol, trehalose, betaine, xylitol, mannitol, and rhamnose;
c) at least one topically acceptable inorganic or organic salt, advantageously chosen from the group comprising a salt of sodium, magnesium, calcium, potassium, phosphorus, copper, zinc, cobalt and their mixtures, in particular the sodium citrate, sodium lactate, magnesium, copper or zinc sulphate, potassium or sodium phosphate, copper or zinc gluconate. Composition, advantageously ecobiological, in particular cosmetic and/or dermatological, comprising at least one ingredient or composition, selected by the selection method according to one of the preceding claims, for its use to maintain and/or restore a healthy skin microbiota. Composition for its use according to claim 11, characterized in that it comprises at least one carbon source, one antioxidant and one inorganic or organic salt, advantageously said carbon source, said antioxidant and/or said salt being selected by selection method according to one of claims 1 to 10. Composition for its use according to one of Claims 11 or 12, characterized in that it is an aqueous composition, comprising at least 60% by weight of the total composition of water, in particular at least 60% , preferably at least 70%, or at least 80% or at least 90% or at least 95%. Composition for its use according to one of Claims 11 to 13, characterized in that it comprises an aqueous phase which is characterized by a resistivity, at room temperature, of between 12.5 and 12,500 Ohms.cm, in particular between 80 and 8000 Ohms.cm. Composition for its use, according to one of Claims 11 to 14, characterized in that the aqueous phase has an osmotic pressure of between 70 and 2500 mOsmol/l and in particular between 100 and 800 mOsmol/l as well as a pH of between between 5.0 and 9.0 and in particular between 6.0 and 8.0. Composition for use according to one of Claims 11 to 15, characterized in that the composition has a pH of between 5.0 and 9.0 and in particular between 6.0 and 8.0. Composition for its use according to one of Claims 12 to 16, characterized in that:
a) the at least one carbon source is capable of being used by bacteria as a growth and multiplication substrate, and, preferably, is chosen from:
- partially or totally hydrolyzed proteins, from cereals (wheat, corn, barley, rye, oats), legumes (soya, fava beans, broad beans, peas), milk (caseins, albumins), yeast, as well as peptides and amino acids alone or as a mixture;
- short-chain oligosaccharides or polysaccharides, such as fructo-oligosaccharides or FOS; hyaluronic acids, pullulan, chitosan, dextran sulphate, galactoarabinan, carrageenan, alginate, cellulose, advantageously microcrystalline cellulose, amylose, amylopectin and their respective hydrolysates;
b) The at least one antioxidant exhibits activity on reactive oxygen species (ROS), advantageously free radicals, and, in particular, is chosen from the group consisting of octadecyl di-t-butyl-4-hydroxyhydrocinnamate ; pentaerythrityl tetra-di-t-butyl hydroxyhydrocinnamate; 2,6-Bis(1,1-dimethylethyl)-4-methylphenol; bis-ethylhexyl hydroxydimethoxy benzylmalonate; tert-butylhydroquinone; tetrabutyl ethylidenebisphenol; thioglycolic acid; thiotaurine; thioctic acid; dilauryl thiodipropionate; sodium erythorbate; sorbityl furfural; erythorbic acid; perillyl alcohol; pyridyloxide t-butylnitrone; ergothioneine; melatonin; acetyl cysteine; cysteine; lysine hydrochloride; carnosic acid; tyrosyl histidine HCl; histidine hydrochloride; pyridoxine serinate; superoxide dismutase; aminopropyl ascorbyl phosphate; ascorbic acid; ascorbyl dipalmitate; ascorbyl glucoside; ascorbyl linoleate; ascorbyl methylsilanol pectinate; ascorbyl palmitate; ascorbyl tetraisopalmitate; ascorbyl tocopheryl maleate; trisodium ascorbyl palmitate phosphate; disodium ascorbyl sulphate; calcium ascorbate; methylsilanol ascorbate; sodium ascorbate; sodium ascorbyl phosphate; sodium ascorbyl/cholesteryl phosphate; tetrahexyldecyl ascorbate; magnesium ascorbyl phosphate; tocopherol; tocopheryl acetate; tocopheryl linoleate; tocopheryl oleate; tocopheryl nicotinate; tocopheryl retinoate; sodium tocopheryl phosphate; dioleyltocopheryl methylsilanol; potassium ascorbyl tocopheryl 5 phosphate; dodecyl gallate; propyl gallate; octyl gallate; epigallocatechin gallate (EGCG); propyl gallate; ethylferulate; ethylhexyl ferulate; chitosan ascorbate; chitosan glycolate; apigenin; tiliroside; alpha-arbutin; arbutin; baicalin; quercetine; quercetin caprylate; isoquercetin; diethylhexyl syringylidenemalonate; dihydroxymethylchromone; dimethoxy di-p-cresol; dimethylmethoxy chromanol; ethylbisiminomethylguaiacol manganese chloride; hesperidin methyl chalcone; kojic acid; kojic dipalmitate; madecassoside; asiaticoside; magnolol (5,5'-diallyl-2,2'-dihydroxybiphenyl); nordihydroguaiaretic acid; phenylethyl resorcinol; resveratrol; troxerutin; glucosylrutin, rutin (4H-1-benzopyran-4-one); disodium rutinyl disulfate; tetrahydrobisdemethoxydiferuloylmethane; tetrahydrodemethoxydiferuloylmethane; tetrahydrodiferuloylmethane; tococysteamide; totarol; hydroxydecyl ubiquinone; ubiquinone; carotenoids; niacinamide or one of its derivatives; creatine; creatinine, carnosine, hypotaurine, inositol, trehalose, betaine, xylitol, mannitol, and rhamnose;
c) The at least one inorganic or organic salt is topically acceptable and, advantageously, is chosen from the group comprising a salt of sodium, magnesium, calcium, potassium, phosphorus, copper, zinc, cobalt and their mixtures, in particular sodium citrate, sodium lactate, magnesium, copper or zinc sulphate, potassium or sodium phosphate, copper or zinc gluconate. Composition for its use according to one of Claims 12 to 17, characterized in that the said at least one antioxidant is chosen from the group comprising tocopherol, carnosine, hypotaurine, inositol, trehalose, betaine, xylitol, mannitol, rhamnose. Composition for its use according to one of Claims 12 to 18, characterized in that it comprises sodium citrate and carnosine. Composition for its use according to one of Claims 12 to 19, characterized in that the composition is applied to a healthy skin area, before or following a skin attack, advantageously the action of bactericides or virucides or soaps or hygiene or care products that are aggressive towards the microbiota.