Method for evaluating the ability of a product to preserve the skin microbiota

Abstract

The invention relates to a method for determining the suitability of a product for use in a cosmetic or pharmaceutical formulation for topical use, the product being a chemical or biochemical substance of any type (S) or a composition of chemical or biochemical substances of any type (C), the method comprising a step of suspending, in a single culture medium suitable for bacterial growth, the following species: C. xerosis, M. luteus, S. epidermidis, C. acnes and S. mitis, in order to form, in the co-culture medium, a co-culture of the consortium of bacterial strains of the five aforementioned species, in proportions close to those found on the skin; followed by a step of bringing the co-culture, in the co-culture medium, into contact with the product for a period of between seven and nine hours, and a step of calculating a score (Sc): Sc = Σ(RL species X), wherein RL species X represents the following logarithmic reduction for a given bacterial species, and a step of determining the suitability of the product for use in a cosmetic or pharmaceutical formulation for topical use, wherein the determination step consists in selecting the substance (S) or the composition (C) when the score (Sc) is less than or equal to 5 and greater than or equal to -5 and rejecting the substance (S) or the composition (C) when the score (Sc) is greater than 5 or less than -5 and the individual RL species X for each species are between -3 and 3.

Description

Process for assessing a product's ability to preserve the skin microbiota Field of invention

[0001] The invention relates to the field of cosmetics. Its subject more particularly is a method for determining the ability of products to modify the microbiota of human skin (cutaneous microbiota), said products being chemical or biochemical substances or compositions of chemical or biochemical substances. It also relates to a method for selecting such products which can be integrated into cosmetic or pharmaceutical formulations intended for the hygiene and / or care of human skin, administered by topical application to said human skin.

[0002] Human skin is a complex organ that covers the entire body. It is composed of three superimposed layers: the epidermis, which is the superficial layer in contact with the exterior of the human body; the dermis, which is the thickest layer, contains a significant proportion of collagen and elastin; it contains blood and lymphatic vessels, hair follicles, sweat glands, sebaceous glands and nerve endings that allow us to feel touch; the hypodermis, which is the deepest layer. It is a loose connective tissue, where blood vessels pass and which may contain adipose tissue. Its function is to protect against shocks and also helps maintain an adequate internal temperature.

[0003] The epidermis is a multi-layered epithelium that is constantly renewing itself. It is mainly composed of keratinocytes (85% to 95%), but also includes melanocytes (2% to 15%), Langerhans cells (1% to 8%), nerve fibers (less than 1%), and Merkel cells (less than 1%). Keratinocytes proliferate from the basal layer and then gradually differentiate to give rise to the different epidermal layers by migrating from the depth of the epidermis to its surface, where desquamation occurs.

[0004] The skin is naturally covered with microorganisms that together make up the skin microbiota. Bacteria represent at least 75% of these microorganisms, while yeasts and viruses represent at most the remaining 25%. The population density of these bacteria present on the skin is estimated at around 10 4 at 10 6 colony forming units (CFU) per square centimeter.

[0005] Moist areas of the skin, such as the armpits or inguinal folds, are colonized by bacteria of the genus Staphylococcus; sebaceous areas, such as the forehead, the sides of the nose or the upper back are colonized to a greater extent by bacteria of the genus Cutibacterium, while dry areas such as the forearms or legs are colonized by a greater variety of bacteria such as those of the genera Staphylococcus, Cutibacterium, Micrococcus, Corynebacterium, Enhydrobacter and Streptococcus.

[0006] Cosmetic or pharmaceutical formulations for topical use for hygiene or therapeutic purposes often contain antimicrobial substances, such as preservatives, which aim to control the unwanted proliferation of pathogenic microorganisms within them. These biocidal active ingredients can potentially reduce the microbial load present at the site of application on the skin. This is the case for hydroalcoholic gels, toothpastes, or soaps. It is therefore useful to be able to assess the safety of the components of cosmetic formulations for topical use with respect to the skin microbiota. State of the art

[0007] There are now a number of commercial processes to certify that a chemical substance or chemical composition does not damage the skin microbiota.

[0008] The MyMicrobiome laboratory process called: “MyMicrobiome Standard 18.10-12 Face and Body Microbiome” consists of four steps: a. A quality test during which the skin care is tested for contamination by aerobic mesophilic microorganisms (bacteria, yeasts, fungi). The acceptable contamination limit is less than or equal to 10 3CFU / g or ml.b. A balance test that verifies that the skin care product does not disturb the balance between the most common skin microorganism, Staphylococcus epidermidis, and the most common pathogen, Staphylococcus aureus. To do this, a coculture of the two microorganisms is mixed with the chemical substance or composition and then incubated for four hours. The ratio between the quantities of the two microbes (in CFU) is compared to the same ratio in the untreated control group. The chemical substance or composition must not have supported the growth of Staphylococcus aureus.c. A diversity test: A coculture of typical facial and body microbes, for dry, moist, or oily skin, is grown and then mixed with the chemical substance or composition to be tested. Changes in the diversity of the coculture are compared to those of the untreated control group. The diversity of the coculture must be preserved.d.A vitality test: A cosmetic formulation suitable for facial or body skin care must not only preserve diversity, but also not impair the growth of microorganisms. To simulate skin contact, the vitality test consists of two subtests. In the first subtest, first batches of each key microorganism are directly exposed to the test formulation; in the second subtest, the test formulation is placed on a layer of agar beneath which are second batches of each key microorganism, thereby simulating the potential penetration of the formulation into deeper layers of the skin. The microbial growth of both batches is compared to the untreated control group. It must not have been significantly inhibited.

[0009] The Labskin laboratory has an in vitro assay in which a coculture of Staphylococcus epidermidis, Cutibacterium acnes, Corynebacterium striatum is placed in contact for twenty-four hours with the chemical substance, chemical composition or formulation to be tested. The results are compared to their untreated references.

[0010] The StratiCELL laboratory's process involves inoculating reconstructed human epidermis (RHE) with microbial strains of Staphylococcus aureus or Staphylococcus epidermidis or Cutibacterium acnes or Malassezia furfur and then monitoring both bacterial growth and the epidermal response to infection. This dual approach makes it possible to objectify the influence of dermocosmetic active substances or compounds such as compositions or chemical substances with anti-acne or anti-inflammatory properties. It also makes it possible to assess the safety of these compositions or chemical substances in dermocosmetic formulations.

[0011] International application published under number WO 2022 / 117422 A1 discloses a method for screening the biological activity of a compound using a synthetic mixed culture, resembling a skin microbiota, by comparing the level of diversity and / or the diversity profile of a mixed culture of synthetic microorganisms and the comparison of the level of diversity and / or the diversity profile of a synthetic mixed culture not exposed to an ingredient (control microbiota). The level of diversity and / or the diversity profile is established by extracting DNA from the cultures, amplifying the gene encoding 16S RNA and analyzing the amplicons to determine the proportion of each species.

[0012] The international application published under number 2023 / 047072 A1 discloses a method for testing and then selecting a substance to be used as an ingredient in a topical formulation that respects the skin microbiota, comprising the selection of a reference microbiota comprising from 70% to 90% of the bacterial genera present on the skin and in particular from 0% to 10% of Corynobacterium, 35% to 85% of Cutibacterium and from 5% to 35% of Staphylococcus.

[0013] Korean patent application published under number KR 2022 0163301 A discloses a method for evaluating the properties of skin-resident microorganisms and a method for selecting prebiotics having the ability to improve the skin, comprising a step of separating skin-resident microorganisms and skin cells, using a semi-permeable membrane.

[0014] In view of this state of the art, there is a need to develop a method for selecting chemical or biochemical substances or compositions capable of maintaining the human skin microbiota stable. This method must be an in vitro method, using wild strains and not collection strains, cultivated together in conditions approaching those of the skin surface; it must be reproducible, sensitive, discriminating, effective and suitable for implementation in the monitoring and development of topical hygiene, cosmetic or pharmaceutical formulations.

[0015] According to a first aspect, the invention relates to a method for determining the suitability of a product for use in a topical cosmetic or pharmaceutical formulation, said product being a chemical or biochemical substance of any nature (S) or a composition of chemical or biochemical substances of any nature (C), characterized in that it comprises the following steps: a. A step a) of providing bacterial strains of the following five species: i) Corynebacterium xerosis, ii) Micrococcus luteus, iii) Staphylococcus epidermidis, iv) Cutibacterium acnes and v) Streptococcus mitis, said provision being carried out in five isolated groups, each consisting of several bacterial strains of one of the five aforementioned species; b.A step b) during which a sample of each of said five isolated groups, each consisting of several bacterial strains of one of the five aforementioned species made available in step a), is taken and then cultured in a suitable specific medium, for twenty-four hours to one hundred and two hours, in particular for twenty-four hours to ninety-six hours, more particularly for twenty-four hours to seventy-two hours, to produce cultures of bacterial strains of each of said aforementioned species; c. A step c) of suspending in a single culture medium suitable for bacterial growth the coculture of said bacterial strains cultured in step b), to form a coculture, in said culture medium, of the consortium of bacterial strains of said five aforementioned species, in proportions close to those found on the skin; d.A step d) of bringing said coculture into contact in said coculture medium obtained in step c), with said product, for a period of between seven and nine hours;e.A step e) of counting the bacteria-forming units of the bacteria remaining at the end of step d);f.A step f) of calculating a score (Sc):[Math 1]Sc = Σ(RL. espèce X ) in which RL espèce X represents the following logarithmic reduction for a given bacterial species:[Math 2]RL espèce X= (log CFU / ml in said coculture medium in the presence of said product) – (log CFU / ml in said coculture medium in the absence of said product); andg.A step g) of determining the suitability of said product for use in a topical cosmetic or pharmaceutical formulation, consisting of selecting said substance (S) or said composition (C), when said score (Sc) is less than or equal to 5 and greater than or equal to -5 and discarding said substance (S) or said composition (C), when said score (Sc) is greater than 5 or less than -5, and the RL espèceX individual values ​​of each species are between -3 and 3.

[0016] According to a particular aspect of the method as defined above, the bacterial strains used in step a) are wild strains taken from human skin.

[0017] According to another particular aspect of the method as defined above, the bacterial strains of the species Staphylococcus epidermidis, Streptococcus mitis, Micrococcus luteus, Corynebacterium xerosis and Cutibacterium acnes are suspended in step c) in proportions such that the initial ratios expressed per 100% of colony-forming units of the total bacteria are as follows: colony-forming units of bacteria of the species Staphylococcus epidermidis to colony-forming units of the total bacteria is between 0.1% and 0.5%; colony-forming units of bacteria of the species Streptococcus mitis to colony-forming units of the total bacteria is between 0.5% and 5.0%; colony-forming units of bacteria of the species Micrococcus luteus to colony-forming units of the total bacteria is between 4% and 9%; colony-forming units of bacteria of the species Corynebacterium xerosis on colony forming units of total bacteria is between 4% and 9%;andcolony forming units of bacteria of the species Cutibacterium acnes on colony forming units of total bacteria is between 80% and 90%.;

[0018] According to another particular aspect of the process as defined above, the culture medium suitable for the bacterial growth of all of said bacterial strains used in step c), is a TSB broth (Tryptic Soy Broth in English) diluted to one tenth in water having a pH of between 5.2 and 7.0 and, preferably between 6.0 and 7.0, and comprising between 0.5% and 5% by weight of Polysorbate 80 (sorbitanethoxylated monolaurate (20 moles) Cas No.: 9005-65-6).

[0019] According to a more particular aspect of the method as defined above, the bacterial strains of the species Staphylococcus epidermidis, Streptococcus mitis, Micrococcus luteus, Corynebacterium xerosis and Cutibacterium acnes are suspended in step c) in proportions such that the initial ratios: colony forming units of bacteria of the species Staphylococcus epidermidis to colony forming units of total bacteria is equal to 0.3%; colony forming units of bacteria of the species Streptococcus mitis to colony forming units of total bacteria is equal to 2.0%; colony forming units of bacteria of the species Micrococcus luteus to colony forming units of total bacteria is equal to 6.4%; colony forming units of bacteria of the species Corynebacterium xerosis to colony forming units of total bacteria is equal to 7.3%; andcolony forming units of bacteria of the species Cutibacterium acnes out of colony forming units of total bacteria is equal to 84%.

[0020] According to another particular aspect of the method as defined above, step a) of providing said five bacterial strains, comprises the following successive sub-steps: a1. A sub-step a1) of sampling wild bacterial strains from the cheeks of a panel consisting of six to twenty healthy subjects; a2. A sub-step a2) of enrichment and then culture of the strains of the following five wild bacterial species: i. Corynebacterium xerosis, ii. Micrococcus luteus, iii. Staphylococcus epidermidis, iv. Cutibacterium acnes and v. Streptococcus mitis, from each of the samples taken in sub-step a1); a3. A sub-step a3) of identification, after sampling and isolation, of the bacterial strains of each of the five aforementioned species and cultured during sub-step a2); a4.A sub-step a4) of gathering the bacterial strains of each of the five aforementioned species, cultivated in sub-step a2) and identified in sub-step a3), to form five isolated groups each consisting of six to twenty bacterial strains of one of the five aforementioned species, and a5. A sub-step a5) of preserving said five isolated groups each consisting of six to twenty bacterial strains of one of the five aforementioned species and formed in sub-step a4), at a temperature below -50°C in a preservation medium consisting of a standard culture medium and a 10% by weight aqueous glycerol solution, for a non-zero preservation period.

[0021] At the end of sub-step a5) as defined above, said five groups, each consisting of six to twenty bacterial strains of one of the five aforementioned species, are used in step b) of the process as defined previously.

[0022] A healthy subject is understood to mean any human being with any skin type, whether phototype or ethnicity, but who does not have skin lesions such as spots or redness and who does not present a recognized pathology such as acne or atopic dermatitis.

[0023] According to another more particular aspect of the method as defined above, in sub-step a2):i. The culture of Corynebacterium xerosisis carried out on Hoyles agar for seventy-two hours at 37°C aerobically,ii. The culture of Micrococcus luteusis carried out on blood agar or Chapman agar for twenty-four hours at 37°C aerobically,iii. The culture of Staphylococcus epidermidisis carried out on Baird Parker agar for twenty-four hours at 37°C aerobically,iv. The culture of Cutibacterium acnesis carried out on blood agar for forty-eight hours at 37°C anaerobically, andv. The culture of Streptococcus mitisis carried out on blood agar, GBS agar or Mitis salivarius agar for forty-eight to seventy-two hours at 37°C aerobically.

[0024] According to another more particular aspect of the method as defined above, sub-step a3) of identification of said bacterial strains of each of the five aforementioned species and cultivated during sub-step a2), is carried out by at least one of the methods chosen from consultation of biochemical identification galleries, mass spectrometry and sequencing of the 16S gene.

[0025] According to another more particular aspect of the process as defined above, sub-step a4) is carried out by collecting colonies from the agar plates and suspending them in the culture medium.

[0026] According to another more particular aspect of the method as defined above, in step b):i) The culture of the sample of Corynebacterium xerosis is carried out in Brain-Heart + Polysorbate 80 (1.5%) medium for at least ninety-six hours and up to one hundred and two hours at 37°C,ii) The culture of the sample of Micrococcus luteus is carried out in TSB liquid medium for twenty-four hours at 37°C,iii) The culture of the sample of Staphylococcus epidermidis is carried out in TSB liquid medium for twenty-four hours at 37°C,iv) The culture of the sample of Cutibacterium acnes is carried out in Brucella + Polysorbate 80 (1.5%) medium for at least ninety-six hours and up to one hundred and two hours at 37°C,v) The culture of the sample of Micrococcus luteus is carried out in TSB liquid medium for twenty-four hours at 37°C,andvi) The culture of the Streptococcus mitis sample is carried out in TSB liquid medium for twenty-four hours at 37°C., Example

[0027] Collection of wild bacterial strains: The sample is taken by lightly rubbing a sterile swab on the left cheek of six healthy volunteers. After enrichment for two hours at 37°C in the media appropriate to the strains of the five selected bacterial species, the samples are spread on the selective agar plates to isolate the selected bacterial strains.i. The culture of the sample of Corynebacterium xerosis (C. xerosis) is carried out on Hoyles agar, for seventy-two hours at 37°C aerobically;ii. The culture of the sample of Micrococcus luteus (M. luteus) is carried out on Chapman agar, for twenty-four hours at 37°C aerobically;iii. The culture of the sample of Staphylococcus epidermidis (S. epidermidis) is carried out on Baird Parker agar, for twenty-four hours at 37°C aerobically;iv. The culture of the sample of Cutibacterium acnes (C.acnes) is carried out on blood agar for forty-eight hours at 37°C in anaerobic conditions. The culture of the Streptococcus mitis (S. mitis) sample is carried out on GBS agar for forty-eight to seventy-two hours at 37°C in anaerobic conditions.

[0028] Identification of bacterial strainsCultured bacterial strains are identified by consulting biochemical identification galleries, by mass spectrometry using a MALDI-TOF mass spectrometer (VITEK® MS Method, bioMérieux) or by sequencing of the 16S gene.

[0029] Species groupingBacterial strains belonging to the same species are grouped together to form homogeneous groups consisting of bacterial strains of a single species; these groups are then distributed into microtubes and stored at -80°C.

[0030] Coculture of bacterial strain groupsa. The culture medium used to simultaneously cultivate a sample of bacterial strain from each group is prepared by diluting the TSB medium tenfold in water, adding 1.5% polysorbate 80 and adjusting the pH of the medium to 6.5.b. The coculture of the strains is carried out at 32°C for eight hours under anaerobic conditions to approach the proportions of bacterial species, representative of those present on the skin of the cheeks after 8 hours of incubation, namely for 100% of colony forming units (CFU), 58% for C. acnes, 18% for S. epidermidis, 14.6% for S. mitis, 4.4% for M. luteus and 5% for C. xerosis. To achieve this result, the coculture is carried out respecting the initial ratios between the following species:- CFU for S. epidermidis / CFU of total bacteria = 0.3%;- CFU for S. mitis / CFU of total bacteria = 2.0%;- CFU for M. luteus / CFU of total bacteria = 6.4%;- CFU for C.xerosis / CFU of total bacteria = 7.3%; - CFU for C. acnes / CFU of total bacteria = 84%.c. After eight hours, the proportions obtained are for 100% of CFU of total bacteria, 52% for C. acnes, 21% for S. epidermidis, 15% for S. mitis, 8% for M. luteus and 4% for C. xerosis. The reproducibility of these results was confirmed by carrying out five independent tests following the same protocol. Statistical analysis of the results of these tests shows that with regard to the bacterial concentrations obtained, the standard deviation to average ratio is less than 3%.

[0031] To measure the relevance of the process, it was implemented on products known to have no effect on the microbiota: glucose, inulin, glycine, proline and glycerol. All the scores obtained show that these products are neutral with respect to commensal bacteria of the skin, as indicated in the following table 1:

[0032] Tested product (concentration % by mass)Score (Sc)Glucose (0.1%, 1%, 2% 5%)Sc between -1.0 and 2.0 depending on the concentrationsInulin (0.1%, 1%)Sc between -1.0 and 1.5 depending on the concentrationsGlycine (0.1%, 1%)Sc between -2.0 and -4.7 depending on the concentrationsProline (0.1%, 1%)Sc between -3.8 and -4.6 depending on the concentrationsGlycerol (0.1%, 1%)Sc between -2.5 and -4.2 depending on the concentrations

[0033] b. and on commonly used antibacterials and solvents: phenoxyethanol, alkyl parahydroxybenzoates or “parabens” and pentylene glycol. The results recorded in Table 2 logically show that the concentration of the product is decisive.

[0034] Tested product (concentration)Score (Sc)Phenoxyethanol (0.5%)Sc = approximately -3Phenoxyethanol (1%)Sc between -15 and -25Methyl- and ethylparabens (0.1%)Sc between -4.5 and -6Methyl- and ethylparabens (1%)S Cless than -40Pentylene glycol (0.1%)Sc = -3.5Pentylene glycol (1%)Sc = approximately -5.7

[0035] All of these results show that the method which is the subject of the present invention makes it possible to effectively and easily detect the safety or aggressiveness of a product with respect to the microbiota present on human skin.

Claims

Method for determining the suitability of a product for use in a topical cosmetic or pharmaceutical formulation, said product being a chemical or biochemical substance of any kind (S) or a composition of chemical or biochemical substances of any kind (C), characterized in that it comprises the following steps: a. Step a) of providing bacterial strains of the following five species: i) Corynebacterium xerosis, ii) Micrococcus luteus, iii) Staphylococcus epidermidis, iv) Cutibacterium acnes and v) Streptococcus mitis, said provision being carried out in five isolated groups, each consisting of several bacterial strains of one of the five aforementioned species; b.A step b) during which a sample of each of said five isolated groups, each consisting of several bacterial strains of one of the five aforementioned species made available in step a), is taken and then cultured in a suitable specific medium, for twenty-four hours to one hundred and two hours, to produce cultures of bacterial strains of each of said aforementioned species; c. A step c) of suspending in a single culture medium suitable for bacterial growth the coculture of said bacterial strains cultured in step b), to form a coculture, in said culture medium, of the consortium of bacterial strains of said five aforementioned species, in proportions close to those found on the skin; d. A step d) of bringing said coculture into contact in said coculture medium obtained in step c), with said product, for a period of between seven and nine hours; e.A step e) of counting the units forming bacteria, the bacteria remaining at the end of step d); f. A step f) of calculating a score (Sc): [Math 1] Sc = Σ(RL. espèce X ) in which RL espèce X represents the following logarithmic reduction for a given bacterial species:[Math 2]RL espèce X = (log CFU / ml in said coculture medium in the presence of said product) – (log CFU / ml in said coculture medium in the absence of said product); andg.A step g) of determining the suitability of said product for use in a topical cosmetic or pharmaceutical formulation, consisting of selecting said substance (S) or said composition (C), when said score (Sc) is less than or equal to 5 and greater than or equal to -5 and discarding said substance (S) or said composition (C), when said score (Sc) is greater than 5 or less than -5, and the RL espèceX individual values ​​of each species are between -3 and 3. Method according to claim 1, characterized in that the bacterial strains used in step a) are wild strains taken from human skin. Method according to any one of claims 1 or 2, characterized in that the bacterial strains of the species Staphylococcus epidermidis, Streptococcus mitis, Micrococcus luteus, Corynebacterium xerosis and Cutibacterium acnes are suspended in step c) in proportions such that the initial ratios expressed per 100% of colony-forming units of the total bacteria are as follows: colony-forming units of bacteria of the species Staphylococcus epidermidis to colony-forming units of the total bacteria is between 0.1% and 0.5%; colony-forming units of bacteria of the species Streptococcus mitis to colony-forming units of the total bacteria is between 0.5% and 5.0%; colony-forming units of bacteria of the species Micrococcus luteus to colony-forming units of the total bacteria is between 4% and 9%; colony-forming units of bacteria of the species Corynebacterium xerosis on colony-forming units of total bacteria is between 4% and 9%;andcolony forming units of bacteria of the species Cutibacterium acnes on colony forming units of total bacteria is between 80% and 90%.; Method according to any one of claims 1 to 3, characterized in that the culture medium suitable for the bacterial growth of all of said bacterial strains used in step c), is a TSB broth (Tryptic Soy Broth in English) diluted to one tenth in water having a pH between 5.2 and 7.0 and, preferably between 6.0 and 7.0, and comprising between 0.5% and 5% by weight of Polysorbate 80. A method according to any one of claims 1 to 4, characterized in that the bacterial strains of the species Staphylococcus epidermidis, Streptococcus mitis, Micrococcus luteus, Corynebacterium xerosis and Cutibacterium acnes are suspended in step c) in proportions such that the initial ratios: colony-forming units of bacteria of the species Staphylococcus epidermidis to colony-forming units of total bacteria is equal to 0.3%; colony-forming units of bacteria of the species Streptococcus mitis to colony-forming units of total bacteria is equal to 2.0%; colony-forming units of bacteria of the species Micrococcus luteus to colony-forming units of total bacteria is equal to 6.4%; colony-forming units of bacteria of the species Corynebacterium xerosis to colony-forming units of total bacteria is equal to 7.3%; andcolony forming units of bacteria of the species Cutibacterium acnes out of colony forming units of total bacteria is equal to 84%.Method according to any one of claims 1 to 5, characterized in that step a) of providing said five bacterial strains, comprises the following successive sub-steps: a1. A sub-step a1) of sampling wild bacterial strains from the cheeks of a panel consisting of six to twenty healthy subjects; a2. A sub-step a2) of enrichment and then culture of the strains of the following five wild bacterial species: i. Corynebacterium xerosis, ii. Micrococcus luteus, iii. Staphylococcus epidermidis, iv. Cutibacterium acnes and v. Streptococcus mitis, from each of the samples taken in sub-step a1); a3. A sub-step a3) of identification, after sampling and isolation, of the bacterial strains of each of the five aforementioned species and cultured during sub-step a2); a4.A sub-step a4) of gathering the bacterial strains of each of the five aforementioned species, cultivated in sub-step a2) and identified in sub-step a3), to form five isolated groups each consisting of six to twenty bacterial strains of one of the five aforementioned species, and a5. A sub-step a5) of preserving said five isolated groups each consisting of six to twenty bacterial strains of one of the five aforementioned species and formed in sub-step a4), at a temperature below -50°C in a preservation medium consisting of a standard culture medium and a 10% by weight aqueous glycerol solution, for a non-zero preservation period. The method of claim 6, characterized in that in sub-step a2):i. The culture of Corynebacterium xerosis is carried out on Hoyles agar for seventy-two hours at 37°C aerobically,ii. The culture of Micrococcus luteus is carried out on blood agar or Chapman agar for twenty-four hours at 37°C aerobically,iii. The culture of Staphylococcus epidermidis is carried out on Baird Parker agar for twenty-four hours at 37°C aerobically,iv. The culture of Cutibacterium acnes is carried out on blood agar for forty-eight hours at 37°C anaerobically, andv. The culture of Streptococcus mitis is carried out on blood agar, GBS agar or Mitis salivarius agar for forty-eight to seventy-two hours at 37°C aerobically. Method according to any one of claims 6 or 7, characterized in that sub-step a3) of identification of the bacterial strains of each of the five aforementioned species and cultivated during sub-step a2), is carried out by at least one of the methods chosen from consultation of biochemical identification galleries, mass spectrometry and sequencing of the 16S gene. Method according to any one of claims 6 to 8, characterized in that sub-step a4) is carried out by taking colonies from the agar plates and suspending them in the culture medium. Method according to any one of claims 1 to 9, characterized in that, in step b): i) The culture of the sample of Corynebacterium xerosis is carried out in Brain-Heart medium + Polysorbate 80 (1.5%) for at least ninety-six hours and up to one hundred and two hours at 37°C, ii) The culture of the sample of Micrococcus luteus is carried out in TSB liquid medium for twenty-four hours at 37°C, iii) The culture of the sample of Staphylococcus epidermidis is carried out in TSB liquid medium for twenty-four hours at 37°C, iv) The culture of the sample of Cutibacterium acnes is carried out in Brucella medium + Polysorbate 80 (1.5%) for at least ninety-six hours and up to one hundred and two hours at 37°C, and v) The culture of the sample of Streptococcus mitisest carried out in TSB liquid medium for twenty-four hours at 37°C.