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

Abstract

Method for assessing the ability of a product to preserve the skin microbiota Method for determining the suitability of a product to be used in a cosmetic or pharmaceutical formulation for topical use, said product being a chemical or biochemical substance of any nature (S) or a composition of chemical or biochemical substances of any nature (C), comprising a step of suspending in a single culture medium suitable for bacterial growth of the following species: C. xerosis, M. luteus, S. epidermidis, C. acnes and S.mitis, to form a coculture in said coculture medium, of the consortium of bacterial strains of said five aforementioned species, in proportions close to those found on the skin; followed by contacting said coculture in said coculture medium with said product for a period of between seven and nine hours, calculating a score (Sc): Sc = Σ(RLpecies X) in which RLpecies X represents the following logarithmic reduction for a given bacterial species and determining the suitability of said product to be used in a cosmetic or pharmaceutical formulation for topical use, 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 individual RLpecies X of each species are between -3 and 3.

Description

Title of the invention: Method for evaluating the capacity of a product 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 consists of three overlapping layers: a. the epidermis which is the superficial layer in contact with the exterior of the human body, b. 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; c. the hypodermis, which is the deepest layer. It is a loose connective tissue, where blood vessels pass and which may contain fatty tissue. Its function is to protect against shocks and also helps maintain an adequate internal temperature.

[0003] The epidermis is a multi-stratified 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, 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 constitute 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 approximately 104 to 106 colony forming units (CFU) per square centimeter.

[0005] Moist areas of the skin, such as the armpits or the inguinal folds, are colonized in particular by bacteria of the genus Staphylococcus; sebaceous areas, such as the forehead, the wings of the nose or the upper back are colonized in greater proportion by bacteria of the genus Cutibacterium, while dry areas such as the forearms or the 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 used for hygiene or therapeutic purposes often contain antimicrobial substances, such as preservatives, which are intended to control the undesirable 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 evaluate 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 for certifying 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” includes four steps: a. A quality test in which the skin care product is tested for contamination by aerobic mesophilic microorganisms (bacteria, yeasts, fungi). The acceptable contamination limit is less than or equal to 103CFU / g or ml. b. A balance test to verify that the skin care product does not disrupt 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 or composition and then incubated for four hours. The ratio of the two microbes (in CFU) is compared to the same ratio in the untreated control group. The chemical or composition must not have promoted the growth of Staphylococcus aureus. c. A diversity test: A coculture of typical facial and body microbes, for dry, wet, or oily skin, is grown and then mixed with the chemical 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 placed second batches of each key microorganism, thereby simulating the potential penetration of the formulation into the 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 test in which a coculture of Staphylococcus epidermidis, Cutibacterium acnes, Corynebacterium striatum. This coculture is placed in contact for twenty-four hours with the chemical substance or chemical composition or formulation to be tested. The results are compared to untreated references.

[0010] The StratiCELL laboratory process consists of inoculating reconstructed human epidermis (RHE) with these 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 the 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 evaluate the safety of these compositions or chemical substances constituting 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 comparing 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 the 16S RNA and analyzing the amplicons to determine the proportion of each species.

[0012] 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. Statement of the invention

[0013] According to a first aspect, the invention relates to a method for determining the suitability of a product to be used 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 (C), characterized in that it comprises the following steps: a. A step a) of making available 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 made 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 an appropriate specific medium, for twenty-four hours to ninety-six 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 cultivated 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 S(RUeSpecex) in which RLespèœ x represents the following logarithmic reduction for a given bacterial species: [Math 2] RLespeœx = (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); and g. 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 individual RLpeciesX of each species are between -3 and 3.

[0014] According to a 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).

[0015] According to another particular aspect of the process 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 on colony forming units of total bacteria is equal to 0.3%; • colony forming units of bacteria of the species Streptococcus mitis on colony forming units of total bacteria is equal to 2.0%; • colony forming units of bacteria of the species Micrococcus luteus on colony forming units of total bacteria is equal to 6.4%; • colony forming units of bacteria of the species Corynebacterium xerosis on colony forming units of total bacteria is equal to 7.3%; and • colony forming units of bacteria of the species Cutibacterium acnes on colony forming units of total bacteria is equal to 84%.

[0016] According to another particular aspect of the method as defined above, step a) of making said five bacterial strains available comprises the following successive sub-steps: al. A sub-step al) of sampling wild bacterial strains on the cheeks of a panel made up of six to twenty healthy subjects; a2. A sub-step a2) of enrichment and then culture of strains of the following five wild bacterial species: i. Corynebacterium xerosis, ii. Micrococcus luteus, iii. Staphylococcus epidermidis, iv. Cutibacterium acnes and y. Streptococcus mitis, from each of the samples taken in sub-step al); a3. A sub-step a3) of identification, after sampling and isolation, of the bacterial strains of each of the five species mentioned above and cultivated 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 an aqueous glycerol solution at 10% by weight, for a non-zero preservation period.

[0017] 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 implemented in step b) of the method as defined previously.

[0018] By healthy subject, we mean any human being with any type of skin, whether phototype or ethnicity, but not having skin lesions such as spots or redness and not presenting a recognized pathology such as acne or atopic dermatitis.

[0019] According to another more particular aspect of the method as defined above, in sub-step a2): i. Corynebacterium xerosis is grown on Hoyles agar for seventy-two hours at 37°C aerobically, ii. Micrococcus luteus is cultured on blood agar or Chapman agar for twenty-four hours at 37°C aerobically, iii. Staphylococcus epidermidis is cultured 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 in anaerobiosis, and v. Streptococcus mitis culture is carried out on blood agar, GBS agar or Mitis salivarius agar for forty-eight to seventy-two hours at 37°C aerobically.

[0020] 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.

[0021] According to another more particular aspect of the method as defined above, sub-step a4) is carried out by collecting colonies from the agar plates and suspending them in the culture medium.

[0022] According to another more particular aspect of the method as defined above, in step b): i) The culture of the Corynebacterium xerosis sample is carried out in Brain-Heart + Polysorbate 80 (1.5%) medium for one hundred and two hours at 37°C, ii) The culture of the Micrococcus luteus sample is carried out in TSB liquid medium for twenty-four hours at 37°C, iii) The culture of the Staphylococcus epidermidis sample is carried out in TSB liquid medium for twenty-four hours at 37°C, iv) The culture of the Cutibacterium acnes sample is carried out in Brucella + Polysorbate 80 (1.5%) medium for one hundred and two hours at 37°C, and (v) The culture of the Streptococcus mitis sample is carried out in TSB liquid medium for twenty-four hours at 37°C. Example

[0023] Sampling of wild bacterial strains: The sample is collected by lightly rubbing a sterile swab on the left cheek of six healthy volunteers. After enrichment for two hours at 37°C in media appropriate for the strains of the five selected bacterial species, the samples are spread on selective agar plates to isolate the selected bacterial strains. i. The culture of the Corynebacterium xerosis (C. xerosis) sample is carried out on Hoyles agar, for seventy-two hours at 37°C in aerobiosis; ii. The culture of the Micrococcus luteus (M. luteus) sample is carried out on Chapman agar, for twenty-four hours at 37°C in aerobiosis; iii. The culture of the Staphylococcus epidermidis (S. epidermidis) sample is carried out on Baird Parker agar, for twenty-four hours at 37°C in aerobiosis; iv. The culture of the Cutibacterium acnes (C. acnes) sample is carried out on blood agar, for forty-eight hours at 37°C in an anaerobic manner and v. 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 under anaerobic conditions.

[0024] Identification of bacterial strains The cultivated bacterial strains are identified by consulting the biochemical identification galleries, by mass spectrometry using a MALDLTOF mass spectrometer (VITEK® MS Method, bioMérieux) or by sequencing of the 16S gene.

[0025] Grouping of species Bacterial 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.

[0026] Coculture of bacterial strain groups a. 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 medium pH 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%.

[0027] To measure the relevance of the process, it was implemented a. 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: [Tables 1] Tested product (concentration % mass) Score (Sc) Glucose (0.1%, 1%, 2% 5%) Sc between -1.0 and 2.0 depending on the concentrations Inulin (0.1%, 1%) Sc between -1.0 and 1.5 depending on the concentrations Glycine (0.1%, 1%) Sc between -2.0 and -4.7 depending on the concentrations Proline (0.1%, 1%) Sc between -3.8 and -4.6 depending on the concentrations Glycerol (0.1%, 1%) Sc between -2.5 and -4.2 depending on the concentrations 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. [Tables 2] Tested product (concentration) Score (Sc) Phenoxyethanol (0.5%) Sc = approximately -3 Phenoxyethanol (1%) Sc between -15 and -25 Methyl- and ethylparabens (0.1%) Sc between -4.5 and -6 Methyl- and ethylparabens (1%) Sc below -40 Pentylene glycol (0.1%) Sc = -3.5 Pentylene glycol (1%) Sc = approximately -5.7

[0028] 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

Claims

1. 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 (S) or a composition of chemical or biochemical substances (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 ninety-six 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 2(RLeSpècex). in which RLspeciesX represents the following logarithmic reduction for a given bacterial species: [Math 2] RLspeciesx = (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); and g. 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 individual RLspeciesX of each species are between -3 and 3.

2. Method according to claim 1, 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.

3. 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: • 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 on colony forming units of total bacteria is equal to 7.3%; and • colony forming units of bacteria of the species Cutibacterium acnes on colony forming units of total bacteria is equal to 84%.

4. Method according to any one of claims 1 to 3, 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 strains of the following five wild bacterial species: i. Corynebacterium xerosis, ii. Micrococcus lut eus, iii. Staphylococcus epidermidis, iv. Cutibacterium acnes and y. 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 species mentioned above and cultivated 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 an aqueous glycerol solution at 10% by weight.

5. Method according to claim 4, characterized in that in sub-step a2): i. Corynebacterium xerosis is cultured on Hoyles agar for seventy-two hours at 37°C aerobically, ii. Micrococcus luteus is cultured on blood agar or Chapman agar for twenty-four hours at 37°C aerobically, iii. Staphylococcus epidermidis is cultured on Baird Parker agar for twenty-four hours at 37°C aerobically, iv. Cutibacterium acnes culture is carried out on blood agar for forty-eight hours at 37°C under anaerobic conditions, and v. Streptococcus mitis culture is carried out on blood agar, GBS agar or Mitis salivarius agar for forty-eight to seventy-two hours at 37°C under aerobic conditions.

6. Method according to any one of claims 4 or 5, 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.

7. Method according to any one of claims 4 to 6, characterized in that sub-step a4) is carried out by taking colonies from the agar plates and suspending them in the culture medium.