Marker combination for skin typing and its application

Abstract

The present invention provides a use of Moraxella osloensis for skin typing, characterizing skin conditions, or for preparing a reagent or kit for characterizing skin conditions. The present invention also provides a marker combination comprising the following markers: Moraxella osloensis and Propionibacterium acnes. The present invention also provides the use of the marker combination for skin typing and / or determining skin conditions.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more specifically to a combination of biomarkers for skin typing and their applications. Background Technology

[0002] With the launch of the microbiome project, it has become increasingly clear that, similar to the gut, the human body surface also harbors a vast number of microorganisms. Gut research has suggested that microbe-host interactions and strategies such as fecal microbiota transplantation, probiotics, and prebiotics can alter the gut microbiota and thus improve gut homeostasis. Research on skin microbiota has lagged behind, particularly lacking large-scale population metagenomic data. Metagenomic data based on North American populations indicate that while skin microbiota exhibits significant individual variability in composition, it also demonstrates high stability; that is, the microbial composition remains relatively stable across time and space, exhibiting strong individual characteristics. This study, based on data from the Han Chinese population, further confirms the existence of individual variability and finds a significant correlation between microbial composition and a range of host skin phenotypes. Skin microbiota can serve as a potential new approach and target for improving skin phenotypes.

[0003] However, the significant individual differences present a severe challenge to the research and design of universally applicable skincare products and pharmaceuticals. Through further statistical analysis, we discovered that despite large individual differences, some key characteristics and patterns still exist at the population level. Combinations of these characteristics can be used to classify populations, thus addressing the issue of large individual differences. Skin typing can effectively meet the needs of low-resolution "precision" / "personalized" care. For example, a commonly used skin typing method—"dry," "normal," and "oily" skin—is based on the water-oil characteristics of the host's skin. Similarly, but unlike this, our study classifies populations based on skin microbiome characteristics, not water-oil characteristics. This helps the industry develop skincare products and pharmaceuticals more suitable for specific populations, achieving a certain degree of "precision" / "personalized" care and effectively improving skin homeostasis.

[0004] Therefore, there is an urgent need in this field to develop new methods for skin typing and skin aging assessment, providing a new diagnostic and typing approach. Summary of the Invention

[0005] The purpose of this invention is to classify people based on skin microbiome characteristics, thereby helping the industry develop skincare products and pharmaceuticals more suitable for specific groups of people with different skin types. It provides a new method for skin classification and assessment of skin condition, offering a novel diagnostic and classification approach.

[0006] This invention focuses on two bacteria that dominate skin typing—Moraxella osloensis and Propionibacterium acnes—as well as Staphylococcus epidermidis, one of the most abundant bacterial colonists on the skin that plays an important role in host health and disease. First, using metagenomic data from a large-scale sample of the Han Chinese population, the correlation between the abundance of the three target skin symbiotic bacteria on the face and the host skin phenotype was determined. Then, qPCR experiments were used to verify this, identifying one bacterium that promotes skin aging—Moraxella osloensis.

[0007] In a first aspect of the invention, there is provided the use of M. osloensis or a detection reagent thereof for (a) skin typing; and / or (b) determining or characterizing skin condition or for preparing a reagent or kit for (a) skin typing; and / or (b) determining or characterizing skin condition.

[0008] In another preferred embodiment, the skin condition includes: skin age, skin moisture content, skin elasticity, skin color, and degree of skin aging.

[0009] In another preferred embodiment, the degree of skin aging is determined based on one or more phenotypes selected from the group consisting of: porphyrins, oil content, water content, luster, pore area, skin yellowness value, pore size, and pigmentation.

[0010] In another preferred embodiment, the reagent or kit further includes a reagent for detecting Propionibacterium acnes (C. acnes).

[0011] In another preferred embodiment, the reagent or kit further includes reagents for detecting Moraxella bovoculi and / or Psychrobacter sp.

[0012] In another preferred embodiment, the reagent or kit further includes reagents for detecting Propionibacterium gravidum, Propionibacterium granulosum, Staphylococcus, Propionibacterium acnes phage and / or Staphylococcus phage.

[0013] In another preferred embodiment, the reagent or kit further includes a reagent for detecting Staphylococcus epidermidis.

[0014] A second aspect of the invention provides a combination of biomarkers comprising M. osloensis and Propionibacterium acnes.

[0015] In another preferred embodiment, the combination of markers further includes Moraxella bovoculi and / or Psychrobacter sp.

[0016] In another preferred embodiment, the combination of markers further includes Propionibacterium gravidum, Propionibacterium granulosum, Staphylococcus, Propionibacterium acnes phage, and / or Staphylococcus phage.

[0017] In another preferred embodiment, the biomarker combination also includes Staphylococcus epidermidis.

[0018] In another preferred embodiment, the combination of markers is used for (a) skin typing; and / or (b) determining skin condition.

[0019] In another preferred embodiment, the skin condition includes: skin age, skin moisture content, skin elasticity, skin color, and degree of skin aging.

[0020] In another preferred embodiment, the degree of skin aging is determined based on one or more phenotypes selected from the group consisting of: porphyrins, oil content, water content, luster, pore area, skin yellowness value, pore size, and pigmentation.

[0021] In another preferred embodiment, the marker or combination of markers is derived from a skin sample, preferably from the whole body skin or facial skin, more preferably from the cheek, forehead, or nostril.

[0022] In another preferred embodiment, the marker or combination of markers is derived from skin samples from Asian populations.

[0023] In another preferred embodiment, the marker or combination of markers is derived from samples of the cheek, forehead, and nostrils.

[0024] In another preferred embodiment, the levels of each marker in the marker combination are detected by one or more of the following methods: sequencing, PCR, and protein quantification.

[0025] In another preferred embodiment, the method for detecting the biomarker group at the level further includes one or more methods selected from the group consisting of: quantitative PCR of characteristic genes, qPCR, real-time quantitative PCR, metagenomic analysis, 16S RNA sequencing, mass spectrometry analysis, and Western blotting.

[0026] In another preferred embodiment, when the ratio (M / C) of the level (e.g., content) of *Moraxella osloensis* (M) to the level (e.g., content) of *Propionibacterium acnes* (C) in the marker combination meets the following conditions: M / C ≤ 1.3, preferably M / C ≤ 0.8, more preferably M / C ≤ 0.4, then the skin type is type C (or type I), whose phenotype includes: high oil content, high water content, good skin elasticity, low degree of skin aging, and bright skin color.

[0027] In another preferred embodiment, when the ratio (M / C) of the level (e.g., content) of *Moraxella osloensis* (M) to the level (e.g., content) of *Propionibacterium acnes* (C) in the marker combination meets the following conditions: 0.3 ≤ M / C ≤ 2.5, preferably 0.3 ≤ M / C ≤ 2.3, and more preferably 0.8 ≤ M / C ≤ 1.8, the skin type is mixed (or type II), and its phenotype includes: moderate oil content, moderate water content, moderate skin elasticity, moderate skin aging, and moderate skin color.

[0028] In another preferred embodiment, when the ratio (M / C) of the level (e.g., content) of the *Moraxella osloensis* in the marker combination to the level (e.g., content) of the *Propionibacterium acnes* (C) meets the following conditions: M / C ≥ 0.5, preferably M / C ≥ 1.8, more preferably M / C ≥ 2.2, then the skin type is type M (type III), whose phenotype includes low skin oil content, low water content, poor skin elasticity, high degree of skin aging, and dull skin color.

[0029] In another preferred embodiment, when the ratio (M / C) of the level (e.g., content) of *Moraxella osloensis* (M) to the level (e.g., content) of *Propionibacterium acnes* (C) obtained from a sample on the cheek meets the following condition: M / C ≤ 0.8, preferably M / C ≤ 0.75, then the skin type is type C (or type I), whose phenotype includes high oil content, high water content, good skin elasticity, low degree of skin aging, and bright skin color.

[0030] In another preferred embodiment, when the ratio (M / C) of the level (e.g., content) of *Moraxella osloensis* (M) to the level (e.g., content) of *Propionibacterium acnes* (C) obtained from a sample on the cheek meets the following conditions: 0.75 ≤ M / C ≤ 2, preferably 0.8 ≤ M / C ≤ 1.7, then the skin type is mixed (or type II), and its phenotype includes: moderate oil content, moderate water content, moderate skin elasticity, moderate skin aging, and moderate skin color.

[0031] In another preferred embodiment, when the ratio (M / C) of the level (e.g., content) of *Moraxella osloensis* (M) to the level (e.g., content) of *Propionibacterium acnes* (C) obtained from the cheek meets the following condition: M / C≥1.7, then the skin type is type M (Type III), whose phenotype includes low skin oil content, low water content, poor skin elasticity, high degree of skin aging, and dull skin color.

[0032] In another preferred embodiment, for a sample from the forehead, if the ratio (M / C) of the level (e.g., content) of *Moraxella osloensis* (M) to the level (e.g., content) of *Propionibacterium acnes* (C) meets the following condition: M / C ≤ 1.5, preferably M / C ≤ 1.25, then the skin type is type C (or type I), whose phenotype includes high oil content, high water content, good skin elasticity, low degree of skin aging, and bright skin color.

[0033] In another preferred embodiment, the sample is taken from the forehead, and the ratio (M / C) of the level (e.g., content) of *Moraxella osloensis* (M) to the level (e.g., content) of *Propionibacterium acnes* (C) meets the following conditions: 1.25 ≤ M / C ≤ 2.5, preferably 1.3 ≤ M / C ≤ 2.2, and the skin type is mixed (or type II), whose phenotype includes: moderate oil content, moderate water content, moderate skin elasticity, moderate skin aging, and moderate skin color.

[0034] In another preferred embodiment, when the ratio (M / C) of the level (e.g., content) of *Moraxella osloensis* (M) to the level (e.g., content) of *Propionibacterium acnes* (C) obtained from the forehead meets the following condition: M / C≥2, preferably ≥2.2, then the skin type is type M (type III), whose phenotype includes low skin oil content, low water content, poor skin elasticity, high degree of skin aging, and dull skin color.

[0035] In another preferred embodiment, for samples derived from the nasal ala, the ratio (M / C) of the level (e.g., content) of *Moraxella osloensis* to the level (e.g., content) of *Propionibacterium acnes* (C) meets the following conditions: M / C ≤ 0.5, preferably M / C ≤ 0.35, the skin type is type C (or type I), and its phenotype includes high oil content, high water content, good skin elasticity, low degree of skin aging, and bright skin color.

[0036] In another preferred embodiment, the sample is taken from the nasal ala, and the ratio (M / C) of the level (e.g., content) of *Moraxella osloensis* (M) to the level (e.g., content) of *Propionibacterium acnes* (C) meets the following conditions: 0.35 ≤ M / C ≤ 0.7, preferably 0.35 ≤ M / C ≤ 0.6, more preferably 0.35 ≤ M / C ≤ 0.55, and the skin type is mixed (or type II), whose phenotype includes: moderate oil content, moderate water content, moderate skin elasticity, moderate skin aging, and moderate skin color.

[0037] In another preferred embodiment, when the ratio (M / C) of the level (e.g., content) of *Moraxella osloensis* (M) to the level (e.g., content) of *Propionibacterium acnes* (C) obtained from the nasal wing sample meets the following conditions: M / C ≥ 0.5, preferably M / C ≥ 0.55, the skin type is type M (type III), whose phenotype includes low skin oil content, low water content, poor skin elasticity, high degree of skin aging, and dull skin color.

[0038] A third aspect of the present invention provides a method for skin typing or determining skin condition, the method comprising:

[0039] (1) Provide a sample from the skin of the subject to be tested, and detect the level (e.g., content) of each marker in the sample marker combination, the combination including the following markers: Oslo Moraxella and Propionibacterium acnes, and obtain the level (e.g., content) of Oslo Moraxella (M) and the level (e.g., content) of Propionibacterium acnes (C) respectively.

[0040] (2) Based on the level (e.g., content) of Moraxella osloensis (M), or by comparing the level (e.g., content) of Moraxella osloensis (M) in the sample with the level (e.g., content) of Propionibacterium acnes (C), the skin of the test subject is classified and / or the skin condition is determined.

[0041] In another preferred embodiment, the subjects to be tested are from an Asian population.

[0042] In another preferred embodiment, in step (2), the skin is classified or the skin condition of the sample is determined based on the relative value (e.g., M / C) of the level (e.g., content) of Moraxella osloensis and the level (e.g., content) of Propionibacterium acnes (C).

[0043] In another preferred embodiment, the skin condition includes skin age, skin moisture content, skin elasticity, skin color, and degree of skin aging.

[0044] In another preferred embodiment, the levels (e.g., content) of Moraxella osloi in the sample of the test subject are determined by one or more methods selected from the group consisting of: sequencing, PCR, and protein quantification.

[0045] In another preferred embodiment, the method for detecting the level (e.g., content) of Moraxella osloensis (M) and the level (e.g., content) of Propionibacterium acnes (C) in the sample further includes one or more methods selected from the group consisting of: quantitative PCR of characteristic genes, qPCR, real-time quantitative PCR, metagenomic analysis, 16S RNA sequencing, mass spectrometry analysis, and Western blotting.

[0046] In another preferred embodiment, when the ratio (M / C) of the Oslo Moraxella acnes (M) to the level (e.g., content) of Propionibacterium acnes (C) in the sample meets the following conditions: M / C ≤ 1.3, preferably M / C ≤ 0.8, more preferably M / C ≤ 0.4, then the skin type is type C (or type I), whose phenotype includes: high oil content, high water content, good skin elasticity, low degree of skin aging, and bright skin color.

[0047] In another preferred embodiment, when the ratio (M / C) of the level (e.g., content) of *Moraxella osloensis* (M) to the level (e.g., content) of *Propionibacterium acnes* (C) in the sample meets the following conditions: 0.3 ≤ M / C ≤ 2.5, preferably 0.3 ≤ M / C ≤ 2.3, and more preferably 0.8 ≤ M / C ≤ 1.8, the skin type is mixed (or type II), and its phenotype includes: moderate oil content, moderate water content, moderate skin elasticity, moderate skin aging, and moderate skin color.

[0048] In another preferred embodiment, when the ratio (M / C) of the level (e.g., content) of *Moraxella osloensis* (M) to the level (e.g., content) of *Propionibacterium acnes* (C) in the sample meets the following conditions: M / C ≥ 0.5, preferably M / C ≥ 1.8, more preferably M / C ≥ 2.2, then the skin type is type M (type III), whose phenotype includes low skin oil content, low water content, poor skin elasticity, high degree of skin aging, and dull skin color.

[0049] In another preferred embodiment, when the ratio (M / C) of the level (e.g., content) of *Moraxella osloensis* (M) to the level (e.g., content) of *Propionibacterium acnes* (C) in the sample from the cheek meets the following condition: M / C ≤ 0.8, preferably M / C ≤ 0.75, then the skin type is type C (or type I), whose phenotype includes high oil content, high water content, good skin elasticity, low degree of skin aging, and bright skin color.

[0050] In another preferred embodiment, when the ratio (M / C) of the level (e.g., content) of *Moraxella osloensis* (M) to the level (e.g., content) of *Propionibacterium acnes* (C) in the sample obtained from the cheek meets the following conditions: 0.75 ≤ M / C ≤ 2, preferably 0.8 ≤ M / C ≤ 1.7, then the skin type is mixed (or type II), and its phenotype includes: moderate oil content, moderate water content, moderate skin elasticity, moderate skin aging, and moderate skin color.

[0051] In another preferred embodiment, when the ratio (M / C) of the level (e.g., content) of *Moraxella osloensis* (M) in the sample to the level (e.g., content) of *Propionibacterium acnes* (C) in the sample meets the following condition: M / C ≥ 1.7, then the skin type is type M (Type III), whose phenotype includes low skin oil content, low water content, poor skin elasticity, high degree of skin aging, and dull skin color.

[0052] In another preferred embodiment, for a sample from the forehead, if the ratio (M / C) of the level (e.g., content) of *Moraxella osloensis* (M) to the level (e.g., content) of *Propionibacterium acnes* (C) in the sample meets the following condition: M / C ≤ 1.5, preferably M / C ≤ 1.25, then the skin type is type C (or type I), whose phenotype includes high oil content, high water content, good skin elasticity, low degree of skin aging, and bright skin color.

[0053] In another preferred embodiment, the sample is derived from the forehead, and the ratio (M / C) of the level (e.g., content) of *Moraxella osloensis* (M) to the level (e.g., content) of *Propionibacterium acnes* (C) in the sample meets the following conditions: 1.25 ≤ M / C ≤ 2.5, preferably 1.3 ≤ M / C ≤ 2.2, and the skin type is mixed (or type II), whose phenotype includes: moderate oil content, moderate water content, moderate skin elasticity, moderate skin aging, and moderate skin color.

[0054] In another preferred embodiment, when the ratio (M / C) of the level (e.g., content) of *Moraxella osloensis* (M) to the level (e.g., content) of *Propionibacterium acnes* (C) in the sample from the forehead meets the following condition: M / C≥2, preferably ≥2.2, then the skin type is type M (Type III), whose phenotype includes low skin oil content, low water content, poor skin elasticity, high degree of skin aging, and dull skin color.

[0055] In another preferred embodiment, for samples derived from the nasal ala, the ratio (M / C) of the level (e.g., content) of *Moraxella osloensis* (M) to the level (e.g., content) of *Propionibacterium acnes* (C) in the sample meets the following conditions: M / C ≤ 0.5, preferably M / C ≤ 0.35, the skin type is type C (or type I), and its phenotype includes high oil content, high water content, good skin elasticity, low degree of skin aging, and bright skin color.

[0056] In another preferred embodiment, the sample is derived from the nasal ala, and the ratio (M / C) of the level (e.g., content) of *Moraxella osloensis* (M) to the level (e.g., content) of *Propionibacterium acnes* (C) in the sample meets the following conditions: 0.35 ≤ M / C ≤ 0.7, preferably 0.35 ≤ M / C ≤ 0.6, more preferably 0.35 ≤ M / C ≤ 0.55, and the skin type is mixed (or type II), whose phenotype includes: moderate oil content, moderate water content, moderate skin elasticity, moderate skin aging, and moderate skin color.

[0057] In another preferred embodiment, the sample is derived from the nasal ala, and the ratio (M / C) of the level (e.g., content) of *Moraxella osloensis* (M) to the level (e.g., content) of *Propionibacterium acnes* (C) in the sample meets the following conditions: M / C ≥ 0.5, preferably M / C ≥ 0.55, and the skin type is type M (type III), whose phenotype includes low skin oil content, low water content, poor skin elasticity, high degree of skin aging, and dull skin color.

[0058] In another preferred embodiment, the relative value meets the following conditions: when M / C ≤ 1.3, the skin is type C; when M / C ≥ 0.5, the skin is type M.

[0059] In another preferred embodiment, the relative value meets the following condition: when 0.3 ≤ M / C ≤ 2.5, the skin is of mixed type.

[0060] In another preferred embodiment, an increase in the level (e.g., content) (M) of the Oslo Moraxella indicates that the skin condition is characterized by increased skin age, darkening or yellowing of the skin, decreased skin moisture content, and decreased sebum and porphyrin content.

[0061] A fourth aspect of the present invention provides a reagent combination for skin typing and / or detecting skin condition, the reagent combination comprising reagents for detecting various markers in the combination described in the second aspect of the present invention.

[0062] In another preferred embodiment, the reagent is used to detect the level (e.g., content) of various markers.

[0063] In another preferred embodiment, the reagent comprises a substance for detecting the levels of each marker in the combination described in the second aspect of the invention using one or more methods selected from the group consisting of sequencing, PCR, and protein quantification.

[0064] In another preferred embodiment, the method for detecting the level of the biomarker further includes: quantitative PCR of characteristic genes, qPCR, real-time quantitative PCR, metagenomics analysis, 16S RNA sequencing, mass spectrometry analysis, and Western blotting.

[0065] In another preferred embodiment, the reagent combination includes:

[0066] A first detection reagent, used to detect the level (M) of *Moraxella osloensis*; and / or

[0067] The second test reagent is used to detect the level of Propionibacterium acnes (C).

[0068] A fifth aspect of the present invention provides a reagent kit comprising the reagent combination described in the second aspect of the present invention.

[0069] In another preferred embodiment, each marker in the combination described in the second aspect of the invention is used as a standard.

[0070] A sixth aspect of the present invention provides a system for classifying and / or determining the skin condition of a test subject, the system comprising:

[0071] (a) A feature receiving module, wherein the feature receiving module is used to receive skin sample feature data; the feature data includes: quantitative information of Moraxella osloi (M) and Propionibacterium acnes (C) in the skin sample;

[0072] (b) A calculation processing module, configured to calculate the feature data from the feature receiving module to obtain the proportion of each feature or the proportional relationship between features; and based on the obtained proportions or proportional relationships between features, compare them with standard values ​​for skin typing or feature representation to obtain a judgment result for skin typing and / or skin condition; and

[0073] (c) Result output module, which is used to receive and output the judgment result.

[0074] In another preferred embodiment, the object is a person.

[0075] In another preferred embodiment, the subject is an Asian population.

[0076] In another preferred embodiment, the objects include men and women.

[0077] In another preferred embodiment, the object includes infants, adolescents, or adults.

[0078] In another preferred embodiment, the quantitative information includes the levels (e.g., content) of each of Moraxella osloi (M) and Propionibacterium acnes (C).

[0079] In another preferred embodiment, the ratio includes the relative values ​​(e.g., content) of the individual levels of Moraxella osloensis (M) and Propionibacterium acnes (C) in the skin sample, such as M / C.

[0080] In another preferred embodiment, the system can classify skin conditions into at least two types.

[0081] In another preferred embodiment, the method for obtaining the quantitative information includes sequencing, PCR, and protein quantification.

[0082] In another preferred embodiment, the method for obtaining the quantitative information further includes: quantitative PCR of characteristic genes, qPCR, real-time quantitative PCR, metagenomics analysis, 16S RNA sequencing, mass spectrometry analysis, and Western blotting.

[0083] In another preferred embodiment, the feature receiving module includes a sample acquisition instrument and a feature signal input terminal.

[0084] In another preferred embodiment, the computational processing module includes a processor and a storage device, wherein the storage device stores threshold information of skin type and / or skin condition.

[0085] In another preferred embodiment, the output module includes any terminal, preferably a monitor, printer, tablet computer (PAD), or smartphone.

[0086] In another preferred embodiment, the modules are connected via wired or wireless means.

[0087] A seventh aspect of the present invention provides a method for screening substances or ingredients that improve skin condition, comprising:

[0088] (a) Provide a screening bacterium, said screening bacterium being Moraxella osloensis (M), Propionibacterium acnes (C), or a screening bacterium containing Moraxella osloensis and / or Propionibacterium acnes (mixed bacterium);

[0089] (b) Co-culture the substance or component to be screened with the screening bacteria and detect the level (e.g., content) of each of the Oslo Moraxella acnes or Propionibacterium acnes; or the relative level (e.g., relative content) between Oslo Moraxella acnes and Propionibacterium acnes (M / C).

[0090] (c) The substance or ingredient to be screened is determined to be a substance or ingredient that improves skin condition based on the individual levels (e.g., content) of Moraxella osloensis or Propionibacterium acnes after culture (b); or the relative levels (e.g., relative content) (M / C) between Moraxella osloensis and Propionibacterium acnes.

[0091] In another preferred embodiment, an increase in the content of *Moraxella osloensis* or the relative level (e.g., relative content) (M / C) between *Moraxella osloensis* and *Propionibacterium acnes* indicates that the substance or ingredient to be screened is a substance for treating acne.

[0092] In another preferred embodiment, a decrease in the level (e.g., content) of *Moraxella osloensis* or a decrease in the relative level (e.g., relative content) (M / C) between *Moraxella osloensis* and *Propionibacterium acnes* indicates that the substance or ingredient to be screened is a skin anti-aging substance.

[0093] In another preferred embodiment, when the level (e.g., content) of Propionibacterium acnes increases or the relative level (e.g., relative content) (C / M) between Propionibacterium acnes and Moraxella osloensis increases, it indicates that the substance or ingredient to be screened is a skin anti-aging substance.

[0094] In another preferred embodiment, a decrease in the level (e.g., content) of Propionibacterium acnes or a decrease in the relative level (e.g., relative content) (C / M) between Propionibacterium acnes and Moraxella osloensis indicates that the substance or ingredient to be screened is a substance for treating acne.

[0095] The eighth aspect of the present invention provides the use of the biomarker combination described in the second aspect of the present invention or the reagent combination described in the fourth aspect of the present invention for preparing a kit for (a) skin typing; and / or (b) determining or characterizing skin condition.

[0096] The ninth aspect of the present invention provides the use of the marker combination described in the second aspect of the present invention or the reagent combination described in the fourth aspect of the present invention for screening substances or ingredients that improve skin condition.

[0097] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0098] Figure 1 A schematic diagram of the skin sampling site is shown.

[0099] Figure 2 This study shows the microbial composition of facial skin in Han Chinese people.

[0100] Figure 3 The optimal clustering number analysis for different locations is shown.

[0101] Figure 4 A and B show the clustering results for the forehead, where the box plots represent the average distance between samples within each group, and the red line represents the average distance between samples from different groups. 4A is the Jensen-Shannon divergence, and 4B is the Bray-Curtis dissimilarity.

[0102] Figure 4 C and D show the relative levels of Propionibacterium acnes or Moraxella osloensis on the forehead, with each point representing one sample.

[0103] Figure 4 E and F show the clustering results for the cheeks, where the box plots represent the average distance between samples within each group, and the red line represents the average distance between samples from different groups. 4E is the Jensen-Shannon divergence, and 4F is the Bray-Curtis dissimilarity.

[0104] Figure 4 G and H show the relative levels of Propionibacterium acnes or Moraxella osloensis on the cheeks, with each point representing one sample.

[0105] Figure 4 I and J show the clustering results for the nasal alar, where the box plots represent the average distance between samples within each group, and the red line represents the average distance between samples from different groups. 4I is the Jensen-Shannon divergence, and 4J is the Bray-Curtis dissimilarity.

[0106] Figure 4 K and L show the relative levels of Propionibacterium acnes or Moraxella osloensis on the nasal ala, with each point representing one sample.

[0107] Figure 5 This displays the differential microbes between different skin types. The color represents the relative level of the microbe, with each column representing one sample and each row representing one microbe.

[0108] Figure 6 The diagram shows the microbial network characteristics of different skin types. The left side shows the microbial species enriched in the M-cutotype, and the right side shows the microbial species enriched in the C-cutotype. Each dot represents a species, and each colored bouquet represents a species. The diagram shows that the enriched microorganisms within one skin type are positively correlated with each other, and negatively correlated with the enriched species in another skin type. Different skin types exhibit different microbial network compositions.

[0109] Figure 7 The study revealed differences in the functional enrichment of skin microbial genes among different skin types.

[0110] Figure 8 It shows the phenotypic differences between different skin types.

[0111] Figure 9 The correlation analysis showed that *Moraxella osloensis* was associated with age and skin phenotype: the adjusted P-value was less than 0.05.

[0112] Figure 10 The results showed that the aceA / aceB genes were enriched in the M-Cutotype.

[0113] Figure 11 The beta-Carotene synthetic pathway was shown to be enriched in M-Cutotype.

[0114] Figure 12 Functional enrichment analysis of differentially expressed genes in human keratinocytes (HaCaT) treated with Oslo Moraxella catarrhalis supernatant and blank control group was presented by RNAseq.

[0115] Figure 13 This demonstrates the utilization of various water-soluble carbon source compounds by *Moraxella osloensis*. The left image shows the results obtained using the CCK-8 kit, and the right image shows the results obtained using Dye mix A.

[0116] Figure 14 The results show the validation of skin types using Singaporean Chinese skin metagenomic data.

[0117] Figure 15 The results show the validation of skin types using metagenomic data from the Philippines and Italy.

[0118] Figure 16 A heatmap showing the correlation between the species level and host phenotype of three skin symbiotic bacteria is presented.

[0119] Figure 17 The image shows the results of the Oslo Moraxella-HaCaT-QPCR.

[0120] Figure 18The image shows the results of Propionibacterium acnes-HaCaT-QPCR.

[0121] Figure 19 The image shows the results of Staphylococcus epidermidis-HaCaT-QPCR. Detailed Implementation

[0122] Through extensive and in-depth research, the inventors have discovered for the first time that *Moraxella osloensis* can be used to characterize skin condition or for skin typing. Furthermore, this invention also discovers for the first time a novel combination of biomarkers: *Moraxella osloensis* and *Propionibacterium acnes*. This biomarker combination can (a) classify skin and / or (b) determine skin condition, exhibiting high sensitivity and specificity, and possessing significant application value. Based on these findings, the inventors completed this invention.

[0123] the term

[0124] The terminology used in this invention has the meanings commonly understood by those skilled in the art. However, for a better understanding of this invention, some definitions and related terms are explained below:

[0125] According to the present invention, the term "combination of markers" refers to a combination of two or more markers.

[0126] According to the present invention, the level of the marker substance is determined by the ratio of the presence and / or expression levels of the two microorganisms.

[0127] According to the present invention, the term "individual" refers to an animal, particularly a mammal such as a primate, and preferably a human.

[0128] According to the present invention, terms such as “a,” “an,” and “this” refer not only to a singular number of individuals, but also to a general class that can be used to describe a particular implementation.

[0129] As used herein, when referring to a specific enumerated value, the term “about” means that the value can vary by no more than 1% from the enumerated values. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0130] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “composed of”.

[0131] It should be noted that the explanations of the terminology provided herein are only to enable those skilled in the art to better understand the invention and are not intended to limit the invention.

[0132] Moraxella osloensis, M. osloensis, hereinafter referred to as M.

[0133] *Moraxolus osloensis*, a bacillus belonging to the genus *Moraxolus*, is a Gram-negative, chemoorganotrophic bacterium. It cannot utilize carbohydrates to produce acid.

[0134] Propionibacterium acnes, C. acnes, hereinafter referred to as C bacteria

[0135] Propionibacterium acnes, a species of Propionibacterium in the family Propionibacteraceae, genus Propionibacterium, phylum Actinomycetes, class Actinomycetes, order Actinomycetes, phylum Actinomycetes, is a Gram-positive bacillus. It is an important colonizing bacterium on human skin, participating in maintaining skin health, and can also be a pathogen of acne vulgaris.

[0136] Staphylococcus epidermidis, S. epidermidis

[0137] It is a Gram-positive coccus that grows on the surface of organisms. It exists on human skin, vagina and other parts of the body. Because it often clusters up in grape-like shapes, it is named Staphylococcus epidermidis.

[0138] Skin type

[0139] In this invention, skin can be further classified into M-Cutotype (M type for short), mixed type, and C-Cutotype (C type for short).

[0140] C-Cutotype

[0141] As used herein, the terms “C-Cutotype” and “C-type” are used interchangeably and refer to a type of skin typing based on microorganisms characterized by high levels of aggregated Propionibacterium acnes (C. acnes).

[0142] M-Cutotype

[0143] As used herein, the terms “M-Cutotype” and “M-type” are used interchangeably and refer to a type of skin typing based on microorganisms characterized by high-level aggregation of M. osloensis.

[0144] Skin elasticity

[0145] As used in this article, skin elasticity depends on, but is not limited to, the sufficiency of skin moisture, collagen, elastin, and natural fats.

[0146] skin color

[0147] As used in this article, skin color depends on, but is not limited to, skin radiance, skin tone, yellowness, etc.

[0148] Skin aging degree

[0149] As used in this article, skin aging depends on, but is not limited to, an increase in skin porphyrins, a decrease in skin moisture and radiance, enlarged and larger pores, an imbalance in skin oil, and dull skin.

[0150] Detection method

[0151] Microbial samples are obtained by repeatedly rubbing the collection site with a sterile swab dipped in bacterial collection fluid using a collection device. The levels (e.g., content) of characterizing M or C bacteria are then examined using common molecular biology techniques. For example, the M / C ratio can be obtained through methods such as: 1. 16sRNA sequencing; 2. Metagenomic sequencing; 3. Designing primers targeting the characteristic sequences of both species and then using qPCR to obtain the M / C ratio; 4. Detecting specific expressed proteins or metabolites of the two bacteria to achieve quantification, such as mass spectrometry and Western blotting.

[0152] Reagent test kit

[0153] In this invention, the reagent kit comprises the combination described in the second aspect of this invention and / or the reagent combination described in the fourth aspect of this invention.

[0154] In another preferred embodiment, each marker in the combination described in the first aspect of the invention is used as a standard.

[0155] The main advantages of this invention include:

[0156] (1) The present invention uses Moraxella osloi and Propionibacterium acnes as markers in combination for (a) skin typing; and / or (b) determining skin condition, such as moisture content, skin elasticity, and / or aging degree. It has the advantages of high sensitivity and high specificity and has important application value.

[0157] (2) This invention is the first to discover in an Asian population that Moraxella osloensis and Propionibacterium acnes can be used as a combination of markers for (a) classifying skin into M-Cutotype, mixed type and / or C-Cutotype; and / or (b) determining skin condition.

[0158] (3) This invention is the first to discover a new classification method that differs from previous classifications based on host physiology (oily skin, dry skin, moist skin). This new method uses skin microbiota as the basis for classification, identifying three skin types with distinct characteristics. Analysis of these three skin types suggests that nutritional differences caused by host physiology may be a driving factor in the development of different skin types. Furthermore, the microbial communities of different skin types may exert specific functions that influence the host skin, affecting skin health and appearance. Therefore, further research on skin types may contribute to the development of personalized medicine to better maintain skin health.

[0159] (4) This invention is the first to discover the correlation between M bacteria and skin phenotype, which is positively correlated with age and related to some skin aging phenotypes, such as as the level of M bacteria increases, skin oil decreases, water content decreases, luster decreases, and yellow value (skin dullness) increases; it is also related to some typical characteristics of acne, such as as M bacteria increase, oil decreases, porphyrins (mostly metabolites of C bacteria, which can promote inflammation) decreases, and pore area decreases.

[0160] (5) This invention is the first to discover that increasing the relative level (e.g., content) of Propionibacterium acnes can adjust M-type skin to C-type skin and can be used for skin anti-aging.

[0161] (6) This invention is the first to discover that increasing the relative level (e.g., content) of Moraxella osloensis can adjust type C skin to type M skin for the treatment of acne.

[0162] (7) This invention is the first to discover that correlation analysis between single bacteria and skin phenotype can be performed to explore whether skin microorganisms may cause changes in the host's skin phenotype, providing new insights and perspectives for the study of the interaction between microorganisms and the host.

[0163] (8) This invention is the first to discover that by using bacterial supernatant to treat host epidermal cells, the interaction between bacteria and the host can be explored at the molecular level, rather than being limited to correlation studies.

[0164] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated.

[0165] Unless otherwise specified, all reagents and materials used in the embodiments of this invention are commercially available products.

[0166] General Method

[0167] 1. Correlation analysis:

[0168] 1) Skin microbiome sample collection and metagenomic sequencing

[0169] Shanghai residents were recruited as volunteers for this study. All volunteers underwent examination by dermatologists at Shanghai Skin Disease Hospital to rule out skin lesions such as dermatitis, eczema, acne, psoriasis, and infections at the test sites, ensuring they had no skin diseases in the past 6 months and excluding volunteers who had used systemic or topical antibiotics in the past 6 months. A total of 294 healthy participants aged 20-65 years were ultimately included, comprising 46 men (M) and 248 women (F) (Table 1).

[0170] Table 1. Demographic data of the participants

[0171]

[0172] Subjects washed their faces only with water on the day of sampling and avoided using any skincare or cosmetic products the day before. The sampling site was maintained at an indoor temperature of 20 degrees Celsius and humidity of 50%. Researchers repeatedly swabbed approximately 4 cm² areas on the subject's forehead (Fh), cheeks (Ch), and sides of the nose (Ns) 20 times using a dedicated sterile swab soaked in 0.15M NaCl and 0.1% Tween 20 solution. The swab was then broken and placed in a 1.5 ml sterile EP tube, frozen at -80°C for later extraction of genomic DNA from the skin microorganisms. A schematic diagram of the sampling process is shown below. Figure 1 As shown.

[0173] The samples were amplified using whole-genome amplification and then metagenomic sequencing was performed, ultimately yielding sequencing data for 822 facial skin microbial samples. The relative abundance of each gene was calculated using SOAP2 (version 2.21), and based on the alignment results of each gene, the sum of the relative abundances of genes from the same species was calculated; this value represents the relative abundance of that species.

[0174] 2) Skin phenotype measurement

[0175] All phenotypic measurements of the 294 Han Chinese subjects were conducted in an indoor environment at 20°C and 50% humidity. Before testing, subjects rested quietly for at least 30 minutes to ensure their blood circulation returned to normal levels after any possible physical activity. The areas for phenotypic measurement coincided with the microbial collection sites. Phenotypic parameters including sebum content, stratum corneum hydration, transepidermal water loss (TEWL), skin pH, porphyrin, skin color (L*a*b), lentigines, pore area, telangiectasia, and elasticity were measured using the instruments listed in Table 2.

[0176] Table 2 Phenotypic Measurement Instruments

[0177]

[0178] 3) Correlation analysis between bacterial abundance and skin phenotype

[0179] The Spearman Rank (SPR) method was used to perform correlation analysis on the species-level distribution of various bacterial communities on the faces of 294 Han Chinese individuals. The analysis considered phenotypic factors such as sebum content, stratum corneum water content, transepidermal water loss, skin pH, pigmentation, porphyrins, skin color, and pore size. This correlation was used to assess the correlation between bacterial strains and phenotypes. The FDR test was performed on the p-values ​​of the correlation analysis results, with a corrected p-value < 0.05 indicating statistical significance. A correlation heatmap (version 1.0.12) was used to create the results. Blue indicates negative correlation; red indicates positive correlation. The significance level of the Spearman correlation is: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

[0180] 2. Matrix metalloproteinase (MMP) expression experiment:

[0181] 1) Preparation of bacterial supernatant

[0182] i. Pick the frozen bacterial suspension obtained from the skin and streak it onto the corresponding petri dishes using the three-zone streak method. Once a single colony has grown on the plate, pick the single colony and transfer it to the corresponding culture medium for liquid culture.

[0183] ii. After 24 hours of culture, genomic DNA was extracted from the bacterial culture medium and 16S rDNA was detected to confirm that the three bacterial cultures were still the target strains and there was no contamination.

[0184] iii. The identified bacteria were cultured in liquid, and a pure culture medium without bacterial inoculation was set up as a control group; iv. The absorbance of the cultured bacterial solution at 600 nm was measured using an ELISA reader and found to be approximately 0.8.

[0185] v. Filter the bacterial cultures of the three strains twice through a 0.22 μm filter to remove bacterial cells and retain the bacterial supernatant. Perform the same procedure on the pure culture medium control group;

[0186] vi. Store the obtained filtered bacterial supernatant and pure culture medium supernatant at -80°C until use.

[0187] 2) HaCaT cells incubated with bacterial supernatant

[0188] i. Culture 1×10 human keratinocytes (HaCaT cells) 6 Three replicates were set up for each group: a bacterial supernatant treatment group (experimental group) and a pure culture medium control group. The culture plates were gently shaken to ensure even cell distribution, and then incubated at 37°C in a 5% CO2 cell culture incubator. Table 3 shows the three types of bacteria used in this invention.

[0189] ii. Continue incubation in the incubator for 24 hours, discard the cell culture medium, and gently rinse with PBS. Then add trypsin to each dish, place in a cell culture incubator for 5 minutes to digest, centrifuge at 800×g for 5 minutes, and collect the cells.

[0190] Table 3 List of bacterial culture media

[0191]

[0192]

[0193] 3) Extracting cellular RNA

[0194] i. Add the collected HaCaT cells or primary fibroblasts to Trizol reagent and incubate at room temperature for 10 min;

[0195] ii. Add chloroform to the centrifuge tube, shake to mix, and let stand for 5 minutes;

[0196] iii. Centrifuge at 13200 rpm, 4℃ for 10 min, and transfer the upper aqueous phase (approximately 200 μL) into another mL centrifuge tube;

[0197] iv. Add an equal volume of isopropanol, mix by inverting the container, and let stand at room temperature for 5 minutes;

[0198] Centrifuge at 13200 rpm, 4℃ for 10 min, discard the supernatant, and observe a white precipitate at the bottom of the tube.

[0199] vi. Add 75% ethanol (prepared with anhydrous ethanol and DEPC water, freshly prepared and cooled to -20°C before use), and gently shake to suspend the RNA precipitate at the bottom of the tube;

[0200] vii. Centrifuge at 13200 rpm, 4℃ for 10 min, and discard the supernatant; repeat the above two steps.

[0201] viii. Centrifuge for 2 minutes, and use a pipette to remove as much of the remaining ethanol as possible. Open the centrifuge tube cap and let it air dry at room temperature for 5 minutes.

[0202] ix. After dissolving the RNA in DEPC water, the purity and concentration of the RNA were determined using NanoDrop, and the RNA concentration of each sample was leveled to 200 ng / μL.

[0203] 4) cDNA synthesis

[0204] i. In this experiment, the Vazyme HiScript III RT SuperMix for qPCR (+gDNAwiper) kit was used to synthesize cDNA via reverse transcription PCR.

[0205] ii. Take 1 μg of total RNA for reverse transcription, and store the remaining RNA at -80℃;

[0206] iii. Before reverse transcription, remove any contaminating genomic DNA from the RNA sample. Prepare the genomic DNA removal reaction mixture (RNA sample: 4×gDNA wiper Mix: RNase-free ddH2O = 5:4:7) in an RNase-free eight-tube strip. Gently mix with a pipette, then add 0.25 times the volume of 5×HiScript qRTSuper Mix.

[0207] iv. Place the aforementioned product in a PCR instrument and incubate at 37°C for 15 min; then at 85°C for 5 s. Once the reverse transcription process is complete, use the product immediately for qPCR or store it at -20°C.

[0208] 5) Real-time quantitative PCR

[0209] i. In this experiment, the QIAGEN QuantiFast SYBR Green PCR Kit was used for real-time quantitative PCR to detect the relative expression level of genes.

[0210] ii. Prepare 2 μM of the primers in Table 4 with DEPC water, mix well, and set aside for later use;

[0211] Table 4. Primer sequences

[0212]

[0213] iii. Prepare a Real-time PCR reaction system in a 384-well plate with Primers (2 μM): cDNA: SYBR Master Mix (2×) = 1:4:5;

[0214] iv. Place the 384-well plate in a centrifuge and centrifuge at 3000 rpm for 3 min at 4°C. Place the 384-well plate in a QuantStudio™ 7Flex Real-Time PCR System for RT-PCR reaction, and the reaction program is shown in Table 5 below;

[0215] Table 5 Real-time PCR Procedure

[0216]

[0217] v. Statistical processing of the experimental results was performed using QuantStudio™ Real-Time PCR Software, and further analyzed according to 2- △△T The relative expression levels of genes in each sample were calculated. The experimental results were then analyzed and plotted using the T-test method in GraphPad Prism 5.0 software. Significance levels: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

[0218] Example 1: Composition of the facial skin microbiome in Han Chinese population

[0219] Study Subjects: This study was approved by the Ethics Committee of the School of Life Sciences, Fudan University, and recruited 294 healthy Shanghai residents as volunteers. Among them, 46 were male (M) and 248 were female (F). Skin microbiota were collected from three areas of the face: forehead (Fh), cheeks (Ch), and sides of the nose (Ns). (Please refer to...) Figure 1 ).

[0220] Using metagenomic sequencing combined with bioinformatics analysis, we systematically described the composition and functional characteristics of the healthy skin microbiome in the Han Chinese population. By comparing data with skin microbiome samples from Americans obtained through the Human Microbiome Project (HMP), we discovered a bacterium, *M. osloensis*, present in the Chinese population at a significantly higher relative level than in Americans. *M. osloensis* was also found to be relatively high in both Chinese and Singaporean populations. This bacterium may be one of the characteristic strains of the skin microbiome in East Asian populations (please refer to...). Figure 2 ).

[0221] Example 2: Population typing analysis based on skin microbiome

[0222] Like the gut microbiome, the skin microbiome is influenced by a variety of factors and exhibits significant individual variability. In this study, we borrow from the gut typing method to classify the population based on skin microbiota and explore the driving factors behind these classifications. This aims to uncover the underlying patterns in the skin microbiome and provide new classification criteria for clinical diagnosis.

[0223] This invention screened 247 subjects whose skin microbiome data from three different sites were not missing.

[0224] Based on skin microbiome data, skin types were identified in these 247 subjects. First, the PAM method was used to cluster samples from three different sites, and the optimal number of clusters was determined using the CH index. Please refer to [link / reference needed]. Figure 3 The CH index results show that the CH index score is highest when the number of clusters is 2, therefore the optimal number of clusters in all three locations is 2.

[0225] Based on these results, this invention performs genotyping analysis on the samples, classifying them into two categories. PCoA analysis is then performed on the genotyping results of the samples from the three locations using JSD distance and Bray-Curtis distance, respectively. Please refer to... Figure 4 The results (A-L) showed that the samples from all three sites could be effectively classified into two categories, with the microorganisms contributing the most to this classification being *Propionibacterium acnes* and *Moraxella osloensis*, respectively. One category was enriched with *Propionibacterium acnes*, and the other with *Moraxella osloensis*. Based on these results, we named the two cutotypes C-Cutotype and M-Cutotype, respectively.

[0226] This invention analyzes the differentially expressed microbiota of C-Cutotype and M-Cutotype based on forehead skin microbiome data. Please refer to [link / reference]. Figure 5 The colors represent the relative levels (e.g., relative abundance) of microorganisms, with each column representing one sample and each row representing one type of microorganism. The differential analysis results show that, possibly due to interactions between microbial communities, some microorganisms are more prevalent in certain skin types. For example, C-Cutotype was enriched in *Propionibacterium greaserii*, *Propionibacterium granulosum*, *Staphylococcus*, *Propionibacterium acnes* phage, and *Staphylococcus* phage. M-Cutotype, on the other hand, was enriched in *Moraxella bovoculi* and *Psychrobacter* sp.

[0227] This invention performs correlation analysis based on the relative levels of differentially expressed microbial communities and presents the results in the form of a network diagram. Please refer to [link / reference]. Figure 6The results showed a strong positive correlation between microorganisms enriched in the same skin type and a strong negative correlation between microorganisms enriched in different skin types. This analysis suggests that microorganisms enriched in the same skin type may occupy different ecological niches, forming a stable ecological network and constructing a robust microbial community to resist the new colonization of other microorganisms, including opportunistic and potentially pathogenic microorganisms.

[0228] Based on these 247 individuals and a total of 741 samples, we present the skin type classification based on the ratio of M bacteria to C bacteria levels (e.g., content) (M / C), as shown in Table 6.

[0229] Table 6

[0230] Type C Hybrid M type cheek ≤0.75 0.75<M / C<1.68 ≥1.68 Forehead ≤1.24 1.24<M / C<2.18 ≥2.18 nose ≤0.34 0.34<M / C<0.55 ≥0.55

[0231] Based on the different sources of microorganisms, the reference values ​​for specific subtyping vary. The principle for skin subtyping sampling in clinical applications is: for facial skin areas that urgently need improvement, the above data should be used for subtyping.

[0232] Furthermore, C bacteria, or Propionibacterium acnes, are closely related to skin acne in terms of their levels (e.g., abundance). Reports on M bacteria are extremely rare; this study is the first to discover a correlation between M bacteria and skin phenotype, a positive correlation with age, and association with some skin aging phenotypes.

[0233] The inventors conducted an experiment on the changes in skin type and M / C ratio of the subjects, and obtained the results in Table 2, which showed that the M / C ratio is related to skin type. As the level of M bacteria increases, skin oil decreases, water content decreases, luster decreases, and yellow value (dull skin) increases; it is related to some typical characteristics of acne, such as as M bacteria increase, oil decreases, porphyrins (mostly metabolites of C bacteria, which can promote inflammation) decreases, and pore area decreases.

[0234] Table 7 lists the correlations between relevant phenotypes and M / C values. All the phenotypes listed are significantly correlated (p<0.05).

[0235] Table 7

[0236]

[0237]

[0238] Example 3: Biological significance of microbial skin types

[0239] Based on gene-level (e.g., abundance) profiling, PCoA analysis was performed on the samples. The results showed that the two skin types could be effectively separated, indicating significant functional differences between them. Specifically, the genes of C-Cutotype were enriched in carbohydrate and sterol metabolism and fatty acid synthesis, while the genes of the microbiome in M-Cutotype were more related to the synthesis of amino acids, aromatic compounds, and some lipids such as inositol. Previous studies have reported that Propionibacterium acnes can utilize carbohydrates as a carbon source, and gene function enrichment results also found that 17 KEGG functional modules in C-Cutotype are related to the phosphotransferase system (PTS). In prokaryotes, this system is known to be responsible for carbohydrate transport and phosphorylation, and is related to the metabolic capacity of glucose, maltose, lactose, fructose, and cellobiose, which may reflect that C-Cutotype depends on carbohydrates as a nutrient source. Conversely, previous studies on Moraxella osloensis found that this microorganism cannot utilize any carbohydrates and relies on fatty acids and alcohols as carbon sources. This further suggests that the two skin types may constitute two communities with different nutritional requirements.

[0240] Since the skin microenvironment is the growth environment for skin microorganisms and determines the nutrients available to them, we further analyzed the phenotypic differences between the two skin types to explore whether skin phenotype is a driving factor for different skin types. Please refer to [link / reference]. Figure 7 The results showed significant differences between the two skin types in terms of stratum corneum moisture, oil content, and skin tone. In contrast, C-Cutotype had higher oil and water content, while M-Cutotype skin was drier. Since oil is a primary nutrient source for microorganisms, these results further suggest that differences in nutritional needs are a driving factor behind the different skin types.

[0241] Furthermore, the skin phenotype of M-Cutotype is similar to that of older adults; therefore, we compared whether there were age differences between the two skin types. Please refer to [link / reference needed]. Figure 8 As expected, the M-Cutotype group was significantly older than the C-Cutotype group. However, further analysis revealed the presence of both C-Cutotype and M-Cutotype across different age groups; that is, C-Cutotype was present in older individuals, while M-Cutotype was present in younger individuals. Therefore, we hypothesize that age is not the true determining factor, but rather nutritional requirements are the direct determinant. The age difference may be due to changes in host physiology during aging affecting skin phenotype, leading to a later age of M-Cutotype.

[0242] Example 4: Correlation between Moraxella osloensis and skin aging phenotypes

[0243] This study collected skin microbiota from three areas—forehead (Fh), cheek (Ch), and nasal side (Ns)—from 248 healthy female residents of Shanghai. Correlation analysis was performed on *Moraxella osloensis* with age and skin phenotype. Spearman coefficients were calculated, and p-values ​​were corrected using the FDR method. A corrected p-value less than 0.05 was used as the screening criterion. Please refer to [reference needed]. Figure 9 The results showed that the levels of *Moraxella osloensis* in all three locations were significantly positively correlated with age and significantly negatively correlated with porphyrins. Furthermore, it was positively correlated with facial spots and negatively correlated with facial stratum corneum moisture and oil content, as well as forehead oil content. Additionally, *Moraxella osloensis* also showed a trend of weak positive correlation with other aging phenotypes.

[0244] Example 5 Potential targets for M bacteria in skin aging

[0245] In M-Cutotype, we observed enrichment of isocitrate lyase (aceA) and malate synthase (aceB) genes in the skin microbiome. The functions of these genes are related to the glyoxylate cycle, which has been shown to participate in the catabolism of ethoxy groups. These results provide a basis for the involvement of skin microbes in the degradation of octylphenol polyoxyethylene ethers (OPEs). The alkylphenols and short polyoxymethylene metabolites formed from OPE degradation have endocrine-disrupting activities. Among them, alkylphenol ethoxylates (APEs) exhibit estrogen-like activity; experiments have shown that these compounds can mimic the effects of estradiol both in vivo and in vitro, and are thus referred to as environmental estrogens. Therefore, skin microbes may interfere with estradiol production, and estradiol is crucial for preventing and treating skin aging (please refer to...). Figure 10 ).

[0246] 1. Please refer to Figure 11 Gene function differential analysis showed that M-Cutotype was enriched in the β-carotene synthesis pathway, suggesting that M-Cutotype may synthesize more β-carotene. β-carotene is associated with yellowing of the skin.

[0247] 2. We used M bacteria culture medium to stimulate keratinocytes and used transcriptomics to explore the potential senescence mechanism of M bacteria.

[0248] RNA-Seq signaling pathway enrichment analysis of differentially expressed genes suggests that *Moraxella osloensis* can affect skin cells through signaling pathways, primarily involving the regulation of collagen synthesis and degradation. Please refer to [link / reference needed]. Figure 12 RNAseq results indicated that differentially expressed genes were highly enriched in biological processes strongly associated with aging phenotypes, such as collagen metabolism and extracellular matrix decomposition.

[0249] Example 6: Method for adjusting the level of Moraxella osloensis

[0250] In the preceding analysis, it was known that *Moraxella osloensis* is associated with age and skin aging phenotypes. This study used a single skin surface compound to incubate *Moraxella osloensis*, and by examining the number of viable bacteria, it reflected the microorganism's utilization of different skin surface compounds and the toxicity of specific compounds to *Moraxella osloensis*. Ultimately, by adjusting the amount of compounds preferred or toxic to *Moraxella osloensis*, its growth can be regulated, thereby achieving the goal of delaying aging.

[0251] Please refer to Figure 13 This study investigated the effects of 32 skin surface compounds on the growth of *Moraxella osloensis* using two methods: CCK-8 and Dye mix A. The compounds were: L-lysine, L-glutamine, L-histidine, L-arginine, taurine, creatine, D-glucose, L-lactic acid, glycerol, 2-carboxybenzaldehyde, urea, SDS, L-threonine, L-tryptophan, glycine, L-methionine, L-serine, L-glutamic acid, L-phenylalanine, L-cysteine, L-tyrosine, L-leucine, and L-isoleucine.

[0252] The compounds included L-ornithine hydrochloride, L-citrulline, L-proline, L-valine, L-alanine, D-aspartic acid, trans-4-hydroxy-L-proline, uric acid, and taurine. Among these, L-glutamine, L-histidine, L-serine, and L-proline significantly promoted the growth of *Moraxella osloensis*, while SDS significantly inhibited its growth.

[0253] Example 7: Detection of populations in other countries

[0254] To verify whether the obtained skin types are widespread, we downloaded multiple publicly available datasets and verified the existence of skin types based on skin microbiome data from different ethnic groups, different body parts, and different health conditions.

[0255] First, we used metagenomic data on the skin of the elbow crease (moist type) of Singaporean Chinese, which included both patients with atopic dermatitis (AD) and healthy individuals. Please refer to [the relevant source]. Figure 14 The results showed that the data samples could be effectively divided into two categories, consistent with previous results: one category was enriched with Propionibacterium acnes, and the other was enriched with Moraxella osloensis. This result suggests that the existence of skin types is not affected by skin health status or location.

[0256] Furthermore, we used publicly available skin metagenomic data on psoriasis in Filipino children (scalp and neck) and Italians, and found consistent results. Please refer to [link / reference needed]. Figure 15The typing results showed that the population could be effectively divided into two categories: C-Cutotype and M-Cutotype.

[0257] In summary, this invention demonstrates that skin types are widespread and are not affected by skin location, race, or health status.

[0258] Example 8: Skin Condition Classification System

[0259] Based on the foregoing embodiments, we have developed a skin condition typing system. The system includes a feature receiving module, a calculation and processing module, and a result output module. These modules are connected via wired or wireless means. The typing steps are as follows:

[0260] (a) Skin microbial samples were collected from the subject's face and tested to generate skin sample characteristic data. The skin sample characteristic data consisted of the levels of *Moraxella osloensis* and *Propionibacterium acnes*, respectively. The collection site was the same as described in Example 1, and the detection method was as described in the detection method.

[0261] (b) Input the skin sample feature data into the system from the feature receiving module.

[0262] (c) The processing module receives skin sample feature data from the feature receiving module, calculates the quantified proportions of *Moraxella osloensis* and *Propionibacterium acnes*, or their relative proportions, and compares these proportions with standard values ​​for skin typing or feature characterization to arrive at a skin typing and / or skin condition determination.

[0263] (d) Result output module, which can be any terminal, such as a monitor, printer, tablet computer (PAD), smartphone, etc., for receiving and outputting the judgment result.

[0264] The system includes a storage device that stores threshold information for standard values.

[0265] The standard values ​​corresponding to the skin type and / or skin condition are:

[0266] Skin type C (or type I): When the ratio (M / C) of the level (e.g., content) of *Moraxella osloensis* to the level (e.g., content) of *Propionibacterium acnes* (C) meets the following conditions: M / C ≤ 1.3, preferably M / C ≤ 0.8, more preferably M / C ≤ 0.4. Skin condition of this phenotype: oily, with higher water content and better skin elasticity.

[0267] Skin type M (Type III): When the ratio (M / C) of the level (e.g., content) of *Moraxella osloensis* (M) to the level (e.g., content) of *Propionibacterium acnes* (C) in the sample meets the following conditions: M / C ≥ 0.5, preferably M / C ≥ 1.8, more preferably M / C ≥ 2.2. Skin condition of this phenotype: poor oiliness, poor water content, poor skin elasticity, and high degree of skin aging.

[0268] Skin type is mixed (or type II): when the ratio (M / C) of the level (e.g., content) of *Moraxella osloensis* (M) to the level (e.g., content) of *Propionibacterium acnes* (C) meets the following conditions: 0.3 ≤ M / C ≤ 2.5, preferably 0.3 ≤ M / C ≤ 2.3, more preferably 0.8 ≤ M / C ≤ 1.8. This phenotype represents a skin condition between type C and type M.

[0269] Example 9: Correlation analysis between bacteria and skin phenotype

[0270] Correlation analysis was performed using R software to assess the correlation between strain level and phenotype of *Moraxella osloensis*, *Propionibacterium acnes*, and *Staphylococcus epidermidis* at the species level and three facial sites (cheek, forehead, and side of the nose) of the host skin, including sebum content, stratum corneum water content, transepidermal water loss, skin pH, pigmentation, porphyrins, skin color, and pores.

[0271] The results showed that Figure 16 The correlations between bacterial abundance and phenotype across the three sites were consistent: *Moraxella osloensis* was positively correlated with age and skin darkening / yellowing, and negatively correlated with skin hydration, sebum, and porphyrin content. *Propionibacterium acnes* showed the opposite trend to *Moraxella osloensis*. *Staphylococcus epidermidis* was negatively correlated with pigmentation and positively correlated with epidermal hydration. These findings suggest that these three bacteria are associated with skin aging.

[0272] Example 10: Molecular-level verification of skin cell treatment with bacterial supernatant

[0273] Supernatants from *Moraxella osloensis*, *Propionibacterium acnes*, and *Staphylococcus epidermidis* were used to treat keratinocytes (HaCaT cell line), the most abundant cells in the host epidermis. qPCR was used to investigate changes in cell expression profiles after incubation, focusing primarily on matrix metalloproteinases (MMPs), genes associated with collagen degradation and extracellular matrix assembly. Notably, MMPs are responsible for degrading extracellular matrix (ECM) proteins, promoting photoaging. Molecular-level validation was used to explore the association between bacteria and host skin phenotype.

[0274] qPCR results showed that Figure 17-19In HaCaT cells treated with *Moraxella osloensis* bacterial supernatant, the expression levels of MMP1, MMP10, MMP12, and MMP13 were significantly increased compared to the control group. However, no significant difference in MMP expression levels was observed after treatment with *Propionibacterium acnes* and *Staphylococcus epidermidis* supernatant. This indicates that *Moraxella osloensis* can promote host skin aging, and also suggests that the skin microbiome's promotion of skin aging is not a universal effect, but rather an effect specific to certain strains.

[0275] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims. sequence list <110> Fudan University <120> Biomarker Combinations for Skin Typing and Their Applications <130> P2021-3155 <150> CN202011444009.3 <151> 2020-12-08 <160> 8 <170> PatentIn version 3.5 <210> 1 <211> twenty one <212> DNA <213> artificial sequence <400> 1 ggggctttga tgtaccctag c 21 <210> 2 <211> twenty one <212> DNA <213> artificial sequence <400> 2 tgtcacacgc ttttggggtt t 21 <210> 3 <211> twenty three <212> DNA <213> artificial sequence <400> 3 ctctggagta atgtcacacc tct 23 <210> 4 <211> twenty two <212> DNA <213> artificial sequence <400> 4 tgttggtcca cctttcatct tc 22 <210> 5 <211> twenty one <212> DNA <213> artificial sequence <400> 5 catgaaccgt gaggatgttg a 21 <210> 6 <211> 20 <212> DNA <213> artificial sequence <400> 6 gcatgggcta ggattccacc 20 <210> 7 <211> twenty one <212> DNA <213> artificial sequence <400> 7 ccagacttca cgatggcatt g 21 <210> 8 <211> twenty two <212> DNA <213> artificial sequence <400> 8 ggcatctcct ccataatttg gc 22

Claims

1. The use of a combination of a test reagent for Moraxella osloi and a test reagent for Propionibacterium acnes, characterized in that, For preparing a reagent or a kit, which is used for (a) skin typing; and / or (b) judging or characterizing skin condition; Among them, the skin is typed according to the relative value of the level of **Moraxella osloensis** (M) to the level of **Propionibacterium acnes** (C), or the skin condition of a sample is judged; the skin condition includes: skin moisture content, skin elasticity, skin color, skin aging degree; the skin typing is to divide the skin into type M, mixed type or type C; among them, the characteristic of type M is a high-level aggregation of **Moraxella osloensis**, and the characteristic of type C is a high-level aggregation of **Propionibacterium acnes**.

2. The use as described in claim 1, characterized in that, For a sample from the cheek, when the ratio (M / C) of the level of **Moraxella osloensis** (M) to the level of **Propionibacterium acnes** (C) meets the following conditions: M / C ≤ 0.75, the skin type is type C, and its phenotype includes high oil content, high water content, good skin elasticity, low skin aging degree, and bright skin color; when the ratio (M / C) of the level of **Moraxella osloensis** (M) to the level of **Propionibacterium acnes** (C) meets the following conditions: 0.75 < M / C < 1.68, the skin type is a mixed type, and its phenotype includes: medium oil content, medium water content, average skin elasticity, medium skin aging degree, and medium skin color; when the ratio (M / C) of the level of **Moraxella osloensis** (M) to the level of **Propionibacterium acnes** (C) meets the following conditions: M / C ≥ 1.68, the skin type is type M, and its phenotype includes low skin oil content, low water content, poor skin elasticity, high skin aging degree, and dull skin color.

3. The use as described in claim 1, characterized in that, For a sample from the forehead, when the ratio (M / C) of the level of **Moraxella osloensis** (M) to the level of **Propionibacterium acnes** (C) meets the following conditions: M / C ≤ 1.24, the skin type is type C, and its phenotype includes high oil content, high water content, good skin elasticity, low skin aging degree, and bright skin color; when the ratio (M / C) of the level of **Moraxella osloensis** (M) to the level of **Propionibacterium acnes** (C) meets the following conditions: 1.24 < M / C < 2.18, the skin type is a mixed type, and its phenotype includes: medium oil content, medium water content, average skin elasticity, medium skin aging degree, and medium skin color; when the ratio (M / C) of the level of **Moraxella osloensis** (M) to the level of **Propionibacterium acnes** (C) meets the following conditions: M / C ≥ 2.18, the skin type is type M, and its phenotype includes low skin oil content, low water content, poor skin elasticity, high skin aging degree, and dull skin color.

4. The use as described in claim 1, characterized in that, When the ratio (M / C) of the level (M) of the Moraxella osloensis to the level (C) of the Propionibacterium acnes in a sample from the nasal alae region meets the following conditions: M / C ≤ 0.34, the skin type is type C, and its phenotypes include high oil content, high water content, good skin elasticity, low skin aging degree, and bright skin color; when the ratio (M / C) of the level (M) of the Moraxella osloensis to the level (C) of the Propionibacterium acnes meets the following conditions: 0.34 < M / C < 0.55, the skin type is mixed, and its phenotypes include: medium oil content, medium water content, average skin elasticity, medium skin aging degree, and medium skin color; when the ratio (M / C) of the level (M) of the Moraxella osloensis to the level (C) of the Propionibacterium acnes meets the following conditions: M / C ≥ 0.55, the skin type is type M, and its phenotypes include low skin oil content, relatively low water content, poor skin elasticity, high skin aging degree, and dull skin color.

5. A system for classifying and / or determining the skin condition of a test subject, characterized in that, The system includes: (a) A feature receiving module for receiving skin sample feature data; the feature data includes: the quantitative information of Moraxella osloensis (M) and Propionibacterium acnes (C) in the skin sample respectively; (b) A calculation and processing module for calculating the feature data from the feature receiving module to obtain the proportional relationship of each feature, where the proportional relationship is the relative value M / C of the level (M) of Moraxella osloensis and the level (C) of Propionibacterium acnes in the skin sample; and based on the obtained proportional relationship, comparing it with the standard value of skin typing or feature characterization to obtain the judgment result of skin typing and / or skin state; and (c) A result output module for receiving and outputting the judgment result; Among them, the skin state includes: skin water content, skin elasticity, skin color, skin aging degree; the skin typing is to classify the skin into type M, mixed type or type C; among them, the characteristics of type M are high-level aggregation of Moraxella osloensis, and the characteristics of type C are high-level aggregation of Propionibacterium acnes.

6. The system as described in claim 5, characterized in that, The methods for obtaining the quantitative information include: sequencing, PCR, and protein quantitative detection.

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