WO2022121864A1

WO2022121864A1 - Marker combination for skin typing and use thereof

Info

Publication number: WO2022121864A1
Application number: PCT/CN2021/135870
Authority: WO
Inventors: 王久存; 夏晶晶; 克鲁德曼·吉恩; 刘晓; 钟倩; 李志明
Original assignee: 复旦大学
Priority date: 2020-12-08
Filing date: 2021-12-06
Publication date: 2022-06-16
Also published as: CN114622024A; KR20230118928A; JP2023552837A

Abstract

Provided in the present invention is the use of Moraxella osloensis, which is used for skin typing, characterizing skin conditions or for preparing a reagent or a kit for characterizing skin conditions. Further provided in the present invention is a marker combination, comprising the following markers: Moraxella osloensis and Propionibacterium acnes. Also provided in the present invention is the use of the marker combination in skin typing and/or judgment of skin conditions.

Description

Marker combination for skin typing and its application

technical field

The present invention relates to the field of biomedicine, in particular to a marker combination for skin typing and its application.

Background technique

With the introduction of the Microbiome Project, people have gradually realized that, similar to the gut, there are also a large number of microorganisms on the surface of the human body. Intestinal studies have suggested that microbiota-host interactions and strategies through fecal transplantation, probiotics, and prebiotics can alter the gut microbiome to improve gut homeostasis. The research progress of skin microecology is relatively lagging behind, especially the metagenomic data of large-scale population is lacking. Metagenomic data based on North American populations suggest that skin microbes have large individual differences in composition but high stability, that is, individuals whose microbial composition changes over time and space are relatively stable and have strong personal characteristics. Based on the data of the Chinese Han population, this study once again confirmed the existence of individual differences, and found that the microbial composition was significantly associated with a series of skin phenotypes of the host. Skin microbes can be used as potential new means and new targets to improve skin phenotypes.

However, the strong individual differences pose severe challenges to the development and design of universal skin care products and medicines. Through further statistical analysis, we found that although there are large individual differences, there are still some main characteristics and laws at the population level. The combination of these characteristics can classify the population and solve the problem of large individual differences. Skin typing is well suited for low resolution "precision"/"personalized" care. For example, one of the commonly used skin classifications - "dry", "normal", "oily" skin, is classified according to the water and oil characteristics of the host skin; similar to this, but the difference is that our Instead of water-oil signatures, the study typed populations based on skin microbiome signatures. This will help the industry to develop skin care products and medicines that are more suitable for specific groups of people for different skin types, achieve a certain degree of "precise"/"personalized" care, and effectively improve skin stability.

Therefore, there is an urgent need in the art to develop new methods for skin typing and judging skin aging, and to provide a new diagnostic and typing method.

SUMMARY OF THE INVENTION

The purpose of the present invention is to classify the population based on the characteristics of the skin microbiome, thereby helping the industry to develop skin care products and medicines that are more suitable for specific populations for different skin types. Provide a new method for skin typing and judging skin state, and provide a new diagnostic and typing method.

The present invention will focus on the two bacteria that dominate skin typing, Moraxella osloensis and Cutibacterium acnes, as well as skin-abundant bacteria that play an important role in host health and disease. on one of the bacterial colonies of the species, Staphylococcus epidermidis. First, in the metagenomic data of large-scale samples of the Han population, the correlation between the distribution abundance of three target skin symbionts on the face and the host skin phenotype was determined. The bacteria that promote skin aging - M. osloensis.

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

In another preferred example, the skin condition includes: skin age, skin moisture content, skin elasticity, skin color, and skin aging degree.

In another preferred embodiment, the aging degree of the skin is judged based on one or more phenotypes selected from the group consisting of porphyrin, oil, water content, gloss, pore area, skin yellowness, pore size, and pigmentation.

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

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

In another preferred embodiment, the reagent or kit further comprises detection of Propionibacterium avidum, Propionibacterium granulosum, Staphylococcus, Propionibacterium acnes and/or Staphylococcus phage reagent.

In another preferred embodiment, the reagent or kit further includes a reagent for detecting Staphylococcus epidermidis (S. epidermidis).

A second aspect of the present invention provides a marker combination comprising M. osloensis and C. acnes.

In another preferred embodiment, the marker combination further includes Moraxella bovoculi and/or Psychrobacter sp..

In another preferred embodiment, the marker combination further includes Propionibacterium avidum, Propionibacterium granulosum, Staphylococcus, Propionibacterium acnes and/or Staphylococcus phage.

In another preferred embodiment, the marker combination further includes Staphylococcus epidermidis (S. epidermidis).

In another preferred embodiment, the marker combination is used for (a) skin typing; and/or (b) judging skin state.

In another preferred embodiment, the marker or marker combination is derived from a skin sample, preferably from whole body skin or facial skin, more preferably from cheek, forehead, and nose.

In another preferred embodiment, the marker or marker combination is derived from skin samples of Asian population.

In another preferred embodiment, the marker or marker combination is derived from cheek, forehead, and alar samples.

In another preferred embodiment, the level of each marker in the marker combination is detected by one or more methods of the following group: sequencing, PCR, protein quantitative detection.

In another preferred embodiment, the method for detecting the level of the marker group further includes one or more methods selected from the group consisting of: quantitative PCR for characteristic genes, qPCR, real-time quantitative PCR, metagenomics analysis, 16s RNA sequencing , mass spectrometry, and western blotting.

In another preferred embodiment, the ratio (M/ C) When the following conditions are met: 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), and its phenotype includes: oil High content, high water content, good skin elasticity, low skin aging, bright skin color.

In another preferred embodiment, the ratio (M/ C) When the following conditions are met: 0.3≤M/C≤2.5, preferably, 0.3≤M/C≤2.3, more preferably, 0.8≤M/C≤1.8, then the skin type is mixed (or type II) , and its phenotypes include: medium oil content, medium water content, average skin elasticity, moderate skin aging, and medium skin color.

In another preferred embodiment, the ratio (M/ C) When the following conditions are met: M/C≥0.5, preferably, M/C≥1.8, more preferably, M/C≥2.2, then the skin type is M type (type III), and its phenotype includes skin oil Low content, low water content, poor skin elasticity, high degree of skin aging, dull skin color.

In another preferred example, from the cheek sample, the ratio (M/C) of the level (eg content) (M) of the Moraxella oslo to the level (eg content) (C) of the P. acnes ) when the following conditions are met: M/C≤0.8, preferably, M/C≤0.75, then the skin type is C type (or type I), and its phenotype includes high oil content, high water content, good skin elasticity, Low level of skin aging, bright skin color.

In another preferred example, from the cheek sample, the ratio (M/C) of the level (eg content) (M) of the Moraxella oslo to the level (eg content) (C) of the P. acnes ) when the following conditions are met: 0.75≤M/C≤2, preferably, 0.8≤M/C≤1.7, the skin type is mixed (or type II), and its phenotype includes: medium oil content, medium water content , The skin elasticity is average, the skin aging degree is moderate, and the skin color is moderate.

In another preferred example, from the cheek sample, the ratio (M/C) of the level (eg content) (M) of the Moraxella oslo to the level (eg content) (C) of the P. acnes ) when the following conditions are met: M/C≥1.7, the skin type is M type (type III), and its phenotypes include low skin oil content, low water content, poor skin elasticity, high skin aging, and dull skin color. .

In another preferred embodiment, for the sample from the forehead, the ratio (M/ C) When the following conditions are met: M/C≤1.5, preferably, M/C≤1.25, the skin type is type C (or type I), and its phenotype includes high oil content, high water content, and good skin elasticity , Low degree of skin aging, bright skin color.

In another preferred example, from the forehead sample, the ratio (M/C) of the level (eg content) (M) of the Moraxella oslo to the level (eg content) (C) of the P. acnes ) when the following conditions are met: 1.25≤M/C≤2.5, preferably, 1.3≤M/C≤2.2, the skin type is mixed (or type II), and its phenotype includes: medium oil content, medium water content, Normal skin elasticity, moderate skin aging, and moderate skin color.

In another preferred example, from the forehead sample, the ratio (M/C) of the level (eg content) (M) of the Moraxella oslo to the level (eg content) (C) of the P. acnes ) when the following conditions are met: M/C≥2, preferably ≥2.2, the skin type is M type (type III), and its phenotype includes low skin oil content, low water content, poor skin elasticity, and skin aging. High degree, dull skin color.

In another preferred example, for the sample derived from the nasal wing, the ratio (M/ C) When the following conditions are met: 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 level of skin aging, bright skin color.

In another preferred example, the ratio (M/C) of the level (such as content) (M) of the Moraxella oslo to the level (such as content) (C) of the P. acnes is derived from a sample from the nose wing ) when the following conditions are met: 0.35≤M/C≤0.7, preferably, 0.35≤M/C≤0.6, more preferably, 0.35≤M/C≤0.55, the skin type is mixed type (or type II), which Phenotypes include: moderate oil content, moderate moisture content, moderate skin elasticity, moderate skin aging, and moderate skin color.

In another preferred example, the ratio (M/C) of the level (such as content) (M) of the Moraxella oslo to the level (such as content) (C) of the P. acnes is derived from a sample from the nose wing ) when the following conditions are met: M/C≥0.5, preferably, M/C≥0.55, the skin type is M type (type III), and its phenotype includes low skin oil content, low water content, poor skin elasticity, High degree of skin aging, dull skin color.

A third aspect of the present invention provides a method for skin typing or judging skin state, the method comprising:

(1) providing a sample derived from the skin of the subject to be tested, and detecting the level (such as content) of each marker in the sample marker combination, the combination including the following markers: Moraxella oslo and Propionibacterium acnes, Obtain the level (such as content) of Moraxella oslo (M) and the level (such as content) of P. acnes (C), respectively;

(2) Based on the level (such as content) (M) of Moraxella oslo, or comparing the level (such as content) (M) of Moraxella oslo in the sample with the level (such as content) of P. acnes ( C) to compare, so as to classify the skin of the object to be tested, and/or to judge the skin state.

In another preferred embodiment, the subject to be tested is from an Asian population.

In another preferred example, in step (2), according to the relative value (such as M/C) of the level (such as content) (M) of Moraxella oslo and the level (such as content) (C) of P. acnes Type the skin, or determine the skin condition of a sample.

In another preferred example, the skin condition includes skin age, skin moisture content, skin elasticity, skin color, and skin aging degree.

In another preferred embodiment, the level (eg content) (M) of Moraxella oslo and the level (eg content) of P. acnes in the sample of the subject to be tested by one or more methods selected from the following group (C) Confirmation: sequencing, PCR, protein quantitative detection.

In another preferred embodiment, the method for detecting the level (eg content) (M) of Moraxella oslo and the level (eg content) (C) of Propionibacterium acnes in the sample further comprises a method selected from the group consisting of: One or more methods: quantitative PCR for signature genes, qPCR, real-time quantitative PCR, metagenomics analysis, 16s RNA sequencing, mass spectrometry, Western blotting.

In another preferred example, the ratio (M/C) of the Moraxella oslo (M) to the level (such as content) (C) of the P. acnes 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 C type (or type I), and its phenotype includes: high oil content, high water content, Good skin elasticity, low skin aging, bright skin color.

In another preferred example, the ratio (M/C) of the level (eg content) (M) of the Moraxella oslo in the sample to the level (eg content) (C) of the P. acnes When the following conditions are met: 0.3≤M/C≤2.5, preferably, 0.3≤M/C≤2.3, more preferably, 0.8≤M/C≤1.8, then the skin type is mixed (or type II), and its Phenotypes include: moderate oil content, moderate moisture content, moderate skin elasticity, moderate skin aging, and moderate skin color.

In another preferred example, the ratio (M/C) of the level (eg content) (M) of the Moraxella oslo in the sample to the level (eg content) (C) of the P. acnes When the following conditions are met: M/C≥0.5, preferably, M/C≥1.8, more preferably, M/C≥2.2, the skin type is M type (type III), and its phenotype includes low skin oil content , low water content, poor skin elasticity, high degree of skin aging, dull skin color.

In another preferred embodiment, the sample is derived from the cheek, and the ratio of the level (such as content) (M) of Moraxella oslo in the sample to the level (such as content) (C) of Propionibacterium acnes ( When M/C) meets the following conditions: M/C≤0.8, preferably, M/C≤0.75, then the skin type is C type (or type I), and its phenotype includes high oil content, high water content, skin Good elasticity, low skin aging, bright skin color.

In another preferred embodiment, the sample is derived from the cheek, and the ratio of the level (such as content) (M) of Moraxella oslo in the sample to the level (such as content) (C) of Propionibacterium acnes ( When M/C) meets the following conditions: 0.75≤M/C≤2, preferably, 0.8≤M/C≤1.7, then the skin type is mixed type (or type II), and its phenotype includes: medium oil content, Moderate moisture content, average skin elasticity, moderate skin aging, and moderate skin tone.

In another preferred embodiment, the sample is derived from the cheek, and the ratio of the level (such as content) (M) of Moraxella oslo in the sample to the level (such as content) (C) of Propionibacterium acnes ( When M/C) meets the following conditions: M/C≥1.7, the skin type is M type (type III), and its phenotypes include low skin oil content, low water content, poor skin elasticity, high skin aging, and skin Dull in color.

In another preferred example, for a sample from the forehead, the ratio of the level (eg content) (M) of Moraxella oslo in the sample to the level (eg content) (C) of Propionibacterium acnes (M/C) When the following conditions are met: M/C≤1.5, preferably, M/C≤1.25, then the skin type is C type (or type I), and its phenotype includes high oil content, high water content, Good skin elasticity, low skin aging, bright skin color.

In another preferred embodiment, the sample is derived from the forehead, the ratio of the level (eg content) (M) of Moraxella oslo in the sample to the level (eg content) (C) of Propionibacterium acnes ( M/C) when the following conditions are met: 1.25≤M/C≤2.5, preferably, 1.3≤M/C≤2.2, the skin type is mixed (or type II), and its phenotype includes: medium oil content, high The amount of water is medium, the skin elasticity is average, the skin aging is medium, and the skin color is medium.

In another preferred embodiment, the sample is derived from the forehead, the ratio of the level (eg content) (M) of Moraxella oslo in the sample to the level (eg content) (C) of Propionibacterium acnes ( When M/C) meets the following conditions: M/C ≥ 2, preferably ≥ 2.2, the skin type is M type (type III), and its phenotype includes low skin oil content, low water content, and poor skin elasticity , High degree of skin aging, dull skin color.

In another preferred example, for a sample derived from the nose wing, the ratio of the level (eg content) (M) of Moraxella oslo in the sample to the level (eg content) (C) of Propionibacterium acnes (M/C) When the following conditions are met: 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, skin Good elasticity, low skin aging, bright skin color.

In another preferred embodiment, it is derived from a sample from the nose wing, and the ratio of the level (such as content) (M) of Moraxella oslo in the sample to the level (such as content) (C) of Propionibacterium acnes ( When M/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, the skin type is mixed (or type II) ), and their phenotypes include: medium oil content, medium water content, average skin elasticity, moderate skin aging, and medium skin color.

In another preferred embodiment, it is derived from a sample from the nose wing, and the ratio of the level (such as content) (M) of Moraxella oslo in the sample to the level (such as content) (C) of Propionibacterium acnes ( M/C) when the following conditions are met: M/C≥0.5, preferably, M/C≥0.55, the skin type is M type (type III), and its phenotype includes low skin oil content, low water content, skin Poor elasticity, high degree of skin aging, dull skin color.

In another preferred example, the relative value meets the following conditions: when M/C≤1.3, the skin is type C, and when M/C≥0.5, the skin is type M.

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

In another preferred example, when the level (such as content) (M) of the Moraxella oslo increases, it indicates that the skin condition is increased skin age, dark and yellow complexion, reduced skin moisture content, sebum and porphyrin content reduce.

The fourth aspect of the present invention provides a reagent combination for skin typing and/or skin condition detection, the reagent combination comprising reagents for detecting each marker in the combination described in the second aspect of the present invention.

In another preferred embodiment, the reagent is used to detect the level (eg content) of each marker.

In another preferred embodiment, the reagent includes a substance for detecting the level of each marker in the combination described in the second aspect of the present invention by one or more methods selected from the group consisting of sequencing, PCR, and quantitative protein detection.

In another preferred embodiment, the method for detecting the marker level further includes: quantitative PCR of characteristic genes, qPCR, real-time quantitative PCR, metagenomic analysis, 16s RNA sequencing, mass spectrometry analysis, and western blotting.

In another preferred embodiment, the reagent combination includes:

A first detection reagent for detecting the level (M) of Moraxella oslo; and/or

A second detection reagent for detecting the level of P. acnes (C).

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

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

A sixth aspect of the present invention provides a system for classifying the skin of an object to be tested and/or judging the skin state of the object to be tested, the system comprising:

(a) a feature receiving module, the feature receiving module is used for receiving skin sample feature data; the feature data includes: the quantitative information of each of Moraxella oslo (M) and P. acnes (C) in the skin sample ;

(b) a calculation processing module for calculating the feature data from the feature receiving module, so as to obtain the respective proportions of the respective features or the proportional relationship between the respective features; and based on the obtained respective proportions or the relationship between the respective features A proportional relationship, compared with a standard value for skin type or characterization, resulting in a judgment of skin type and/or skin condition; and

(c) a result output module, the output module is used for receiving and outputting the judgment result.

In another preferred embodiment, the subject is a human.

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

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

In another preferred embodiment, the subject includes infants, adolescents or adults.

In another preferred embodiment, the quantitative information includes the respective levels (eg content) of Moraxella oslo (M) and Propionibacterium acnes (C).

In another preferred embodiment, the proportional relationship includes the relative values of the respective levels (eg contents) of Moraxella oslo (M) and Propionibacterium acnes (C) in the skin sample, such as M/C.

In another preferred embodiment, the system can be divided into at least two types according to the skin condition.

In another preferred embodiment, the method for obtaining the quantitative information includes: sequencing, PCR, and quantitative protein detection.

In another preferred embodiment, the method for obtaining the quantitative information further includes: quantitative PCR for characteristic genes, qPCR, real-time quantitative PCR, metagenomics analysis, 16s RNA sequencing, mass spectrometry analysis, and western blotting.

In another preferred embodiment, the feature receiving module includes a sample collector and a feature signal input end.

In another preferred embodiment, the calculation processing module includes a processor and a storage, wherein the storage stores threshold information of skin type and/or skin state.

In another preferred embodiment, the output module includes any terminal, preferably a display, a printer, a tablet computer (PAD), and a smart phone.

In another preferred embodiment, the modules are connected in a wired or wireless manner.

A seventh aspect of the present invention provides a method for screening substances or ingredients for improving skin condition, comprising:

(a) providing a screening bacteria, the screening bacteria is Moraxella oslo (M), P. acnes (C), or screening bacteria (mixed bacteria) comprising Moraxella oslo and/or P. acnes;

(b) co-culturing the substance or component to be screened with the screening bacteria, and detecting the respective levels (eg content) of Moraxella oslo or P. acnes; or between Moraxella oslo and P. acnes The relative level (such as relative content) (M/C);

(c) According to (b) the respective levels (such as content) of Moraxella oslo or P. acnes after culture; or the relative level (such as relative content) between Moraxella oslo and P. acnes (M/C ), thereby judging that the substance or ingredient to be screened is a substance or ingredient that improves skin condition.

In another preferred example, when the content of Moraxella oslo or the relative level (such as relative content) (M/C) between Moraxella oslo and P. acnes increases, it indicates the substance to be screened Or the ingredients are substances used to treat acne.

In another preferred example, when the level (such as content) of Moraxella oslo decreases or the relative level (such as relative content) (M/C) between Moraxella oslo and P. acnes decreases, it indicates that the The substances or ingredients to be screened are skin anti-aging substances.

In another preferred example, when the level (such as content) of P. acnes increases or the relative level (such as relative content) (C/M) between P. acnes and Moraxella oslo increases, it indicates that the The substances or ingredients to be screened are skin anti-aging substances.

In another preferred example, when the level (such as content) of P. acnes decreases or the relative level (such as relative content) (C/M) between P. acnes and Moraxella oslo decreases, it indicates that the The substance or ingredient to be screened is a substance for treating acne.

The eighth aspect of the present invention provides a use of the marker combination according to the second aspect of the present invention or the reagent combination according to the fourth aspect of the present invention, for preparing a kit for use in (a) skin typing; and/or (b) judging or characterizing skin condition.

The ninth aspect of the present invention provides the use of the combination of markers described in the second aspect of the present invention or the combination of reagents described in the fourth aspect of the present invention for screening substances or components that improve skin condition.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (eg, the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, it is not repeated here.

Description of drawings

Figure 1 shows a schematic diagram of the sampled skin site.

Figure 2 shows the microbial composition of the facial skin of the Han population.

Figure 3 shows the analysis of the optimal number of clusters for different parts.

Figure 4A and B show the clustering results of the forehead, where the box plots represent the average distance between samples in the two groups, respectively, and the red line represents the average distance between samples in different groups. 4A is the JS divergence (Jensen-Shannon divergence), 4B is the Bray-Curtis dissimilarity (Bray-Curtis dissimilarity).

Figure 4C,D show the relative levels of P. acnes or M. oslo on the forehead, with each dot representing a sample.

Figure 4E and F show the clustering results of cheeks, where the box plots represent the average distance between samples in the two groups, respectively, and the red line represents the average distance between samples in different groups. 4E is JS divergence (Jensen-Shannon divergence), 4F is Bray-Curtis dissimilarity (Bray-Curtis dissimilarity).

Figures 4G,H show relative levels of P. acnes or M. oslo in cheeks, with each dot representing a sample.

Figure 4I and J show the clustering results of the nose ala, where the box plots represent the average distance between samples in the two groups, respectively, and the red line represents the average distance between samples in different groups. 4I is the JS divergence (Jensen-Shannon divergence), 4J is the Bray-Curtis dissimilarity (Bray-Curtis dissimilarity).

Figure 4K,L show the relative levels of P. acnes or M. oslo in the nasal ala, with each dot representing a sample.

Figure 5 shows the differential microorganisms between different skin types, with colors representing the relative levels of microorganisms, one sample per column and one microorganism per row.

Figure 6 shows the microbial network characteristics of different skin types, with M-cutotype-enriched microbial species on the left and C-cutotype-enriched microbial species on the right. Each dot represents a species, and each color of the bouquet represents a species. It can be seen from the figure that the enriched microorganisms in one skin type are positively correlated with each other and negatively correlated with the enriched species of another skin type. Different skin types show different microbial network compositions.

Figure 7 shows the differences in functional enrichment of skin microbial genes for different skin types.

Figure 8 shows phenotypic differences between different skin types.

Figure 9 shows the correlation analysis between Moraxella oslo and age and skin phenotype: adjusted P value is less than 0.05.

Figure 10 shows that aceA/aceB genes are enriched in M-Cutotype.

Figure 11 shows that the beta-Carotene synthesis pathway is enriched for M-Cutotype.

Figure 12 shows the functional enrichment analysis of RNAseq differentially expressed genes in human keratinocyte HaCaT treated with Moraxella oslo supernatant and blank control group.

Figure 13 shows the utilization of various water-soluble carbon source compounds by Moraxella oslo. The left picture is measured by CCK-8 kit, and the right picture is measured by Dye mix A.

Figure 14 shows the results of skin type validation using Singapore Chinese skin metagenomic data.

Figure 15 shows the results of skin type validation using Philippine and Italian skin metagenomic data.

Figure 16 shows a heatmap of the correlation between species level and host phenotype of three dermatosymbionts.

Figure 17 shows a graph of Moraxella oslo-HaCaT-QPCR results.

Figure 18 shows a graph of P. acnes-HaCaT-QPCR results.

Figure 19 shows a graph of S. epidermidis-HaCaT-QPCR results.

Detailed ways

After extensive and in-depth research, the inventors found that Moraxella oslo can be used to characterize skin conditions or for skin typing for the first time, and the present invention also discovered a new marker combination for the first time: Moraxella oslo and C. acnes Acidobacter. The marker combination of the present invention can (a) classify the skin; and/or (b) judge the skin state, has the advantages of high sensitivity and high specificity, and has important application value. On this basis, the inventors have completed the present invention.

the term

Terms used herein have the meanings commonly understood by those of ordinary skill in the relevant art. However, for a better understanding of the present invention, some definitions and related terms are explained as follows:

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

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

According to the present invention, the term "individual" refers to animals, especially mammals, such as primates, preferably humans.

In accordance with the present invention, terms such as "a," "an," and "the" do not refer only to the singular, but include the general class that may be used to describe particular embodiments.

As used herein, when used in reference to a specifically recited value, the term "about" means that the value may vary by no more than 1% from the recited value. For example, as used herein, the expression "about 100" includes all values between 99 and 101 and (eg, 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the terms "containing" or "including (including)" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of," or "consisting of."

It should be noted that the explanations of terms provided here are only for the purpose of enabling those skilled in the art to better understand the present invention, rather than limiting the present invention.

Moraxella Osloensis, M.osloensis, hereinafter referred to as M bacteria

Moraxella oslo, Moraxella spp., bacilli, gram-negative, chemoorganotrophic bacteria. Can not use carbohydrates to produce acid.

Propionibacterium acnes, Cutibacterium acnes, C. acnes, hereinafter referred to as C bacteria

Propionibacterium acnes, a gram-positive bacillus, is a genus of Propionibacterium in the Actinomycete Actinomycete Actinomycetales. Important colonizer of human skin, involved in maintaining skin health, and can also act as a causative agent of acne vulgaris.

Staphylococcus epidermidis, Staphylococcus epidermidis, S. epidermidis

It is a gram-positive coccus that breeds on the epidermis of organisms. It exists in the skin, vagina and other parts of the human body. Because it often accumulates into grape clusters, it is named Staphylococcus epidermidis.

skin type

In the present invention, the skin can be further classified into M-Cutotype type (referred to as M type), mixed type, and C-Cutotype type (referred to as C type).

C-Cutotype

As used herein, the terms "C-Cutotype", "C-type" are used interchangeably and refer to a type of microbe-based skin typing characterized by high levels of aggregation of P. acnes (C. acnes).

M-Cutotype

As used herein, the terms "M-Cutotype", "M-type" are used interchangeably and refer to a type of microbe-based skin typing characterized by high levels of aggregation of M. osloensis.

skin elasticity

As used herein, skin elasticity depends on, but is not limited to, the adequacy of skin moisture, collagen, elastin, and natural fats, among others.

skin color

As used herein, skin color depends on, but is not limited to, skin radiance, skin tone, yellowness, and the like.

degree of skin aging

As used herein, skin aging depends on, but is not limited to, increased skin porphyrins, decreased skin moisture content and radiance, enlarged and enlarged pores, skin oil imbalance, and dull skin.

Detection method

Use a collector, such as a sterile cotton swab, to dip the bacterial solution, and repeatedly rub the bacterial sampling site to obtain a microbial sample. The level (such as content) that can characterize M bacteria or C bacteria is checked by common molecular biology methods. For example, the M/C ratio can be obtained in the following ways: 1. 16sRNA sequencing; 2. Metagenome sequencing; 3. Characteristics of two species Sequence design primers, and then use qPCR to obtain the M/C ratio; 4. Detect the specific expressed proteins or metabolites of the two bacteria for quantitative purposes, such as mass spectrometry analysis, Western blotting, etc.

Reagent test kit

In the present invention, the kit of the present invention includes the combination described in the second aspect of the present invention and/or the reagent combination described in the fourth aspect of the present invention.

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

The main advantages of the present invention include:

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

and /or (b) determine skin condition.

(3) The present invention discovers for the first time a new classification method that is different from the previous classification based on the host physiological drive (oily skin, dry skin, wet skin). The new classification method uses skin microorganisms as the classification basis. Three skin types with different characteristics. The analysis of these three skin types suggests that nutritional differences caused by our host physiology may be the driving factors for the different skin types. Moreover, the microbiome communities of different skin types may react against the host skin by exerting specific functions, which may have certain effects on skin health and skin appearance. Therefore, further research on skin type may contribute to the development of personalized medicine to better maintain skin health.

(4) The present invention finds for the first time the correlation between M bacteria and skin phenotype, which is positively correlated with age, and is related to some skin aging phenotypes, such as with the increase of the level of M bacteria, the skin oil decreases, the moisture content decreases, and the luster decreases. It is associated with some typical features of acne, such as increased M bacteria, decreased oil, decreased porphyrins (mostly metabolites of C bacteria, which can be pro-inflammatory), and decreased pore area .

(5) The present invention finds for the first time that by increasing the relative level (eg content) of P. acnes, M-type skin can be adjusted to C-type skin, which can be used for skin anti-aging.

(6) The present invention finds for the first time that by increasing the relative level (such as content) of Moraxella oslo, the C-type skin can be adjusted to the M-type skin, which is used for the treatment of acne.

(7) The present invention finds for the first time that the correlation analysis between single bacteria and skin phenotypes is carried out to explore whether skin microorganisms may cause changes in the skin phenotype of the host, and to provide new insights and new perspectives for the study of the interaction between microorganisms and the host.

(8) The present invention finds for the first time that the bacterial supernatant is used to treat the host epidermal cells to explore the interaction relationship between the bacteria and the host at the molecular level, and is not limited to correlation research.

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. In the following examples, the experimental methods without specific conditions are usually in accordance with conventional conditions, or in accordance with the conditions suggested by the manufacturer. Percentages and parts are by weight unless otherwise indicated.

Unless otherwise specified, the reagents and materials used in the examples of the present invention are all commercially available products.

general approach

1. Correlation analysis:

1) Skin microbial sample collection and metagenomic sequencing

The Shanghai resident population was recruited as volunteers for this study. All volunteers participating in this study will be examined by a dermatologist at Shanghai Dermatology Hospital to rule out skin lesions such as dermatitis, eczema, acne, psoriasis, infection, etc. Skin disease, while excluding volunteers treated with systemic or topical antibiotics within the past 6 months. Finally, 294 healthy subjects aged 20-65 were included, including 46 males (M) and 248 females (F) (Table 1).

Table 1 Subject demographic data

Subjects only washed their faces with water on the day of sampling, and avoided using any skin care products or cosmetics the day before sampling. The sampling site was maintained at an indoor temperature of 20 degrees Celsius and a humidity of 50%. The experimenter used a special sterile swab dipped in 0.15M NaCl and 0.1% Tween20 solution to repeatedly wipe the subject's forehead (Fh), cheeks (Ch) and paranasal (Ns) three parts about 4cm2 area, back and forth 20 times . Then, the swab was broken, placed in a 1.5ml sterile EP tube, and frozen at -80°C until the skin microorganism genomic DNA was extracted. The schematic diagram of the sampling part is shown in Figure 1.

The samples were amplified by the whole genome amplification method, followed by metagenomic sequencing, and finally the sequencing data of 822 facial skin microbial samples were obtained. The relative abundance of each gene was calculated by SOAP2 (version 2.21), and then the sum of the relative abundances of genes from the same strain was calculated according to the alignment result of each gene, which represented the relative abundance of the strain.

2) Skin phenotypic measurements

All phenotypic measurements on 294 Han subjects were performed in an indoor environment with a temperature of 20 degrees Celsius and a humidity of 50%. Before taking the test, the test subject sits and rests for more than 30 minutes to ensure that his blood circulation can return to normal levels after possible physical activity. The area where the phenotype was measured coincided with the microbial collection site. The following instruments (Table 2) were used to measure skin sebum content (sebum), stratum corneum water content (hydration), transepidermal water loss (TEWL), skin pH, porphyrin, skin color (L*a*) b) Phenotypes such as lentigines, pores_area, telangiectasia, and elasticity were measured.

Table 2 Phenotypic Measurement Instruments

3) Correlation analysis between bacterial abundance and skin phenotype

Based on the R software psych package (version 2.0.12), the species level and sebum content, stratum corneum water content, transepidermal water loss rate, skin pH value, pigmentation, Porphyrin, skin color, pores and other phenotypes were subjected to correlation analysis (Spearman Rank method) to evaluate the correlation between strain levels and phenotypes; FDR test was performed on the P value of the correlation analysis results, and the corrected P value <0.05 indicated The difference is statistically significant. Correlation heatmaps were drawn using the R package pheatmap (version 1.0.12) as the results. Blue, negative correlation; red: positive correlation; significant level of Spearman correlation: *, p<0.05; **, p<0.01; ***, p<0.001.

2. Matrix metalloproteinase (MMP) expression experiment:

1) Preparation of strain supernatant

i. Pick the frozen bacterial solution obtained from the skin, and use the three-zone streak method to plate to the corresponding petri dish for cultivation. When a single colony grows on the plate, pick a single colony to the corresponding medium for liquid culture;

ii. After culturing for 24 hours, extract genomic DNA from the bacterial culture solution, and perform 16S rDNA detection to confirm that the three bacterial culture solutions are still the target strains, and no contamination exists;

iii. The confirmed bacteria are cultured in liquid, and the pure medium without bacteria inoculation is set as the control group;

iv. Use a microplate reader to detect the absorbance at 600 nm of the cultured bacterial liquid at about 0.8;

v. Filter the three bacterial strains twice with a 0.22 μm filter to remove the bacterial cells and retain the bacterial supernatant. The same operation was performed on the pure medium control group;

vi. The obtained filtered bacterial supernatant and pure medium supernatant were stored at -80°C until use.

2) Incubation of HaCaT cells with bacterial supernatant

i. Culture human keratinocytes (HaCaT cells) 1×10 ⁶ . The bacterial liquid supernatant treatment group (experimental group) and the pure medium control group were set, and each group was repeated three times. Gently shake the culture plate to distribute the cells evenly, and place it into a 37°C, 5% CO ₂ cell incubator for culture. Table 3 is three kinds of bacteria used in the present invention;

ii. Continue to incubate in the incubator for 24 hours, discard the cell culture medium, and add PBS to rinse slightly. Then, trypsin was added to each dish, placed in a cell incubator for digestion for 5 min, centrifuged at 800 × g for 5 min, and the cells were collected.

Table 3 List of bacterial culture media

3) Extract cellular RNA

i. Add the collected HaCaT cells or primary fibroblasts to Trizol reagent and place at room temperature for 10 minutes;

ii. Add chloroform into the centrifuge tube, shake and mix, and let stand for 5 minutes;

iii. 13200rpm, 4°C, centrifuge for 10min, suck the upper aqueous phase (about 200μL) into another mL centrifuge tube;

iv. Add an equal volume of isopropanol, invert up and down to mix, and let stand for 5 minutes at room temperature;

v. 13200rpm, 4℃, centrifuge for 10min, discard the supernatant, white precipitate can be observed at the bottom of the tube;

vi. Add 75% ethanol (prepared with absolute ethanol and DEPC water, ready to use, and should be placed at -20°C to cool before use), shake gently to suspend the RNA precipitation at the bottom of the tube;

vii.13200rpm, 4℃, centrifuge for 10min, discard the supernatant; repeat the above two steps;

viii. Evacuate for 2 minutes, and use a pipette to absorb the residual ethanol as much as possible, open the lid of the centrifuge tube, and dry at room temperature for 5 minutes;

ix. After adding DEPC water to dissolve the RNA, use NanoDrop to determine the purity and concentration of RNA, and the RNA concentration of each sample is adjusted to 200ng/μL.

4) cDNA synthesis

i. In this experiment, the HiScript III RT SuperMix for qPCR (+gDNA wiper) kit of Vazyme Company was used for reverse transcription PCR to synthesize cDNA.

ii. Take a total of 1 μg of total RNA for reverse transcription reaction, and store the remaining RNA at -80°C;

iii. Prior to reverse transcription, possible confounding genomic DNA in the RNA sample will be removed first. The genomic DNA removal reaction mixture (RNA sample: 4×gDNA wiper Mix: RNase-free ddH ₂ O = 5:4:7) was prepared in an RNase-free eight-row tube, and mixed with a pipette. , and then add 5×HiScript qRT Super Mix with a volume ratio of 0.25 to mix.

iv. Place the aforementioned product in a PCR machine, 37°C, 15min; 85°C, 5s. When the reverse transcription process is complete, the product is used immediately in the qPCR reaction, or stored at -20°C.

5) Real-time quantitative PCR (Real-time PCR)

i. In this experiment, the QuantiFast SYBR Green PCR Kit of QIAGEN company was used for real-time quantitative PCR (Real-time PCR) to detect the relative expression level of the gene.

ii. Prepare the primers in Table 4 below with DEPC water to 2 μM, mix well, and set aside;

Table 4 Human primer sequences

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

iv. Place the 384-well plate in a centrifuge, 4°C, 3000 rpm, and centrifuge for 3 minutes. The 384-well plate was placed in the QuantStudioTM 7 Flex Real-Time PCR System for RT-PCR reaction, and the reaction program was as follows in Table 5;

Table 5 Real-time PCR program

v. Use QuantStudioTM Real-Time PCR Software to perform statistical processing on the experimental results, and further calculate the relative expression of genes in each sample according to the 2- ^△△T method. Finally, in the GraphPad Prism 5.0 software, the T-test test method is used to detect The experimental results were analyzed and plotted. Significance level: *, p<0.05; **, p<0.01; ***, p<0.001.

Example 1 Composition of the facial skin microbiome of the Han population

Subjects: This study was approved by the Ethics Committee of the School of Life Sciences, Fudan University, and 294 Shanghai resident healthy subjects were recruited as volunteers for this study. Among them, 46 were male (M) and 248 were female (F). The skin microbes were collected from three parts of the head, forehead (Fh), cheeks (Ch), and paranasal (Ns) (please refer to Figure 1).

Through metagenomic sequencing, combined with bioinformatics analysis, the composition and functional characteristics of the healthy skin microbiome of the Chinese Han population were systematically described. By comparing with the American skin microbiome sample data from the Human Microbiome Project (HMP), we found that there is a bacterium with a significantly higher relative level in the Chinese population than Americans - M. osloensis, Oslo Mora The level of La. spp. was relatively high among Chinese Singaporeans, and this strain may be one of the characteristic strains of the skin flora of East Asian populations (please refer to Figure 2).

Example 2 Population typing analysis based on skin microbiome

The skin microbiome, like the gut microbiome, is affected by a variety of factors, and there are significant individual differences. In the research, the present invention draws on the method of enterotype to classify the population based on skin microbes and explore the driving factors of the type, so as to find the potential rules behind the skin microbiome, and provide a new classification standard for clinical diagnosis.

The present invention screened subjects with no missing skin microbiome data in three sites, a total of 247 subjects.

Skin type identification was performed on these 247 subjects based on skin microbiome data. First, the samples from the three parts were clustered using the PAM method, and the CH index was used to determine the optimal number of clusters. Please refer to Figure 3, the results of the CH index show that when the number of clusters is 2, the CH index has the highest score, so the optimal number of clusters in the three parts is 2.

Based on the result, the present invention performs genotyping analysis on the samples, which are divided into two categories, and respectively use the JSD distance and the Bray-Curtis distance to perform PCoA analysis on the genotyping results of the samples in the three parts. Please refer to Figures 4A-L. The results show that the sample typing results of the three parts can be effectively divided into two categories, and the microorganisms that contribute the most to the classification are Propionibacterium acnes and Moraxella oslo, namely a The class enriches P. acnes and the other class enriches Moraxella oslo. Based on this result, we named the two cutotypes as C-Cutotype and M-Cutotype, respectively.

The present invention analyzes the differential flora of C-Cutotype and M-Cutotype based on the skin microbiome data of the forehead. Referring to Figure 5, the colors represent relative levels (eg, relative abundance) of microorganisms, with one sample per column and one microorganism per row. The results of the differential analysis showed that some microbes prefer a certain skin type, possibly due to the interaction between the flora. For example, P. greedy, P. granulosum, staphylococcus, P. acnes and staphylococcus phage were enriched in C-Cutotype. In M-Cutotype, Moraxella bovoculi and Psychrobacter sp. were enriched.

In the present invention, correlation analysis is performed based on the relative levels of the differential flora, and is displayed in the form of a network diagram. Referring to Figure 6, the results show that there is a strong positive correlation between microorganisms enriched in the same skin type, while a strong negative correlation exists between microorganisms enriched between different skin types. The results of this analysis indicate that microorganisms enriched in the same skin type may occupy different ecological niches, form a stable ecological network with each other, and build a strong microbial community to resist other microorganisms including opportunistic and New colonization of potentially pathogenic microorganisms.

Based on this 247 people, a total of 741 samples, we give the skin type based on the ratio of M bacteria to C bacteria levels (such as content) (M/C) as shown in Table 6.

Table 6

	C型Type C	混合型Hybrid	M型M type
面颊cheek	≤0.75≤0.75	0.75<M/C<1.680.75<M/C<1.68	≥1.68≥1.68
额头forehead	≤1.24≤1.24	1.24<M/C<2.181.24<M/C<2.18	≥2.18≥2.18
鼻翼Nose	≤0.34≤0.34	0.34<M/C<0.550.34<M/C<0.55	≥0.55≥0.55

Based on the different source parts of microorganisms, the reference values for specific classification are different. The skin classification sampling principle in clinical application: the facial skin parts that need to be improved, and then refer to the above data classification.

In addition, the full name of C bacteria is Propionibacterium acnes, and its level (eg abundance) is closely related to skin acne. There are very few related reports on M bacteria. The present study finds for the first time that M bacteria are correlated with skin phenotypes, are positively correlated with age, and are associated with some skin aging phenotypes.

The inventors conducted experiments on changes in skin texture and M/C of subjects, and obtained the results in Table 2, which showed that the M/C ratio was related to skin texture. As the level of M bacteria increased, skin oil decreased, moisture content decreased, gloss decreased, and yellow value (skin dullness) increased; associated with some typical features of acne, such as increased M bacteria, decreased oil, porphyrin Morphine (mostly C bacteria metabolites, which can promote inflammation) decreased, and the pore area decreased.

Table 7 lists the correlation between related phenotypes and M/C value, and all phenotypes are significantly correlated (p<0.05).

Table 7

位置Location	变量1variable 1	变量2variable 2	相关系数Correlation coefficient	P值P value
Fh额头Fh forehead	卟啉Porphyrin	M/C ratioM/C ratio	-0.4788-0.4788	2.30E-162.30E-16
	油脂grease	M/C ratioM/C ratio	-0.2592-0.2592	2.24E-052.24E-05

	含水量water content	M/C ratioM/C ratio	-0.1782-0.1782	3.87E-033.87E-03
	光泽度Gloss	M/C ratioM/C ratio	-0.1585-0.1585	1.03E-021.03E-02
	毛孔面积Pore area	M/C ratioM/C ratio	-0.1405-0.1405	2.32E-022.32E-02

Ck面颊Ck cheeks	卟啉Porphyrin	M/C ratioM/C ratio	-0.4103-0.4103	1.65E-121.65E-12
	含水量water content	M/C ratioM/C ratio	-0.2876-0.2876	1.35E-061.35E-06
	油脂grease	M/C ratioM/C ratio	-0.2217-0.2217	2.22E-042.22E-04

Ns鼻旁Ns beside the nose	卟啉Porphyrin	M/C ratioM/C ratio	-0.3722-0.3722	1.35E-101.35E-10
	皮肤黄值skin yellowness	M/C ratioM/C ratio	0.19930.1993	8.16E-048.16E-04
	毛孔大小Pore size	M/C ratioM/C ratio	-0.1383-0.1383	2.09E-022.09E-02
	毛孔面积Pore area	M/C ratioM/C ratio	-0.1320-0.1320	2.75E-022.75E-02
	油脂grease	M/C ratioM/C ratio	鼻旁未测Nasal not tested
	含水量water content	M/C ratioM/C ratio	鼻旁未测Nasal not tested

Example 3 Biological significance of microbial skin type

Based on gene-level (eg, abundance) profiles, PCoA analysis of the samples showed that the two skin types could be effectively separated, indicating that there are significant differences in function between the two skin types. Specifically, the genes of C-Cutotype are enriched in the metabolism of carbohydrates and sterols and the synthesis of fatty acids, while the genes of the microbiome in M-Cutotype are more related to the synthesis of amino acids, aromatic compounds and some lipids such as inositol, etc. . Previous studies reported that P. acnes could utilize carbohydrates as a carbon source, and the results of gene function enrichment also found that 17 KEGG functional modules in C-Cutotype were all related to the phosphotransferase system (PTS). In prokaryotes, this system is known to be responsible for carbohydrate transport and phosphorylation and is associated with the ability to metabolize glucose, maltose, lactose, fructose and cellobiose, possibly reflecting C-Cutotype's dependence on carbohydrates as a nutrient source. Conversely, previous research on Moraxella oslo found that the microbe was unable to utilize any carbohydrates and relied on fatty acids and alcohols as carbon sources. This further suggests that the two skin types may constitute two communities with different nutritional requirements.

Since the skin microenvironment is the growth environment of skin microorganisms, it determines the nutrients available to the microorganisms. Therefore, to explore whether the skin phenotype is the driving factor for the different skin types, we further analyzed the phenotypic differences between the two skin types. Referring to Figure 7, it was found that the two skin types had significant differences in stratum corneum moisture, oil, and skin color. In contrast, C-Cutotype has higher oil and moisture content, while M-Cutotype has drier skin. Since oil is the main source of nutrients for microbes, this result further suggests that differences in our nutritional requirements are the driving factors for different skin types.

In addition, the skin phenotypic characteristics of M-Cutotype were similar to those of the elderly, therefore, we compared whether there were differences in age between the two skin types. Please refer to Figure 8. As expected, the age of the M-Cutotype group was significantly larger than that of the C-Cutotype group, but further analysis found that both C-Cutotype and M-Cutotype existed in different age groups, that is, C-Cutotype in the elderly , M-Cutotype is present in young adults. Therefore, we guess that age is not the real determinant, and nutritional needs are the direct determinant. The difference in age may be due to changes in host physiology during aging that affect the skin phenotype, resulting in an older M-Cutotype.

Example 4 Correlation of Moraxella oslo with skin aging phenotype

In this study, the skin microbiota of three parts of the forehead (Fh), cheeks (Ch) and paranasal (Ns) of 248 healthy female subjects living in Shanghai were collected, and the correlation between Moraxella oslo and age and skin phenotype was analyzed. For correlation analysis, the Spearman coefficient was calculated and the P value was corrected by the FDR method. The corrected P value was less than 0.05 as the screening standard. Please refer to Figure 9. It was found that the levels of Moraxella oslo in the three parts were significantly positively correlated with age, and significantly negatively correlated with porphyrin. In addition, there was a positive correlation with cheek spots, and a negative correlation with moisture, oil in the stratum corneum of the cheeks, and oil content in the forehead. In addition, Moraxella oslo also showed a weak positive correlation trend with other aging phenotypes.

Example 5 Potential targets for skin aging by M bacteria

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 glyoxylic acid cycle. Studies have shown that the glyoxylic acid cycle is involved in the catabolism of ethoxylates. These results provide a basis for skin microorganisms to participate in the degradation of octylphenol polyoxyethylene ethers (OPEs). . The alkylphenols and short polyoxy metabolites formed by the degradation of OPE have endocrine disrupting activity. Among them, alkylphenol ethoxylates (APE) have estrogen-like activities, and it has been proved that these compounds can mimic the effect of estradiol in vivo and in vitro, and are called environmental estrogens. Therefore, skin microbes have the potential to interfere with the production of estradiol, which is essential for preventing skin aging (see Figure 10).

1. Please refer to Figure 11. The results of gene function difference analysis show that M-Cutotype is enriched in the pathway of β-carotene synthesis, which indicates that M-Cutotype may synthesize more β-carotene. Beta-carotene is associated with yellowing of the skin.

2. We stimulated keratinocytes with the culture medium of M. spp. and explored the potential aging mechanism of M. spp. using transcriptomic methods.

Signal pathway enrichment analysis of differentially expressed genes by RNA-Seq suggested that Moraxella oslo could affect skin cells through signaling pathways, mainly including regulation of collagen synthesis and decomposition. Please refer to Figure 12. The RNAseq results indicated that the differential genes were abundantly enriched in biological processes such as collagen metabolism and extracellular matrix decomposition, which were strongly related to the aging phenotype.

Example 6 Methods of Regulating Moraxella Oslo Levels

In the previous analysis, Moraxella oslo was known to be associated with age and skin aging phenotypes. In this study, a single skin surface compound was used to incubate Moraxella oslo, and the number of viable bacteria was examined to reflect the microbial utilization of different skin surface compounds and the toxicity of specific compounds to Moraxella oslo. Finally, by adjusting the amount of the preferred compounds or toxic compounds of Moraxella oslo, the growth of Moraxella oslo can be regulated, so as to achieve the purpose of delaying aging.

Please refer to Figure 13. In this study, CCK-8 and Dye mix A were used to explore the effects of 32 skin surface compounds on the growth of Moraxella oslo. The compounds are: L-lysine, L-glutamine, L-histidine, L-arginine, taurocholic acid, creatinine, 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-cystine, L-tyrosine Acid, L-Leucine, L-Isoleucine

, L-ornithine hydrochloride, L-citrulline, L-proline, L-valine, L-alanine, D-aspartic acid, trans-4-hydroxy-L- Proline, uric acid, taurine. Among them, L-glutamine, L-histidine, L-serine, and L-proline significantly promoted the growth of Moraxella oslo, while SDS had a significant inhibitory effect.

Example 7 Population detection in other countries

In order to verify whether the obtained skin types are widespread, we downloaded multiple public data and verified the existence of skin types based on skin microbiome data of different races, different parts, and different health conditions.

We first used skin metagenomic data from the cubital fossa site (wet type) in Singapore Chinese in patients with atopic dermatitis (AD) and healthy individuals. Please refer to Figure 14. It is found that the samples of this data can be effectively divided into two categories, which are the same as the previous results. One is enriched with P. acnes and the other is enriched with Moraxella oslo. This result suggests that our skin The presence of the type is not affected by skin health and location.

Further, we used publicly available skin metagenomic data of psoriasis in Filipino children (scalp and neck) and Italians and found consistent results, please refer to Figure 15. The typing results show that the population can be effectively divided into two categories, One is C-Cutotype and the other is M-Cutotype.

In conclusion, the present invention proves that skin types exist widely and are not affected by skin location, race, health status, and the like.

Example 8 Skin condition typing system

According to the foregoing embodiment, we have developed a skin condition typing system, the system includes a feature receiving module, a calculation processing module and a result output module, each module is connected by wire or wireless, and the typing steps are as follows:

(a) Collect and detect skin microbe samples from the subject's face to further generate skin sample feature data. The skin sample characteristic data is the respective levels of Moraxella oslo and P. acnes. The collection site is the same as that described in Embodiment 1, and the detection method is as described in the detection method.

(b) inputting the skin sample feature data into the system from the feature receiving module.

(c) the processing module receives the skin sample characteristic data from the characteristic receiving module, calculates the respective ratios of the quantified Moraxella oslo and P. acnes or the relative ratios between each other, and based on the obtained respective ratios or the relative proportions of each other, compared with the standard value of skin type or characteristic characterization, so as to obtain the judgment result of skin type and/or skin condition

(d) A result output module, the output module can be any terminal, such as a display, a printer, a tablet computer (PAD), a smart phone, etc., for receiving and outputting the judgment result.

The system includes a storage, wherein threshold information of standard values is stored in the storage.

The standard values corresponding to the skin type and/or skin state are:

Skin type is C (or type I): when the ratio (M/C) of the level (eg content) (M) of the Moraxella oslo to the level (eg content) (C) of the P. acnes When the following conditions are met: M/C≤1.3, preferably, M/C≤0.8, more preferably M/C≤0.4. Skin state of this phenotype: oily, higher water content, good skin elasticity.

The skin type is M type (type III): when the ratio of the level (eg content) (M) of the Moraxella oslo to the level (eg content) (C) of the P. acnes in the sample ( M/C) meets the following conditions: M/C≥0.5, preferably, M/C≥1.8, more preferably, M/C≥2.2. The skin condition of this phenotype: poor oil and water content, poor skin elasticity, and high degree of skin aging.

The skin type is mixed (or type II): when the ratio (M/C) of the level (eg content) (M) of the Moraxella oslo to the level (eg content) (C) of the P. acnes When the following conditions are met: 0.3≤M/C≤2.5, preferably, 0.3≤M/C≤2.3, more preferably, 0.8≤M/C≤1.8. The skin state of this phenotype is between C and M types.

Example 9 Correlation analysis between bacteria and skin phenotype

Species levels of Moraxella oslo, Propionibacterium acnes and Staphylococcus epidermidis based on R software and sebum content, stratum corneum water content, transepidermal water loss rate, skin Correlation analysis was performed on host skin phenotypes such as pH, pigmentation, porphyrin, skin color, pores, etc., to evaluate the correlation between strain levels and phenotypes.

The results show that Figure 16: The correlation between bacterial abundance and phenotype in the three parts is consistent, that is, Moraxella oslo is positively correlated with age, skin color black and yellow, and skin water content, sebum and porphyrin The content of morpholino was negatively correlated. Propionibacterium acnes and Moraxella oslo showed opposite trends. Staphylococcus epidermidis was negatively correlated with pigmentation and positively correlated with epidermal water content. The above suggests that the three kinds of bacteria are related to skin aging.

Example 10 Molecular-level verification of skin cells treated with bacterial supernatant

The bacterial supernatants of Moraxella oslo, Propionibacterium acnes and Staphylococcus epidermidis were treated with the most abundant cells in the host epidermis-keratinocytes (HaCaT cell line), and the expression profiles of the incubated cells were explored by QPCR. The changes focused on genes involved in collagen degradation and extracellular matrix assembly—matrix metalloproteinases (MMPs). In particular, MMPs are responsible for degrading extracellular matrix (ECM) proteins and promoting photoaging. Through molecular-level verification, the association between bacteria and host skin phenotypes was explored.

The QPCR results show that, as shown in Figure 17-19, the expressions of MMP1, MMP10, MMP12 and MMP13 in HaCaT cells treated with Moraxella oslo supernatant were significantly higher than those in the control group, while P. acnes and There was no significant difference in the expression of MMPs in HaCaT cells treated with Staphylococcus epidermidis supernatant. It is shown that Moraxella oslo can promote host skin aging, and it also shows that skin microbes promote skin aging, not a general effect, but an effect of specific strains.

All documents mentioned herein are incorporated by reference in this application as if each document were individually incorporated by reference. In addition, it should be understood that after reading the above-mentioned teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims

A use of Moraxella oslo or a detection reagent thereof, characterized in that, for (a) skin typing; and/or (b) judging or characterizing skin condition; or for preparing a reagent or kit, the The reagents or kits are used to (a) type skin; and/or (b) determine or characterize skin conditions.
The use of claim 1, wherein the skin condition comprises: skin age, skin moisture content, skin elasticity, skin color, and skin aging degree.
A marker combination, characterized in that the marker combination comprises Moraxella oslo and Propionibacterium acnes.
The marker combination of claim 3, wherein the marker combination further comprises (a) Moraxella bovoculi and/or Psychrobacter sp.; and/or (b) Propionibacterium avidum, Propionibacterium granulosum, Staphylococcus, Propionibacterium acnes and/or Staphylococcus phage.
A method for skin typing or judging skin state, characterized in that the method comprises:

(1) Provide a sample derived from the skin of the subject to be tested, and detect the level of each marker in the sample marker combination, where the combination includes the following markers: Moraxella oslo and Propionibacterium acnes, obtained respectively The level of Lageria (M) and the level of P. acnes (C);

(2) based on the level (such as content) (M) of Moraxella oslo, or by comparing the level (M) of Moraxella oslo with the level (C) of P. acnes in the sample, so as to be tested. The subject's skin is typed, and/or the skin condition is judged.
The method according to claim 5, wherein in step (2), according to the relative value (such as M/C) of the level (M) of Moraxella oslo and the level (C) of Propionibacterium acnes (such as M/C) Type, or judge the skin condition of the sample.
A reagent combination for skin typing and/or skin condition detection, characterized in that, the reagent combination includes a reagent for detecting each marker in the combination according to claim 3.
A kit, characterized in that the kit comprises the reagent combination of claim 7 .
A system for classifying the skin of an object to be tested and/or judging the state of the skin of the object to be tested, wherein the system comprises:

(a) a feature receiving module, the feature receiving module is used for receiving skin sample feature data; the feature data includes: the quantitative information of each of Moraxella oslo (M) and P. acnes (C) in the skin sample ;

(b) a calculation processing module for calculating the feature data from the feature receiving module, so as to obtain the respective proportions of the respective features or the proportional relationship between the respective features; and based on the obtained respective proportions or the relationship between the respective features A proportional relationship, compared with a standard value for skin type or characterization, resulting in a judgment of skin type and/or skin condition; and

(c) a result output module, the output module is used for receiving and outputting the judgment result.
The system of claim 9, wherein the method for obtaining the quantitative information comprises: sequencing, PCR, and quantitative protein detection.
A method for screening skin condition-improving substances or ingredients, comprising:

(a) providing a screening bacteria, the screening bacteria is Moraxella oslo (M), P. acnes (C), or screening bacteria comprising Moraxella oslo and/or P. acnes;

(b) co-culturing the substance or component to be screened with the screening bacteria, and detecting the respective levels of Moraxella oslo or P. acnes; or the relative level between Moraxella oslo and P. acnes ( M/C);

(c) according to (b) the respective levels of Moraxella oslo or P. acnes after culture; or the relative level (M/C) between Moraxella oslo and P. acnes, thereby judging the to-be-screened The substance or ingredient is a skin condition-improving substance or ingredient.
The method of claim 11, wherein in step (c), the level of Moraxella oslo is increased, or the relative level (M/C) between Moraxella oslo and P. acnes is increased, or acne When the level of Propionibacterium is decreased, or the relative level (C/M) between Propionibacterium acnes and Moraxella oslo is decreased, the substance or ingredient to be screened is a substance for treating acne; and/or step (c) decreased levels of Moraxella oslo, or decreased relative levels (M/C) between Moraxella oslo and P. acnes, or increased levels of P. acnes, or increased levels of P. acnes and P. acnes The relative level (C/M) of Moraxella was increased, and the substance or ingredient to be screened was a skin anti-aging substance.
A use of the combination of markers of claim 3 or the combination of reagents of claim 7, wherein (a) is used to prepare a kit for skin typing, and/or Assessing or characterizing skin condition; and/or (b) screening for substances or ingredients that improve skin condition.