Composition for inhibiting acne bacteria and Staphylococcus aureus

Abstract

The objective is to identify ingredients that can suppress acne and sensitive skin, and to provide compositions for cosmetic or beauty use. [Solution] It has been found that Alteromonas ferment filtrate acts only on Propionibacterium acnes that possess the lanthobiotic biosynthesis gene, and does not act on Propionibacterium acnes that do not possess the lanthobiotic biosynthesis gene. Alteromonas ferment filtrate acts on Propionibacterium acnes and / or Staphylococcus aureus that possess the lanthobiotic biosynthesis gene, thereby increasing the diversity of the skin microbiota and enabling the treatment, prevention, and / or reduction of acne-prone and / or sensitive skin. Therefore, a cosmetic or beauty composition containing Alteromonas ferment filtrate is provided.

Description

[Technical Field]

[0001] The present invention relates to a composition for inhibiting Cutibacterium acnes and / or Staphylococcus aureus, particularly a composition for selectively inhibiting Cutibacterium acnes harboring a lantibiotic biosynthetic gene cluster, and a composition for treating, improving or preventing skin conditions such as sensitive skin, acne, atopic dermatitis and folliculitis. The present invention also relates to a method for screening a drug capable of inhibiting Cutibacterium acnes harboring a lantibiotic biosynthetic gene cluster. [Background Art]

[0002] Cutibacterium acnes is an indigenous bacterium that inhabits human skin, particularly sites rich in sebaceous glands. An increase in its proportion among skin indigenous bacteria becomes a factor that causes acne (acne vulgaris). On the other hand, Cutibacterium acnes is a major component of the skin indigenous flora and is mainly classified into three phylogenetic groups. Among these, type IA Cutibacterium acnes is closely associated with acne, while type IB, type II and type III Cutibacterium acnes have low pathogenicity and low relevance to acne. There are also reports that type II Cutibacterium acnes enhances the skin barrier function as an indigenous bacterium, and it has been pointed out that it may contribute to the maintenance of healthy skin. Microbiome analyses of sensitive skin and non-sensitive skin have been performed, and it has been shown that the proportion of Cutibacterium acnes is relatively higher in sensitive skin compared to non-sensitive skin (Non-Patent Document 1: Annual Meeting of the Bacteriological Society of Japan 2024 Shibagaki et al., Non-Patent Document 2: Oleoscience, Vol. 23, No. 11, pp. 569-574). Antibiotics that kill Cutibacterium acnes are sometimes used as a countermeasure for acne, but the use of antibiotics is undesirable because they inhibit not only highly pathogenic Cutibacterium acnes, but also less pathogenic Cutibacterium acnes and other skin indigenous bacteria, and antibiotic-resistant bacteria emerge. Furthermore, while Staphylococcus aureus is also a type of commensal bacterium on the skin, it is known to produce toxins and, when the balance of the skin flora is disrupted, can exhibit pathogenicity, and is known to be involved in the onset and exacerbation of inflammatory skin diseases and skin infections. On the other hand, because it is a component of the skin's commensal flora, in recent years, it has been considered important to suppress the excessive colonization and proliferation of Staphylococcus aureus rather than to completely eliminate it.

[0003] Lanthionine is a type of sulfur-containing amino acid and is known as a component of lantibiotics, which are antimicrobial peptides. Some strains of Propionibacterium acnes possess the lantibiotics biosynthesis gene, while others do not. It is believed that strains possessing the lantibiotics biosynthesis gene establish an advantage over other competing microorganisms in the skin environment by producing lantibiotics (Non-patent Literature 3: Claesen J et al., Sci Transl Med, 12, (2020)). [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Special Publication No. 2016-525884 [Patent Document 2] Japanese Patent Publication No. 2019-187322 [Non-patent literature]

[0005] [Non-Patent Document 1] 2024 Annual Meeting of the Japanese Society for Bacteriology, Shibagaki et al., “Characterization of the sensitive skin microbiome of Japanese women” https: / / doi.org / 10.3412 / jsb.79.162 [Non-Patent Document 2] Nakako Shibagaki, Oleoscience Vol. 23, No. 11, pp. 569-574 [Non-Patent Document 3] Claesen J et al., Sci Transl Med, 12, (2020). [Overview of the project] [Problems that the invention aims to solve]

[0006] The objective is to identify ingredients that can treat, improve, or prevent sensitive skin and to provide compositions for cosmetic or beauty use. [Means for solving the problem]

[0007] The inventors focused on the fact that acne bacteria possessing the lanchiobiotic biosynthesis gene are particularly abundant in skin sensitive to irritation, and devised a method for screening components that act only on acne bacteria possessing the lanchiobiotic biosynthesis gene and not on acne bacteria lacking the gene. As a result of implementing this screening method, the inventors were able to select Alteromonas ferment filtrate as a component that acts only on acne bacteria possessing the lanchiobiotic biosynthesis gene and not on acne bacteria lacking the gene. Furthermore, they found that Alteromonas ferment filtrate also exhibits an inhibitory effect on the growth of Staphylococcus aureus, in addition to acne bacteria. Therefore, the present invention relates to the following: [1-1] A composition containing Alteromonas ferment filtrate for inhibiting either or both Propionibacterium acnes and Staphylococcus aureus. [1-2] A method for inhibiting either or both Propionibacterium acnes and Staphylococcus aureus, comprising administering Alteromonas ferment filtrate to a subject suffering from acne or sensitive skin. [1-3] Use of Alteromonas ferment broth for the suppression of either or both Propionibacterium acnes and Staphylococcus aureus. [1-4] Use of Alteromonas ferment filtrate for the production of compositions for inhibiting either or both Propionibacterium acnes and Staphylococcus aureus. [1-5] A cosmetic method comprising applying an Alteromonas ferment filtrate to the skin for the suppression of either or both Propionibacterium acnes and Staphylococcus aureus. [2] The invention described in items 1-1 to 1-5, wherein the composition is for selective suppression of Propionibacterium acnes possessing the lanthobiotic biosynthesis gene group. [3] The invention described in item 1-1 to 1-5 or 2, wherein the composition inhibits the growth of Propionibacterium acnes possessing the lanthobiotics biosynthesis gene group, but does not inhibit the growth of Propionibacterium acnes that does not possess the lanthobiotics biosynthesis gene group. [4] The invention according to any one of items 1-1 to 1-5 to 3, wherein the group of lanthobiotic biosynthesis genes is determined by the presence or absence of a gene having the sequence of SEQ ID NO: 3 or SEQ ID NO: 4, or a sequence that is at least 90% identical thereto. [5-1] Compositions comprising Alteromonas ferment filtrate for the treatment, improvement or prevention of acne, atopic dermatitis, folliculitis, or sensitive skin. [5-2] A method for treating, improving or preventing acne, atopic dermatitis, folliculitis or sensitive skin, comprising administering Alteromonas ferment filtrate to a subject suffering from acne or sensitive skin. [5-3] Use of Alteromonas ferment filtrate for the treatment, improvement or prevention of acne, atopic dermatitis, folliculitis or sensitive skin. [5-4] Use of Alteromonas ferment filtrate for the manufacture of compositions for the treatment, improvement or prevention of acne, atopic dermatitis, folliculitis or sensitive skin. [5-5] A cosmetic method comprising applying Alteromonas ferment filtrate to the skin for the treatment, improvement or prevention of acne, atopic dermatitis, folliculitis or sensitive skin. [6] A method for screening for acne bacteria inhibitors that selectively act on lanthobiotic biosynthesis gene-possessing acne bacteria, comprising culturing lanthobiotic biosynthesis gene-possessing acne bacteria in the presence of a candidate drug. [7] A step of culturing Propionibacterium acnes possessing the lanthobiotics biosynthesis gene group and Propionibacterium acnes not possessing the lanthobiotics biosynthesis gene group, respectively, in the presence of a candidate drug. The number of cells of acne bacteria possessing the control lantibiotics biosynthesis gene group and the number of cells of acne bacteria without the control lantibiotics biosynthesis gene group were measured in the absence of the candidate drug. The screening method described in item 6, which selects candidate components that do not suppress the growth of Propionibacterium acnes that do not possess the lanthobiotics biosynthesis gene group, but suppress the growth of Propionibacterium acnes that possess the lanthobiotics biosynthesis gene group, as Propionibacterium acnes inhibitors that selectively act on Propionibacterium acnes that possess the lanthobiotics biosynthesis gene group. [8] The screening method according to item 6 or 7, wherein the presence or absence of lanthobiotic biosynthesis gene groups is determined by the presence or absence of the sequence of SEQ ID NO: 3 or SEQ ID NO: 4, or a sequence that is at least 90% identical thereto. [Effects of the Invention]

[0008] The screening method of the present invention makes it possible to select components that act only on Propionibacterium acnes possessing the lanthobiotic biosynthesis gene, and do not act on Propionibacterium acnes that do not possess the lanthobiotic biosynthesis gene. Such components are useful in suppressing Propionibacterium acnes, which causes sensitive skin, and can be used for the treatment, improvement, or prevention of sensitive skin. In addition, Alteromonas ferment filtrate has an inhibitory effect not only on Propionibacterium acnes but also on the growth of Staphylococcus aureus. [Brief explanation of the drawing]

[0009] [Figure 1] Figure 1(a) shows the cell number ratio of Propionibacterium acnes when Alteromonas fermentation broth (0.005%, 0.01%, 0.02%) was added, compared to when Alteromonas fermentation broth was not added. Figure 1(b) shows the average cell number ratio compared to the control without Alteromonas fermentation broth, for strains possessing the lanchiobiotic gene and strains without the lanchiobiotic gene. [Figure 2]Figure 2 shows the ratio of the number of Cutibacterium acnes cells when cosmetic raw materials other than Alteromonas fermentation broth ((a) Phellodendron amurense extract, (b) Sapindus saponaria extract, (c) Hibiscus fermentation broth) are added, compared to the case where no raw material is added. [Figure 3] Figure 3 shows changes in OD600 when Staphylococcus aureus ((a) clinical strain 1, (b) clinical strain 2) is cultured in a medium supplemented with Alteromonas fermentation broth (0% (no addition), 0.01%, 0.02%).

Mode for Carrying Out the Invention

[0010] One aspect of the present invention relates to a composition for inhibiting lantibiotic biosynthetic gene cluster-carrying Cutibacterium acnes and / or Staphylococcus aureus, which comprises an Alteromonas fermentation broth. By applying a composition containing the Alteromonas fermentation broth, it becomes possible to inhibit lantibiotic biosynthetic gene cluster-carrying Cutibacterium acnes and / or Staphylococcus aureus on the skin, and enables treatment, alleviation or prevention of acne, atopic dermatitis, folliculitis, or sensitive skin.

[0011] Propionibacterium acnes possessing the lantibiotics biosynthesis gene (Lan gene) group refers to Propionibacterium acnes that possesses at least one gene from the lantibiotics biosynthesis gene (Lan gene) group. Propionibacterium acnes possessing the lantibiotics biosynthesis gene (Lan gene) group usually produces lantibiotics, which are antimicrobial peptides. Examples of Lan genes include the LanA gene which encodes a precursor peptide, the LanB gene which encodes a dehydrating enzyme, the LanC gene which encodes a cyclase, the LanT gene which performs extracellular transport, the LanP gene which performs processing, the LanM gene which simultaneously catalyzes dehydration and cyclization reactions, and the LanI, LanFEG, and LanH genes which are involved in self-resistance. Whether or not a strain of Propionibacterium acnes possesses the lantibiotics biosynthesis gene group can be confirmed by a PCR reaction targeting at least one of the above-mentioned genes. Alternatively, it can be confirmed using antibodies that can detect the proteins encoded by the above-mentioned genes. For example, whether or not a bacterium possesses the Lan gene group can be determined by whether or not it has the nucleotide sequence of Sequence ID No. 3 and a nucleotide sequence that has at least 95% sequence homology or identity with said sequence. Another example is that the presence or absence of the Lan gene group can be determined by an amplification reaction using primers consisting of the following sequences (Non-Patent Literature 3: Claesen J et al., Sci Transl Med, 12, (2020)). Those skilled in the art can design primers that specifically target any sequence of the Lan gene group. [Table 1] In the PCR reaction using such primers, it is determined whether a gene encoding Lantibiotic dehydratase (LanB) is contained in the Lan gene cluster. In the present specification, Cutibacterium acnes containing the LanB gene may be defined as Cutibacterium acnes harboring the Lan gene cluster, and Cutibacterium acnes not containing the LanB gene may be defined as Cutibacterium acnes not harboring the Lan gene cluster. In the present invention, it has been confirmed by inhibition zone assay that Cutibacterium acnes containing the LanB gene, which has been verified to carry the LanB gene, inhibits other skin-resident bacteria, and it has been confirmed by GCMS that lanthionine is produced by the Cutibacterium acnes.

[0012] As a result of determining the presence or absence of the Lan gene cluster in deposited strains, among Cutibacterium acnes strains available from NITE, strains NBRC113815, NBRC113816, NBRC1139, and NBRC113869 do not have the Lan gene cluster, while strains NBRC113817 and NBRC113818 have the Lan gene cluster (Example 1). In addition, among clinically obtained clinical strains, clinical strain 1, clinical strain 2, clinical strain 4, and clinical strain 5 do not have the Lan gene cluster, while clinical strain 3 has the Lan gene cluster (Example 1). The composition according to the present invention exerts a growth inhibitory effect on NBRC113817, NBRC113818, and clinical strain 3 that all have the Lan gene cluster (Figure 1). It is preferable that the composition according to the present invention acts on Cutibacterium acnes harboring the Lan gene cluster, while it does not act or acts only weakly on Cutibacterium acnes not having the Lan gene cluster. More preferably, the composition acts selectively on Cutibacterium acnes harboring the Lan gene cluster. The phrase "acts on Cutibacterium acnes harboring the Lan gene cluster" can be defined as meaning that when the target ingredient is applied, the number of cells becomes 70% or less, preferably 60% or less, more preferably 50% or less, compared to when the target ingredient is not applied (control). The composition according to the present invention preferably acts on Cutibacterium acnes harboring the Lan gene cluster in a dose-dependent manner.

[0013] The Lan gene in the present invention may have a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% sequence homology and / or identity with, for example, SEQ ID NO: 3 and SEQ ID NO: 4.

[0014] The "homology" of two sequences is the ratio of identical or similar amino acid residues that appear at corresponding locations when the two sequences are aligned, while the "identity" of two sequences is the ratio of identical amino acid residues or bases that appear at corresponding locations when the two sequences are aligned. The homology of amino acid sequences and base sequences can be determined using algorithms such as BLAST (Karlin, S. and Altschul, SF 1993. Pro.Natl.Acad.Sci.USA90:5873) by Karlin and Altschul, or FASTA (Pearson W.R. 1990. MethodsEnzymol. 183:63-98). Based on the BLAST algorithm, programs called BLASTN and BLASTX have been developed (see http: / / www.ncbi.nlm.nih.gov).

[0015] While not intended to be limited to theory, *Propionibacterium acnes* strains possessing the *Lanthiotics* biosynthesis gene group produce *Lanthiotics*, which inhibit the growth of other strains. This leads to a loss of bacterial diversity in the skin's microbiome, particularly deep within pores, resulting in the dominance of certain *Propionibacterium acnes* strains and ultimately causing acne. By suppressing *Propionibacterium acnes* strains possessing the *Lanthiotics* biosynthesis gene group in the skin's microbiome, bacterial diversity can be maintained, thereby enabling the treatment, improvement, or prevention of acne, as well as improving overall skin condition. It has been reported that commensal skin bacteria contribute to the repair of damaged epidermal barrier by activating the AhR receptor expressed in keratinocytes and promoting epidermal differentiation, that sphingomyelinase secreted extracellularly by Staphylococcus epidermidis promotes ceramide biosynthesis, and that the lower the proportion of Staphylococcus epidermidis in the commensal skin flora, the more sensitive the skin (Non-Patent Literature 2: Oleoscience Vol. 23 No. 11 pp. 569-574). Therefore, suppressing Propionibacterium acnes possessing the lanthobiotics biosynthesis gene group can maintain bacterial diversity in the flora and further improve skin barrier function and sensitive skin. Consequently, inhibitors of Propionibacterium acnes possessing the lanthobiotics biosynthesis gene group can also be called skin flora diversity improvers, skin barrier function improvers, and / or sensitive skin improvers, or acne treatment, prevention, or improvement agents.

[0016] Propionibacterium acnes, possessing the lanthobiotics biosynthesis gene, produces lanthobiotics containing lanthionine and 3-methyllanthionine, which are abnormal amino acids resulting from intramolecular cross-linking (monosulfide). Lanthobiotics are a type of bacteriocin, an antimicrobial peptide produced by bacteria, and are defined by containing the abnormal amino acids lanthionine and 3-methyllanthionine. Lanthionine has the following formula: [ka] It has the following formula: 3-methyllanthionine [ka] It has the following formula: [ka] It is represented as a compound (Claesen et al., (2020)). Lanthionine and 3-methyllanthionine are types of amino acids that are widely found in hair, hair, feathers, etc., and are also components of lanthibiotics. Lanthionine and 3-methyllanthionine are produced by a condensation reaction between cysteine ​​and dehydroalanine and dehydrobutyrin, which are produced by subjecting serine and threonine to a dehydration reaction, respectively. These reactions occur during the production and secretion of lanthibiotics. Specifically, certain serine and threonine of gene-expressed lanthibiotic precursor peptides are converted to dehydroalanine and dehydrobutyrin through a dehydration reaction, and these are then used in the condensation reaction. Furthermore, lanthionine and 3-methyllanthionine are formed through a condensation reaction with cysteine. Since cyclization occurs within the peptide molecule during this condensation reaction, it can also be called a cyclization reaction. The dehydration and condensation reactions occur within the bacterial cell and are transported outside the cell by a transporter encoded by LanT. The transported precursor peptide is processed to produce lanthibiotics. In each of the dehydration, condensation, and processing reactions, dehydrating enzymes (LanB gene or LanM gene), cyclizing enzymes (LanC gene or LanM gene), and processing enzymes (LanP gene), encoded by the Lan gene family, are active.

[0017] Lanthibiotics, when multiplied, can form pores in lipid bilayers. Lanthibiotics, in particular, have a pore-forming effect on bacterial cell membranes. Furthermore, when LipidII is present in the lipid bilayer, their activity is enhanced, and along with pore formation, they exert a cell division inhibitory effect, acting as an antibiotic. Therefore, when the proportion of Propionibacterium acnes possessing lanthibiotic biosynthesis genes increases in the skin microbiota, the influence of the produced lanthibiotics increases their dominance in the skin microbiota, while the diversity of the skin microbiota decreases.

[0018] Alteromonas ferment filtrate refers to a fermented liquid containing components secreted into the culture medium when microorganisms of the genus Alteromonas, which are Gram-negative bacteria, are cultured. In the labeling names used for the full ingredient list of cosmetics as defined by the Japan Cosmetic Industry Association, and in the international labeling names according to INCI (International Nomenclature for Cosmetic Ingredients), it is expressed as: Labeling Name / INCI Name: Alteromonas Ferment Extract / Alteromonas Ferment Extract. Alteromonas microorganisms are common bacteria widely distributed in marine ecosystems and can be isolated from coastal and estuary seawater, deep sea, sea ice, organic sediments, plant and animal surfaces, and sludge in aquaculture. Alteromonas microorganisms are characterized by their ability to form biofilms in seawater environments and can be easily isolated from biofilms formed in seawater. Biofilms are extracellular macromolecules secreted by microorganisms, mainly composed of polysaccharides, and exhibit a slimy texture. More preferably, Alteromonas fermentation extract is a fermentation extract obtained using microorganisms originating from the deep sea off the coast of Mexico. Alteromonas fermentation liquid contains components of biofilms and is sold as a cosmetic ingredient for its water retention and moisturizing properties, while it has also been reported to possess various physiological activities (Patent Document 1: JP 2016-525884, Patent Document 2: JP 2019-187322). For example, the reported benefits include suppressing inflammation by inhibiting ICAM-1, improving skin metabolism, suppressing the formation of age spots and melasma caused by inflammation, and improving dullness of the skin tone. Alteromonas antarctica, a species of the Alteromonas genus, can be used.

[0019] Alteromonas ferment filtrate can be obtained by methods well known in the art, or commercially available Alteromonas ferment filtrate can be used. More specifically, Alteromonas ferment filtrate can be obtained by culturing Alteromonas ferment filtrate in any medium such as seawater, artificial seawater, PTM medium (0.5% Bacto tryptone, 0.5% yeast extract (Difco), 80% artificial seawater (Marine Art, Tomita Pharmaceutical), pH 7.2), Marine Broth medium, F29 medium, etc., and removing the microbial cells. Alteromonas ferment filtrate can be incorporated into a composition at a concentration of 0.001% to 5% by mass using commercially available cosmetic raw material Alteromonas ferment filtrate. More specifically, any range selected from 0.001 to 0.005% by mass, 0.005 to 0.05% by mass, 0.05% to 0.5% by mass, and 0.5 to 1.0% by mass, and consecutive ranges thereof, can be selected.

[0020] Alteromonas ferment filtrate exhibits not only an inhibitory effect on the growth of Propionibacterium acnes, particularly Propionibacterium acnes possessing the Lanthibiotics biosynthesis gene group, but also an inhibitory effect on the growth of Staphylococcus aureus (Figure 3). Staphylococcus aureus produces toxins and exhibits pathogenicity when the balance of the bacterial flora is disrupted. Skin conditions in which an increase in Staphylococcus aureus is involved include inflammatory skin diseases such as atopic dermatitis and folliculitis. Therefore, the composition containing Alteromonas ferment filtrate of the present invention, particularly the topical skin composition, can treat, prevent, or improve skin conditions in which an increase in Staphylococcus aureus and / or Propionibacterium acnes is involved. Such skin conditions include acne, atopic dermatitis, folliculitis, or sensitive skin. Therefore, for individuals with an imbalance in their skin flora, such as acne bacteria, particularly acne bacteria possessing the Lanthibiotics biosynthesis gene group, and / or an increase in Staphylococcus aureus, and a reduced diversity of skin flora, a cosmetic product containing Alteromonas ferment filtrate can be provided to maintain an appropriate skin condition.

[0021] The composition containing Alteromonas ferment filtrate of the present invention can be administered to any subject for the treatment, prevention, or improvement of sensitive skin, but in particular, by applying it to subjects suffering from sensitive skin or subjects with reduced diversity of skin flora, it can suppress Propionibacterium acnes possessing the Lanthibiotics biosynthesis gene group. By suppressing Propionibacterium acnes possessing the Lanthibiotics biosynthesis gene group, skin flora diversity is increased, and sensitive skin can be treated, prevented, or improved. The concentration and dosage form can be arbitrarily selected from the viewpoint of exerting these effects. The inhibitor of Propionibacterium acnes possessing the Lanthibiotics biosynthesis gene group can be incorporated into cosmetics, pharmaceuticals, or quasi-drugs. When incorporated into cosmetics, it can be incorporated into facial or body cosmetics such as lotions, emulsions, serums, creams, packs, essences, and gels, as well as makeup cosmetics such as foundations, makeup bases, and concealers, and even bath additives. When incorporated into pharmaceuticals, it may be administered orally or parenterally, for example, transdermally. When the composition of the present invention is administered transdermally, it can be formulated into a topical skin preparation. An inhibitor of Propionibacterium acnes possessing the Lanthibiotics biosynthesis gene group can be administered over a long period of time as a cosmetic or pharmaceutical. From the viewpoint of improving acne-prone and dry skin, the composition of the present invention may be administered for several days or more, one week or more, two weeks or more, one month or more, three months or more, or six months or more. There is no particular upper limit, but it may be several years or less, for example, one year or less.

[0022] The inhibitor of Propionibacterium acnes possessing the Lanthobiotic biosynthesis gene group according to the present invention is preferably applied to the skin and can be incorporated into topical skin preparations. From the viewpoint of incorporation into topical skin preparations, Alteromonas ferment filtrate can be incorporated at a concentration of 0.001% to 5% by mass. Alteromonas ferment filtrate can also be combined with other cosmetic ingredients. From the viewpoint of fully exhibiting its effects, it can be incorporated into a composition as a topical skin preparation at a concentration of preferably 0.001% to 5% by mass. More specifically, any range selected from 0.001 to 0.005% by mass, 0.005 to 0.05% by mass, 0.05% to 0.5% by mass, and 0.5 to 1.0% by mass, and a continuous range therebetween, are preferred.

[0023] Topical skin preparations are not particularly limited as long as they can be applied to the skin, and any dosage form can be used, such as solution, emulsion, solid, semi-solid, powder, powder dispersion, water-oil two-layer separation, water-oil-powder three-layer separation, ointment, gel, aerosol, mousse, stick, etc. When formulated as a topical skin preparation, bases and excipients commonly used in topical skin preparations, such as preservatives, emulsifiers, and pH adjusters, may be used.

[0024] Another aspect of the present invention may relate to a beauty method comprising applying a cosmetic containing Alteromonas ferment filtrate to the skin, particularly the skin of the face. Such a beauty method may be used on acne-prone skin, sensitive skin, or skin of a person with reduced diversity of the skin's resident flora. Mechanical stimulation, such as vibration, or electrical stimulation, such as iontophoresis, may be applied with the intention of the extract's components reaching deep into the pores. The beauty method according to the present invention may be offered as a service in beauty salons, beauty clinics, etc., or may be offered together with the sale of cosmetics in cosmetic retail stores. The beauty method is a non-therapeutic or non-medical method that can be distinguished from treatment or preventive methods performed by doctors or medical professionals, and is provided by a practitioner who is not a doctor or other medical professional.

[0025] Another aspect of the present invention relates to a screening method for acne bacteria inhibitors that act on lanchiobiotic biosynthesis gene-possessing acne bacteria, comprising culturing the lanchiobiotic biosynthesis gene-possessing acne bacteria in the presence of a candidate component. More preferably, it is preferable to screen for candidate components that do not inhibit the growth of acne bacteria that do not possess the lanchiobiotic biosynthesis gene-possessing acne bacteria, but inhibit the growth of acne bacteria that possess the lanchiobiotic biosynthesis gene-possessing acne bacteria. The screening method according to the present invention can increase the diversity of the skin microbiota and allows for the selection of components useful for the treatment, alleviation, or prevention of acne-prone and / or sensitive skin.

[0026] The screening method according to the present invention is, more specifically, as follows: A process of culturing *Propionibacterium acnes* possessing the *Lanthiotics* biosynthesis gene group and *Propionibacterium acnes* not possessing the *Lanthiotics* biosynthesis gene group, respectively, in the presence of a candidate drug. A process of measuring the number of cells of acne bacteria possessing the lantibiotic biosynthesis gene group and the number of cells of acne bacteria without the lantibiotic biosynthesis gene group, compared to the number of cells of control acne bacteria without the lantibiotic biosynthesis gene group, cultured in the absence of the candidate drug. A process to select candidate components that do not inhibit the growth of Propionibacterium acnes that do not possess the lanthobiotics biosynthesis gene group, but inhibit the growth of Propionibacterium acnes that possess the lanthobiotics biosynthesis gene group, as Propionibacterium acnes inhibitors that selectively act on Propionibacterium acnes that possess the lanthobiotics biosynthesis gene group. This includes [the following]. As for the cell number, an index corresponding to the cell number can also be used. As an index corresponding to the cell number, absorbance, for example, the OD600 value may be used.

[0027] Candidate components may be added to the culture medium at a predetermined timing, or the medium may be replaced with a medium containing the candidate components. As a control, the number of cells obtained by culturing cultures containing *Propionibacterium acnes* possessing the lantibiotics biosynthesis gene group and *Propionibacterium acnes* without the lantibiotics biosynthesis gene group, respectively, in a medium that differs only in that it does not contain the candidate components, can be used. The control group may have experiments conducted in advance to set a threshold, or the culture and measurement steps may be performed in parallel. If the number of cells decreases compared to the control cell count, it can be screened as an *Propionibacterium acnes* inhibitor. Therefore, candidate components that reduce the number of *Propionibacterium acnes* possessing the lantibiotics biosynthesis gene group, while not reducing the number of *Propionibacterium acnes* without the lantibiotics biosynthesis gene group, can be screened as *Propionibacterium acnes* inhibitors that act on *Propionibacterium acnes* possessing the lantibiotics biosynthesis gene group, compared to the control.

[0028] The candidate components used in the screening method of the present invention can be any library, such as cosmetic materials, food materials, or pharmaceutical materials. Such libraries may include compound libraries, extract libraries, etc. The compounds and extracts contained in each library may be commercially available compounds and extracts, or synthesized compounds and prepared extracts.

[0029] All references made herein are incorporated herein by citation in their entirety.

[0030] The embodiments of the present invention described below are for illustrative purposes only and do not limit the technical scope of the invention. The technical scope of the invention is limited solely by the claims. Modifications to the invention, such as additions, deletions, and substitutions of constituent elements of the invention, can be made without departing from the spirit of the invention. [Examples]

[0031] Example 1: Confirmation of the presence or absence of lanthobiotic biosynthesis genes in Propionibacterium acnes We obtained NBRC113815, NBRC113816, NBRC113817, NBRC113819, and NBRC113869 from NITE. In addition, bacteria were collected from the facial skin of healthy Japanese women using cotton swabs, cultured on modified GAM agar medium (manufactured by Nissui Pharmaceutical Co., Ltd.), and isolated from colonies. These clinical strains were designated as strains A to G. For these strains, PCR was performed using the primer set in Table 2 below, according to the PCR reaction conditions in Non-Patent Literature 1, to confirm the presence or absence of lanchiobiotic biosynthesis genes. Strains NBRC113815, NBRC113816, NBRC1139, NBRC113869, A, B, C, D, and E did not possess the Lan gene group, while NBRC113817, NBRC113818, and F did not possess the Lan gene group. Subsequently, each strain of *Propionibacterium acnes* was pre-cultured in modified GAM liquid medium under anaerobic conditions at 37°C. The increased bacteria were collected by centrifugation, washed twice with physiological saline, and then reinoculated into phosphate-buffered saline (PBS) to which *Lucas Meyer Cosmetics SAS* was added at concentrations of 0% (no addition), 0.005% (no addition), 0.01% (no addition), and 0.02% (no addition). The cultures were then incubated at 37°C under anaerobic conditions for 24 hours, and the OD600 was measured. The OD600 values ​​of the cultures with *Lucas Meyer Cosmetics SAS* added were calculated relative to the OD600 values ​​of the cultures without *Lucas Meyer Cosmetics SAS* added, and are shown in Figure 1(a). The average ratio of OD600 values ​​to the control without *Lucas Meyer Cosmetics SAS* added, for strains possessing and not possessing the lanthobiotics biosynthesis gene, is shown in Figure 1(b). Figure 2 shows the ratio of OD600 values ​​to the target when other cosmetic ingredients besides Alteromonas ferment filtrate were used in the same way: Phellodendron amurense bark extract (cosmetic name: Phellodendron amurense bark extract), Sapindus mukorossi extract (cosmetic name: Sapindus mukorossi extract), and hibiscus ferment filtrate (cosmetic name: Lactobacillus hibiscus flower ferment filtrate). [Table 2]

[0032] Example 2: Screening Method NBRC113815, which lacks the Lan gene cluster, and NBRC113817, which possesses the Lan gene cluster, were pre-cultured in modified GAM liquid medium under anaerobic conditions at 37°C. NBRC113815 and NBRC113817 were inoculated into PBS containing candidate components and control PBS without candidate components, respectively. The OD600 values ​​of the cultures containing candidate components were measured relative to the control cultures. Candidate components that did not reduce the growth rate of NBRC113815 but reduced the growth rate of NBRC113817 were screened and obtained as acne bacteria inhibitors that act on Propionibacterium acnes possessing the lanthobiotic biosynthesis gene cluster.

[0033] Example 3: Suppression of Staphylococcus aureus Clinical strains 1 and 2 of Staphylococcus aureus were inoculated into M9 minimal medium supplemented with 0.5% glycerin and 0.1% yeast extract, with Alteromonas ferment broth (Lucas Meyer Cosmetics SAS) added to concentrations of 0%, 0.01%, and 0.02% by mass, respectively. The cultures were incubated with shaking at 37°C and 140 rpm, and the change in OD600 over time was measured. The results are shown in Figure 3. The addition of Alteromonas ferment broth inhibited the growth of Staphylococcus aureus.

Claims

1. A composition containing Alteromonas ferment filtrate for inhibiting either or both Propionibacterium acnes and Staphylococcus aureus.

2. The composition according to claim 1, wherein the composition is for selective suppression of Propionibacterium acnes possessing a group of lanthobiotic biosynthesis genes.

3. The composition according to claim 1, wherein the composition suppresses the growth of Propionibacterium acnes possessing the lanthobiotics biosynthesis gene group, but does not suppress the growth of Propionibacterium acnes that does not possess the lanthobiotics biosynthesis gene group.

4. The composition according to claim 1, wherein the group of lanthobiotic biosynthesis genes is determined by the presence or absence of a gene having the sequence of SEQ ID NO: 3 or SEQ ID NO: 4, or a sequence that is at least 90% identical thereto.

5. A composition containing Alteromonas ferment filtrate for the treatment, improvement, or prevention of acne, atopic dermatitis, folliculitis, or sensitive skin.

6. A method for screening acne bacteria inhibitors that selectively act on lanchiobiotic biosynthesis gene-containing acne bacteria, comprising culturing the acne bacteria containing lanchiobiotic biosynthesis gene clusters in the presence of a candidate drug.

7. A process of culturing *Propionibacterium acnes* possessing the *Lanthiotics* biosynthesis gene group and *Propionibacterium acnes* not possessing the *Lanthiotics* biosynthesis gene group, respectively, in the presence of a candidate drug. The number of cells of acne bacteria possessing the control lantibiotics biosynthesis gene group and the number of cells of acne bacteria without the control lantibiotics biosynthesis gene group were measured in the absence of the candidate drug. The screening method according to claim 6, wherein a candidate component that does not suppress the growth of Propionibacterium acnes that does not possess the lanthobiotics biosynthesis gene group, but suppresses the growth of Propionibacterium acnes that possesses the lanthobiotics biosynthesis gene group, is selected as a Propionibacterium acnes inhibitor that selectively acts on Propionibacterium acnes that possesses the lanthobiotics biosynthesis gene group.

8. The screening method according to claim 6 or 7, wherein the presence or absence of a group of lanthobiotic biosynthesis genes is determined by the presence or absence of the sequence of SEQ ID NO: 3 or SEQ ID NO: 4, or a sequence that is at least 90% identical thereto.

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