Lytic agent for controlling skin microbiota

Abstract

To provide a novel lytic agent enabling effective lysis of Staphylococcus hominis without substantially killing resident bacteria that are beneficial for the skin's barrier function, such as Staphylococcus epidermidis.SOLUTION: A lytic agent for controlling the skin microbiota involved in body odor production is provided, the lytic agent comprising a lytic enzyme derived from bacteriophage infectious to Staphylococcus hominis or an active fragment thereof, a polynucleotide encoding the lytic enzyme or the active fragment thereof, or the bacteriophage.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a bacteriolytic agent for controlling skin microbiota involved in body odor generation, a bacteriolytic agent for Staphylococcus hominis, and a composition, a cosmetic composition, a pharmaceutical composition, etc. for controlling skin microbiota involved in body odor generation.

Background Art

[0002] In recent years, awareness of body odor has been increasing, and the need for cosmetics and quasi-drugs for controlling one's own body odor so as not to give discomfort to those around has been increasing.

[0003] It is known that body odor is generated by the decomposition / metabolism of sweat, sebum, etc. secreted from the human body by skin commensal bacteria. Among the human body, especially the armpits where apocrine glands are developed may emit a strong odor (bromhidrosis), which may give a great deal of discomfort to those around. Bromhidrosis varies greatly among individuals and is classified into 7 types including A type (acid type), C type (curry spice type), K type (mold type), E type (steamed meat type), W type (dry type), F type (iron type), and Other type (others) excluding M type (milk type) (Non-Patent Document 1).

[0004] Among the above 7 types, C-type bromhidrosis contains 3-hydroxy-3-methylhexanoic acid as an odor component, has a high odor intensity, and gives a strong discomfort to those around. It is known that this odor component is produced by the decomposition / metabolism of the conjugate of 3-hydroxy-3-methylhexanoic acid and glutamine (for example, 3-hydroxy-3-methylhexanoyl-glutamine) contained in the sweat secreted from the apocrine glands by the armpit commensal bacteria.

[0005] Currently, as methods for suppressing axillary osmidrosis, bactericides, astringents, deodorants (adsorbents for axillary osmidrosis components), antioxidants, etc. are used, and in particular, research is actively being conducted on methods for suppressing the production of axillary osmidrosis causative substances by bactericides and the like. As substances for suppressing the production of axillary osmidrosis causative substances, substances that kill axillary osmidrosis causative bacteria and substances that inhibit the metabolic pathway of axillary osmidrosis causative bacteria have been studied. However, those having a high axillary osmidrosis suppression effect have not been found so far.

Prior Art Documents

Non-Patent Documents

[0006]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0007] An object of the present invention is to provide a new bacteriolysin that can effectively lyse Staphylococcus hominis without substantially killing the resident bacteria beneficial to the skin barrier function such as Staphylococcus epidermidis.

Means for Solving the Problems

[0008] There are various types of resident bacteria on the skin, and these resident bacteria are called the skin microbiota (skin microbiome). It is known that the skin microbiota differs in composition depending on the physiological state and site of the skin, and also differs depending on the health status and age of an individual.

[0009] The inventors considered that the bactericides and the like tried in the past kill all the bacteria in the skin microbiota, so there is a risk of not only reducing the causative bacteria of axillary osmidrosis but also killing the resident bacteria beneficial to the skin barrier function and inducing skin troubles. In addition, since they do not specifically kill the causative bacteria of axillary osmidrosis, it is considered that when the skin microbiota recovers after sterilization, the ratio of the causative bacteria of axillary osmidrosis also recovers in the same way, so it was considered that a long-term axillary osmidrosis inhibitory effect could not be obtained.

[0010] In order to identify the bacteria involved in type C axillary osmidrosis showing strong axillary osmidrosis, the inventors collected sweat from the axilla of type C subjects and performed metabolome analysis, and also collected bacteria from the axilla and performed whole-genome analysis, and compared the results with those of type M subjects who do not have axillary osmidrosis. As a result, it was found that in the axilla of type C subjects, bacteria of the family Staphylococcaceae including Staphylococcus hominis (S. hominis) were significantly more present compared to the axilla of type M subjects.

[0011] Furthermore, as a result of performing whole-genome analysis on the Staphylococcus hominis strain, the inventors found a prophage sequence in the genomic sequence of the Staphylococcus hominis strain, and found a gene sequence encoding a plurality of endolysins (lysozymes) in the prophage sequence. The inventors synthesized this endolysin and measured the lytic activity against various skin resident bacteria including Staphylococcus hominis. As a result, the found endolysin showed lytic activity only against Staphylococcus hominis and showed no lytic activity against skin resident bacteria other than Staphylococcus hominis. For example, it was found that it also showed no lytic activity against Staphylococcus epidermidis which is beneficial for maintaining the skin barrier function. The endolysin of the present invention can specifically lyse only specific resident bacteria that can be involved in axillary osmidrosis without lysing the beneficial resident bacteria in the skin microbiota. The present invention is based on the above findings and provides the following.

[0012] (1) A bacteriolytic agent for controlling the skin microbiota involved in body odor generation, comprising a lytic enzyme derived from a Staphylococcus hominis infectious bacteriophage or an active fragment thereof, a polynucleotide encoding the lytic enzyme or an active fragment thereof, or the bacteriophage wherein the bacteriolytic agent contains the above components. (2) The bacteriolytic agent according to (1), wherein the lytic enzyme or an active fragment thereof contains an N-acetylmuramoyl-L-alanine amidase domain. (3) The lytic enzyme is (a) an amino acid sequence represented by any one of SEQ ID NOs: 1 to 8, (b) an amino acid sequence in which one or more amino acids are added, deleted, and / or substituted in the amino acid sequence represented by any one of SEQ ID NOs: 1 to 8, or (c) an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by any one of SEQ ID NOs: 1 to 8 and the bacteriolytic agent according to (2) is composed of the above components. (4) The polynucleotide is (d) a nucleotide sequence represented by any one of SEQ ID NOs: 9 to 16, (e) a nucleotide sequence in which one or more bases are deleted, substituted or added in the nucleotide sequence represented by any one of SEQ ID NOs: 9 to 16, (f) a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by any one of SEQ ID NOs: 9 to 16, or (g) a nucleotide sequence that hybridizes under highly stringent conditions with a nucleotide sequence complementary to the nucleotide sequence represented by any one of SEQ ID NOs: 9 to 16 and the bacteriolytic agent according to any one of (1) to (3) contains the above components. (5) The bacteriophage is (h) a nucleotide sequence represented by any one of SEQ ID NOs: 17 to 24, (i) a nucleotide sequence in which one or more bases are deleted, substituted or added in the nucleotide sequence represented by any one of SEQ ID NOs: 17 to 24, (j) a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by any one of SEQ ID NOs: 17 to 24 The bacteriolytic agent according to any one of (1) to (4), having a genomic DNA sequence containing (6) The bacteriolytic agent according to any one of (1) to (5), wherein the bacteriolytic activity against Staphylococcus hominis is higher than the bacteriolytic activity against Staphylococcus epidermidis. (7) A bacteriolytic agent for Staphylococcus hominis, a bacteriolytic enzyme derived from a Staphylococcus hominis-infectious bacteriophage or an active fragment thereof, a polynucleotide encoding the bacteriolytic enzyme or an active fragment thereof, or the bacteriophage containing, wherein the bacteriolytic enzyme is (a) an amino acid sequence represented by any one of SEQ ID NOs: 1 to 8, (b) an amino acid sequence in which one or more amino acids are added, deleted, and / or substituted in the amino acid sequence represented by any one of SEQ ID NOs: 1 to 8, or (c) an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by any one of SEQ ID NOs: 1 to 8 and consisting of, wherein the bacteriophage is (h) a base sequence represented by any one of SEQ ID NOs: 17 to 24, (i) a base sequence in which one or more bases are deleted, substituted, or added in the base sequence represented by any one of SEQ ID NOs: 17 to 24, (j) a base sequence having 90% or more identity with the base sequence represented by any one of SEQ ID NOs: 17 to 24 The bacteriolytic agent having a genomic DNA sequence containing (8) A composition for controlling the skin microbiota involved in body odor generation, containing the bacteriolytic agent according to any one of (1) to (6) as an active ingredient. (9) A cosmetic composition containing the composition according to (8). (10) The cosmetic composition according to (9), further containing a deodorant, an antiperspirant, and / or a fragrance. (11) The cosmetic composition according to (10) for reducing or removing a curry spice-like odor or preventing the generation of a curry spice-like odor. A cosmetic composition according to any one of (9) to (11), which is a liquid, gel, cream, lotion, spray, mist, stick, or patch. (13) A pharmaceutical composition comprising the composition according to (8). (14) The pharmaceutical composition according to (13), which is used for the treatment or prevention of bromhidrosis. [Advantages of the Invention]

[0013] According to the present invention, there is provided a new bacteriolysin that can effectively lyse Staphylococcus hominis without substantially killing the resident bacteria beneficial to the skin barrier function such as Staphylococcus epidermidis. [Brief Description of the Drawings]

[0014]

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Mode for Carrying Out the Invention

[0015] 1. Bacteriolytic agent 1-1. Overview The first aspect of the present invention is a bacteriolytic agent (hereinafter abbreviated as "the bacteriolytic agent of the present invention") for controlling the skin microbiota involved in body odor generation. The bacteriolytic agent of the present invention contains a bacteriolytic enzyme derived from a Staphylococcus hominis infectious bacteriophage and can specifically lyse Staphylococcus hominis. Different from a bactericidal agent that kills all bacteria in the skin microbiota, the bacteriolytic agent of the present invention does not kill commensal bacteria beneficial to the skin barrier function such as Staphylococcus epidermidis, so there is no risk of inducing skin trouble and it has high safety.

[0016] 1-2. Definitions of terms The terms frequently used in this specification are defined below.

[0017] In this specification, "skin microbiota" means an aggregate of microorganisms including bacteria, fungi, etc. present on the body surface such as the skin, sweat glands (including apocrine glands and eccrine glands), sebaceous glands, and hair follicles. In this specification, the skin microbiota in the armpit is particularly referred to as "axillary microbiota".

[0018] As used herein, the term "resident skin bacteria" refers to the general term for various bacteria and fungi that normally exist in the skin, sweat glands (including apocrine glands and eccrine glands), sebaceous glands, hair follicles, and the like. Resident skin bacteria include various bacteria and fungi, some of which have beneficial functions in maintaining the skin's barrier function, while others can cause skin diseases. Examples of resident skin bacteria include the families Staphylococcaceae, Corynebacteriaceae, and Propionibacteriaceae. Examples of resident skin bacteria belonging to the family Staphylococcaceae include Staphylococcus hominis, S. epidermidis, and Staphylococcus haemolyticus. Examples of resident skin bacteria belonging to the family Corynebacteriaceae include C. tuberculostearicum and C. ureicelerivorans. An example of a resident skin bacterium belonging to the family Propionibacteriaceae is Propionibacterium acnes.

[0019] "Staphylococcus hominis" is a type of gram-positive coagulase-negative staphylococcus and is often abbreviated as "S. hominis" in this specification. Staphylococcus hominis is known as a resident skin bacterium and is commonly found on the human body, particularly in areas with apocrine glands such as the armpits and genitals.

[0020] "Staphylococcus epidermidis" is a type of Gram-positive coagulase-negative staphylococcus, and is often abbreviated as "S. epidermidis" in this specification. Staphylococcus epidermidis produces glycerin and fatty acids from sweat and sebum. Fatty acids keep the skin weakly acidic and produce antibacterial peptides to prevent the growth of Staphylococcus aureus, while glycerin is known to function in maintaining the skin's barrier function.

[0021] In this specification, "body odor" means all odors emitted from the body. Body odor can be classified into axillary osmidrosis (odor emitted from the armpit), foot odor, scalp odor, hair odor, etc. according to the site of occurrence. It can also be classified into sweat odor, sebum odor, acid odor, oxidation odor, aging odor, etc. according to the type of odor. Generally, body odor is known to be caused in part by substances produced by the metabolism of substances present on the body surface such as sweat, sebum, and keratin by microorganisms such as skin commensal bacteria. For example, lipids (e.g., steroid hormones), fatty acids (e.g., short-chain fatty acids; medium-chain fatty acids such as HMHA-Gln, 3M2H-Gln), proteins, lactic acid, pyruvic acid, and components such as Cys-Gly-3M3SH in sweat are decomposed by commensal bacteria to produce body odor substances such as isovaleric acid (odor of the sole of the foot), 3-hydroxy-3-methyl-hexanoic acid (HMHA; spicy odor like cumin), 3-methyl-2-hexanoic acid (3M2H; odor like a rag), 3-methyl-3-sulfanylhexan-1-ol (3H3SH; sulfur-like odor), androsteroid (musk or urine odor) (hereinafter referred to as "body odor substances").

[0022] As used herein, "bromhidrosis" refers to a condition in which an unpleasant odor emanates from the armpits. Bromhidrosis can be classified into seven types: type A (acid type), type C (curry spice type), type K (mold type), type E (steamed meat type), type W (dry type), type F (iron type), and Other type, excluding type M (milk type) as described above. Among them, type C exhibits strong bromhidrosis and thus falls under the category of bromhidrosis, while type M does not. For types A, K, E, W, F, and Other type, they are considered to have bromhidrosis when an unpleasant odor is emitted.

[0023] As used herein, "treating bromhidrosis" means reducing the odor (bromhidrosis) emitted from the armpits or decreasing the intensity of bromhidrosis. Also, "preventing bromhidrosis" means reducing bromhidrosis to such an extent that no measures, including treatment, are required.

[0024] As used herein, "bacteriophage" (often abbreviated simply as "phage" in this specification) refers to a general term for viruses that infect bacteria. Phages have extremely high host specificity and do not infect eukaryotes. Therefore, drugs using phages are harmless to humans, animals, and plants. Note that phages are broadly classified into "lytic cycle", "lysogenic cycle", and "lytic / lysogenic cycle" based on the infection mode. In the lysogenic cycle, the phage integrates its own DNA into the bacterial chromosome without lysing the target bacteria and proliferates along with the growth of the bacteria. Phages in the lysogenic cycle are called "prophages". On the other hand, in the lytic cycle, after self-proliferating inside the host bacteria, the host bacteria are lysed to release a large number of daughter phages.

[0025] As used herein, "lysis" refers to the phenomenon of destroying the cell membrane of bacteria. Bacteria can be killed by lysis.

[0026] As used herein, "lytic agent" refers to a drug containing a lytic enzyme or its active fragment having lytic activity against target bacteria, a drug containing a polynucleotide encoding the lytic enzyme, or a drug containing an organism or virus containing the lytic enzyme or its active fragment or the polynucleotide.

[0027] As used herein, the term "lysozyme" refers to an enzyme having lytic activity against Staphylococcus hominis, unless otherwise specified. Specifically, it refers to any endolysin (hereinafter referred to as "wild-type endolysin") found in the prophage sequence of Staphylococcus hominis, and a mutant endolysin derived from the wild-type endolysin and having lytic activity against Staphylococcus hominis. In the present invention, it is preferred that the lysozyme can selectively lyse Staphylococcus hominis. For example, it is preferred that Staphylococcus hominis can be selectively lysed as compared with bacteria other than Staphylococcus hominis (e.g., resident bacteria beneficial to the skin barrier function such as Staphylococcus epidermidis) in the skin microbiota.

[0028] As used herein, the term "endolysin" refers to an enzyme derived from a bacteriophage and having the activity of degrading the cell wall of a host bacterium. Endolysin usually has the activity of hydrolyzing peptidoglycan. A bacteriophage can exist as a part of the bacterial genome or an extrachromosomal plasmid without destroying the bacterial cell, and this state is called a prophage. When a bacteriophage is released outside the bacterial cell through or without passing through a prophage, it is known that endolysin contributes to the release process by cleaving the bacterial cell wall.

[0029] As used herein, the term "a plurality" refers to, for example, 2 to 40, 2 to 30, 2 to 20, 2 to 15, 2 to 10, 2 to 7, 2 to 5, 2 to 4, 2 to 3, or 2.

[0030] As used herein, "amino acid identity" refers to the percentage (%) of identical amino acid residues in the total number of amino acid residues when two polypeptides to be compared are aligned by appropriately inserting gaps into one or both of them as necessary so that the number of matching amino acid residues is maximized in the amino acid sequences of the two polypeptides. The alignment of two amino acid sequences for calculating amino acid identity can be performed using known programs such as Blast, FASTA, ClustalW, etc. "Nucleotide identity" is calculated in the same manner.

[0031] As used herein, "(amino acid) substitution" refers to substitution within a group of conservative amino acids with similar properties such as charge, side chain, polarity, aromaticity, etc. among the 20 types of amino acids that make up natural proteins. For example, substitution within the group of uncharged polar amino acids with low-polarity side chains (Gly, Asn, Gln, Ser, Thr, Cys, Tyr), the group of branched-chain amino acids (Leu, Val, Ile), the group of neutral amino acids (Gly, Ile, Val, Leu, Ala, Met, Pro), the group of neutral amino acids with hydrophilic side chains (Asn, Gln, Thr, Ser, Tyr, Cys), the group of acidic amino acids (Asp, Glu), the group of basic amino acids (Arg, Lys, His), and the group of aromatic amino acids (Phe, Tyr, Trp). Substitution of amino acids within these groups is preferred because it is known that such substitution is less likely to cause a change in the properties of the polypeptide.

[0032] 1-3. Composition Hereinafter, the composition of the bacteriolytic agent of the present invention will be specifically described.

[0033] The bacteriolytic agent for controlling the skin microbiota involved in the generation of body odor of the present invention contains, as an active ingredient, (1) a bacteriolytic enzyme derived from a Staphylococcus hominis-infectious bacteriophage or an active fragment thereof, or consists of the same, (2) a polynucleotide encoding a bacteriolytic enzyme derived from a Staphylococcus hominis-infectious bacteriophage or an active fragment thereof, or contains the same or consists of the same, or (3) a Staphylococcus hominis-infectious bacteriophage, or consists of the same.

[0034] (1) Lysozyme or its active fragment In one embodiment, the lytic agent of the present invention consists of or contains lysozyme or its active fragment.

[0035] The lysozyme (hereinafter referred to as "the lysozyme of the present invention") constituting the lytic agent of the present invention is an endolysin derived from a Staphylococcus hominis infectious bacteriophage and having lytic activity against Staphylococcus hominis. The lysozyme is, for example, an endolysin derived from a prophage of Staphylococcus hominis.

[0036] The lysozyme of the present invention may be either an endolysin (hereinafter referred to as "wild-type endolysin") consisting of a wild-type amino acid sequence encoded by a Staphylococcus hominis infectious bacteriophage, for example, a prophage of Staphylococcus hominis, or an endolysin (hereinafter referred to as "mutant endolysin") consisting of a mutant amino acid sequence derived from the wild-type endolysin.

[0037] The type based on the amino acid sequence of the lysozyme of the present invention is not particularly limited, and it may be, for example, an endolysin containing an N-acetylmuramoyl-L-alanine amidase domain. The N-acetylmuramoyl-L-alanine amidase domain has an activity of hydrolyzing the amide bond between N-acetylmuramic acid and L-alanine of peptidoglycan. Specific examples of the wild-type endolysin containing the N-acetylmuramoyl-L-alanine amidase domain include endolysins consisting of the amino acid sequences shown in any of SEQ ID NOs: 1 to 8. The endolysin consisting of the amino acid sequence shown in SEQ ID NO: 8 corresponds to endolysin 3 in the examples described later.

[0038] The lysozyme of the present invention may be a wild-type endolysin consisting of the amino acid sequence represented by any of SEQ ID NOs: 1 to 8. The endolysin consisting of the amino acid sequence represented by SEQ ID NOs: 1 to 8 is an endolysin encoded by a prophage sequence identified by genomic analysis of eight types of Staphylococcus hominis strains in this example.

[0039] Further, the lysozyme of the present invention may be a mutant endolysin derived from the above wild-type endolysin. Examples of the mutant endolysin include an amino acid sequence in which one or more (for example, 1 to 3 or 1 to 2) amino acids are deleted, substituted, or added in the amino acid sequence represented by any of SEQ ID NOs: 1 to 8 (for example, the amino acid sequence represented by SEQ ID NO: 8), or an amino acid sequence having 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 82% or more, 85% or more, 87% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 99.9% or more identity with the amino acid sequence represented by any of SEQ ID NOs: 1 to 8 (for example, the amino acid sequence represented by SEQ ID NO: 8). The mutant endolysin preferably has 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more of the activity of the wild-type endolysin, or activity equivalent thereto or higher. Examples of the mutant endolysin having such activity include those containing an N-acetylmuramoyl-L-alanine amidase domain derived from the wild-type endolysin.

[0040] In one embodiment, the lysozyme of the present invention consists of (a) the amino acid sequence represented by any of SEQ ID NOs: 1 to 8 (for example, the amino acid sequence represented by SEQ ID NO: 8), (b) an amino acid sequence in which one or more (for example, 1 to 3 or 1 to 2) amino acids are added, deleted, and / or substituted in the amino acid sequence represented by any of SEQ ID NOs: 1 to 8 (for example, the amino acid sequence represented by SEQ ID NO: 8), or (c) an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by any of SEQ ID NOs: 1 to 8 (for example, the amino acid sequence represented by SEQ ID NO: 8).

[0041] As used herein, the "active fragment" of a lysozyme refers to a fragment having lytic activity against Staphylococcus hominis in any of the above lysozymes, for example, a fragment having an activity of 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more of the activity of the above wild-type endolysin, or a fragment having an activity equivalent thereto or more. As an example of an active fragment, a fragment containing an N-acetylmuramoyl-L-alanine amidase domain can be mentioned. The amino acid length of the polypeptide constituting this fragment is not particularly limited, but for example, in wild-type endolysin, it may be a continuous region of at least 50, 100, 150, 200, 250, or 300 amino acids.

[0042] (2) A polynucleotide encoding a lysozyme or an active fragment thereof In one embodiment, the bacteriolytic agent of the present invention contains, or consists of, a polynucleotide encoding a lysozyme derived from a Staphylococcus hominis-infectious bacteriophage or an active fragment thereof.

[0043] The polynucleotide constituting the bacteriolytic agent of the present invention (hereinafter referred to as "the polynucleotide of the present invention") encodes the above lysozyme or an active fragment thereof. The polynucleotide of the present invention is not particularly limited as long as it is a polynucleotide encoding any of the above lysozymes or an active fragment thereof. For example, a polynucleotide encoding a wild-type endolysin consisting of the amino acid sequence shown in any of SEQ ID NOs: 1 to 8 (for example, a polynucleotide consisting of the nucleotide sequence shown in any of SEQ ID NOs: 9 to 16, or a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 16) can be mentioned. The polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 16 encodes endolysin 3 in the examples described later.

[0044] In one embodiment, the polynucleotide of the present invention comprises any one of the following: (a) a nucleotide sequence represented by any of SEQ ID NOs: 9 to 16 (for example, the nucleotide sequence represented by SEQ ID NO: 16); (b) a nucleotide sequence in which one or more (for example, 1 to 3 or 1 to 2) nucleotides are deleted, substituted, or added in the nucleotide sequence represented by any of SEQ ID NOs: 9 to 16 (for example, the nucleotide sequence represented by SEQ ID NO: 16); (c) a nucleotide sequence having 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 82% or more, 85% or more, 87% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 99.9% or more identity with the nucleotide sequence represented by any of SEQ ID NOs: 9 to 16 (for example, the nucleotide sequence represented by SEQ ID NO: 16); or (d) a nucleotide sequence that hybridizes under highly stringent conditions with a nucleotide sequence complementary to the nucleotide sequence represented by any of SEQ ID NOs: 9 to 16 (for example, the nucleotide sequence represented by SEQ ID NO: 16).

[0045] In one embodiment, the nucleotide sequence of the polynucleotide of the present invention may be a codon-optimized nucleotide sequence according to the codon usage frequency in the cells into which the polynucleotide is introduced.

[0046] Further, the polynucleotide of the present invention may be DNA or RNA such as mRNA.

[0047] When the polynucleotide of the present invention is mRNA, the nucleotide sequence thereof can be an mRNA containing, as a coding region, a nucleotide sequence in which thymine (T) is substituted with uracil (U) in any of the nucleotide sequences exemplified above. The mRNA corresponding to the polynucleotide of the present invention may contain, in addition to the coding region, a 5'-terminal cap structure, a 3'-terminal poly-A chain, a 5' untranslated region (5' UTR) upstream of the start codon, and / or a 3' untranslated region (3' UTR) downstream of the stop codon. The 5' UTR and / or 3' UTR may contain sequences for regulating the translation amount from the mRNA.

[0048] In a further embodiment, the bacteriolytic agent of the present invention comprises, or consists of, an expression vector containing a polynucleotide encoding a bacteriolytic enzyme capable of lysing Staphylococcus hominis or an active fragment thereof.

[0049] The expression vector of the present invention contains, in an expressible state, a polynucleotide encoding the bacteriolytic enzyme of the present invention or a fragment thereof. As used herein, the term "expressible state" means that the gene to be expressed is located in the downstream region of the promoter under the control of the promoter.

[0050] The expression vector of the present invention essentially comprises a promoter and any of the above-described polynucleotides.

[0051] Vectors that can be used as the expression vector of the present invention are, for example, expression vectors using plasmids or viruses. As used herein, the term "expression vector" includes plasmid vectors, viral vectors, and recombinant vectors.

[0052] As the promoter, various promoters can be used, such as overexpression promoters, constitutive promoters, site-specific promoters, time-specific promoters, and / or inducible promoters. Examples include the CMV promoter (CMV-IE promoter), SV40 early promoter, RSV promoter, HSV-TK promoter, EF1α promoter, Ub promoter, metallothionein promoter, SRα promoter, or CAG promoter.

[0053] The expression vector may also contain a terminator, enhancer, polyA addition signal, 5'-UTR (untranslated region) sequence, intron sequence, ribosome binding sequence, label or selectable marker gene, multiple cloning site, nuclease recognition sequence, and / or replication origin. Each type is not particularly limited as long as it can function in the host cell.

[0054] (3) Staphylococcus hominis infectious bacteriophage In one embodiment, the bacteriolytic agent of the present invention contains or consists of a Staphylococcus hominis infectious bacteriophage.

[0055] The phage constituting the bacteriolytic agent of the present invention (hereinafter referred to as "the phage of the present invention") is not particularly limited as long as it is a phage that infects Staphylococcus hominis. Examples of Staphylococcus hominis infectious bacteriophages include phages containing the gene encoding the above-described bacteriolytic enzyme of the present invention in their genomes. Examples of such phages include phages having a genomic DNA sequence containing the base sequence shown by any one of SEQ ID NOs: 17 to 24 (for example, the base sequence shown by SEQ ID NO: 24). These phages correspond to eight prophage sequences containing an endolysin gene, which were identified by genome analysis of eight strains of Staphylococcus hominis in this example. Among these, the base sequence shown by SEQ ID NO: 24 is the genomic sequence of a phage containing the gene encoding endolysin 3 in the examples described later.

[0056] In one embodiment, the phage of the present invention has a genomic DNA sequence containing (h) the base sequence shown by any one of SEQ ID NOs: 17 to 24 (for example, the base sequence shown by SEQ ID NO: 24), (i) a base sequence in which one or more (for example, 1 to 3 or 1 to 2) bases are deleted, substituted or added in the base sequence shown by any one of SEQ ID NOs: 17 to 24 (for example, the base sequence shown by SEQ ID NO: 24), and (j) a base sequence having 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, 99.8% or more, or 99.9% or more identity with the base sequence shown by any one of SEQ ID NOs: 17 to 24 (for example, the base sequence shown by SEQ ID NO: 24).

[0057] 1-4. Effects According to the bacteriolytic agent of the present invention, Staphylococcus hominis can be selectively lysed. The bacteriolytic agent of the present invention can selectively lyse Staphylococcus hominis in the skin microbiota, and does not substantially lyse beneficial bacteria such as Staphylococcus epidermidis. Therefore, instead of killing the entire skin resident bacteria including beneficial bacteria like conventional bactericides, the composition can effectively change the composition of the skin microbiota involved in the production of body odor substances by selectively lysing Staphylococcus hominis.

[0058] According to the bacteriolytic agent of the present invention, axillary osmidrosis caused by Staphylococcus hominis (e.g., type C (curry spice type)) can be treated or prevented, and body odor caused by Staphylococcus hominis can be reduced or prevented. This effect is different from bactericides that kill all bacteria in the skin microbiota. Since it selectively kills the causative bacteria of axillary osmidrosis, there is no risk of killing beneficial resident bacteria and inducing skin problems, and the safety is extremely high.

[0059] According to the present invention, host cells into which polynucleotides or expression vectors are introduced are also provided. Also provided is a method for lysing Staphylococcus hominis.

[0060] Also, according to the present invention, there are provided a method for treating or preventing axillary osmidrosis (e.g., type C (curry spice type)) and a method for reducing or preventing body odor, which include the step of administering the bacteriolytic agent of the present invention to a subject.

[0061] The use of the bacteriolytic agent of the present invention in the manufacture of a medicament for treating or preventing axillary osmidrosis is also provided.

[0062] 2. Composition 2-1. Overview The second aspect of the present invention is a composition (hereinafter referred to as "the composition of the present invention") for controlling the skin microbiota involved in the production of body odor. The composition of the present invention includes the bacteriolytic agent of the first aspect, and can treat or prevent axillary osmidrosis and reduce or prevent body odor such as curry spice-like odor.

[0063] 2-2. Composition The composition of the present invention includes an active ingredient as an essential constituent, and a pharmaceutically acceptable carrier or other components as optional components. The composition of the present invention can also be composed of only the active ingredient. However, in order to facilitate the formation of a dosage form and maintain the pharmacological effect and / or dosage form of the active ingredient, it is preferably composed as a composition containing a pharmaceutically acceptable carrier described below.

[0064] The composition of the present invention may be a cosmetic composition for controlling body odor or a pharmaceutical composition for use in the treatment or prevention of bromhidrosis according to the intended use, and can also be a quasi-drug. Any of the compositions can be configured according to the constituent components, dosage forms, administration methods, and target diseases described below.

[0065] 2-2-1. Constituent components Each component constituting the composition of the present invention will be specifically described. (1) Active ingredient The active ingredient in the composition of the present invention is the bacteriolytic agent of the present invention. Since its configuration has already been described in detail in the first aspect, the specific description thereof is omitted here. The number of bacteriolytic agents contained in the composition of the present invention is not limited and may be one or more. When the composition of the present invention contains a plurality of bacteriolytic agents, for example, it can contain any combination of the wild-type endolysin and / or mutant endolysin described in the first aspect.

[0066] (2) Pharmaceutically acceptable carrier "Pharmaceutically acceptable carrier" refers to a solvent and / or additive that can be usually used in the pharmaceutical technology field and has little or no harm to the living body.

[0067] Pharmaceutically acceptable solvents include, for example, water, ethanol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters, and the like. These are preferably sterilized and, if necessary, are preferably isotonic with body fluids such as sweat and blood.

[0068] In addition, pharmaceutically acceptable additives include, for example, excipients, binders, disintegrants, fillers, emulsifiers, flow additive regulators, lubricants, and the like.

[0069] Examples of excipients include sugars such as monosaccharides, disaccharides, cyclodextrins, and polysaccharides (more specifically, but not limited to, glucose, sucrose, lactose, raffinose, mannitol, sorbitol, inositol, dextrin, maltodextrin, starch, and cellulose), metal salts (e.g., sodium chloride, sodium phosphate or calcium phosphate, calcium sulfate, magnesium sulfate, calcium carbonate), citric acid, tartaric acid, glycine, low, medium, or high molecular weight polyethylene glycol (PEG), pluronic (registered trademark), kaolin, silicic acid, or combinations thereof.

[0070] Examples of binders include starch paste using starch from corn, wheat, rice, or potato, simple syrup, glucose solution, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, shellac, and / or polyvinylpyrrolidone, and the like.

[0071] Examples of disintegrants include the aforementioned starch, lactose, carboxymethyl starch, crosslinked polyvinylpyrrolidone, agar, laminaran powder, sodium hydrogen carbonate, calcium carbonate, alginic acid or sodium alginate, polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, monoglyceride stearate, or salts thereof.

[0072] Examples of the filler include the sugar and / or calcium phosphate (e.g., tricalcium phosphate or calcium hydrogen phosphate).

[0073] Examples of the emulsifier include sorbitan fatty acid ester, glycerin fatty acid ester, sucrose fatty acid ester, and propylene glycol fatty acid ester.

[0074] Examples of the flow additive regulator and lubricant include silicate, talc, stearate, or polyethylene glycol.

[0075] In addition to the above additives, dissolution aids (solubilizers), suspending agents, diluents, surfactants (cationic surfactants, anionic surfactants, amphoteric surfactants, etc.), stabilizers, extenders, humectants (e.g., glycerin, starch), adsorbents (e.g., starch, lactose, kaolin, bentonite, colloidal silica), ultraviolet absorbers, coating agents, colorants, preservatives, antioxidants, fragrances, cooling agents, buffers, chelating agents, thickeners, vitamins, neutralizing agents, amino acids, pH adjusters, whitening agents, anti-inflammatory agents, deodorants, animal and plant extracts, sequestering agents, essential oils, and other additives can also be included as needed.

[0076] (3) Other components The composition of the present invention can also contain other components as long as the pharmacological effects of the above active ingredients are not lost. Here, the "other components" include agents having bacteriolytic activity against Staphylococcus hominis similar to the bacteriolytic agent of the present invention, other body odor control substances, and the like. For example, known therapeutic agents for preventing body odor such as bromhidrosis, antibacterial agents, or germicides can be mentioned. Examples of other components include deodorant agents, antiperspirants, fragrances, germicides, and the like. The antiperspirant may be, for example, chlorhydroxyaluminum, zinc paraphenolsulfonate, or alum. The germicide may be, for example, isopropylmethylphenol, benzalkonium chloride, hinokitiol, triclosan, or salicylic acid. Further, it may be a drug having no direct action on body odor or an action unrelated to the bacteriolytic activity against Staphylococcus hominis. For example, from the viewpoint of flavoring, the composition of the present invention may contain essential oils. Examples of essential oils include rosemary leaf oil, lavender oil, eucalyptus oil, spearmint oil, and the like.

[0077] When the composition of the present invention is a compound preparation containing other components, it is convenient because synergistic effects such as the ability to comprehensively suppress body odor such as bromhidrosis can be expected.

[0078] 2-2-2. Dosage form The dosage form of the composition of the present invention is not particularly limited as long as it does not inactivate or hardly inactivates the bacteriolytic agent of the present invention, which is the active ingredient, and can sufficiently exhibit its pharmacological effects after administration.

[0079] The dosage form can be classified into a liquid dosage form or a solid dosage form (including semi-solid dosage forms such as gels) depending on its form, but the pharmaceutical composition of the present invention can be any of them. Further, the dosage form can be roughly classified into an oral dosage form and a parenteral dosage form depending on the administration method, and either of them is acceptable in this regard, but the parenteral dosage form is more preferable.

[0080] Specific parenteral dosage forms include, for example, liquid dosage forms such as suspensions, emulsions, and injections, and solid dosage forms such as creams, ointments, plasters, gels, lotions, sprays (e.g., aerosol sprays, mists), sticks, patches, and suppositories. Preferred dosage forms are liquids, gels, creams, lotions, sprays, mists, sticks, or patches. Gels and creams have advantages such as good adhesion to the skin, no dripping or scattering, and good usability.

[0081] 2-2-3. Application method / Administration method For the composition of the present invention, any method known in the art can be applied as long as it can administer an effective amount of the bacteriolysin of the present invention, which is an active ingredient, to a living body for body odor control or for the treatment or prevention of bromhidrosis.

[0082] As used herein, "effective amount" refers to the amount necessary for the active ingredient to exert its function, that is, the amount necessary for the composition of the present invention to control body odor or to treat or prevent bromhidrosis, and that causes little or no harmful side effects to the living body to which it is applied. This effective amount may vary depending on conditions such as the information of the subject, the administration route, and the number of administrations. "Subject" or "object" refers to an animal individual to which the composition of the present invention is applied. A preferred subject is a human, for example, an adult male. "Subject information" refers to various individual information of the subject, including, for example, the subject's age, weight, gender, overall health status, drug sensitivity, and the presence or absence of drugs being taken. The effective amount and the dosage calculated based on it are determined by the judgment of a doctor or veterinarian according to the information of each subject.

[0083] The administration method of the composition of the present invention may be either systemic administration or local administration, but local administration to the skin, for example, the armpit, is more preferred. Examples of local administration include application or spraying on the skin, transdermal administration, intradermal administration, subcutaneous administration, and implants.

[0084] When administering or ingesting the composition of the present invention, the dosage or ingestion amount is appropriately selected according to the age, weight, symptoms, health status, type of the composition (cosmetic composition, pharmaceutical composition, quasi-drug), etc. of the subject. For example, it may be 0.001 mg / kg / day to 1000 mg / kg / day, 0.01 mg / kg / day to 500 mg / kg / day, 0.1 mg / kg / day to 200 mg / kg / day, 1 mg / kg / day to 100 mg / kg / day, 5 mg / kg / day to 50 mg / kg / day, or 10 mg / kg / day.

[0085] 2-2-4. Target body odor / target disease The body odor such as bromhidrosis targeted by the composition of the present invention is not particularly limited as long as it is caused by Staphylococcus hominis. Bromhidrosis may be any type other than the M type, and may be any of the A type (acid type), C type (curry spice type), K type (mold type), E type (steamed meat type), W type (dry type), F type (iron type), or Other type (others), but preferably the C type. Each type constituting bromhidrosis can be identified by an odor appraiser.

[0086] 2-3. Effects According to the pharmaceutical composition of the present invention, bromhidrosis can be treated or prevented. Further, according to the cosmetic composition of the present invention, body odor can be controlled. For example, the curry spice-like odor can be reduced or removed, and the generation of the curry spice-like odor can be prevented.

Examples

[0087] <Example 1: Metabolome analysis of axillary skin and whole genome analysis of axillary microbiota> (Purpose) Male axillary osmidrosis is classified into seven types, namely, type A (acid type), type C (curry spice type), type K (mold type), type E (steamed meat type), type W (dry type), type F (iron type), and Other type (others), excluding type M (milk type). Among them, type C corresponds to osmidrosis, and type M does not correspond to osmidrosis (Figure 1A). In this example, in order to analyze the odor substances and skin microbiota involved in type C, sweat was collected from the axillae of subjects corresponding to type C and type M, and metabolome analysis was performed, and bacteria were recovered from the axillae and whole-genome analysis was performed (Figure 1B).

[0088] (Methods and Results) (1) Metabolome analysis Samples were collected from the axillae of 11 male subjects classified as type C and 9 male subjects classified as type M by an odor appraiser, and metabolome analysis by CE-FTMS was commissioned to Human Metabolome Technologies Co., Ltd. (HMT). Specifically, a sterilized glass tube was applied to the axilla of the subject 24 hours after washing with fragrance-free soap, 5 mL of a 5% ethanol solution was added to the axilla, and then the axilla was rubbed for 1 minute using a glass rod to obtain an extract. The obtained extract was passed through a 0.22 μm membrane filter, stored at -80°C, centrifuged to remove the residue, 20 μL of Milli-Q water containing an internal standard substance (manufactured by Human Metabolome Technologies (HMT)) was added to 4,000 μL of the supernatant, and the mixture was concentrated under reduced pressure to 50 μL. Centrifugal filtration was performed at 9,100×g and 4°C for 120 minutes using a 5 kDa filtration filter (ULTRAFREE MC PLHCC, HMT), the filtrate was subjected to capillary electrophoresis-time of flight mass spectrometer (CE-TOFMS), and the obtained peaks were analyzed using automatic integration software MasterHands (Keio University).

[0089] For each substance identified by metabolome analysis, the average value of the detected amount was calculated for each of the M-type subject group and the C-type subject group, and the ratio of the average value of the M-type to the average value of the C-type was obtained. Further, based on the detected amount of each substance, the statistical significance between the M-type subject group and the C-type subject group was calculated. By plotting the logarithmic value (log2(M / C)) of the above ratio on the horizontal axis and the calculated value (-log 10 (q-value of Wilcoxon rank sum test)) based on the above statistical significance on the vertical axis, a volcano plot was created (Figure 2).

[0090] From the volcano plot shown in Figure 2, it was found that in C-type subjects, various odor substances derived from apocrine glands and their precursors, such as 3-hydroxy-3-methyl-hexanoic acid (HMHA), were detected in large amounts.

[0091] (2) Whole genome analysis Samples containing cutin, sweat, sebum, etc. on the skin surface were collected from the axillae of 11 male subjects classified as C-type and 9 male subjects classified as M-type described above by the following scraping method.

[0092] A sterilized glass tube was pressed against the axilla of the subject 24 hours after washing with fragrance-free soap. After adding 2 mL of phosphate buffer containing 0.5% Tween20, the axilla was gently rubbed with a glass rod for 1 minute, and the collected liquid was recovered. The collected liquid was stored at -80°C until DNA extraction. Bacterial DNA extraction from the collected sample was performed by the following method. The axilla sample stored in phosphate buffer was centrifuged at 16,000×g for 5 minutes, and the supernatant was removed. DNA was extracted from the precipitate using the DNeasy PowerSoil Kit (QIAGEN) according to the attached instructions.

[0093] Subsequently, whole-genome analysis was performed. Specifically, the DNA library was prepared according to the attached instructions using the KAPA HyperPlus Kit (KAPA Biosystems, Indianapolis, IN, USA), except that NEBNext Multiplex Oligos (New England BioLabs, Ipswich, MA, USA) was used in the adapter ligation and barcoding steps. The libraries were pooled and sequenced using a HiSeq2500 sequencer (2×250 paired end reads, HiSeq Rapid SBS Kit v2 (Illumina, San Diego, CA, USA)).

[0094] The results of shotgun sequencing-based microbial composition analysis are shown in Figure 3. Bacterial families with an average abundance exceeding 1% in either the C-type or M-type were identified as Propionibacteriaceae, Corynebacteriaceae, Porphyromonadaceae, Staphylococcaceae, Clostridiales family XI Incertae sedis, and Enterobacteriaceae.

[0095] In the axillae of C-type subjects, significantly more bacteria of the family Staphylococcaceae were found compared to those of M-type subjects. The Staphylococcaceae bacteria detected from the axillae of C-type subjects included Staphylococcus hominis, which has a highly active 3M3SH synthase, and Staphylococcus epidermidis, which is known as a commensal bacterium beneficial to the skin barrier function.

[0096] On the one hand, in the axilla of the M-type subjects, it was revealed that there were more bacteria of the families Corynebacteriaceae and Propionibacteriaceae than in the axilla of the C-type subjects. The Corynebacteriaceae bacteria detected from the axilla of the M-type subjects included C. granulosum and C. acnes.

[0097] (3) Pathway analysis of the C-type axillary microbiota Using KEGG pathway analysis, the microbiota detected in the axilla of the C-type subjects (C-type axillary microbiota) was analyzed. Specifically, gene set enrichment analysis (GSEA) was performed on the gene sets that make up the KEGG pathways, and gene sets that were specifically different between the C-type or M-type with significant differences were detected.

[0098] The analysis results are shown in Figure 4. In the C-type axillary microbiota, it was revealed that the pathways related to the survival of Staphylococcus bacteria were specifically different with significant differences. Specifically, the pathways related to resistance to antimicrobial peptides, the pathway related to uric acid metabolism (ensuring nitrogen reduction), and the pathway related to the synthesis of bacterial cell wall components were specifically different in the C-type with significant differences.

[0099] <Example 2: Whole-genome analysis of Staphylococcus hominis and identification of phage-derived endolysin> (Objective) Staphylococcus hominis is a type of Gram-positive coagulase-negative staphylococcus. It is a resident bacterium on the skin of sites with apocrine glands such as the axilla and genitals in the human body, but it may cause diseases in humans with a weak immune system. In this example, in order to find a method for specifically killing Staphylococcus hominis without killing other resident bacteria on the skin, whole-genome analysis of Staphylococcus hominis was performed, and endolysin encoded by phage was identified.

[0100] (Methods and Results) Eight strains of Staphylococcus hominis (ATCC25615, ATCC27844T, ATCC27845, ATCC27847, ATCC35981, ATCC51624, ATCC700236, and ATCC700586) were obtained from ATCC (American Type Culture Collection), and whole-genome analysis was performed for each strain. Specifically, the DNA library was prepared using the KAPA HyperPlus Kit (KAPA Biosystems, Indianapolis, IN, USA) according to the attached instructions, except that NEBNext Multiplex Oligos (New England BioLabs, Ipswich, MA, USA) was used in the adapter ligation and barcoding steps. The libraries were pooled and sequenced using the MiSeq v3 Reagent kit and 15% PhiX spike (Illumina) on a MiSeq instrument (Illumina, San Diego, CA). The open reading frames (ORFs) identified as a result of the sequencing of each sample were annotated according to the KEGG prokaryotic genes and the corresponding KOs. Also, prophage sequences in the bacterial contigs were predicted by VirSorter (v1.0.3). Twenty-two types of prophage sequences were identified from the sequence data of each recent strain.

[0101] As a result of analyzing 22 types of prophage sequences, 8 types of prophages containing an endolysin (lysozyme) gene were found. The genomic sequences of these 8 types of prophages are shown by SEQ ID NOs: 17 to 24. The prophages having a phage genomic sequence consisting of the nucleotide sequences shown by SEQ ID NOs: 17 to 24 are derived from the following Staphylococcus hominis strains: ATCC700236, ATCC700586, ATCC27847, ATCC700586, ATCC25615, ATCC27844, ATCC27847, and ATCC700236, respectively. Further, the amino acid sequences of the 8 types of endolysins encoded by the prophages having a phage genomic sequence consisting of the nucleotide sequences shown by SEQ ID NOs: 17 to 24 are shown by SEQ ID NOs: 1 to 8, respectively, and the nucleotide sequences encoding the endolysins consisting of the amino acid sequences shown by SEQ ID NOs: 1 to 8 are shown by SEQ ID NOs: 9 to 16, respectively.

[0102] The results of classifying the above 8 types of endolysins together with 4 known endolysin sequences (YP_009226740.1, YP_239818.1, YP_007236686.1, and YP_007236621.1) based on the amino acid sequence are shown in FIG. 5. The endolysins consisting of the amino acid sequences shown by SEQ ID NOs: 1 to 8 all have an N-acetylmuramoyl-L-alanine amidase domain (in the classification results shown in FIG. 5, the Amidase_2 domain and the Amidase_3 domain belonging to the N-acetylmuramoyl-L-alanine amidase domain are shown). In the present specification, the endolysins consisting of the amino acid sequences shown by SEQ ID NOs: 2, 3, and 8 are referred to as "endolysin 1", "endolysin 2", and "endolysin 3", respectively. In Example 3 below, the enzyme activity was verified using endolysin 3 as an example.

[0103] <Example 3: Lytic activity of endolysin 3> (Objective) Synthesize the endolysin 3 identified in Example 2 and verify its in vitro lytic activity against Staphylococcus hominis (S. hominis).

[0104] (Method and Results) (1) Protein synthesis The phage-derived endolysin gene was artificially synthesized (Invitrogen) and ligated to the BamHI / SalI site of the pCold-SUMO expression vector (Creative Biogene, Shirley, NY) to generate a His-SUMO-tagged endolysin expression vector. This expression vector was transformed into BL21(DE3) cells, and the target protein was purified using a His-Trap HP column (cytiva). The obtained protein was loaded onto an Amicon (registered trademark) Ultra-15 10K (Millipore) and centrifuged at 5,000×g for 20 minutes at 4°C. The concentration of the target protein was measured using Protein Assay CBB Solution (Nacalai Tesque). The obtained endolysin 3 was confirmed by SDS polyacrylamide gel electrophoresis (SDS-PAGE).

[0105] (2) Bacteriolytic activity against Staphylococcus hominis strains Staphylococcus hominis strain ATCC27844 was aerobically cultured in tryptic soy broth medium and recovered by centrifugation at 3,000×g for 15 minutes. The cell pellet was washed and resuspended in HiTrap buffer. Endolysin 3 was added to the cell resuspension at a final concentration of 50 μg / mL. The bacteriolytic activity was measured by measuring the turbidity (OD 600 ) every minute using a TVS062CA BioPhoto recorder (ADVANTEC, Tokyo, Japan).

[0106] In addition, as skin commensal bacteria other than Staphylococcus hominis, the bacterial strains NBRC 100911 of Staphylococcus epidermidis and NBRC 109768 of S. haemolyticus cultured in the L-drying sample restoration medium "Dai-go" 802 medium, and the bacterial strains JCM 13389 of C. tuberculostearicum and JCM 15295 of C. ureicelerivorans cultured in R medium were used for the measurement of bacteriolytic activity by endolysin 3.

[0107] The measurement results of lytic activity are shown in FIGS. 6 and 7. Endolysin 3 showed lytic activity only against Staphylococcus hominis and did not show lytic activity against skin commensal bacteria other than Staphylococcus hominis. Therefore, it was revealed that endolysin 3 has lytic activity specific to Staphylococcus hominis.

[0108] <Example 4: Sequence analysis> (Objective) Perform BLAST analysis on the amino acid sequences of the eight types of endolysins identified in Example 2, the nucleotide sequences encoding the eight types of endolysins, and the phage genome sequences of the eight types of prophages.

[0109] (Method and results) As a result of performing BLAST analysis on the amino acid sequences (SEQ ID NOs: 1 to 8) of the eight types of endolysins, sequence identity of 90% or more was detected between the sequences of the SEQ ID NOs shown below. SEQ ID NO: 1 vs SEQ ID NO: 2: Sequence identity 100% (Query cover: 100%) SEQ ID NO: 3 vs SEQ ID NO: 4: Sequence identity 97.13% (Query cover: 100%) SEQ ID NO: 3 vs SEQ ID NO: 6: Sequence identity 97.84% (Query cover: 57%) SEQ ID NO: 4 vs SEQ ID NO: 6: Sequence identity 96.76% (Query cover: 57%) SEQ ID NO: 6 vs SEQ ID NO: 3: Sequence identity 97.84% (Query cover: 100%) SEQ ID NO: 6 vs SEQ ID NO: 4: Sequence identity 96.76% (Query cover: 100%)

[0110] As a result of performing BLAST analysis on the nucleotide sequences (SEQ ID NOs: 9 to 16) encoding the eight types of endolysins, sequence identity of 90% or more was detected between the sequences of the SEQ ID NOs shown below. Array No. 9 vs Array No. 10: Sequence identity 100% (Query cover 100%) Array No. 11 vs Array No. 12: Sequence identity 93.92% (Query cover: 100%) Array No. 11 vs Array No. 14: Sequence identity 95.58% (Query cover: 57%) Array No. 12 vs Array No. 14: Sequence identity 94.03% (Query cover: 57%) Array No. 14 vs Array No. 11: Sequence identity 95.58% (Query cover: 100%) Array No. 14 vs Array No. 12: Sequence identity 94.03% (Query cover: 100%)

[0111] As a result of performing BLAST analysis on the phage genome sequences (Array Nos. 17 to 24) of 8 types of prophages, identities of 90% or more were detected between the sequences of the following array numbers. Array No. 17 vs Array No. 18: Sequence identity 99.99% (Query cover: 83%) Array No. 18 vs Array No. 17: Sequence identity 99.99% (Query cover: 100%) Array No. 19 vs Array No. 23: Sequence identity 94.28% (Query cover: 3%) Array No. 19 vs Array No. 20: Sequence identity 93.40% (Query cover: 3%) Array No. 19 vs Array No. 22: Sequence identity 92.83% (Query cover: 5%) Array No. 20 vs Array No. 24: Sequence identity 99.42% (Query cover: 33%) Array No. 20 vs Array No. 22: Sequence identity 97.56% (Query cover: 60%) Array No. 20 vs Array No. 21: Sequence identity 94.38% (Query cover: 1%) Array No. 20 vs Array No. 17: Sequence identity 99.99% (Query cover: 100%) Array No. 21 vs Array No. 24: Sequence identity 95.42% (Query cover: 65%) Array No. 21 vs Array No. 20: Sequence identity 94.20% (Query cover: 2%) Array No. 21 vs Array No. 23: Sequence identity 90.73% (Query cover: 1%) Array No. 22 vs Array No. 24: Sequence identity 97.47% (Query cover: 19%) Array No. 22 vs Array No. 20: Sequence identity 97.56% (Query cover: 34%) Array No. 22 vs Array No. 23: Sequence identity 93.00% (Query cover: 3%) Array No. 23 vs Array No. 19: Sequence identity 94.28% (Query cover: 5%) Array No. 23 vs Array No. 24: Sequence identity 91.89% (Query cover: 0%) Array No. 23 vs Array No. 21: Sequence identity 90.73% (Query cover: 1%) Array No. 24 vs Array No. 21: Sequence identity 95.42% (Query cover: 31%) Array No. 24 vs Array No. 22: Sequence identity 97.47% (Query cover: 24%) Array No. 24 vs Array No. 20: Sequence identity 99.42% (Query cover: 24%) Array No. 24 vs Array No. 23: Sequence identity 91.39% (Query cover: 0%)

Claims

1. A bacteriolytic agent for controlling the skin microbiota involved in body odor generation, comprising: a lytic enzyme derived from a Staphylococcus hominis-infecting bacteriophage or an active fragment thereof, a polynucleotide encoding the lytic enzyme or an active fragment thereof, or the bacteriophage The bacteriolytic agent as described above.

2. The bacteriolytic agent according to claim 1, wherein the lytic enzyme or an active fragment thereof contains an N-acetylmuramoyl-L-alanine amidase domain.

3. The lytic enzyme is (a) an amino acid sequence represented by any one of SEQ ID NOs: 1 to 8, (b) an amino acid sequence in which one or more amino acids are added, deleted, and / or substituted in the amino acid sequence represented by any one of SEQ ID NOs: 1 to 8, or (c) an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by any one of SEQ ID NOs: 1 to 8 The bacteriolytic agent according to claim 2, which consists of.

4. The polynucleotide is (d) a nucleotide sequence represented by any one of SEQ ID NOs: 9 to 16, (e) a nucleotide sequence in which one or more bases are deleted, substituted, or added in the nucleotide sequence represented by any one of SEQ ID NOs: 9 to 16, (f) a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by any one of SEQ ID NOs: 9 to 16, or (g) a nucleotide sequence that hybridizes under highly stringent conditions with a nucleotide sequence complementary to the nucleotide sequence represented by any one of SEQ ID NOs: 9 to 16 The bacteriolytic agent according to claim 1, which contains.

5. The bacteriophage is (h) a nucleotide sequence represented by any one of SEQ ID NOs: 17 to 24, (i) a nucleotide sequence in which one or more bases are deleted, substituted, or added in the nucleotide sequence represented by any one of SEQ ID NOs: 17 to 24, (j) a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by any one of SEQ ID NOs: 17 to 24 The bacteriolytic agent according to claim 1, which has a genomic DNA sequence containing.

6. The bacteriolytic agent according to claim 1, wherein the lytic activity against Staphylococcus hominis is higher than the lytic activity against Staphylococcus epidermidis.

7. A bacteriolytic agent for Staphylococcus hominis, comprising: a lytic enzyme derived from a Staphylococcus hominis-infecting bacteriophage or an active fragment thereof, a polynucleotide encoding the lytic enzyme or an active fragment thereof, or the bacteriophage including The lytic enzyme is (a) an amino acid sequence represented by any one of SEQ ID NOs: 1 to 8, (b) an amino acid sequence in which one or more amino acids are added, deleted, and / or substituted in the amino acid sequence represented by any one of SEQ ID NOs: 1 to 8, or (c) an amino acid sequence having 90% or more sequence identity with the amino acid sequence represented by any one of SEQ ID NOs: 1 to 8 and comprising the bacteriophage is (h) a nucleotide sequence represented by any one of SEQ ID NOs: 17 to 24, (i) a nucleotide sequence in which one or more nucleotides are deleted, substituted or added in the nucleotide sequence represented by any one of SEQ ID NOs: 17 to 24, (j) a nucleotide sequence having 90% or more identity with the nucleotide sequence represented by any one of SEQ ID NOs: 17 to 24 and having a genomic DNA sequence containing the same, the lysing agent.

8. A composition for controlling the skin microbiota involved in the production of body odor, comprising the lysing agent according to any one of Claims 1 to 6 as an active ingredient.

9. A cosmetic composition comprising the composition according to Claim 8.

10. The cosmetic composition according to Claim 9, further comprising a deodorant, an antiperspirant, and / or a fragrance.

11. The cosmetic composition according to Claim 10, for reducing or removing a curry spice-like odor or for preventing the generation of a curry spice-like odor.

12. The cosmetic composition according to Claim 9, which is a liquid, a gel, a cream, a lotion, a spray, a mist, a stick, or a patch.

13. A pharmaceutical composition comprising the composition according to Claim 8.

14. The pharmaceutical composition according to Claim 13, for use in the treatment or prevention of bromhidrosis.

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