Anti-biofilm and Anti-microbial composition

Abstract

The present invention provides a more effective use of protamine or a protamine degradation product. Provided is a composition for anti-biofilm and / or anti-microbial use, the composition comprising at least two components selected from protamine, protamine degradation products, basic polyamino acids (e.g., polylysine, polyornithine, and polyarginine), and salts thereof. Also provided is a composition for inhibiting production of an extracellular polysaccharide (EPS), the composition containing a protamine degradation product or a salt thereof.

Description

Anti-biofilm and anti-microbial compositions

[0001] The present invention relates to a composition for at least one of antibiofilm and antibacterial properties, comprising protamine or a degradation product of protamine. The present invention is useful in the fields of development and manufacturing of oral care products.

[0002] Protamine is a protein extracted from fish testes and is rich in the basic amino acid arginine. Protamine is known to have antibacterial activity. Because protamine is a food-derived component, its use in foods and oral compositions has been investigated for its antibacterial activity. For example, Patent Document 1 describes a food material and a composition thereof, containing an extract of the whole plant of Magnolia obovata, an extract of the whole plant of Kahlo (Chinese laurel), and licorice extract as essential ingredients, with polylysine and / or protamine as other essential ingredients. Patent Document 2 also describes a method for stabilizing a carbohydrate-rich food, which comprises adding protamine or a salt thereof and / or ε-polylysine or a salt thereof to a carbohydrate-rich food containing 10% to less than 70% by weight of carbohydrates by solid content and having a water activity of 0.81 to 0.97, and further adding sodium acetate. Furthermore, Patent Document 3 describes an oral composition containing (A) an organic acid calcium salt, (B) fluoride, and (C) a basic peptide and / or a basic protein in which 60% or more of the constituent amino acid residues are basic amino acid residues, wherein the component (C) is at least one selected from the group consisting of ε-polylysine, α-polylysine, and protamine. Patent Document 4 also describes an oral composition containing protamine and arginine for improving the balance of oral bacterial flora.

[0003] Protamine is also used as a degradation product. It is known that degradation products of protamine have antifungal activity (Patent Document 5). Regarding protamine degradation products, Patent Document 6 describes an agent for preventing or ameliorating dry mouth or xerostomia. Patent Document 7 describes a periodontal disease treatment material comprising an edible sheet containing protamine degradation peptides. Furthermore, Patent Document 8 describes an oral antibacterial composition containing, as Component A, at least one selected from the group consisting of protamine, protamine degradation products having a molecular weight of 500 to 4000 Da, and pharmaceutically acceptable salts thereof, and as Component B, 3-methyl-4-isopropylphenol. Patent Document 9 describes an oral cavity-retaining composition that contains at least one compound selected from the group consisting of shellac and zein, a functional substance, and an acidic polysaccharide, in which the functional substance coated with at least one compound selected from the group consisting of shellac and zein is mixed with the acidic polysaccharide, and the basic substance is at least one selected from the group consisting of protamine degradation products, arginine, lysine, glucosamine, spermine, spermidine, putrescine, piperine, and chitosan.

[0004] Meanwhile, the use of basic amino acids and their polypeptides for oral care has been investigated. Patent Document 10 describes an anti-caries and anti-periodontal disease agent containing ε-poly-L-lysine or a salt thereof as an active ingredient. Patent Document 11 describes dental wipes combined with or associated with a basic amino acid, wherein the basic amino acid is arginine, lysine, citrulline, ornithine, creatine, histidine, diaminobutanoic acid, diaminopropionic acid, salts thereof, and / or combinations thereof. Patent Document 12 further describes an oral care composition comprising (A) a polycationic substance having two or more cationic functional groups in the molecule, and (B) laccase, wherein the polycationic substance is a basic peptide, and the basic peptide is one or more selected from polylysine, polyarginine, and polyhistidine.

[0005] JP 2001-321123 A JP 2014-143946 A (Patent No. 6188008) International Publication WO2018 / 168997 JP 2023-91841 A JP 2008-133253 A (Patent No. 4520477) JP 2010-265217 A (Patent No. 5467800) JP 2011-2 25490 (Patent No. 4520477) International Publication WO2021-132180 International Publication WO2022 / 019290 JP 5-310544 A (Patent No. 3114359) International Publication WO2009 / 100270 (Patent No. 2011-511796) JP 2012-25695 A (Patent No. 5703617)

[0006] It would be desirable to have a more effective method for using protamine or protamine degradation products.

[0007] The present invention provides the following: [1] A composition for at least one of antibacterial, antibiofilm, and antimicrobial properties, comprising at least two components selected from protamine, a protamine degradation product, a basic polyamino acid, and a salt thereof. [2] The composition according to 1, wherein the at least two components comprise a protamine degradation product or a salt thereof and a basic polyamino acid or a salt thereof. [3] The composition according to 1, wherein the at least two components are a protamine degradation product and polylysine, or polylysine and polyornithine. [4] The composition according to 1, wherein the at least two components are a protamine degradation product and polylysine, a protamine degradation product and polyarginine, a protamine degradation product and polyornithine, or polylysine and polyornithine. [5] The composition according to 1, wherein the composition is for at least one of antibacterial, antibiofilm, and antibacterial properties against at least one selected from Streptococcus mutans and Candida albicans. [6] The composition according to 1, wherein the composition is for oral care or for preventing oral biofilm infections. [7] The composition according to any one of 1 to 6, which is a health care composition or a food composition. [8] A composition for producing an oral care composition, comprising (A) a protamine degradation product or a salt thereof; and (B) at least one selected from polylysine, polyarginine, polyornithine, and salts thereof, wherein the mass ratio of (A) to (B) ((A):(B)) is 1 to 9:9 to 1. [9] A composition for producing an oral care composition, comprising (A) polyornithine or a salt thereof; and (B) polylysine or a salt thereof, wherein the mass ratio of (A) to (B) ((A):(B)) is 1 to 9:9 to 1.

[10] A composition for inhibiting extracellular polysaccharide (EPS) production, comprising a protamine degradation product or a salt thereof.

[0008] The present invention provides the following: [1] A composition for at least one of antibacterial and antibiofilm properties, comprising at least two components selected from protamine, a protamine degradation product, polylysine, polyornithine, polyarginine, and salts thereof. [2] The composition according to 1, comprising a protamine degradation product, wherein the protamine degradation product comprises a peptide of (1) or (2) below: (1) A peptide consisting of any one of the sequences of SEQ ID NOs: 1 to 6, or (2) A peptide consisting of any one of the sequences of SEQ ID NOs: 1 to 6, with 1 to 3 amino acids deleted or added at one or both termini. [3] The composition according to 1 or 2, which is for antibacterial use and wherein the at least two components are a protamine degradation product and polylysine, or polylysine and polyornithine. [4] The composition according to any one of 1 to 3, which is for antibiofilm use and wherein the at least two components are a protamine degradation product and polylysine, a protamine degradation product and polyarginine, a protamine degradation product and polyornithine, or polylysine and polyornithine. [5] The composition according to any one of 1 to 4, which has at least one of antibacterial and antibiofilm properties against Streptococcus mutans. [6] The composition according to any one of 1 to 5, which is for preventing oral biofilm infections. [7] The composition according to any one of 1 to 6, which is a health care composition or a food composition. [8] A composition comprising (A) a protamine degradation product or a salt thereof; and (B) at least one selected from polylysine, polyarginine, polyornithine, and salts thereof, wherein the mass ratio of (A) to (B) ((A):(B)) is 1 to 9:9 to 1. [9] A composition comprising (A) polyornithine or a salt thereof; and (B) polylysine or a salt thereof, wherein the mass ratio of (A) to (B) ((A):(B)) is 1 to 9:9 to 1.

[0009] The present invention provides a composition having at least one of antibacterial activity and antibiofilm activity. In an embodiment in which active ingredients are used in combination, a synergistic effect can be achieved.

[0010] Protamine hydrolysate-ε-poly-L-lysine combination treatment (24-hour treatment). A significant reduction in viable bacterial counts was confirmed with a 1 / 2 MIC combination treatment compared to the viable bacterial counts with polylysine and protamine hydrolysate alone. Polylysine-polyornithine combination treatment. A significant reduction in bacterial counts was confirmed with a 1 / 2 MIC combination treatment compared to polylysine or polyornithine alone. Antibiofilm investigation - total biofilm volume. The concentration-dependent inhibitory effect of biofilm formation by antimicrobial peptides was confirmed. The inhibitory effect of protamine hydrolysate was lower than the other compounds. Polylysine, polyarginine, and polyornithine inhibited biofilm formation to a similar extent when used alone. Protamine hydrolysate: protamine hydrolysate. Antibiofilm investigation - total biofilm volume. The biofilm formation inhibitory effect of a 1 / 2 MIC combination treatment was equal to or greater than that of MIC treatment alone, confirming that the combination of protamine hydrolysate and antimicrobial peptides was equally effective. The greatest antibiofilm effect was confirmed with the combination of protamine hydrolysate and polyarginine. Pro: protamine degradation product, Pro: protamine, Lys: polylysine, Arg: polyarginine, Orni: polyornithine. Antibacterial effect under C. albicans biofilm formation conditions. Effect on the total amount of C. albicans biofilm. Effect on the amount of EPS in S. mutans. SEM image of S. mutans biofilm. Bar indicates 5 μm. Molecular weight distribution of HAP-100.

[0011] 1. Anti-biofilm and anti-bacterial compositions This embodiment provides a composition for at least one of anti-biofilm and anti-bacterial properties, which comprises, as active ingredients, at least two components selected from protamine, protamine degradation products, polylysine, polyornithine, polyarginine, and salts thereof.

[0012] [Active ingredient] (Protamine) Protamine exists in fish testes (milt) bound to DNA and is a strongly basic protein generally composed of 27 to 65 amino acid residues, of which more than two-thirds are arginine residues, depending on the fish from which it is derived. Depending on the source material, it is called salmine (salmon), clupein (herring), etc., and its structure varies slightly.

[0013] The method for producing protamine used in this embodiment is not particularly limited. The fish used as the raw material are not particularly limited, but are preferably fish of the Salmonidae, Clupeidae, or Gadidae families. The Salmonidae family includes the genus Salmonella, Salmognathus, Salmognathus, and Salmognathus, with Salmognathus or Salmognathus being more preferred. Examples of fish of the Salmonidae genus include Chum salmon, Coho salmon (Coho salmon, Silver salmon), Pink salmon, Cherry salmon, Yamame salmon, Taiwan salmon, Satsukimasu, Amago salmon, Biwa salmon (Ame nou), Rainbow trout (Steelhead), Chinook salmon (Chinook salmon), Sockeye salmon, Kokanee salmon, and Kunimasu. Examples of fish of the Salmonidae genus include Atlantic salmon and brown trout. The Clupeidae family includes the Clupeinae, Clupeinae, Scutellinae, and Ehiravinae subfamilies, with Clupeinae and Clupeinae fish being preferred. Examples of Clupeidae fish include Pacific herring and Atlantic herring. The Gadidae family includes the Gadus, Micromesistius, Gadiculus, Trisopterus, Microgadus, Eleginus, Merlangius, Melanogrammus, Pollachius, Boreogadus, and Arctogadus genera, with Gadus being preferred. Examples of Gadus fish include Alaska pollock, Pacific cod, Pacific cod, and Greenland cod. From the perspective of easy availability, Salmonidae and Clupeidae fish are preferred. The extraction method is not particularly limited, and known methods can be appropriately adopted. Protamine may be purified after extraction.

[0014] In this embodiment, the protamine may be derived from one type of fish, or may contain a combination of protamines derived from two or more types of fish.

[0015] In one embodiment, protamine derived from salmon milt is used, while in another embodiment, protamine derived from herring milt is used.

[0016] In one embodiment, protamine may include any of the following: (a) a protein consisting of the amino acid sequence of any one of SEQ ID NOs: 7 to 10; (b) a protein consisting of the amino acid sequence of any one of SEQ ID NOs: 7 to 10 in which one or more amino acids have been deleted, substituted, added, and / or inserted, and which has antibacterial activity; (c) a protein consisting of an amino acid sequence having at least 90% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 7 to 10, and which has antibacterial activity.

[0017] The individual sequences are as follows: (SEQ ID NO: 7) PRRRRRSSSRPIRRRRRPRASRRRRR-GGRRRR (SEQ ID NO: 8) PRRRR-SSRRPVRRRRRPRVSRRRRRRGGRRRR (SEQ ID NO: 9) PRRRR-SSSRPVRRRRRPRVSRRRRRRGGRRRR (SEQ ID NO: 10) PRRRR-ASRR-IRRRRRPRVSRRRRR-GGRRRR

[0018] Furthermore, in the present invention, when a protein or amino acid sequence is referred to as an amino acid sequence in which one or more amino acids have been deleted, substituted, added, and / or inserted, the number of amino acids to be substituted, etc. is not particularly limited, unless otherwise specified, in any protein, as long as the protein consisting of that amino acid sequence has the intended function, but may be about 1-15, 1-9, or 1-4 amino acids, or even more if the substitutions are with amino acids with similar properties.

[0019] In the present invention, unless otherwise specified, the term "sequence identity" used with respect to amino acid sequences refers to the percentage of identical amino acids shared between two sequences when the two sequences are aligned in an optimal manner. Searches and analyses of amino acid sequence identity can be performed using algorithms or programs well known to those skilled in the art (e.g., BLASTN, BLASTP, BLASTX, ClustalW). When using a program, parameters can be appropriately set by those skilled in the art, or the default parameters of each program may be used. Specific techniques for these analysis methods are also well known to those skilled in the art. Genetic information processing software may be used to calculate identity. Note that if the target sequence for which percent identity is to be determined contains an additional sequence, such as a tag sequence at the end, that is not present in the sequence being compared, the additional sequence portion is not included in the calculation of percent identity.

[0020] In the present invention, when referring to amino acid sequences, sequence identity refers to sequence identity of at least 70% or more, preferably 80% or more, more preferably 85% or more, even more preferably 90% or more, even more preferably 95% or more, even more preferably 97.5% or more, and even more preferably 99% or more, unless otherwise specified.

[0021] (Protamine Degradation Product) In this embodiment, a product obtained by decomposing the above-mentioned protamine, i.e., a protamine degradation product, may be used. The protamine degradation product may be composed of one peptide or may be a composition containing multiple peptides.

[0022] In one embodiment, the protamine degradation product is an enzymatic hydrolysis product of protamine. Examples of proteolytic enzymes that can be used for hydrolysis include enzymes produced by the genus Bacillus (e.g., Bacillus subtilis, Bacillus thermoproteolyticus, Bacillus licheniformis, etc.), enzymes produced by the genus Aspergillus (e.g., Aspergillus oryzae, Aspergillus niger, Aspergillus mellens, etc.), enzymes produced by the genus Rhizopus (e.g., Rhizopus niveus, Rhizopus deremar), and the like. Examples of enzymes that can be used include enzymes produced by bacteria such as cereals (e.g., cereals containing glutamic acid), pepsin, pancreatin, and papain. These enzymes can be used alone or in combination. Proteolytic enzymes are further classified into endopeptidases, which specifically recognize and cleave the internal sequence of proteins, and exopeptidases, which cleave one to two amino acid residues at a time from the end. Therefore, various peptide chains can be produced by combining endopeptidases and exopeptidases as needed. When hydrolyzing with an enzyme, 0.001 to 10% of the enzyme is added to the substrate, and the solution is hydrolyzed at the optimal pH for the enzyme used.

[0023] The molecular weight of the protamine degradation product is not particularly limited as long as it has the desired activity. In one embodiment, the protamine degradation product has a molecular weight of less than 1,000, 1,000 to less than 2,000, 2,000 to less than 3,000, 3,000 to less than 4,000, 4,000 to less than 5,000, 5,000 to less than 6,000, 6,000 to less than 7,000, 7,000 to less than 8,000, 8,000 to less than 9,000, or 9,000. The protamine degradation products may include all or part of those having a molecular weight of 2,000 or more but less than 3,000, 3,000 or more but less than 4,000, 4,000 or more but less than 5,000, 5,000 or more but less than 6,000, 6,000 or more but less than 7,000, 7,000 or more but less than 8,000, and 8,000 or more but less than 9,000 (expressed as peak area percentage (%)). In one embodiment, the peak area in the molecular weight range of 1,000 or more is 70% or more, preferably 80% or more, more preferably 90% or more, and preferably 95% or less. The peak area in the molecular weight range of less than 9,000 is 70% or more, preferably 80% or more, more preferably 90% or more, and preferably 98% or less. In one embodiment, the protamine degradation products may include those having a molecular weight of 500 to 4,000 Da when analyzed by GPC.

[0024] In one embodiment, the protamine digest is derived from salmon milt, hi another embodiment, the protamine digest is derived from herring milt.

[0025] In one embodiment, the protamine degradation product contains at least one peptide selected from the following: a peptide consisting of the sequence of SEQ ID NO: 1, and a peptide consisting of the sequence of SEQ ID NO: 1 with 1 to 4 amino acids deleted or added at one or both termini, a peptide consisting of the sequence of SEQ ID NO: 2, and a peptide consisting of the sequence of SEQ ID NO: 2 with 1 to 4 amino acids deleted or added at one or both termini, a peptide consisting of the sequence of SEQ ID NO: 3, and a peptide consisting of the sequence of SEQ ID NO: 3 with 1 to 4 amino acids deleted or added at one or both termini, a peptide consisting of the sequence of SEQ ID NO: 4, and a peptide consisting of the sequence of SEQ ID NO: 4 with 1 to 4 amino acids deleted or added at one or both termini, a peptide consisting of the sequence of SEQ ID NO: 5, and a peptide consisting of the sequence of SEQ ID NO: 5 with 1 to 4 amino acids deleted or added at one or both termini, a peptide consisting of the sequence of SEQ ID NO: 6, and a peptide consisting of the sequence of SEQ ID NO: 6 with 1 to 4 amino acids deleted or added at one or both termini, provided that the peptides have a length of at least 2 amino acids.

[0026] The individual sequences are as follows: (SEQ ID NO: 1) IRRRRPRR (SEQ ID NO: 2) SRRRRRRGGRRRR (SEQ ID NO: 3) VSRRRRRRGGRRRR (SEQ ID NO: 4) RRRRRRGGRRRR (SEQ ID NO: 5) RRRRRGGRRRR (SEQ ID NO: 6) RRRRGGRRRR

[0027] In the sequence of SEQ ID NO: 1 (the same applies to sequences 2 to 6), a sequence in which 1 to 4 amino acids are deleted or added at one or both termini is a continuous portion of the protamine sequence, since protamine degradation products are derived from protamine. The number of amino acids deleted or added is 1 to 4, and may be 3, 1 or 2, or even 1. The terminus of the deleted or added amino acid is preferably the N-terminus. More preferably, one amino acid is deleted at the N-terminus.

[0028] In a preferred embodiment, the protamine degradation product comprises at least one selected from the group consisting of a peptide having the sequence of SEQ ID NO: 1, a peptide having the sequence of SEQ ID NO: 2, a peptide having the sequence of SEQ ID NO: 3, a peptide having the sequence of SEQ ID NO: 4, a peptide having the sequence of SEQ ID NO: 5, and a peptide having the sequence of SEQ ID NO: 6, and preferably comprises five of the peptides other than the peptide having the sequence of SEQ ID NO: 1, and more preferably comprises all of them.

[0029] (Basic Polyamino Acid) In this embodiment, a basic polyamino acid may be used. In the present invention, the term "basic polyamino acid" refers to a polymer of a basic amino acid, preferably a homopolymer of a basic amino acid, unless otherwise specified. Examples of basic amino acids are lysine, ornithine, arginine, and histidine. The degree of polymerization of the basic polyamino acid is not particularly limited as long as the desired activity is obtained, but is preferably 10 to 200.

[0030] (Polylysine) In this embodiment, polylysine may be used. In the present invention, unless otherwise specified, the term "polylysine" refers to a homopolypeptide of L-lysine in which the peptide bond of each lysine residue is between the ε-amino group and the carboxyl group of the side chain (ε-poly-L-lysine). The degree of polymerization is not particularly limited as long as the desired activity is obtained, but a degree of polymerization of 25 to 35 is preferred.

[0031] (Polyornithine) In this embodiment, polyornithine may be used. In the present invention, unless otherwise specified, the term "polyornithine" refers to a homopolypeptide of L-ornithine. The degree of polymerization is not particularly limited as long as the desired activity is obtained, but is preferably 90 to 180.

[0032] (Polyarginine) In this embodiment, polyarginine may be used. In the present invention, unless otherwise specified, the term "polyarginine" refers to a homopolypeptide of L-arginine. The degree of polymerization is not particularly limited as long as the desired activity is obtained, but is preferably 24 to 80.

[0033] (Salts Acceptable for Foods, Pharmaceuticals, etc.) In this embodiment, protamine, protamine degradation products, and basic polyamino acids such as polylysine, polyornithine, or polyarginine may be used as salts thereof. Preferably, the salts are acceptable for foods, pharmaceuticals, cosmetics, quasi-drugs, medical devices, dental materials, etc. Examples of such acceptable salts include inorganic and organic acids for forming acid addition salts, such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, methanesulfonic acid, p-toluenesulfonic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, and dicarboxylic acids, and monocarboxylic acids, such as acetic acid, propionic acid, and butyric acid; inorganic bases for forming salts, such as hydroxides, carbonates, and bicarbonates of ammonia, sodium, lithium, calcium, magnesium, and aluminum; and organic bases for forming salts, such as mono-, di-, and tri-alkylamines, mono-, di-, and tri-hydroxyalkylamines, such as methylamine, dimethylamine, and triethylamine, guanidine, and N-methylglucosamine. These salts may be used alone or in combination of two or more kinds, as required.

[0034] (Combination) This embodiment relates to the use of at least two active ingredients selected from a predetermined group. When referring to at least two active ingredients, a certain active ingredient and its salt (e.g., protamine and protamine sodium) are considered to be one active ingredient.

[0035] In one embodiment, the at least two components are a protamine degradation product and a basic amino acid, for example, a protamine degradation product and polylysine, or polylysine and polyornithine, because a combination of these components is expected to exhibit synergistic antibacterial activity compared to when each component is used alone.

[0036] In another embodiment, the at least two components are a protamine degradation product and a basic amino acid, for example, a protamine degradation product and polylysine, a protamine degradation product and polyarginine, a protamine degradation product and polyornithine, or polylysine and polyornithine, because these combinations are expected to exhibit synergistic antibiofilm activity compared to when each component is used alone.

[0037] [Use] The composition of this embodiment has at least one of anti-biofilm and anti-bacterial properties.

[0038] In the present invention, the term "antibacterial" refers to at least inhibiting the growth of bacteria. As long as the growth is inhibited, the substance may also have a bactericidal or disinfecting effect.

[0039] The composition of this embodiment may exhibit antibacterial activity against Gram-positive bacteria, such as microorganisms belonging to the genera Bacillus, Staphylococcus, Streptococcus, Lactobacillus, Clostridium, Listeria, Micrococcus, and Propionibacterium, preferably Bacillus subtilis, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus mutans, Lactobacillus plantarum, and Propionibacterium acnes.

[0040] The antibacterial activity can also be exerted against Gram-negative bacteria, such as bacteria of the genus Escherichia, Pseudomonas, Enterobacter, Klebsiella, Citrobacter, Proteus, Serratia, Erwinia, Vibrio, Acinetobacter, and Salmonella, preferably Escherichia coli.

[0041] Furthermore, the antibacterial activity can be exerted against fungi. Examples of fungi include the genera Candida, Saccharomyces, Schizosaccharomyces, Pichia, Kluyveromyces, Williopsis, Debaryomyces, Galactomyces, Torulaspora, and Rhodotorula. rula), Yarrowia, Zygosaccharomyces, Aspergillus, Cryptococcus, Trichophyton, Microsporum, and Pneumocystis, and more specific examples include Candida albicans. albicans), Candida glabrata, Candida tropicalis, Candida kurse, Candida zubriniensis, Candida lypolitica, Candida utilis, Candida sake, Saccharomyces cerevisiae, Saccharomyces sake, Saccharomyces beticus, Aspergillus fumigatus, Aspergillus flavus, Aspergillus nigerniger), Cryptococcus neoformans, Cryptococcus albidus, Cryptococcus gattii, Cryptococcus laurentii, Trichophyton rubrum, Trichophyton mentagrophytes, Trichophyton tonsurans, Trichophyton verrucosum, Microsporum canis, Microsporum gypseum, and Pneumocystis jiroveci.

[0042] When antibacterial activity against fungi is particularly desired, the composition may be formulated to contain a protamine degradation product, because although protamine has antibacterial activity against fungi, protamine degradation products have a stronger antibacterial effect against fungi.

[0043] In the present invention, anti-biofilm activity may be active against biofilm-forming bacteria or against the biofilm itself. Anti-biofilm compositions may inhibit or eliminate the growth of bacteria within a biofilm, or may reduce or disrupt the biofilm itself. Alternatively, they may inhibit the process of bacterial biofilm formation. Biofilms are aggregates composed of bacteria / microorganisms and their metabolic products, exopolysaccharides (EPS). To evaluate anti-biofilm activity, for example, sucrose can be added to an experimental system (e.g., J Dent Res. 2006 October; 85(10): 878-887. FEMS (Fed Eur Microbiol Soc) Microbiol Ecol, 94 (7) (2018), pp. 1-9). Sucrose not only promotes the colonization and growth of mutans streptococci, such as S. mutans, in oral (dental) biofilms, but also serves as a substrate for the synthesis of exopolysaccharides (EPS) in biofilms, particularly cariogenic biofilms. S. mutans can also use sucrose to synthesize EPS using glucosyltransferases (GTFs). The antibiofilm activity of a substance can be evaluated by measuring the amount of biofilm formed and the inhibition of biofilm formation by the substance using crystal violet staining. Because crystal violet is a positively charged stain, it stains negatively charged substances, such as bacteria, EPS, and extracellular DNA, purple.

[0044] The composition of this embodiment may also be a composition for inhibiting the production of EPS from microorganisms, i.e., a composition for inhibiting EPS production. EPS is a general term for polysaccharides secreted and produced extracellularly by microorganisms, and plays a role in protecting the microorganisms from environmental stresses and the like.

[0045] The inhibitory activity against EPS production can be evaluated by measuring the amount of EPS produced in a system treated with the agent to be evaluated and in an untreated system. The amount of EPS produced by the microorganism in the system can be indirectly measured as fluorescence intensity, for example, using an appropriately labeled dextran.

[0046] In a preferred embodiment of the anti-biofilm composition and in a preferred embodiment of the composition for inhibiting EPS production, the active ingredient is a protamine degradation product or a salt thereof. Studies by the present inventors have confirmed that the protamine degradation product has anti-biofilm activity and EPS production inhibitory activity even when used alone.

[0047] This application is the first to disclose the anti-biofilm activity and EPS production inhibitory activity of protamine degradation products. According to the inventors' studies, combining protamine degradation products with specific components exhibits higher anti-biofilm activity and EPS production inhibitory activity.

[0048] In one aspect, the composition is suitable for use against Streptococcus mutans for antibiofilm and / or antibacterial purposes, and in another aspect, the composition is suitable for inhibiting EPS production by Streptococcus mutans.

[0049] In one aspect, the composition is suitable for treating oral biofilm infections, where treatment includes reducing the risk of developing, preventing, curing, halting or inhibiting the progression of the disease or condition.

[0050] In one aspect, the composition is suitable for antibiofilm and / or antibacterial use against Candida albicans, hi another aspect, the composition is suitable for inhibiting EPS production by Candida albicans.

[0051] In one embodiment, the composition is suitable for treating candidiasis, which is an infection of the skin, oral mucosa, etc. caused by fungi of the genus Candida, such as Candida albicans.

[0052] [Composition] The composition of the present embodiment can be a pharmaceutical composition, a health care composition, a cosmetic composition, a quasi-drug, a medical device, a dental material, a food composition, or the like. Unless otherwise specified, these include not only those for humans but also those for non-human animals, such as pets or companion animals such as dogs and cats. In addition, the composition of the present embodiment can be used to produce an oral care composition.

[0053] In one aspect, the composition is used for its antibacterial effect against oral microorganisms (including gram-positive and gram-negative bacteria and fungi) that cause undesirable oral diseases or conditions, such as dental and gum diseases such as dental caries and periodontal disease, and their associated or precursor symptoms, such as oral malodor and plaque formation. The composition may be an oral care composition selected from, for example, mouthwash, mouth spray, toothpaste, tooth powder, tooth cream, gel, chewing gum, liquid center-filled gums, mints, lozenges, chewable tablets, gummies, jellies, oral films, etc.

[0054] In addition to the oral care applications described above, the composition of the present embodiment can also be used as a composition for preventing bacterial infections or suppressing causative bacteria. For example, it can be used in face care products for acne care, body (foot and armpit) deodorants, body care products for preventing impetigo in children, and eye care products such as eye drops and contact lens cleaners.

[0055] Depending on the form, the composition may contain other bases or additives acceptable for pharmaceuticals, cosmetics, or foods. For example, in the case of a toothpaste product, additives may include abrasives such as calcium hydrogen phosphate, calcium carbonate, and aluminum hydroxide, foaming agents such as sodium lauroyl sarcosine, sodium lauryl sulfate, and sucrose fatty acid esters, humectants such as sorbitol, glycerin, and propylene glycol, binders such as xanthan gum, sodium alginate, and carboxymethylcellulose, sweeteners such as xylitol, flavors such as menthol, flavoring agents such as calcium lactate, auxiliary agents such as ethanol, and medicinal ingredients such as fluoride, dextranase, glucose oxidase, chlorhexidine, and lysozyme chloride.

[0056] In the case of mouthwash, water such as purified water is used as a base, and other ingredients may include humectants such as glycerin and sorbitol, sweeteners such as hydrogenated starch and xylitol, solubilizers such as polyoxyethylene-polyoxypropylene block copolymers (poloxamer, etc.), binders such as hydroxyethyl cellulose, fragrances such as peppermint and aloe vera juice, thymol, menthol, methyl salicylate (wintergreen oil), eucalyptol, carvacrol, camphor, anethole, carvone, eugenol, isoeugenol, limonene, ocimene, n-decyl alcohol, citronellol, α-salpineol, methyl acetate, citronellyl acetate, methyleugenol, cineole, linalool, ethyl linalaol, safrola vanillin, and the like. Other examples of the additives that may be used include essential oils such as vanillin, spearmint oil, peppermint oil, lemon oil, orange oil, sage oil, rosemary oil, cinnamon oil, pimento oil, laurel oil, thuja leaf oil, gerianol, verbenone, anise oil, bay oil, benzaldehyde, bergamot oil, bitter almond, chlorothymol, cinnamaldehyde, citronella oil, clove oil, coal tar, eucalyptus oil, guaiacol, lavender oil, mustard oil, phenol, phenyl salicylate, pine oil, pine needle oil, sassafras oil, spike lavender oil, storax, thyme oil, tolu balsam, turpentine, and clove oil; ethanol; lysozyme chloride; lactoferrin; glucose oxidase; and other auxiliary agents; and flavoring agents such as calcium lactate.

[0057] Bacteria that are the target of the antibacterial or antibiofilm activity of the antibacterial care composition include Streptococcus mutans, Streptococcus sanguis, Fusobacterium nucleatum, Prevotella intermedia, Actinomyces viscosus, Campylobacter rectus, Porphyromonas gingivalis, Actinobacillus, Bacteroides, Capnocytophaga, Eikenella, Propionibacterium, and Candida albicans. Examples of the bacteria include, but are not limited to, Streptococcus mutans, Tannerella forsythia, Treponema denticola, etc. Preferred are Streptococcus mutans, Tannerella forsythia, Treponema denticola, and Porphyromonas gingivalis, which are said to be the main causative bacteria of periodontal disease, and particularly preferred is Streptococcus mutans.

[0058] The content of the active ingredient (the total amount when two or more types are contained) in the composition is not particularly limited as long as the desired activity is obtained, but for example, the composition can be contained so that the concentration at the time of use is 0.001 to 500,000 μg / mL, or alternatively, 0.005 to 50,000 μg / mL, 0.1 to 1,000 μg / mL, 0.2 to 500 μg / mL, 0.5 to 300 μg / mL, 1 to 100 μg / mL, or 5 to 30 μg / mL.

[0059] 2. Composition Comprising At Least Two Components This embodiment provides a composition comprising at least two components selected from protamine, protamine degradation products, polylysine, polyornithine, polyarginine, and salts thereof, wherein the mass ratio ((A):(B)) of the two components (A) and (B) is a specific ratio. Note that in this embodiment, the mass ratio (A):(B) of the two components (A) and (B) is expressed so that the sum of (A):(B) is 10, unless otherwise specified.

[0060] In one embodiment, the at least two components are a protamine degradation product and a basic polyamino acid. In a more specific embodiment, the at least two components are a protamine degradation product and polylysine, or polylysine and polyornithine. Such a combination is suitable for antibacterial use. In another embodiment, the at least two components are (A) a protamine degradation product and (B) polylysine, (A) a protamine degradation product and (B) polyarginine, (A) a protamine degradation product and (B) polyornithine, or (A) polyornithine and (B) polylysine. Such a combination is suitable for antibiofilm use.

[0061] The mass ratio ((A):(B)) of the two components (A) and (B) contained in the composition can be 0.8-9.2:9.2-0.8, or 1-9:9-1, 2-8:8-2, 2.5-7.5:7.5-2.5, 3-7:7-3, or 3.5-6.5:6.5-3.5. According to the inventors' studies, a higher effect can be achieved when the amount of (A) pro-component is greater than that of (B) amino acid. From this perspective, (A) > (B) is preferred. Specifically, the mass ratio ((A):(B)) of (A) to (B) is 5.5-9.9:4.5-0.1, preferably 5.6-9.5:4.4-0.5, more preferably 5.7-9.0:4.3-1.0, and even more preferably 5.8-8.8:4.2-1.2.

[0062] The ratio of the two components (A) and (B) contained in the composition can also be determined based on the minimum inhibitory concentration (MIC) of each component (MIC ratio). For example, the MIC ratio ((A):(B)) can be 2-8:8:2, or 3-7:7-3, preferably 3.5-6.5:3.5-6.5, more preferably 4.0-6.0:6.0-4.0, and even more preferably 4.5-5.5:5.5-4.5.

[0063] Throughout this specification, descriptions of certain embodiments, aspects, and examples apply to other embodiments, aspects, and examples where the same terminology is used, unless otherwise stated.

[0064] [Production 1: Production of protamine digest] 50 g of protamine (Proserve; Maruha Nichiro Corporation) derived from the milt of chum salmon (Oncorhynchus keta) was added to 80 mL of deionized water, and sodium hydroxide was added to adjust the pH to 8.0. After heating to 65°C, 1.5 mg of thermolysin (Nacalai Tesque, Inc., derived from Bacillus thermoproteolyticus) was added, and the enzymatic reaction was carried out with stirring for 2 hours. After completion of the reaction, the reaction solution was heated to 95°C and inactivated by heat for 30 minutes, and the pH was adjusted to 8.5. The reaction solution was then lyophilized to obtain a protamine digest peptide mixture.

[0065] [Experiment 1: Antibacterial Activity Investigation] 1. Experimental Method 1-1. Pre-culture and Preparation of Bacterial Solution S. mutans NBRC13955 (distributed by NBRC) was anaerobically cultured in 10 mL of medium in an incubator at 37°C for approximately 17 hours. After cultivation, the culture was centrifuged at 3000 rpm for 4 minutes, the supernatant was removed, and the mixture was resuspended in 5 mL of fresh medium and further incubated for approximately 5 hours. The final concentration of S. mutans was 4.0 × 10 6 The medium was prepared so that the concentration of CFU / mL was 100%. TM Brain heart infusion broth (BHI medium) (Shimadzu Diagnostics Co., Ltd.) was used.

[0066] 1-2. Treatment Solutions of each treatment concentration were prepared by mixing the prepared bacterial suspension and each drug in combination in a 96-well plate, and treatment was carried out by culturing under anaerobic conditions for 24 hours under the following treatment conditions. Experiments were carried out with each drug alone and in combination with other drugs.

[0067] Treatment conditions Test bacteria: S. mutans Medium: BHI medium (medium concentration: 10%) Bacterial concentration: 4 x 10 5 CFU / mL Incubation temperature: 37°C Incubation equipment: 96-well plate Solution volume: 0.2 mL Agents used: Protamine hydrolysate (HAP-100, Maruha Nichiro), polylysine (ε-poly-L-lysine, JNC Corporation. Produced by fermentation using microorganisms, this polymerizes 25-35 L-lysines), polyarginine (poly-L-arginine hydrochloride, Sigma-Aldrich®, molecular weight 5,000-15,000), polyornithine (poly-L-ornithine hydrochloride, Sigma-Aldrich®, molecular weight 15,000-30,000)

[0068] 1-3. Addition of alamar Blue® reagent After 24 hours of treatment, 20 μL of alamar Blue® reagent was added and the mixture was left to stand for 3 hours under aerobic conditions at 37°C. The point where no color change was observed was taken as the minimum inhibitory concentration (MIC).

[0069] 1-4. Colony Count After 0.1 mL of the solution was taken from the wells containing the treatment concentrations (MIC, 1 / 2 MIC, etc.) determined in 1-3, the solution was spread on agar and cultured at 37°C under anaerobic conditions for 2 days. After that, the number of colonies was counted to determine the number of viable bacteria at that treatment concentration.

[0070] 2. Results In the experiment, treatment was performed using protamine hydrolysate, polylysine, polyarginine, and polyornithine, either alone or in combination of two types. As a result, a significant decrease in the number of live bacteria was confirmed when protamine hydrolysate and polylysine were used in combination compared to treatment with either drug alone, demonstrating an increased antibacterial effect when used in combination (Figure 1-1). Furthermore, a significant increase in the antibacterial effect was confirmed when polylysine and polyornithine were used in combination compared to treatment with either drug alone (Figure 1-2).

[0071] [Experiment 2: Anti-biofilm investigation] 1. Experimental method 1-1. Pre-culture and bacterial solution preparation S. mutans was anaerobically cultured in 10 mL of medium in an incubator at 37°C for approximately 17 hours. After cultivation, the culture was centrifuged at 3000 rpm for 4 minutes, the supernatant was removed, and the mixture was resuspended in 5 mL of fresh medium and further incubated for approximately 5 hours. The final concentration of S. mutans was 8.0 × 10 6 The culture medium was adjusted to give a concentration of CFU / mL.

[0072] 1-2. Treatment Solutions of each treatment concentration were prepared by combining and mixing the prepared bacterial suspension, sucrose solution, and drug in a 48-well plate. Treatment was carried out under anaerobic conditions for 24 hours under the treatment conditions shown below. Drugs were used alone or in combination (with other drugs at a concentration of 1 / 2 the MIC).

[0073] Treatment conditions Test bacteria: S. mutans Medium: BHI medium (medium concentration: 25%) Bacterial concentration: 2.0 x 10 6 CFU / mL Incubation temperature: 37°C Incubation equipment: 48-well plate Solution volume: 1 mL Sucrose concentration: 1% Drugs used: Protamine (Proserve; Maruha Nichiro Co., Ltd.), protamine hydrolysate (HAP-100; Maruha Nichiro Co., Ltd.), polylysine (same as in Experiment 1), polyarginine (same as in Experiment 1), polyornithine (same as in Experiment 1)

[0074] The concentrations of each drug (single or combined) are as follows:

[0075]

[0076] 1-3. After washing and staining, 1 mL of medium was removed from each well of the 48-well plate and washed with 0.4 mL of phosphate-buffered saline (PBS). After washing, 0.4 mL of 0.1% Crystal Violet reagent was added to each well and the plate was left standing in the dark for 15 minutes. After staining, the Crystal Violet reagent was removed from the wells.

[0077] 1-4. Washing and Extraction: After washing twice with 0.4 mL of PBS and removing the PBS, 1 mL of 33% acetic acid was added to each well and allowed to stand for 10 minutes to extract the pigment. This procedure was repeated twice to obtain a total of 2 mL of extract. This solution was centrifuged at 8000 rpm for 10 minutes and then diluted appropriately. 0.1 mL of the supernatant from each system was dispensed into a 96-well plate, and the absorbance at 595 nm was measured using a microplate reader.

[0078] 2. Results: Concentration-dependent reductions in biofilm formation were confirmed for all antimicrobial peptides (Figure 2-1). Focusing on protamine hydrolysates, it was confirmed that the antibiofilm effect was enhanced when combined with polylysine, polyornithine, and polyarginine compared to the results for protamine hydrolysates alone. In particular, the combination of protamine hydrolysates and polyarginine resulted in the lowest amount of biofilm formation compared to the single or combined treatments of other drugs, suggesting that the combination enhances the antibiofilm effect (Figure 2-2).

[0079] [Experiment 3: Antibacterial effect under biofilm formation conditions of C. albicans] Pre-cultured Candida albicans (provided by NITE, National Institute of Technology and Evaluation) was added to a final concentration of 8 × 10 6The 108 medium (10 g glucose, 5 g peptone, 3 g yeast extract, 3 g malt extract, 1 L distilled water, 15 g agar (for agar medium)), pH unadjusted, was prepared to achieve a CFU / mL concentration. 100 μL of 10% sucrose solution, 100 μL of one of the antimicrobial agents (50 μL each of two agents for combined treatment), and 800 μL of bacterial solution containing 108 medium were added to a 48-well plate, and the plates were cultured at 37°C for 24 hours. The final concentration of the 108 medium was adjusted with sterile water to 10%.

[0080] After treatment, the biofilm surface was washed once with PBS. After washing, 1 mL of PBS was added to each well, and the biofilm was scratched with a pipette tip to recover biofilm cells (cells that form biofilms, i.e., cells inside the biofilm; the same applies below). The recovered biofilm suspension was homogenized by vortexing. The homogenized suspension was serially diluted with PBS, and 100 μL of each sample was plated on a 108 agar plate and cultured at 37°C for 48 hours. After culture, the number of colonies formed on the agar was counted.

[0081] Treatment conditions Test bacteria: C. albicans Medium: 108 medium (medium concentration: 10%) Bacterial concentration: 8.0 x 10 6 CFU / mL Incubation temperature: 37°C Incubation equipment: 48-well plate Solution volume: 1 mL Sucrose concentration: 1% Drugs used: Protamine (Proserve; Maruha Nichiro Co., Ltd.), protamine digest (HAP-100, Maruha Nichiro Co., Ltd.), polylysine (same as in Experiment 1), polyornithine (same as in Experiment 1)

[0082] The concentrations of each drug (single or combined) are as follows:

[0083]

[0084] As a result, although the antibacterial effect of each drug may be reduced in the presence of sucrose, a tendency for bacterial counts to decrease was confirmed for all antibacterial peptides when treated alone at MIC. Furthermore, it was confirmed that the number of live bacteria was greater than the initial number after MIC treatment. Furthermore, compared to treatment with each drug alone at MIC, the combined use of protamine digests and polylysine, and polylysine and polyornithine, confirmed a decrease in bacterial counts under biofilm formation. These results suggest that the antibacterial effect of drugs is enhanced by their combination (Figure 3).

[0085] [Experiment 4: Effect on the total amount of C. albicans biofilm] After pre-culture, C. albicans was incubated at a final concentration of 8 × 10 6 The concentration of CFU / mL was adjusted to 108 medium. 100 μL of 10% sucrose solution, 100 μL of one of the antimicrobial agents (for combined treatment, 50 μL of both agents) and 800 μL of the bacterial solution containing 108 medium were added to a 48-well plate, and the plate was cultured at 37°C for 24 hours. The final concentration of the 108 medium was adjusted to 10% with sterile water.

[0086] After incubation, the supernatant was removed from each well of the 48-well plate, and the formed biofilm was washed once with PBS. After washing, 1000 μL of 0.1% Crystal Violet reagent was added to each well and stained in the dark for 15 minutes. Crystal Violet reagent is positively charged, so it stains negatively charged substances such as bacteria, EPS, and extracellular DNA purple. In this experiment, it was used to quantify the total amount of biofilm. After treatment, the Crystal Violet reagent was removed from the wells and washed three times with PBS. After washing, 1000 μL of 33% acetic acid was added to each well and the dye was extracted for 10 minutes. The extracted solution was transferred to a 2 mL tube, and this extraction procedure was repeated to obtain 2000 μL of extract. The extract was then centrifuged (10,000 rpm, 5 minutes). The centrifuged extract was diluted appropriately and 100 μL was added to a 96-well plate. The absorbance was measured at 595 nm using a microplate reader (MULTISKAN FC, Thermo Fisher Scientific).

[0087] Treatment conditions Test bacteria: S. mutans Medium: BHI medium (medium concentration: 25%) Bacterial concentration: 8.0 x 10 6 CFU / mL Incubation temperature: 37°C Incubation equipment: 48-well plate Solution volume: 1 mL Sucrose concentration: 1% Drugs used: Protamine (Proserve; Maruha Nichiro Co., Ltd.), protamine digest (HAP-100, Maruha Nichiro Co., Ltd.), polylysine (same as in Experiment 1), polyornithine (same as in Experiment 1)

[0088] The concentrations of each drug (single or combined) are as follows:

[0089]

[0090] As a result, we confirmed that the biomass was reduced when protamine digests and polylysine, and when polylysine and polyornithine were used in combination, compared to when each drug was used alone at its MIC. These results suggest that the anti-biofilm effect of drugs is enhanced by their combination (Figure 4).

[0091] [Experiment 5: Effect of S. mutans on EPS content] After pre-culture, S. mutans was cultured at a final concentration of 8 × 10 6 Biofilms were prepared in BHI medium at a concentration of CFU / mL. A 48-well plate was used for culture, and biofilms were formed on glass coverslips placed at the bottom of the wells. 100 μL of protamine digest or PL (50 μL each of the two drugs for combined treatment), 100 μL of 10% sucrose solution, and 800 μL of the prepared bacterial solution were added and cultured at 37°C under anaerobic conditions in the dark for 24 hours. The final concentration of BHI medium was adjusted to 25% with sterile water. The concentrations of each drug (single and combined) were as follows. To label EPS, 1.0 mM Alexa Fluor 594 dextran conjugate (Thermo Fisher Scientific) was added before culture. The dextran fluorescence in the solution in the wells was measured and compared before and after treatment, and the amount of EPS in the biofilm was calculated using the following formula:

[0092]

[0093] The concentrations of each drug (single or combined) are as follows: The drugs used were the same as in Experiment 4.

[0094]

[0095] The results confirmed that when protamine digests and polylysine were treated alone at 1 / 2 MIC, the amount of EPS in the biofilm was significantly reduced, and the thickness of the biofilm itself was also reduced. Furthermore, it was confirmed that the amount of EPS in the biofilm was significantly reduced even when treated with 1 MIC of protamine digests and polylysine. The highest inhibition of EPS production was observed when treated with both protamine digests and polylysine in combination. These results suggest that bacterial growth may be significantly inhibited not only by the EPS production inhibitory effect of protamine digests, but also by the improved antibacterial activity of combined use with polylysine (Figure 5).

[0096] [Experiment 6: SEM observation of biofilm] After pre-culture, S. mutans was cultured at a final concentration of 8 × 10 6 The biofilm was prepared in BHI medium at a concentration of CFU / mL. A 48-well plate was used for culture, and a biofilm was formed on a glass coverslip placed at the bottom of the well. 100 μL of protamine digest or PL (50 μL each of the two drugs for combined treatment), 100 μL of 10% sucrose solution, and 800 μL of the prepared bacterial solution were added and cultured at 37°C under anaerobic conditions for 24 hours. The final concentration of BHI medium was adjusted to 25% with sterile water.

[0097] After treatment, the coverslips were washed once with PBS to remove planktonic and loosely bound bacteria. Then, 1 mL of a 2.5% solution of 25% glutaraldehyde in PBS was added to each well. The biofilms were fixed by leaving the wells at 4°C for 1 hour in the dark. After fixation, the 2.5% glutaraldehyde was removed and the wells were washed with PBS. The wells were then dehydrated in 50, 70, 80, 90, and 95% ethanol for 20 minutes each, then in 100% ethanol for 1 hour, and then air-dried overnight. For observation, the samples were gold-coated for 90 seconds and observed under a scanning electron microscope (VE-9800, KEYENCE) at an accelerating voltage of 10 kV.

[0098] The concentrations of each drug (single or combined) are as follows: The drugs used were the same as in Experiment 4.

[0099]

[0100] In the untreated control, microcolonies were bound together by EPS, and EPS was observed to coat the bacterial surface. EPS coating of the bacterial surface was also observed with 1 / 2 MIC of polylysine. On the other hand, with 1 / 2 MIC of protamine degradation product, a decrease in EPS covering the bacterial body, microcolonies, and bonds connecting microcolonies were confirmed. Furthermore, with the combined treatments at 1 / 4 MIC each, areas where bacteria were exposed and areas where the bacterial surface was coated with EPS were similarly observed (Figure 6).

[0101] The molecular weight distribution of HAP-100 used as a protamine digest (analyzed by size exclusion chromatography using a TSK gel G2500PWXL column (Tosoh Corporation) (analysis institution: Japan Food Research Laboratories (JFRL))) is shown ( FIG. 7 ).

[0102] [Sequences listed in the sequence listing] SEQ ID NO: 1 Protamine digest SEQ ID NO: 2 Protamine digest SEQ ID NO: 3 Protamine digest SEQ ID NO: 4 Protamine digest SEQ ID NO: 5 Protamine digest SEQ ID NO: 6 Protamine digest SEQ ID NO: 7 Protamine SEQ ID NO: 8 Protamine SEQ ID NO: 9 Protamine SEQ ID NO: 10 Protamine

Claims

1. A composition for at least one of anti-biofilm and anti-bacterial properties, comprising at least two components selected from protamine, protamine degradation products, basic polyamino acids, and salts thereof.

2. The composition according to claim 1, wherein the at least two components comprise a protamine degradation product or a salt thereof and a basic polyamino acid or a salt thereof.

3. The composition according to claim 1, which is for antibacterial use and wherein the at least two components are a protamine degradation product and polylysine, or polylysine and polyornithine.

4. The composition according to claim 1, which is for anti-biofilm use and comprises at least two components selected from the group consisting of a protamine hydrolysate and polylysine, a protamine hydrolysate and polyarginine, a protamine hydrolysate and polyornithine, or polylysine and polyornithine.

5. The composition according to claim 1, which has at least one of antibiofilm and antibacterial properties against at least one selected from Streptococcus mutans and Candida albicans.

6. The composition according to claim 1 for oral care or for preventing oral biofilm infections.

7. The composition according to any one of claims 1 to 6, which is a health care composition or a food composition.

8. A composition for producing an oral care composition, comprising: (A) a protamine degradation product or a salt thereof; and (B) at least one selected from polylysine, polyarginine, polyornithine, and salts thereof, wherein the mass ratio of (A) to (B) ((A):(B)) is 1-9:9-1.

9. A composition for producing an oral care composition, comprising: (A) polyornithine or a salt thereof; and (B) polylysine or a salt thereof, wherein the mass ratio of (A):(B) is 1-9:9-1.

10. A composition for inhibiting extracellular polysaccharide (EPS) production, comprising a protamine degradation product or a salt thereof.

Images