Antiviral composition and method for producing same

Abstract

The present invention provides: a composition containing a naturally derived peptide having antiviral activity against non-enveloped viruses; and a method for producing the composition. Specifically, the present invention provides an antibacterial and antiviral composition containing a bacteria-derived polypeptide having antibacterial activity against at least one bacterium selected from the group consisting of Escherichia coli and Staphylococcus aureus and also having antiviral activity against non-enveloped viruses.

Description

Antiviral composition and method for producing same

[0001] This application claims priority to and the benefit of Japanese Patent Application No. 2023-130801, filed on August 10, 2023, the entire contents of which are incorporated herein by reference. The present invention relates to an antiviral composition and a method for producing the same.

[0002] Viruses are divided into enveloped and non-enveloped types. While alcohol disinfectants are effective against enveloped viruses, they are less effective against non-enveloped viruses (Non-Patent Document 1). Furthermore, to inactivate them with heat, they must be heated at 85°C to 90°C for at least 90 seconds. Therefore, chlorine-based disinfectants such as sodium hypochlorite are used to disinfect non-enveloped viruses, but they are unsafe and can cause dermatitis or blindness. Furthermore, sodium hypochlorite is corrosive to metals and has a bleaching effect on leather products. Meanwhile, natural materials such as saxifrage, persimmon tannin, and grapefruit seed extract have also been reported to be effective against non-enveloped viruses (e.g., Patent Document 1), but many issues remain, such as limited production volumes. Therefore, existing antiviral ingredients currently have limited uses. Given the above background, there is a need for antiviral materials that are both effective and safe against non-enveloped viruses.

[0003] Patent application 2021-081943

[0004] Soap&Chemical Specialties. 1963(39):70-72:95-97 Antiviral action of germicides. Klein M, Deforest A.

[0005] Under these circumstances, the present invention aims to provide a composition containing a naturally occurring peptide having antiviral activity against non-enveloped viruses (hereinafter also referred to as "non-enveloped viruses") and a method for producing the same.

[0006] To solve this problem, the present inventors conducted extensive research and found that antimicrobial peptides produced by bacteria have antiviral activity against non-enveloped viruses. The present inventors also found that these peptides have antiviral activity against enveloped viruses (hereinafter also referred to as "enveloped viruses").

[0007] The present invention encompasses the following embodiments. [1] An antibacterial and antiviral composition comprising a polypeptide derived from bacteria that has antibacterial activity against at least one bacterium selected from the group consisting of Escherichia coli and Staphylococcus aureus and antiviral activity against non-enveloped viruses (hereinafter also referred to as the "composition of the present invention"). [2] The antibacterial and antiviral composition according to [1], wherein the polypeptide further has antiviral activity against enveloped viruses. [3] The antibacterial and antiviral composition according to [1] or [2], wherein the polypeptide has an isoelectric point of 9.9 or higher and a molecular weight of 2,000 to 6,000 Da. [3'] The antibacterial and antiviral composition according to [1] or [2], wherein the polypeptide has an isoelectric point of 11 or higher and a molecular weight of 2,000 to 5,000 Da. [4] The antibacterial and antiviral composition according to any one of [1] to [3'], wherein the bacterium is a lactic acid bacterium. [5] The antibacterial and antiviral composition according to any one of [1] to [4], wherein the polypeptide is: a polypeptide consisting of an amino acid sequence derived from a lactic acid bacterium of the order Lactobacillales; a polypeptide consisting of an amino acid sequence derived from a lactic acid bacterium of the order Lactobacillales, in which one or several amino acid residues have been substituted, deleted, inserted, and / or added; or a polypeptide consisting of an amino acid sequence having 85% or more sequence identity to an amino acid sequence derived from a lactic acid bacterium of the order Lactobacillales. [5'] The antibacterial and antiviral composition according to any one of [1] to [4], wherein the polypeptide is: a polypeptide consisting of an amino acid sequence derived from lactic acid bacteria of the genus Lactiplantibacillus; a polypeptide consisting of an amino acid sequence derived from lactic acid bacteria of the genus Lactiplantibacillus, in which one or several amino acid residues have been substituted, deleted, inserted, and / or added; or a polypeptide consisting of an amino acid sequence having 85% or more sequence identity to an amino acid sequence derived from lactic acid bacteria of the genus Lactiplantibacillus.[6] The antibacterial and antiviral composition according to [5], wherein the lactic acid bacterium of the Lactobacillales order is Lactiplantibacillus plantarum, Lactobacillus sp. SC-2001, or Lactococcus lactis. [6'] The antibacterial and antiviral composition according to [5'], wherein the lactic acid bacterium of the genus Lactiplantibacillus is Lactiplantibacillus plantarum. [7] The antibacterial and antiviral composition according to [5] or [6], wherein the amino acid sequence derived from a lactic acid bacterium of the Lactobacillales order is any one of the amino acid sequences of SEQ ID NOs: 1 and 5 to 10. [7'] The antibacterial and antiviral composition according to [5'] or [6'], wherein the amino acid sequence derived from a lactic acid bacterium of the genus Lactiplantibacillus is the amino acid sequence of SEQ ID NO: 1. [8] The antibacterial and antiviral composition according to any one of [1] to [7'], wherein the polypeptide is a combination of two or more polypeptides. [9] The antibacterial and antiviral composition according to any one of [1] to [8], further comprising an additive.

[10] The antibacterial and antiviral composition according to any one of [1] to [9], which has antiviral activity against one or more viruses selected from the group consisting of feline calicivirus, norovirus, rotavirus, adenovirus, and poliovirus.

[11] The antibacterial and antiviral composition according to

[10] , which further has antiviral activity against one or more viruses selected from the group consisting of influenza virus, hepatitis B virus, hepatitis C virus, Zika virus, novel coronavirus, SARS coronavirus, MERS coronavirus, varicella-zoster virus, rubella virus, AIDS virus, Ebola virus, and rabies virus.

[12] A nucleic acid comprising a nucleotide sequence encoding a polypeptide defined in any one of [1] to [7'].

[13] The nucleic acid according to

[12] , wherein the nucleotide sequence encoding the polypeptide is one or more nucleotide sequences selected from SEQ ID NOs: 2, 3, and 16 to 27. [13'] The nucleic acid according to

[12] , wherein the nucleotide sequence encoding the polypeptide is the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 3.

[14] A vector comprising the nucleic acid according to any one of

[12] to [13'].

[15] An in vitro translation template molecule comprising the nucleic acid according to any one of

[12] to [13'].

[16] A transformant comprising the vector according to

[14] .

[17] A method for producing the antibacterial and antiviral composition according to any one of [1] to

[11] , comprising any of the following steps: (A) culturing bacteria to produce a polypeptide; (B) synthesizing the polypeptide by chemical synthesis; (C) producing the polypeptide from an organism into which the vector according to

[14] has been introduced; (D) producing the polypeptide by translation using the in vitro translation template molecule according to

[15] ; or (E) producing the polypeptide from the transformant according to

[16] .

[18] A method for inhibiting the growth of or removing at least one bacterium selected from the group consisting of E. coli and Staphylococcus aureus and non-enveloped viruses, comprising applying the antibacterial and antiviral composition according to any one of [1] to

[11] to at least one bacterium selected from the group consisting of E. coli and Staphylococcus aureus and non-enveloped viruses.

[19] A method for inhibiting the growth of or removing at least one bacterium selected from the group consisting of E. coli and Staphylococcus aureus and non-enveloped viruses and enveloped viruses, comprising applying the antibacterial and antiviral composition according to any one of [1] to

[11] to at least one bacterium selected from the group consisting of E. coli and Staphylococcus aureus and non-enveloped viruses and enveloped viruses.

[0008] According to the present invention, it is possible to provide a composition containing a naturally occurring peptide having antiviral activity against non-enveloped viruses and a method for producing the same. Furthermore, according to the present invention, it is possible to provide a composition containing a naturally occurring peptide having antiviral activity against enveloped viruses and a method for producing the same. The peptides according to the present invention are highly safe and stable, and furthermore, because of their linear structure, they can be chemically synthesized, making it easy to ensure a stable supply. Furthermore, they can be processed into metal products and animal fibers, and are thought to be useful for a variety of applications.

[0009] Hereinafter, embodiments for carrying out the present invention will be described. Note that the present invention is not limited to the following embodiments. Furthermore, the present invention also encompasses embodiments in which one or more of the embodiments described in this specification are combined in any manner.

[0010] Compositions of the Invention In one embodiment, the present invention provides an antibacterial and antiviral composition comprising a polypeptide derived from bacteria that has antibacterial activity against at least one bacterium selected from the group consisting of Escherichia coli and Staphylococcus aureus, and has antiviral activity against non-enveloped viruses.

[0011] In this embodiment, the polypeptide is derived from bacteria, including gram-positive bacteria (e.g., lactic acid bacteria, bifidobacteria, and staphylococci) and gram-negative bacteria (e.g., Escherichia coli), preferably gram-positive bacteria, more preferably lactic acid bacteria. Examples of lactic acid bacteria include Lactobacillales lactic acid bacteria, such as Lactiplantibacillus lactic acid bacteria, Lactobacillus lactic acid bacteria, and Lactococcus lactic acid bacteria. Examples of Lactiplantibacillus lactic acid bacteria include Lactiplantibacillus plantarum (e.g., NITE BP-03198, BP-03200, or NITE BP-03201), more specifically NITE BP-03198. Examples of Lactobacillus lactic acid bacteria include Lactobacillus sp. Examples of Lactococcus lactic acid bacteria include Lactococcus lactis (for example, NITE ABP-03536).

[0012] In this embodiment, the polypeptide may be a linear polypeptide, a branched polypeptide, or a cyclic polypeptide, and in this embodiment, the polypeptide may be modified or unmodified.

[0013] In this embodiment, "antibacterial activity" refers to the property of inhibiting the growth of or eliminating a target microorganism, i.e., at least one bacterium selected from the group consisting of Escherichia coli and Staphylococcus aureus. The antibacterial activity can be evaluated by the antibacterial activity evaluation test described in the Examples below.

[0014] In this embodiment, the term "non-enveloped virus" refers to a virus that does not have a membrane (i.e., an envelope) made of lipids, proteins, and glycoproteins. Examples of non-enveloped viruses include feline calicivirus, norovirus, rotavirus, adenovirus, and poliovirus.

[0015] In this embodiment, the term "antiviral activity" refers to the property of suppressing the proliferation of a target virus or eliminating the virus. The antiviral activity can be evaluated by the antiviral activity evaluation test described in the Examples below.

[0016] In one embodiment, the invention provides a composition of the invention according to any of the above embodiments, wherein the polypeptide further has antiviral activity against enveloped viruses.

[0017] In this embodiment, the term "enveloped virus" refers to a virus having a membrane (i.e., an envelope) made of lipids, proteins, and glycoproteins. Examples of enveloped viruses include influenza virus, hepatitis B virus, hepatitis C virus, Zika virus, novel coronavirus, SARS coronavirus, MERS coronavirus, varicella-zoster virus, rubella virus, AIDS virus, Ebola virus, and rabies virus.

[0018] In one embodiment, the present invention provides a composition of the present invention according to any one of the above embodiments, wherein the polypeptide has an isoelectric point of 9.9 or higher. In one embodiment, the present invention provides a composition of the present invention according to any one of the above embodiments, wherein the polypeptide has an isoelectric point of 11 or higher. In one embodiment, the present invention provides a composition of the present invention according to any one of the above embodiments, wherein the polypeptide has an isoelectric point of 11 to 13. In one embodiment, the present invention provides a composition of the present invention according to any one of the above embodiments, wherein the polypeptide has an isoelectric point of 11 to 12. In one embodiment, the present invention provides a composition of the present invention according to any one of the above embodiments, wherein the polypeptide has a molecular weight of 2000 to 6000 Da. In one embodiment, the present invention provides a composition of the present invention according to any one of the above embodiments, wherein the polypeptide has a molecular weight of 2000 to 5000 Da. In one embodiment, the present invention provides a composition of the present invention according to any one of the above embodiments, wherein the polypeptide has a molecular weight of 2500 to 4000 Da. In one embodiment, the present invention provides a composition of the present invention according to any one of the above embodiments, wherein the polypeptide has a molecular weight of 3000 to 3500 Da. In one embodiment, the present invention provides a composition of the present invention according to any one of the above embodiments, wherein the polypeptide has an isoelectric point of 9.9 or higher and a molecular weight of 2000 to 6000 Da. In one embodiment, the present invention provides a composition of the present invention according to any one of the above embodiments, wherein the polypeptide has an isoelectric point of 11 or higher and a molecular weight of 2000 to 5000 Da. In one embodiment, the present invention provides a composition of the present invention according to any one of the above embodiments, wherein the polypeptide has an isoelectric point of 11 to 13 and a molecular weight of 2500 to 4000 Da. In one embodiment, the present invention provides a composition of the present invention according to any one of the above embodiments, wherein the polypeptide has an isoelectric point of 11 to 12 and a molecular weight of 3000 to 3500 Da.

[0019] In one embodiment, the present invention provides a composition of the present invention according to any of the above embodiments, wherein the polypeptide is: a polypeptide consisting of an amino acid sequence derived from a lactic acid bacterium of the order Lactobacillales; a polypeptide consisting of an amino acid sequence derived from a lactic acid bacterium of the order Lactobacillales, in which one or more amino acid residues have been substituted, deleted, inserted, and / or added; or a polypeptide consisting of an amino acid sequence having 85% or more sequence identity to an amino acid sequence derived from a lactic acid bacterium of the order Lactobacillales.

[0020] In one embodiment, the present invention provides the composition of the present invention according to any one of the above embodiments, wherein the polypeptide is: a polypeptide consisting of an amino acid sequence derived from lactic acid bacteria of the genus Lactiplantibacillus; a polypeptide consisting of an amino acid sequence derived from lactic acid bacteria of the genus Lactiplantibacillus, in which one or several amino acid residues have been substituted, deleted, inserted, and / or added; or a polypeptide consisting of an amino acid sequence having 85% or more sequence identity to an amino acid sequence derived from lactic acid bacteria of the genus Lactiplantibacillus.

[0021] In one embodiment, the present invention provides the composition of the present invention according to any one of the above embodiments, wherein the polypeptide is a polypeptide consisting of an amino acid sequence derived from a lactic acid bacterium of the genus Lactiplantibacillus.

[0022] In one embodiment, the present invention provides a composition of the present invention according to any of the above embodiments, wherein the polypeptide is a polypeptide consisting of an amino acid sequence derived from a lactic acid bacterium of the genus Lactiplantibacillus, in which one or several amino acid residues have been substituted, deleted, inserted, and / or added.

[0023] In one embodiment, the present invention provides the composition of the present invention according to any one of the above embodiments, wherein the polypeptide is: a polypeptide consisting of an amino acid sequence derived from a lactic acid bacterium of the genus Lactobacillus; a polypeptide consisting of an amino acid sequence derived from a lactic acid bacterium of the genus Lactobacillus, in which one or several amino acid residues have been substituted, deleted, inserted, and / or added; or a polypeptide consisting of an amino acid sequence having 85% or more sequence identity to an amino acid sequence derived from a lactic acid bacterium of the genus Lactobacillus.

[0024] In one embodiment, the present invention provides the composition of the present invention according to any one of the above embodiments, wherein the polypeptide is a polypeptide consisting of an amino acid sequence derived from a lactic acid bacterium of the genus Lactobacillus.

[0025] In one embodiment, the present invention provides the composition of the present invention according to any of the above embodiments, wherein the polypeptide is a polypeptide consisting of an amino acid sequence derived from a lactic acid bacterium of the genus Lactobacillus, in which one or several amino acid residues have been substituted, deleted, inserted, and / or added.

[0026] In one embodiment, the present invention provides the composition of the present invention according to any of the above embodiments, wherein the polypeptide is: a polypeptide consisting of an amino acid sequence derived from a lactic acid bacterium of the genus Lactococcus; a polypeptide consisting of an amino acid sequence derived from a lactic acid bacterium of the genus Lactococcus, in which one or several amino acid residues have been substituted, deleted, inserted, and / or added; or a polypeptide consisting of an amino acid sequence having 85% or more sequence identity to an amino acid sequence derived from a lactic acid bacterium of the genus Lactococcus.

[0027] In one embodiment, the present invention provides the composition of the present invention according to any one of the above embodiments, wherein the polypeptide is a polypeptide consisting of an amino acid sequence derived from a lactic acid bacterium of the genus Lactococcus.

[0028] In one embodiment, the present invention provides a composition of the present invention according to any of the above embodiments, wherein the polypeptide is a polypeptide consisting of an amino acid sequence derived from a Lactococcus lactic acid bacterium in which one or more amino acid residues have been substituted, deleted, inserted, and / or added.

[0029] The term "one or several" varies depending on the position and type of amino acid residue in the three-dimensional structure of the protein, but may be, for example, 1 to 10. The polypeptide according to this embodiment may be a polypeptide comprising an amino acid sequence in which, for example, one to ten amino acid residues have been substituted, deleted, inserted, and / or added to an amino acid sequence derived from a lactic acid bacterium of the genus Lactiplantibacillus. The upper limit of the number of substituted, deleted, inserted, and / or added amino acid residues may be 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less.

[0030] In one aspect of this embodiment, an "amino acid sequence in which one or several amino acid residues have been substituted, deleted, inserted, and / or added" can include, for example, an amino acid sequence that, due to the substitution, deletion, insertion, and / or addition, has a sequence identity of 80% or more, 85% or more, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more to the amino acid sequence before the substitution, deletion, insertion, and / or addition.

[0031] An example of the substitution, deletion, insertion, and / or addition of amino acid residues is a conservative mutation that maintains normal polypeptide function. A representative conservative mutation is a conservative substitution. Examples of conservative substitutions include the following: When the substitution site is an aromatic amino acid, it is a mutation in which Phe, Trp, and Tyr are substituted with each other; when the substitution site is a hydrophobic amino acid, it is a mutation in which Leu, Ile, and Val are substituted with each other; when the substitution site is a polar amino acid, it is a mutation in which Gln and Asn are substituted with each other; when the substitution site is a basic amino acid, it is a mutation in which Lys, Arg, and His are substituted with each other; when the substitution site is an acidic amino acid, it is a mutation in which Asp and Glu are substituted with each other; and when the substitution site is an amino acid with a hydroxyl group, it is a mutation in which Ser and Thr are substituted with each other. Substitutions that are considered conservative substitutions include substitutions of Ala with Ser or Thr, substitutions of Arg with Gln, His, or Lys, substitutions of Asn with Glu, Gln, Lys, His, or Asp, substitutions of Asp with Asn, Glu, or Gln, substitutions of Cys with Ser or Ala, substitutions of Gln with Asn, Glu, Lys, His, Asp, or Arg, substitutions of Glu with Gly, Asn, Gln, Lys, or Asp, substitutions of Gly with Pro, substitutions of His with Asn, Lys, Gln, Arg, or Tyr, substitutions of Ile with L substitution of Lys with Asn, Glu, Gln, His or Arg; substitution of Met with Ile, Leu, Val or Phe; substitution of Phe with Trp, Tyr, Met, Ile or Leu; substitution of Ser with Thr or Ala; substitution of Thr with Ser or Ala; substitution of Trp with Phe or Tyr; substitution of Tyr with His, Phe or Trp; and substitution of Val with Met, Ile or Leu.

[0032] In one embodiment, the present invention provides a composition of the present invention according to any one of the above embodiments, wherein the polypeptide is a polypeptide consisting of an amino acid sequence having 85% or more sequence identity to an amino acid sequence derived from a lactic acid bacterium of the genus Lactiplantibacillus.

[0033] In one embodiment, the present invention provides the composition of the present invention according to any one of the above embodiments, wherein the polypeptide is a polypeptide consisting of an amino acid sequence having 85% or more sequence identity to an amino acid sequence derived from a lactic acid bacterium of the genus Lactobacillus.

[0034] In one embodiment, the present invention provides a composition of the present invention according to any one of the above embodiments, wherein the polypeptide is a polypeptide consisting of an amino acid sequence having 85% or more sequence identity to an amino acid sequence derived from a Lactococcus lactic acid bacterium.

[0035] In one aspect of this embodiment, the lower limit of the sequence identity may be 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0036] In this embodiment, "sequence identity" refers to the percentage (%) of identical amino acid residues relative to the total overlapping amino acid sequence in the optimal alignment of two amino acid sequences when the sequences are aligned using a mathematical algorithm known in the art (preferably, the algorithm can consider the introduction of gaps into one or both of the sequences for optimal alignment). The "sequence identity" of amino acid sequences can be easily confirmed by those skilled in the art. For example, NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) can be used. Sequence identity of nucleotide sequences can also be confirmed by the same method as above.

[0037] The polypeptide according to this embodiment may contain a signal peptide (leader peptide) or may be a precursor polypeptide of the above-mentioned polypeptide. The precursor polypeptide may be cleaved by a peptidase intracellularly or extracellularly to become the above-mentioned polypeptide (mature polypeptide). The precursor polypeptide usually contains an amino acid sequence for cleavage by a specific peptidase. An example of a precursor polypeptide of the polypeptide consisting of the amino acid sequence of SEQ ID NO: 1 is a polypeptide consisting of the amino acid sequence of SEQ ID NO: 4. The polypeptide according to this embodiment may be a secreted polypeptide or a membrane-bound polypeptide.

[0038] In another aspect of this embodiment, the polypeptide may further comprise a peptide that serves as a tag for purification, such as a polyhistidine sequence (6xHis).

[0039] In one embodiment, the present invention provides a composition of the present invention according to any of the above embodiments, wherein the amino acid sequence derived from a lactic acid bacterium belonging to the order Lactobacillales is the amino acid sequence of any one of SEQ ID NOs: 1 or 5 to 10.

[0040] In one embodiment, the present invention provides the composition of the present invention according to any one of the above embodiments, wherein the amino acid sequence derived from a lactic acid bacterium belonging to the order Lactobacillales is the amino acid sequence of SEQ ID NO: 1. The amino acid sequence of SEQ ID NO: 1 is the amino acid sequence of the mature form of the polypeptide predicted from the genome sequence of the lactic acid bacterium Lactiplantibacillus plantarum (NITE BP-03198).

[0041] The amino acid sequence of SEQ ID NO: 1 is shown below: MSKKQWYRLGERVGRVAVFIGGGLLFK (SEQ ID NO: 1) The polypeptide consists of 27 amino acid residues, has a molecular weight of approximately 3110.7 (measured and theoretical), and an isoelectric point of approximately 11.1 (theoretical).

[0042] In one embodiment, the present invention provides the composition of the present invention according to any one of the above embodiments, wherein the amino acid sequence derived from a lactic acid bacterium belonging to the order Lactobacillales is the amino acid sequence of SEQ ID NO: 5. The amino acid sequence of SEQ ID NO: 5 is a partial amino acid sequence consisting of residues 1 to 17 of the mature form of the polypeptide predicted from the genome sequence of the lactic acid bacterium Lactobacillus sp. SC-2001 (NITE BP-03197).

[0043] The amino acid sequence of SEQ ID NO: 5 is shown below: KIIRISKWSYYNTKTKK (SEQ ID NO: 5) The polypeptide consists of 17 amino acid residues, has a molecular weight of approximately 2157.6 (measured and theoretical), and an isoelectric point of approximately 10.4 (theoretical).

[0044] In one embodiment, the present invention provides the composition of the present invention according to any one of the above embodiments, wherein the amino acid sequence derived from a lactic acid bacterium belonging to the order Lactobacillales is the amino acid sequence of SEQ ID NO: 6. The amino acid sequence of SEQ ID NO: 6 is the amino acid sequence of the mature form of the polypeptide predicted from the genome sequence of the lactic acid bacterium Lactiplantibacillus plantarum (NITE BP-03200).

[0045] The amino acid sequence of SEQ ID NO: 6 is shown below: WLQYLIPVVVSGLNHTDQIMRGWKKGYHRHH (SEQ ID NO: 6) The polypeptide consists of 31 amino acid residues, has a molecular weight of approximately 3770.4 (measured and theoretical), and an isoelectric point of approximately 10.0 (theoretical).

[0046] In one embodiment, the present invention provides the composition of the present invention according to any one of the above embodiments, wherein the amino acid sequence derived from a lactic acid bacterium belonging to the order Lactobacillales is the amino acid sequence of SEQ ID NO: 7. The amino acid sequence of SEQ ID NO: 7 is the amino acid sequence of the mature form of the polypeptide predicted from the genome sequence of the lactic acid bacterium Lactiplantibacillus plantarum (NITE BP-03200).

[0047] The amino acid sequence of SEQ ID NO: 7 is shown below: SSSLLNTAWRKFGQIAGTVTSAGLDAGWIRAHKYGHGRSH (SEQ ID NO: 7) The polypeptide consists of 40 amino acid residues, has a molecular weight of approximately 4295.8 (measured and theoretical), and an isoelectric point of approximately 11.0 (theoretical).

[0048] In one embodiment, the present invention provides the composition of the present invention according to any one of the above embodiments, wherein the amino acid sequence derived from a lactic acid bacterium belonging to the order Lactobacillales is the amino acid sequence of SEQ ID NO: 8. The amino acid sequence of SEQ ID NO: 8 is the amino acid sequence of the mature form of the polypeptide predicted from the genome sequence of the lactic acid bacterium Lactiplantibacillus plantarum (NITE BP-03200).

[0049] The amino acid sequence of SEQ ID NO: 8 is shown below: DLTTKLWSSWGYYLGKKARWNLKHPYVQF (SEQ ID NO: 8) The polypeptide consists of 29 amino acid residues, has a molecular weight of approximately 3587.1 (measured and theoretical), and an isoelectric point of approximately 9.9 (theoretical).

[0050] In one embodiment, the present invention provides the composition of the present invention according to any one of the above embodiments, wherein the amino acid sequence derived from a lactic acid bacterium belonging to the order Lactobacillales is the amino acid sequence of SEQ ID NO: 9. The amino acid sequence of SEQ ID NO: 9 is the amino acid sequence of the mature form of the polypeptide predicted from the genome sequence of the lactic acid bacterium Lactiplantibacillus plantarum (NITE BP-03200).

[0051] The amino acid sequence of SEQ ID NO: 9 is shown below: SVPTSVYTLGIKILWSAYKHRKTIEKSFNKGFYH (SEQ ID NO: 9) The polypeptide consists of 34 amino acid residues, has a molecular weight of approximately 4000.7 (measured and theoretical), and an isoelectric point of approximately 10.0 (theoretical).

[0052] In one embodiment, the present invention provides the composition of the present invention according to any one of the above embodiments, wherein the amino acid sequence derived from a lactic acid bacterium belonging to the order Lactobacillales is the amino acid sequence of SEQ ID NO: 10. The amino acid sequence of SEQ ID NO: 10 is the amino acid sequence of the mature form of the polypeptide predicted from the genome sequence of the lactic acid bacterium Lactococcus lactis subsp. lactis (NITE ABP-03536).

[0053] The amino acid sequence of SEQ ID NO: 10 is shown below: MAGFLKVVQLLAKYGSKAVQWAWANKGKILDWLNAGQAIDWVVSKIKQILGIK (SEQ ID NO: 10) The polypeptide consists of 53 amino acid residues, has a molecular weight of approximately 5898.1 (measured and theoretical), and an isoelectric point of approximately 10.1 (theoretical).

[0054] In one embodiment, the present invention provides a polypeptide comprising: (1a) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1; (1b) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 1, wherein one or several amino acid residues have been substituted, deleted, inserted, and / or added; (1c) a polypeptide consisting of an amino acid sequence having 85% or more sequence identity to the amino acid sequence of SEQ ID NO: 1; (5a) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 5; (5b) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 5, wherein one or several amino acid residues have been substituted, deleted, inserted, and / or added; (5c) a polypeptide consisting of an amino acid sequence having 85% or more sequence identity to the amino acid sequence of SEQ ID NO: 5; (6a) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 6; (6b) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 6, wherein one or several amino acid residues have been substituted, deleted, inserted, and / or added; (6c) a polypeptide consisting of an amino acid sequence having 85% or more sequence identity to the amino acid sequence of SEQ ID NO: 6; (7a) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 7; (7b) a polypeptide consisting of an amino acid sequence in which one or several amino acid residues in the amino acid sequence of SEQ ID NO: 7 have been substituted, deleted, inserted, and / or added; (7c) a polypeptide consisting of an amino acid sequence having 85% or more sequence identity to the amino acid sequence of SEQ ID NO: 7; (8a) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 8; (8b) a polypeptide consisting of the amino acid sequence in which one or several amino acid residues in the amino acid sequence of SEQ ID NO: 8 have been substituted, deleted, inserted, and / or added; (8c) a polypeptide consisting of an amino acid sequence having 85% or more sequence identity to the amino acid sequence of SEQ ID NO: 8; (9a) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 9; (9b) a polypeptide consisting of the amino acid sequence in which one or several amino acid residues in the amino acid sequence of SEQ ID NO: 9 have been substituted, deleted, inserted, and / or added; (9c) a polypeptide consisting of an amino acid sequence having 85% or more sequence identity to the amino acid sequence of SEQ ID NO: 9; (10a) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 10;(10b) a polypeptide consisting of the amino acid sequence of SEQ ID NO: 10 in which one or several amino acid residues have been substituted, deleted, inserted, and / or added; or (10c) a polypeptide consisting of an amino acid sequence having 85% or more sequence identity to the amino acid sequence of SEQ ID NO: 10.

[0055] In one embodiment, the present invention provides a composition of the present invention according to any one of the above embodiments, in which two or more polypeptides are used in combination, wherein at least one of the polypeptides in the combination does not have antibacterial or antiviral activity by itself, but when used together with another polypeptide, exhibits an antibacterial or antiviral activity that is greater than the antibacterial or antiviral activity of the other polypeptide alone.

[0056] Here, when two or more polypeptides are used in combination, they may be in the form of a mixture of the individual polypeptides, or each polypeptide may be independent and used together when used. Furthermore, when a combination of polypeptides is used, the polypeptides may be used simultaneously or sequentially. For example, when a first polypeptide and a second polypeptide are used, the first polypeptide and the second polypeptide may be used simultaneously, or the first polypeptide may be used followed by the second polypeptide, or the second polypeptide may be used followed by the first polypeptide.

[0057] In one embodiment, the present invention provides a composition of the present invention according to any one of the above embodiments for use in combination with two or more polypeptides selected from the polypeptides described in (1a) to (10c) above. In one embodiment, the present invention provides a composition of the present invention according to any one of the above embodiments for use in combination with two or more polypeptides selected from (1a), (5a), (6a), (7a), (8a), (9a), and (10a) above. In one embodiment, the present invention provides a composition of the present invention according to any one of the above embodiments for use in combination with the polypeptides (6a) and (7a) above. In one embodiment, the present invention provides a composition of the present invention according to any one of the above embodiments for use in combination with the polypeptides (8a) and (9a) above.

[0058] In one embodiment, the present invention provides a composition of the present invention according to any one of the above embodiments, further comprising an additive. Examples of the additive include a surfactant, a preservative, a pH adjuster, a thickener, an excipient, and a flavoring agent. The additive may be a single additive or a combination of two or more additives.

[0059] The surfactant may be an anionic surfactant, a nonionic surfactant, or an amphoteric surfactant. Examples of anionic surfactants include N-acylamino acid salts, α-olefin sulfonates, N-acylsulfonates, alkyl sulfates (e.g., sodium lauryl sulfate), and sulfates of glycerin fatty acid esters. Examples of nonionic surfactants include polyoxyethylene alkyl ethers, polyoxyethylene-polyoxypropylene block copolymers, polyoxyethylene hydrogenated castor oil, polyoxyethylene ethers of glycerin esters, alkylolamides, sucrose fatty acid esters, glycerin fatty acid esters, sorbitan fatty acid esters, propylene glycol fatty acid esters, glycerin organic acid fatty acid esters, polyglycerin fatty acid esters, calcium stearoyl lactylate, sodium stearoyl lactylate, polyoxyethylene sorbitan fatty acid esters, and alkyl glycosides. Examples of amphoteric surfactants include alkyl betaine surfactants, amine oxide surfactants, and imidazolinium betaine surfactants. Specific examples thereof include 2-alkyl-N-carboxymethyl-N-hydroxyethylimidazolinium betaine, lauryl dimethylaminoacetic acid betaine, coconut oil alkyl betaine (coconut oil alkyl dimethylaminoacetic acid betaine), stearyl dimethylaminoacetic acid betaine, sodium stearyl dimethyl betaine, coconut oil fatty acid amido alkyl betaine, palm oil fatty acid amidopropyl betaine, lauric acid amidopropyl betaine, ricinoleic acid amidopropyl betaine, and stearyl dihydroxyethyl betaine.

[0060] Examples of preservatives or antiseptics include paraoxybenzoic acid esters such as sodium benzoate, methylparaben, ethylparaben, butylparaben, isopropylparaben, propylparaben, isobutylparaben, and benzylparaben; alcohols such as phenoxyethanol and ethanol; sorbic acid, benzoic acid, dehydroacetic acid, propionic acid, or salts thereof; salts such as sodium chloride; ethylenediaminetetraacetate, benzalkonium chloride, benzethonium chloride, cetylpyridinium chloride, and alkyldiaminoethylglycine hydrochloride.

[0061] Examples of pH adjusters include acids and alkalis such as acetic acid, hydrochloric acid, sulfuric acid, nitric acid, citric acid, phosphoric acid, malic acid, gluconic acid, maleic acid, succinic acid, glutamic acid, pyrophosphoric acid, tartaric acid, sodium hydroxide acetate, potassium hydroxide, sodium hydroxide, sodium acetate, sodium carbonate, potassium carbonate, sodium citrate, sodium hydrogen citrate, phosphoric acid, sodium phosphate, sodium monohydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, and buffers.

[0062] Examples of thickeners include organic binders such as methyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, carboxymethyl cellulose or salts thereof, pullulan, gelatin, carrageenan, sodium alginate, xanthan gum, sodium polyacrylate, gum arabic, guar gum, locust bean gum, polyvinyl alcohol, polyvinylpyrrolidone, and carboxyvinyl polymers; and inorganic binders such as thickening anhydrous silicic acid and bentonite.

[0063] Examples of excipients include crystalline cellulose, powdered cellulose, potato starch, corn starch, light anhydrous silicic acid, hydrous silicon dioxide, silicon dioxide, precipitated calcium carbonate, anhydrous calcium hydrogen phosphate, magnesium oxide, calcium lactate, calcium silicate, magnesium aluminometasilicate, synthetic hydrotalcite, synthetic aluminum silicate, lactose, sucrose, D-mannitol, erythritol, glucose, fructose, talc, dextrin, and cyclodextrin.

[0064] Examples of the fragrance include peppermint and aloe vera juice.

[0065] The polypeptide contained in the composition of the present invention has antibacterial activity against at least one bacterium selected from the group consisting of Escherichia coli and Staphylococcus aureus. The antibacterial activity of the polypeptide can be measured, for example, according to the antibacterial activity test method in Experimental Example 3 described below. The polypeptide of this embodiment has high pH stability and has antibacterial activity not only in the acidic range (e.g., pH 4.0) but also in the neutral range (e.g., pH 7.4) to alkaline range (e.g., pH 10.0). The polypeptide of this embodiment has high thermal stability and retains antibacterial activity, for example, even after heat treatment in the neutral range (temperatures of 80°C, 100°C, 121°C, etc.). As described above, the polypeptide of this embodiment is a naturally occurring polypeptide with high antibacterial activity, has a wide usable pH range, and is stable to heat, etc., making it highly versatile as an antibacterial component.

[0066] The polypeptide contained in the composition of the present invention has antiviral activity against a wide range of non-enveloped viruses. The antiviral activity of the polypeptide can be measured, for example, according to the antiviral activity test method described below.

[0067] In one embodiment, the present invention provides a composition of the present invention according to any one of the above embodiments, which has antiviral activity against one or more viruses selected from the group consisting of feline calicivirus, norovirus, rotavirus, adenovirus, and poliovirus. In one embodiment, the present invention provides a composition of the present invention according to any one of the above embodiments, which has antiviral activity against feline calicivirus.

[0068] The polypeptide contained in the composition of the present invention has antiviral activity against a wide range of enveloped viruses. The antiviral activity of the polypeptide can be measured, for example, according to the antiviral activity test method described below.

[0069] In one embodiment, the present invention provides a composition of the present invention according to any one of the above embodiments, which has antiviral activity against one or more viruses selected from the group consisting of influenza virus, hepatitis B virus, hepatitis C virus, Zika virus, novel coronavirus, SARS coronavirus, MERS coronavirus, varicella-zoster virus, rubella virus, AIDS virus, Ebola virus, and rabies virus. In one embodiment, the present invention provides a composition of the present invention according to any one of the above embodiments, which has antiviral activity against influenza virus.

[0070] In one embodiment, the present invention provides a method for inhibiting the growth of or removing at least one bacterium selected from the group consisting of Escherichia coli and Staphylococcus aureus and non-enveloped viruses, comprising applying a polypeptide described in any of the above embodiments to at least one bacterium selected from the group consisting of Escherichia coli and Staphylococcus aureus and non-enveloped viruses.

[0071] In one embodiment, the present invention provides a method for inhibiting the growth of or removing at least one bacterium selected from the group consisting of Escherichia coli and Staphylococcus aureus, and non-enveloped viruses and enveloped viruses, comprising applying a polypeptide described in any of the above embodiments to at least one bacterium selected from the group consisting of Escherichia coli and Staphylococcus aureus, and non-enveloped viruses and enveloped viruses.

[0072] In one embodiment, the present invention provides a method for inhibiting the growth of or eliminating non-enveloped viruses and enveloped viruses, comprising applying a polypeptide described in any of the above embodiments to the non-enveloped viruses and enveloped viruses.

[0073] In one embodiment, the present invention provides a method for inhibiting the growth of or eliminating a non-enveloped virus, comprising applying a polypeptide according to any of the above embodiments to the non-enveloped virus.

[0074] In one embodiment, the present invention provides a method for inhibiting the growth of or eliminating an enveloped virus, comprising applying a polypeptide according to any of the above embodiments to the enveloped virus.

[0075] In one embodiment, the present invention provides a method for inhibiting the growth of or removing at least one bacterium selected from the group consisting of Escherichia coli and Staphylococcus aureus and non-enveloped viruses, comprising applying the composition of the present invention described in any of the above embodiments to at least one bacterium selected from the group consisting of Escherichia coli and Staphylococcus aureus and non-enveloped viruses.

[0076] In one embodiment, the present invention provides a method for inhibiting the growth of or removing at least one bacterium selected from the group consisting of Escherichia coli and Staphylococcus aureus, and non-enveloped viruses and enveloped viruses, comprising applying the composition of the present invention described in any of the above embodiments to at least one bacterium selected from the group consisting of Escherichia coli and Staphylococcus aureus, and non-enveloped viruses and enveloped viruses.

[0077] In one embodiment, the present invention provides a method for inhibiting the growth of or removing non-enveloped viruses and enveloped viruses, comprising applying a composition of the present invention described in any of the above embodiments to the non-enveloped viruses and enveloped viruses.

[0078] In one embodiment, the present invention provides a method for inhibiting the growth of or removing a non-enveloped virus, comprising applying a composition of the present invention described in any of the above embodiments to the non-enveloped virus.

[0079] In one embodiment, the present invention provides a method for inhibiting the growth of or eliminating an enveloped virus, comprising applying a composition of the present invention described in any of the above embodiments to the enveloped virus.

[0080] In one embodiment, the present invention provides the use of a polypeptide according to any of the above embodiments in the manufacture of a composition of the invention according to any of the above embodiments.

[0081] Nucleic Acids Encoding Polypeptides with Antiviral Activity The nucleic acid of this embodiment is a nucleic acid comprising a nucleotide sequence encoding the above-described polypeptide with antiviral activity. The nucleic acid may be DNA or RNA. The nucleic acid may or may not comprise an initiation codon at the 5'-end and a stop codon at the 3'-end. The nucleic acid may or may not comprise an intron sequence. An example of a nucleic acid of this embodiment comprises one or more nucleotide sequences selected from SEQ ID NOs: 2, 3, and 16-27. Each of the nucleotide sequences of SEQ ID NOs: 2-3 comprises a sequence encoding the amino acid of SEQ ID NO: 1, each of the nucleotide sequences of SEQ ID NOs: 16-17 comprises a sequence encoding the amino acid of SEQ ID NO: 5, each of the nucleotide sequences of SEQ ID NOs: 18-19 comprises a sequence encoding the amino acid of SEQ ID NO: 6, each of the nucleotide sequences of SEQ ID NOs: 20-21 comprises a sequence encoding the amino acid of SEQ ID NO: 7, each of the nucleotide sequences of SEQ ID NOs: 22-23 comprises a sequence encoding the amino acid of SEQ ID NO: 8, each of the nucleotide sequences of SEQ ID NOs: 24-25 comprises a sequence encoding the amino acid of SEQ ID NO: 9, and each of the nucleotide sequences of SEQ ID NOs: 26-27 comprises a sequence encoding the amino acid of SEQ ID NO: 10. Needless to say, when the nucleic acid is RNA, the base sequence of the RNA is a base sequence in which thymine in the base sequence of the corresponding DNA is replaced with uracil.

[0082] The base sequence (DNA sequence) of SEQ ID NO: 2 is shown below: ATGAGTAAAAAAGCAATGGTATCGTTTAGGAGAACGTGTCGGTCGTGTAGCAGTTTTTATCGGTGGAGGATTACTTTTTAAATAA (SEQ ID NO: 2) The nucleic acid is composed of 84 bases.

[0083] The base sequence (RNA sequence) of SEQ ID NO: 3 is shown below: AUGAGUAAAAAAGCAAUGGUAUCGUUUAGGAGAACGUGUCGGUCGUGUAGCAGUUUUUAUCGGUGGAGGAUUACUUUUUAAAUAA (SEQ ID NO: 3) The nucleic acid is composed of 84 bases.

[0084] The base sequence (DNA sequence) of SEQ ID NO: 16 is shown below: AAGATTATCCGTATTTCAAAATGGAGTTACTATAACACAAAGACAAAAAAAAA (SEQ ID NO: 16) The nucleic acid is composed of 51 bases.

[0085] The base sequence (RNA sequence) of SEQ ID NO: 17 is shown below: AAGAUUAUCCGUAUUUCAAAAUGGAGUUACUAUAACACAAAGACAAAAAAAAA (SEQ ID NO: 17) The nucleic acid is composed of 51 bases.

[0086] The base sequence (DNA sequence) of SEQ ID NO: 18 is shown below: TGGCTACAATACCTGATCCCTGTTGTGGTTAGCGGCCTCAATCATACAGACCAAATTATGCGTGGTTGGAAAAAAGGATACCATCGTCATCATTAG (SEQ ID NO: 18) The nucleic acid is composed of 96 bases.

[0087] The base sequence (RNA sequence) of SEQ ID NO: 19 is shown below: UGGCUACAAUACCUGAUCCCUGUUGUGGUUAGCGGCCUCAAUCAUACAGACCAAAUUAUGCGUGGUUGGAAAAAAAGGAUACCAUCGUCAUCAUUAG (SEQ ID NO: 19) The nucleic acid is composed of 96 bases.

[0088] The base sequence (DNA sequence) of SEQ ID NO: 20 is shown below: TCTAGCAGTCTTTTAAATACTGCATGGCGGAAATTGGTCAAATAGCTGGTACCGTTACTTCTGCAGGATTGGATGCAGGTTGGATCAGGGCTCATAAGTATGGACATGGTAGGAGTCACTAG (SEQ ID NO: 20) The nucleic acid is composed of 123 bases.

[0089] The base sequence (RNA sequence) of SEQ ID NO: 21 is shown below: UCUAGCAGUCUUUUAAAUACUGCAUGGCGGAAAUUUGGUCAAAUAGCUGGUACCGUUACUUCUGCAGGAUUGGAUGCAGGUUGGAUCAGGGGCUCAUAAGUAUGGACAUGGUAGGAGUCACUAG (SEQ ID NO: 21) The nucleic acid is composed of 123 bases.

[0090] The base sequence (DNA sequence) of SEQ ID NO: 22 is shown below: GATTTAACAACCAAGTTATGGAGCTCTTGGGGATATTATCTTGGCAAGAAAGCACGTTGGAATTTAAAGCACCCATATGTTCAATTTTAG (SEQ ID NO: 22) The nucleic acid is composed of 90 bases.

[0091] The base sequence (RNA sequence) of SEQ ID NO: 23 is shown below: GAUUUAAACAACCAAGUUAUGGAGCUCUUGGGGAUAUUAUCUUGGCAAGAAAGCACGUUGGAAUUUAAAGCACCCAUAUGUUCAAUUUUAG (SEQ ID NO: 23) The nucleic acid is composed of 90 bases.

[0092] The base sequence (DNA sequence) of SEQ ID NO: 24 is shown below: TCAGTCCCAACTTCAGTATATACGCTTGGAATTAAAATTCTATGGTCTGCGTATAAGCATCGCAAACGATTGAAAAAAGTTTTAATAAAGGCTTTTATCAT (SEQ ID NO: 24) The nucleic acid is composed of 102 bases.

[0093] The base sequence (RNA sequence) of SEQ ID NO: 25 is shown below: UCAGUCCCAACUUCAGUAUAUACGCUUGGAAUUAAAAUUCUAUGGUCUGCGUAUAAGCAUCGCAAACGAUUGAAAAAAGUUUUAAUAAAGGCUUUUAUCAU (SEQ ID NO: 25) The nucleic acid is composed of 102 bases.

[0094] The base sequence (DNA sequence) of SEQ ID NO: 26 is shown below: ATGGCAGGGTTTTTTAAAAGTAGTTCAATTACTAGCTAAATATGGTTCTAAAAGCTGTACAATGGGCTTGGGCAAACAAGGGTAAGATTTTAGATTGGCTTAATGCAGGTCAGGCTATTGATTGGGTAGTTTCGAAAATTAAGCAAATTTTAGGTATTAAG (SEQ ID NO: 26) The nucleic acid is composed of 159 bases.

[0095] The base sequence (RNA sequence) of SEQ ID NO: 27 is shown below: AUGGCAGGGUUUUUAAAGUAGUUCAAUUACUAGCUAAAUAUGGUUCUAAAGCUGUACAAUGGGCUUGGGCAAACAAGGGUAAGAUUUUAGAUUGGCUUAAUGCAGGUCAGGCUAUUGAUUGGGUAGUUUCGAAAAUUAAGCAAAUUUUAGGUAUUAAG (SEQ ID NO: 27) The nucleic acid is composed of 159 bases.

[0096] In one aspect of this embodiment, the base sequences of SEQ ID NOs: 2 and 3 are sequences derived from the genome sequence of the lactic acid bacterium Lactiplantibacillus plantarum (NITE BP-03198) and encode the polypeptide of SEQ ID NO: 1.

[0097] In one aspect of this embodiment, the nucleotide sequences of SEQ ID NOs: 16 and 17 are sequences derived from the genome sequence of lactic acid bacteria Lactobacillus sp. SC-2001 (NITE BP-03197) and encode the polypeptide of SEQ ID NO: 5.

[0098] In one aspect of this embodiment, the nucleotide sequences of SEQ ID NOs: 18 and 19 are sequences derived from the genome sequence of the lactic acid bacterium Lactiplantibacillus plantarum (NITE BP-03200) and encode the polypeptide of SEQ ID NO: 6.

[0099] In one aspect of this embodiment, the nucleotide sequences of SEQ ID NOs: 20 and 21 are sequences derived from the genome sequence of the lactic acid bacterium Lactiplantibacillus plantarum (NITE BP-03200) and encode the polypeptide of SEQ ID NO: 7.

[0100] In one aspect of this embodiment, the base sequences of SEQ ID NOs: 22 and 23 are sequences derived from the genome sequence of the lactic acid bacterium Lactiplantibacillus plantarum (NITE BP-03200) and encode the polypeptide of SEQ ID NO: 8.

[0101] In one aspect of this embodiment, the base sequences of SEQ ID NOs: 24 and 25 are sequences derived from the genome sequence of the lactic acid bacterium Lactiplantibacillus plantarum (NITE BP-03200) and encode the polypeptide of SEQ ID NO: 9.

[0102] In one aspect of this embodiment, the base sequences of SEQ ID NOs: 26 and 27 are sequences derived from the genome sequence of the lactic acid bacterium Lactococcus lactis subsp. lactis (NITE ABP-03536) and encode the polypeptide of SEQ ID NO: 10.

[0103] The nucleic acid is not limited to the base sequence of the above-mentioned lactic acid bacteria NITE BP-03198, NITE BP-03197, NITE BP-03200, or NITE ABP-03536, and may be a nucleic acid containing a base sequence in which a codon encoding each amino acid in the coding region is replaced with another equivalent codon encoding the same amino acid. The nucleic acid according to this embodiment may also be a nucleic acid containing a base sequence in which the codon usage has been changed so as to improve the expression of the polypeptide according to this embodiment.

[0104] In one aspect of this embodiment, the nucleic acid may further comprise a base sequence encoding a peptide that serves as a tag for purification.

[0105] The nucleic acid according to this embodiment can be obtained by chemical synthesis or by PCR using the genome sequence of lactic acid bacteria NITE BP-03198, NITE BP-03197, NITE BP-03200, or NITE ABP-03536 as a template.

[0106] The nucleic acid may be a combination of two or more nucleic acids each containing a base sequence encoding the polypeptide having antiviral activity. The nucleic acid combination can produce a combination of polypeptides having antibacterial and antiviral activities.

[0107] Here, the "combination of nucleic acids" may be in the form of a mixture of two or more nucleic acids, or two or more nucleic acids that are independent and used together. Furthermore, the combination of nucleic acids may be nucleic acids obtained from a microorganism, and the combination of nucleic acids may be contained in a single nucleic acid derived from the microorganism.

[0108] Vector The vector according to this embodiment includes the nucleic acid described above. In the vector, the nucleic acid may be a single nucleic acid or a combination of nucleic acids. The vector is a nucleic acid molecule capable of amplifying and maintaining DNA, and examples thereof include expression vectors and cloning vectors. In one example, the nucleic acid described above is inserted into an expression vector and introduced into a host cell or the like, where it expresses a polypeptide having antibacterial and antiviral activity. The vector can express a polypeptide in the host cell by being introduced into the host cell. The expression vector may have a promoter sequence and a terminator sequence for expressing the incorporated gene. The vector can be constructed by incorporating the nucleic acid described above into a parent vector according to conventional genetic engineering techniques. The vector may also have a selection marker sequence.

[0109] In one embodiment, when the vector contains the combination of nucleic acids, a single vector may contain both of the two nucleic acids constituting the combination. Alternatively, the vector may include a first vector and a second vector, where the first vector contains the first nucleic acid and the second vector contains the second nucleic acid.

[0110] The vector may be, for example, a bacterial plasmid-derived vector, a yeast plasmid-derived vector, a viral vector, a cosmid vector, a phagemid vector, an artificial chromosome vector, etc. Examples include pBR322, pUC plasmid vectors, pET-based plasmid vectors, etc. Specifically, when Escherichia coli is used as the host cell, examples of the vector include pUC19, pUC18, pUC119, pBluescript II, pET32, etc. When mammalian cells are used as the host cell, examples of the vector include pRc / RSV, pRc / CMV, retroviral vectors, adenoviral vectors, adeno-associated viral vectors, etc.

[0111] The nucleic acid to be incorporated into the vector is inserted downstream of the promoter in a functional state. Any promoter may be used as long as it is appropriate for the host used to express the gene. For example, when the host is an animal cell, the SRα promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, MoMuLV (Moloney murine leukemia virus) LTR, HSV-TK (herpes simplex virus thymidine kinase) promoter, etc. are used. Among these, the CMV promoter and the SRα promoter are preferred. When the host is Escherichia coli, the T7 promoter, cspA promoter, trp promoter, lac promoter, recA promoter, λPL promoter, lpp promoter, tac promoter, etc. are preferred. When the host is a Bacillus bacterium, the SPO1 promoter, SPO2 promoter, penP promoter, etc. are preferred. When the host is yeast, the Gal1 / 10 promoter, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, AOX1 promoter, etc. are preferred. When the host is Aspergillus oryzae, the ENOA promoter, TAA promoter, GLA promoter, AGL promoter, etc. are preferred. When the host is an insect cell, the polyhedrin promoter, P10 promoter, etc. are preferred. When the host is a plant cell, the CaMV35S promoter, CaMV19S promoter, NOS promoter, etc. are preferred.

[0112] In addition to the above, expression vectors that can be used include those that have, depending on the purpose, an enhancer, a splicing signal, a polyA addition signal, a selection marker such as a drug resistance gene or an auxotrophy complementing gene, a replication origin, etc.

[0113] In vitro translation template molecule The in vitro translation template molecule according to this embodiment includes the nucleic acid described above. In the in vitro translation template molecule, the nucleic acid may be a single nucleic acid or a combination of nucleic acids. Here, "in vitro translation template molecule" refers to a nucleic acid molecule that serves as a translation template in a cell-free protein synthesis system. The in vitro translation template molecule may be DNA or RNA. The in vitro translation template molecule may be single-stranded or double-stranded. The in vitro translation template molecule may have a promoter sequence and a terminator sequence for expressing the incorporated gene. Hereinafter, the in vitro translation template molecule may be referred to as a "translation template molecule."

[0114] In one embodiment, when the in vitro translation template molecule contains a combination of nucleic acids, a single in vitro translation template molecule may contain both of the nucleic acids constituting the combination, or the in vitro translation template molecule may contain a first translation template molecule and a second translation template molecule, where the first translation template molecule contains the first nucleic acid and the second translation template molecule contains the second nucleic acid.

[0115] The nucleic acid to be incorporated into the in vitro translation template molecule is inserted downstream of a promoter in a state in which the promoter is operable. Examples of the promoter include the promoters mentioned above.

[0116] Transformant The transformant according to this embodiment is a cell into which the above-described nucleic acid or vector has been introduced. In the transformant, the nucleic acid may be a single nucleic acid or a combination of nucleic acids. The transformant can express the polypeptide according to this embodiment.

[0117] The host cells into which the vector is introduced can be eukaryotic or prokaryotic cells. More specifically, examples include bacteria, fungi, plant cells, animal cells, and insect cells. The host cells may be yeast, Escherichia coli, Aspergillus oryzae, or mammalian cells. Examples of mammalian cells include humans, cows, horses, sheep, monkeys, pigs, mice, rats, hamsters, guinea pigs, rabbits, and dogs. In one aspect of this embodiment, the host cells may be, for example, yeast (BG10, BG11, etc.), Escherichia coli (BL21, DH5α, etc.), HEK 293T cells, CHO cells, Expi293F cells, or ExpiCHO cells.

[0118] Examples of methods for introducing the nucleic acid or vector into host cells include chemical methods such as the calcium phosphate method, the DEAE-dextran method, and the cationic liposome method; biological methods such as adenovirus vectors, vaccinia virus vectors, retrovirus vectors, and HVJ liposomes; and physical methods such as electroporation, direct DNA injection, and a gene gun. An appropriate introduction method can be selected depending on the host cells into which the nucleic acid or vector is to be introduced.

[0119] The vector may be maintained extrachromosomally within the host cell, or may be integrated into the chromosome.

[0120] Transformants into which the nucleic acid or vector has been introduced may be selected using a selection marker. For example, a selection marker gene is introduced into host cells simultaneously with the nucleic acid according to this embodiment, and the cells are cultured by a method appropriate for the properties of the selection marker. If the selection marker gene is a gene that confers drug resistance to a selection drug that exhibits lethal activity on the host cells, the host cells may be cultured in a medium containing the selection drug after the nucleic acid introduction operation.

[0121] Method for Producing the Composition of the Present Invention The method for producing the composition of the present invention according to this embodiment comprises any of the following steps: (A) culturing bacteria to produce a polypeptide; (B) synthesizing a polypeptide by chemical synthesis; (C) producing a polypeptide from an organism into which the vector described above has been introduced; (D) producing a polypeptide by translation using the in vitro translation template molecule described above; or (E) producing a polypeptide from the transformant described above.

[0122] In producing a combination of polypeptides, the polypeptides can be produced by producing each polypeptide of the combination of polypeptides individually according to the above-mentioned polypeptide production method, or by producing the combination of polypeptides at the same time. In producing the polypeptides, two or more polypeptides may be obtained separately, or a mixture of two or more polypeptides may be obtained. Furthermore, a polypeptide produced using nucleic acids may be combined with a chemically synthesized polypeptide.

[0123] In step (A) according to this embodiment, the polypeptide can also be obtained by culturing bacteria in an appropriate medium such as MRS liquid medium. The polypeptide according to this embodiment can be recovered, extracted, and purified from the bacterial culture by the same method as for the culture of the transformant.

[0124] Examples of bacteria having the gene for the polypeptide according to this embodiment include NITE BP-03198, NITE BP-03197, NITE BP-03200, and NITE ABP-03536. NITE BP-03198 and NITE BP-03200 are bacteria belonging to Lactiplantibacillus plantarum, a type of lactic acid bacteria, and NITE BP-03197 is a type of Lactobacillus sp. SC-2001 (Lactobacillus sp. SC-2001), and NITE ABP-03536 is a bacterium belonging to Lactococcus lactis, a type of lactic acid bacteria.

[0125] In step (B) according to this embodiment, the polypeptide can be produced using a general protein chemical synthesis method, such as a liquid phase method or a solid phase method.

[0126] In step (C) according to this embodiment, the polypeptide can be produced by producing the polypeptide from an organism into which the vector has been introduced. Examples of the organism include a microorganism. The organism into which the vector has been introduced may be a transformant. Production of the polypeptide using the transformant is described below.

[0127] In step (D) according to this embodiment, the polypeptide can be produced by translating the polypeptide from an in vitro translation template molecule using a general cell-free protein synthesis system, such as a wheat germ cell-free protein synthesis system.

[0128] In step (E) according to this embodiment, the polypeptide can be produced by culturing the transformant.

[0129] The transformant can be cultured by the same method as for culturing the host cell from which it was derived. When the transformant is a microorganism, it can be cultured using various media containing, for example, carbon sources, nitrogen sources, organic and inorganic salts, etc., commonly used in microbial culture. Examples of carbon sources include glucose, dextrin, soluble starch, sucrose, etc. Examples of nitrogen sources include inorganic substances such as ammonium salts and nitrates, as well as organic substances such as corn steep liquor, peptone, casein, meat extract, soybean meal, and potato extract. Examples of inorganic substances include calcium chloride, sodium dihydrogen phosphate, and magnesium chloride. The medium may further contain yeast extract, vitamins, growth-promoting factors, etc.

[0130] A culture containing the polypeptide of this embodiment can be obtained by culturing the transformant. The polypeptide of this embodiment can accumulate, for example, inside and / or outside the transformant (e.g., in the culture supernatant of the transformant). The polypeptide of this embodiment can be obtained from the transformant and / or its culture supernatant. The polypeptide of this embodiment may be obtained by appropriately disrupting, lysing, extracting, and purifying the transformant. Disruption, lysis, extraction, etc. can be performed by known methods. Examples of such methods include ultrasonic disruption, Dynomill method, bead disruption, French press disruption, and lysozyme treatment. These methods may be used alone or in appropriate combinations of two or more. Purification can be performed by known methods used for polypeptide purification. Examples of such methods include ammonium sulfate fractionation, ion exchange chromatography, hydrophobic chromatography, affinity chromatography, gel filtration chromatography, and isoelectric precipitation. These methods may be used alone or in appropriate combinations of two or more. The transformant may be recovered from the culture by centrifugation or other methods. When the polypeptide of the present invention accumulates in the culture supernatant, the culture supernatant can be collected by centrifugation or the like, and the polypeptide of the present invention can be recovered from the culture supernatant. The polypeptide of this embodiment may be any fraction of the culture supernatant of the transformant.

[0131] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0132] Experimental Example 1: Estimation of antibacterial peptides by genome analysis of lactic acid bacteria Lactiplantibacillus plantarum (NITE BP-03198) For the lactic acid bacteria Lactiplantibacillus plantarum (NITE BP-03198), long-read sequence data was obtained using a PacBio RSII sequencer (Pacific Biosciences) and short-read sequence data was obtained using a NovaSeq6000 sequencer (Illumina). The acquired short sequence data and long sequence data were subjected to hybrid assembly processing using Unicycler to obtain the genome sequence of Lactiplantibacillus plantarum (NITE BP-03198). Subsequently, gene prediction was performed on the obtained genome sequence using DFAST, and sequences homologous to known antimicrobial peptides were searched for using HMMER and secondary metabolite-related genes were searched for using antiSMASH, resulting in the amino acid sequence of SEQ ID NO: 4, which is a candidate antimicrobial peptide.

[0133] The amino acid sequence of SEQ ID NO: 4 is shown below: MNKYVELDLEQLQQISGGMSKKQWYRLGERVGRVAVFIGGGLLFK (SEQ ID NO: 4) The polypeptide consists of 45 amino acid residues.

[0134] Using the corresponding lactic acid bacteria, the amino acid sequences of SEQ ID NOs: 10 to 15, which are candidates for antimicrobial peptides, were obtained in the same manner as above. The amino acid sequence of SEQ ID NO: 10 is as described above.

[0135] The amino acid sequence of SEQ ID NO: 11 is shown below: MASLTSFSNISEDVLSKVKGGKIIRISKWSYYNTKTKKYYAADNSAIMSTLGHTVVNGWVEHFPY (SEQ ID NO: 11) The polypeptide consists of 64 amino acid residues.

[0136] The amino acid sequence of SEQ ID NO: 12 is shown below: MKNLSSLNQFKTIDAKQQANISGGWLQYLIPVVVSGLNHTDQIMRGWKKGYHRHH (SEQ ID NO: 12) The polypeptide consists of 55 amino acid residues.

[0137] The amino acid sequence of SEQ ID NO: 13 is shown below: MQNFSSIKTEELQAIVGGSSSSLLNTAWRKFGQIAGTVTSAGLDAGWIRAHKYGHGRSH (SEQ ID NO: 13) The polypeptide consists of 58 amino acid residues.

[0138] The amino acid sequence of SEQ ID NO: 14 is shown below: MDKFEKISTSNLEKISGGDLTTKLWSSWGYYLGKKARWNLKHPYVQF (SEQ ID NO: 14) The polypeptide consists of 47 amino acid residues.

[0139] The amino acid sequence of SEQ ID NO: 15 is shown below: MNNLNKFSTLGKSSLSQIEGGSVPTSVYTLGIKILWSAYKHRKTIEKSFNKGFYH (SEQ ID NO: 15) The polypeptide consists of 55 amino acid residues.

[0140] Experimental Example 2: Isolation and Purification of Peptides Purification of Active Ingredients The active ingredient was purified as follows. NITE BP-03198 was inoculated into MRS medium and cultured for 24 hours at 30°C to obtain a culture solution. The resulting culture solution was centrifuged at 6,000 g for 10 minutes to obtain a culture supernatant. The resulting culture supernatant was further filtered through a cellulose acetate membrane with a pore size of 0.22 μm to remove the bacterial cells. Ammonium sulfate was added to 500 mL of the lactic acid bacteria culture supernatant to a final concentration of 80%, and the mixture was stirred overnight at 6°C to precipitate proteins containing antimicrobial peptides. The precipitated proteins were collected by centrifugation at 13,000 g for 60 minutes and redissolved in 50 mL of 50 mM sodium phosphate buffer (pH 5.6; buffer A). The reconstituted protein solution was then applied to an AKTA pure Fraction Collector (GE Healthcare) connected to a Hitrap SP FF 1 mL (GE Healthcare) for fractionation. The mobile phases used were buffer A as mobile phase A and 50 mM sodium phosphate buffer (pH 5.6) containing 1 M NaCl as mobile phase B. The flow rate during fractionation was 1 mL / min. The gradient conditions were an initial mobile phase composition of 0% mobile phase B and a final mobile phase composition of 100% mobile phase B, with a gradient time of 10 minutes. Fractions were collected in 1 mL increments.

[0141] The active fractions were further purified by applying them to an AKTA pure Fraction Collector (GE Healthcare) connected to a 1 mL RESOURCE PRC (GE Healthcare). The mobile phases used were 0.1% formic acid aqueous solution as mobile phase A, and 0.1% formic acid and ethanol as mobile phase B. The flow rate during purification was 1 mL / min. The gradient conditions were an initial mobile phase composition of 0% mobile phase B and a final composition of 100% mobile phase B, with a gradient time of 10 minutes. Fractions were collected in 1 mL increments. The purified active fractions were stored at -30°C. The antibacterial activity of the fractions obtained in each purification step was evaluated as follows.

[0142] Experimental Example 3: Antibacterial Activity Test 1 The antibacterial activity of the peptide was evaluated using the spot-on-lawn method (also called the "growth inhibition method"), in which 10 μL of the test solution was spotted on a lawn of test bacteria. Lactiplantibacillus sakei (NBRC15893) was used as the test bacteria. 4 × 10 test bacteria cultured overnight were placed on a 2% agar-containing MRS plate. ７ The test solution was spotted onto Lactobacillus AOAC agar and layered to a density of 100 CFU / mL. After overnight incubation at 30°C, the size of the growth inhibition zone was evaluated. The larger the growth inhibition zone, the higher the antibacterial activity. The fraction with the highest antibacterial activity was used as the purified active fraction in Experimental Examples 4 and 5 described below.

[0143] Experimental Example 4: Structural Determination of Antimicrobial Peptides 1. 30 μL of an ethanol solution (purified active fraction) suspected to contain the target antimicrobial peptide was evaporated to dryness and then redissolved in 50% methanol to a final volume of 10 μL. The redissolved purified active fraction was measured by liquid chromatography-tandem mass spectrometry (LC / MS / MS) using electrospray ionization as the interface. An Ultimate 3000 RSLC nano (Thermo Fisher Scientific) was used for the liquid chromatograph and autosampler. Mobile phase A consisted of an aqueous solution of 0.5% formic acid and 0.05% trifluoroacetic acid (TFA), and mobile phase B consisted of an acetonitrile solution of 0.5% formic acid and 0.05% TFA. The flow rate during analysis was 400 nL / min. The gradient conditions were an initial mobile phase composition of 5% mobile phase B and a final composition of 90% mobile phase B, with a gradient time of 40 minutes. An NTCC-360 / 100-3-125 (Nikkyo Technos) was used as the analytical column. An Orbitrap Lite (Thermo Fisher Scientific) was used as the mass spectrometer, and MS spectra of the full-length peptide were acquired in full-scan mode. Separately, the peptide mass (theoretical value) calculated in Experimental Example 1 above was compared with the spectral data (measured value) acquired by MS analysis, and the sequence of SEQ ID NO: 1 was estimated as the sequence of the mature polypeptide.

[0144] Experimental Example 5: Structural Determination of Antimicrobial Peptides 2 Trypsin Digestion Digestion of the target peptide was performed according to standard methods. Specifically, 10 μL of the fraction with antimicrobial activity (purified active fraction) was mixed with 10 μL of 2x sample buffer, and the mixture was separated by Tris-Tricin SDS Page (both from Invitrogen). A band with a molecular weight of approximately 3,000 was excised. The 2x sample buffer used was "Novex Tricine SDS Sample Buffer." The gel used for SDS Page was "Novex 10 to 20%, Tricine, 1.0 mm, Mini Protein Gels 15 well." The excised gel was subjected to reductive alkylation using Iodoacetamide (Thermo Fisher Scientific) followed by trypsin digestion at 37°C for 16 hours. Sequencing Grade Modified Trypsin (Promega) was used for digestion. The gel was then shrunk with acetonitrile to extract digested peptides. The extract was desalted and analyzed by MS. MS analysis was performed under the same conditions as in Experimental Example 4 above, except that the gradient time was changed to 30 minutes and the data acquisition method was changed to Data Dependent Acquisition (DDA) mode, and MS and MS / MS spectra were acquired.

[0145] Mascot search The protein frames obtained from genome analysis were converted to FASTA files and loaded into MASCOT (Matrix Science) for each strain. The database was collated with the MS / MS spectral data and MS spectral data obtained from the measurements (tolerance: precursor 30 ppm, fragment 0.8 Da, maximum charge 2+). Antimicrobial peptide candidates were selected based on criteria such as high reliability based on scores calculated using Proteome Discoverer (Thermo Fisher Scientific), peptides that met the criteria (approximately 50 amino acid residues or less), and those that were not nuclear proteins. As a result, an antimicrobial peptide with an internal sequence matching SEQ ID NO: 1 was detected.

[0146] Experimental Example 6: Chemical Synthesis of Peptides Starting from TentaGel S PHB resin, the peptide chain was sequentially elongated using the 9-fluorenylmethoxycarbonyl method (Fmoc method) using a Gyros Protein Technologies automated peptide synthesizer, PurePepChorus, to construct the desired protected peptide resin. The resin was then deprotected and deprotected by trifluoroacetic acid treatment to obtain the crude peptide. The resulting crude peptide was purified using a Shimadzu LC-8A high-performance liquid chromatograph on a YMC-Pack ODS-A (30 x 250 mm) column, yielding the desired product as a lyophilized powder. The product was confirmed to be the polypeptide of SEQ ID NO: 1 by HPLC, amino acid analysis, and mass spectrometry.

[0147] Using the corresponding lactic acid bacteria, peptides were identified in the same manner as in Experimental Examples 1 to 5, and polypeptides of SEQ ID NOs: 5 to 10 were produced in the same manner as in Experimental Example 6.

[0148] Experimental Example 7: Evaluation of antiviral activity Experimental Example 7-1 Activity of the polypeptide of SEQ ID NO: 1 against feline calicivirus CRFK cells (feline kidney-derived cells, ATCC CCL-94 strain) were infected with feline calicivirus (F-9) (Feline calicivirus Strain, F-9 (ATCC VR-782)), EMEM (Minimum Essential Medium Eagle) medium was added, and the cells were cultured at 37°C. After centrifugation at 1000 x g for 15 minutes at 4°C, the supernatant was collected to obtain a test virus suspension. Separately, the polypeptide of SEQ ID NO: 1 synthesized in Experimental Example 6 was diluted with sterile purified water to 100 ppm, 200 ppm, 400 ppm, or 800 ppm to obtain test samples. Sterile purified water was used as a control sample. 0.1 mL of the test virus suspension was added to 0.9 mL of the test sample or control sample and thoroughly stirred. The mixture was left to stand at 25°C for 24 hours to obtain a test solution. 0.1 mL of the suspension was taken out and mixed with 0.9 mL of EMEM to inactivate the test solution, thereby obtaining a reaction stop solution. The reaction stop solution was diluted to 10 mL. ０ A 10-fold serial dilution was prepared with EMEM, the virus infectivity per 0.1 mL of the reaction stop solution was measured by plaque assay, and the virus infectivity per 1 mL of the test solution was calculated.

[0149] The test virus suspension concentration was 4.5 x 10 ８ The results in terms of PFU / mL are shown in the following Table 1. As is clear from the results in Table 1, the polypeptide of SEQ ID NO: 1 exhibited antiviral activity at all concentrations.

[0150] Experimental Example 7-2 Activity of the polypeptide of SEQ ID NO: 5 against feline calicivirus The polypeptide of SEQ ID NO: 5 was used instead of the polypeptide of SEQ ID NO: 1, and the test virus suspension concentration was 3.3 x 10 ８ The test was carried out in the same manner as in Experimental Example 7-1, except that the concentration was changed to PFU / mL.

[0151] The results are shown in the following Table 2. As is clear from the results in Table 2, the polypeptide of SEQ ID NO: 5 exhibited antiviral activity at all concentrations.

[0152] Experimental Example 7-3 Activity of a mixture of the polypeptide of SEQ ID NO: 6 and the polypeptide of SEQ ID NO: 7 against feline calicivirus A mixture containing equimolar amounts of the polypeptide of SEQ ID NO: 6 and the polypeptide of SEQ ID NO: 7 was used instead of the polypeptide of SEQ ID NO: 1, and the test virus suspension concentration was 4.5 x 10 ８ The test was carried out in the same manner as in Experimental Example 7-1, except that the concentration was changed to PFU / mL.

[0153] The results are shown in the following Table 3. As is clear from the results in Table 3, the mixture of the polypeptide of SEQ ID NO: 6 and the polypeptide of SEQ ID NO: 7 exhibited antiviral activity at all concentrations.

[0154] Experimental Example 7-4 Activity of a mixture of the polypeptide of SEQ ID NO: 8 and the polypeptide of SEQ ID NO: 9 against feline calicivirus A mixture containing equimolar amounts of the polypeptide of SEQ ID NO: 8 and the polypeptide of SEQ ID NO: 9 was used instead of the polypeptide of SEQ ID NO: 1, and the test virus suspension concentration was 3.7 x 10 ８ The test was carried out in the same manner as in Experimental Example 7-1, except that the concentration was changed to PFU / mL.

[0155] The results are shown in the following Table 4. As is clear from the results in Table 4, the mixture of the polypeptide of SEQ ID NO: 8 and the polypeptide of SEQ ID NO: 9 exhibited antiviral activity at all concentrations.

[0156] Experimental Example 7-5 Activity of the polypeptide of SEQ ID NO: 10 against feline calicivirus The polypeptide of SEQ ID NO: 10 was used instead of the polypeptide of SEQ ID NO: 1, and the test virus suspension concentration was 3.3 x 10 ８ The test was carried out in the same manner as in Experimental Example 7-1, except that the concentration was changed to PFU / mL.

[0157] The results are shown in the following Table 5. As is clear from the results in Table 5, the polypeptide of SEQ ID NO: 10 exhibited antiviral activity at concentrations of 400 ppm or more.

[0158] Experimental Example 7-6: Activity of the Polypeptide of SEQ ID NO: 1 Against Influenza Virus MDCK cells (canine kidney-derived cells, ATCC CCL-34 strain) were infected with influenza A virus (H3N2) (A / Hong Kong / 8 / 68; TC adapted (ATCC VR-1679)), EMEM medium was added, and the cells were cultured at 34°C. After centrifugation at 1000 x g for 15 minutes at 4°C, the supernatant was collected to obtain a test virus suspension. Separately, the polypeptide of SEQ ID NO: 1 synthesized in Experimental Example 6 was diluted with sterile purified water to 100 ppm, 200 ppm, 400 ppm, or 800 ppm to obtain test samples. Sterile purified water was used as a control sample. 0.1 mL of the test virus suspension was added to 0.9 mL of the test sample or control sample, and the mixture was thoroughly stirred. The mixture was allowed to stand at 25°C for 24 hours to obtain a test solution. 0.1 mL of the mixture was taken out and mixed with 0.9 mL of EMEM to inactivate the test solution, thereby obtaining a reaction stop solution. ０ A 10-fold serial dilution was prepared with EMEM, the virus infectivity per 0.1 mL of the reaction stop solution was measured by plaque assay, and the virus infectivity per 1 mL of the test solution was calculated.

[0159] The test virus suspension concentration was 3.8 x 10 ８ The results in terms of PFU / mL are shown in Table 6 below. As is clear from the results in Table 6, the polypeptide of SEQ ID NO: 1 exhibited antiviral activity at concentrations of 400 ppm or more.

[0160] Experimental Example 7-7 Activity of the polypeptide of SEQ ID NO: 5 against influenza virus The polypeptide of SEQ ID NO: 5 was used instead of the polypeptide of SEQ ID NO: 1, and the test virus suspension concentration was 4.2 × 10 ８ The test was carried out in the same manner as in Experimental Example 7-6, except that the concentration was changed to PFU / mL.

[0161] The results are shown in the following Table 7. As is clear from the results in Table 7, the polypeptide of SEQ ID NO: 5 exhibited antiviral activity at concentrations of 800 ppm or more.

[0162] Experimental Example 7-8: Activity of a mixture of the polypeptide of SEQ ID NO: 6 and the polypeptide of SEQ ID NO: 7 against influenza virus A test was carried out in the same manner as in Experimental Example 7-6, except that a mixture containing equimolar amounts of the polypeptide of SEQ ID NO: 6 and the polypeptide of SEQ ID NO: 7 was used instead of the polypeptide of SEQ ID NO: 1.

[0163] The results are shown in the following Table 8. As is clear from the results in Table 8, the mixture of the polypeptide of SEQ ID NO: 6 and the polypeptide of SEQ ID NO: 7 exhibited antiviral activity at all concentrations.

[0164] Experimental Example 7-9 Activity of a mixture of the polypeptide of SEQ ID NO: 8 and the polypeptide of SEQ ID NO: 9 against influenza virus A mixture containing equimolar amounts of the polypeptide of SEQ ID NO: 8 and the polypeptide of SEQ ID NO: 9 was used instead of the polypeptide of SEQ ID NO: 1, and the test virus suspension concentration was 3.2 x 10 ８ The test was carried out in the same manner as in Experimental Example 7-6, except that the concentration was changed to PFU / mL.

[0165] The results are shown in the following Table 9. As is clear from the results in Table 9, the mixture of the polypeptide of SEQ ID NO: 8 and the polypeptide of SEQ ID NO: 9 exhibited antiviral activity at all concentrations.

[0166] Experimental Example 7-10 Activity of the polypeptide of SEQ ID NO: 10 against influenza virus The polypeptide of SEQ ID NO: 10 was used instead of the polypeptide of SEQ ID NO: 1, and the test virus suspension concentration was 4.2 x 10 ８ The test was carried out in the same manner as in Experimental Example 7-6, except that the concentration was changed to PFU / mL.

[0167] The results are shown in the following Table 10. As is clear from the results in Table 10, the polypeptide of SEQ ID NO: 10 exhibited antiviral activity at all concentrations.

[0168] Experimental Example 8: Antibacterial Activity of Synthetic Peptides The polypeptides of SEQ ID NOS: 1 and 5-10 synthesized in Experimental Example 6 were subjected to an antibacterial activity test in the same manner as in Experimental Example 3 above, and the minimum concentration at which a growth inhibition zone was formed was determined. Test solutions were prepared by dissolving the polypeptides in water to the desired concentrations. As a result, the polypeptide of SEQ ID NOS: 1 exhibited a minimum concentration of 10 μM. The polypeptide of SEQ ID NOS: 5 exhibited a minimum concentration of 1 mM. The mixture of the polypeptides of SEQ ID NOS: 6 and 7 exhibited a minimum concentration of 0.15 μM. The mixture of the polypeptides of SEQ ID NOS: 8 and 9 exhibited a minimum concentration of 0.15 μM. The polypeptide of SEQ ID NOS: 10 exhibited a minimum concentration of 0.1 μM.

[0169] Experimental Example 9: Wide pH Range Stability Test The polypeptide of SEQ ID NO: 1 was evaluated for its antibacterial activity over a wide pH range. First, the polypeptide of SEQ ID NO: 1 was redissolved in the following buffers to prepare a concentration of 100 μM, i.e., 10 times the minimum concentration of Experimental Example 8: sodium phosphate buffer (50 mM, pH 4), 1x phosphate buffer saline (pH 7.4), and ammonium chloride-ammonia buffer (50 mM, pH 10.0). Each test solution prepared at each pH was incubated at 27°C for 24 hours. Next, an antibacterial activity test was performed using Lactiplantibacillus sakei (NBRC15893) as the test strain, using the same method as in Experimental Example 3. The antibacterial activity of each test solution incubated at pH 4, 7, or 10 was measured. A test solution prepared by diluting the target peptide in water was used as a control. Antibacterial activity was evaluated based on spot size (size of growth inhibition zone). As a result, the polypeptide of SEQ ID NO: 1 exhibited spot sizes equivalent to those of the control at all pH values ​​(spot diameters of 80% or more). This indicates that the polypeptide of SEQ ID NO: 1 is stable and has antibacterial activity over a wide pH range. Using a similar method, the polypeptide of SEQ ID NO: 5, a mixture of the polypeptides of SEQ ID NO: 6 and SEQ ID NO: 7, a mixture of the polypeptides of SEQ ID NO: 8 and SEQ ID NO: 9, and the polypeptide of SEQ ID NO: 10 were also found to be stable and have antibacterial activity over a wide pH range.

[0170] Experimental Example 10: Thermal Stability Test The polypeptide of SEQ ID NO: 1 was evaluated for its antibacterial activity after heat treatment. First, the polypeptide of SEQ ID NO: 1 was redissolved in water to a concentration 10 times the minimum concentration in Experimental Example 8, i.e., 100 μM. The prepared test solution was heat-treated at 80°C for 30 minutes, 100°C for 30 minutes, or 121°C for 15 minutes. The heat treatment at 121°C was performed using an autoclave "LSX-700" manufactured by Tomy Seiko Co., Ltd. Next, an antibacterial activity test was performed using Lactiplantibacillus sakei (NBRC15893) as the test bacterium in the same manner as in Experimental Example 3, and the antibacterial activity of the test solution after each heat treatment was measured. A sample solution that was not heat-treated served as a control. Antibacterial activity was evaluated based on the spot size (size of the growth inhibition zone). As a result, the polypeptide of SEQ ID NO: 1 showed a spot size equivalent to that of the control at all temperatures (spot diameter was 80% or more). That is, it was found that the polypeptide of SEQ ID NO: 1 has antibacterial activity even in the temperature range of 80°C to 121°C. By a similar method, it was found that the polypeptide of SEQ ID NO: 5, a mixture of the polypeptides of SEQ ID NO: 6 and SEQ ID NO: 7, a mixture of the polypeptides of SEQ ID NO: 8 and SEQ ID NO: 9, and the polypeptide of SEQ ID NO: 10 also have antibacterial activity in the temperature range of 80°C to 121°C.

[0171] Experimental Example 11: Antibacterial Activity Test 2 The polypeptide of SEQ ID NO: 1, the polypeptide of SEQ ID NO: 5, a mixture of the polypeptide of SEQ ID NO: 6 and the polypeptide of SEQ ID NO: 7, a mixture of the polypeptide of SEQ ID NO: 8 and the polypeptide of SEQ ID NO: 9, and the polypeptide of SEQ ID NO: 10 were tested to determine the types of microorganisms against which they exhibit antibacterial activity. First, each polypeptide or mixture was dissolved in MRS medium to a concentration of 20 μM to 1000 μM. 10 μL of this solution was taken, mixed with various test bacteria and 90 μL of test bacteria culture medium, and cultured for 24 to 48 hours. The test bacteria used were Lactobacillus sakei (lactic acid bacteria), Streptococcus uberis (streptococcus), and Staphylococcus aureus (Staphylococcus aureus). After culture, the number of bacteria was determined by OD ６３０ The OD was measured compared with the culture solution before the culture. ６３０ When an increase in the concentration or visual turbidity is observed, it can be determined that the test bacteria are growing. As a result, the polypeptide of SEQ ID NO: 1, the polypeptide of SEQ ID NO: 5, a mixture of the polypeptide of SEQ ID NO: 6 and the polypeptide of SEQ ID NO: 7, a mixture of the polypeptide of SEQ ID NO: 8 and the polypeptide of SEQ ID NO: 9, and the polypeptide of SEQ ID NO: 10 were found to have an inhibitory effect on the growth of all test bacteria at concentrations of 100 μM or less, thereby demonstrating antibacterial activity. Furthermore, the polypeptide of SEQ ID NO: 1 was found to have an inhibitory effect on the growth of Escherichia coli (E. coli), thereby demonstrating antibacterial activity.

[0172] The present invention provides a composition containing a naturally occurring peptide having antiviral activity against non-enveloped viruses and a method for producing the same. The present invention also provides a composition containing a naturally occurring peptide having antiviral activity against enveloped viruses and a method for producing the same.

Claims

1. An antibacterial and antiviral composition comprising a polypeptide derived from bacteria having antibacterial activity against at least one bacterium selected from the group consisting of Escherichia coli and Staphylococcus aureus, and having antiviral activity against non-enveloped viruses.

2. The antibacterial and antiviral composition of claim 1, wherein the polypeptide further has antiviral activity against enveloped viruses.

3. The antibacterial and antiviral composition according to claim 1 or 2, wherein the polypeptide has an isoelectric point of 9.9 or higher and a molecular weight of 2000 to 6000 Da.

4. An antibacterial and antiviral composition according to any one of claims 1 to 3, wherein the bacterium is a lactic acid bacterium.

5. An antibacterial and antiviral composition according to any one of claims 1 to 4, wherein the polypeptide is: a polypeptide consisting of an amino acid sequence derived from a lactic acid bacterium of the order Lactobacillales; a polypeptide consisting of an amino acid sequence in which one or several amino acid residues have been substituted, deleted, inserted and / or added in an amino acid sequence derived from a lactic acid bacterium of the order Lactobacillales; or a polypeptide consisting of an amino acid sequence having 85% or more sequence identity to an amino acid sequence derived from a lactic acid bacterium of the order Lactobacillales.

6. The antibacterial and antiviral composition according to claim 5, wherein the Lactobacillales lactic acid bacterium is Lactiplantibacillus plantarum, Lactobacillus sp. SC-2001, or Lactococcus lactis.

7. The antibacterial and antiviral composition according to claim 5 or 6, wherein the amino acid sequence derived from a lactic acid bacterium of the order Lactobacillales is any one of the amino acid sequences of SEQ ID NO: 1 or 5 to 10.

8. An antibacterial and antiviral composition according to any one of claims 1 to 7, wherein the polypeptide is a combination of two or more polypeptides.

9. The antibacterial and antiviral composition of any one of claims 1 to 8, further comprising an additive.

10. The antibacterial and antiviral composition according to any one of claims 1 to 9, having antiviral activity against one or more viruses selected from the group consisting of feline calicivirus, norovirus, rotavirus, adenovirus, and poliovirus.

11. The antibacterial and antiviral composition of claim 10, further having antiviral activity against one or more viruses selected from the group consisting of influenza virus, hepatitis B virus, hepatitis C virus, Zika virus, novel coronavirus, SARS coronavirus, MERS coronavirus, varicella-zoster virus, rubella virus, AIDS virus, Ebola virus, and rabies virus.

12. A nucleic acid comprising a base sequence encoding a polypeptide as defined in any one of claims 1 to 7.

13. The nucleic acid according to claim 12, wherein the base sequence encoding the polypeptide is one or more base sequences selected from SEQ ID NOs: 2, 3, and 16 to 27.

14. A vector comprising the nucleic acid according to claim 12 or 13.

15. An in vitro translation template molecule comprising the nucleic acid according to claim 12 or 13.

16. A transformant comprising the vector according to claim 14.

17. A method for producing the antibacterial and antiviral composition according to any one of claims 1 to 11, comprising any of the following steps: (A) culturing a bacterium to produce a polypeptide; (B) synthesizing the polypeptide by chemical synthesis; (C) producing the polypeptide from an organism into which the vector according to claim 14 has been introduced; (D) producing the polypeptide by translation using the in vitro translation template molecule according to claim 15; or (E) producing the polypeptide from the transformant according to claim 16.

18. A method for inhibiting the growth of or removing at least one bacterium selected from the group consisting of Escherichia coli and Staphylococcus aureus and non-enveloped viruses, comprising applying an antibacterial and antiviral composition according to any one of claims 1 to 11 to at least one bacterium selected from the group consisting of Escherichia coli and Staphylococcus aureus and non-enveloped viruses.

19. A method for inhibiting the growth of or removing at least one bacterium selected from the group consisting of Escherichia coli and Staphylococcus aureus, and non-enveloped viruses and enveloped viruses, comprising applying an antibacterial and antiviral composition according to any one of claims 1 to 11 to at least one bacterium selected from the group consisting of Escherichia coli and Staphylococcus aureus, and non-enveloped viruses and enveloped viruses.

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