NOVEL ANTIBACTERIAL PEPTIDE AND USE THEREOF
20230287044 · 2023-09-14
Inventors
- Jae Il KIM (Gwangju, KR)
- Jae Ha RYU (Gwangju, KR)
- Shang Hyeon KIM (Daejeon, KR)
- Sol LEE (Suwon-si, Gyeonggi-do, KR)
Cpc classification
A23K20/147
HUMAN NECESSITIES
A01P1/00
HUMAN NECESSITIES
A61K8/64
HUMAN NECESSITIES
International classification
A61K8/64
HUMAN NECESSITIES
A61Q17/00
HUMAN NECESSITIES
A01P1/00
HUMAN NECESSITIES
A23K20/147
HUMAN NECESSITIES
Abstract
The present invention relates to a novel antibacterial peptide and a composition comprising the same as an active ingredient. The novel antibacterial peptide of the present invention exhibits excellent antibacterial activity against antibiotic-resistant bacteria as well as Gram-positive bacteria and Gram-negative bacteria and is low in cytotoxicity and, as such, can be advantageously utilized as an active ingredient in an antibiotic, a cosmetic composition, a food additive, a feed additive, a biotic pesticide, a quasi-drug product, and the like.
Claims
1. An antibacterial peptide represented by Structural Formula I or Structural Formula II below or a salt thereof: ##STR00013## ##STR00014## in which, N′ is the N-terminus of the antibacterial peptide, C′ is the C-terminus of the antibacterial peptide, the C-terminus is one in which a carboxy group or hydroxy (-OH) of the carboxy group is substituted with an amine group (-NH.sub.2), A consists of the amino acid sequence of (X.sub.1).sub.a-(X.sub.2).sub.b-(X.sub.3).sub.c-(X.sub.4).sub.d, wherein a, b, c and d are each independently 0 or 1, and X.sub.1 to X.sub.4 are each independently any one amino acid selected from the group consisting of Arg, Lys, Asn, Gln, Asp, Val, Leu, Ser, His, Gly and Tyr, and B consists of the amino acid sequence of Trp-(Z.sub.1).sub.e-(Z.sub.2).sub.f-Trp-(Z.sub.3).sub.g-(Z.sub.4).sub.h-Trp, wherein e, f, g and h are each independently 0 or 1, and Z.sub.1 to Z.sub.4 are each independently any one amino acid selected from the group consisting of Val, Leu, Ile, Gly, Ala, Ser, Phe, Tyr, Trp, Lys and His, and C consists of the amino acid sequence of (X.sub.5).sub.i-(X.sub.6).sub.j-(X.sub.7).sub.k-(X.sub.8).sub.1, wherein i, j, k and l are each independently 0 or 1, and X.sub.5 to X.sub.8 are each independently any one amino acid selected from the group consisting of Arg, Lys, Asn, Gln, Asp, Val, Leu, Ser, His, Gly and Tyr, and wherein Trp may be substituted with C.sub.1-6 alkoxy or halogen, or nitrogen (N) in the indole ring of Trp may be modified with sulfur (S), and when an amino acid at the N-terminus is Lys, a C.sub.3 to C.sub.10 fatty acid may be further bound to an amine group, Tyr may be substituted with halogen, and Asn may be glycosylated.
2. The antibacterial peptide or salt thereof according to claim 1, wherein any one of e, f, g and h is 0, and the remainder is 1.
3. The antibacterial peptide or salt thereof according to claim 1, wherein B consists of the amino acid sequence of Trp-Leu-Val-Trp-Ile-Trp (SEQ ID NO: 1).
4. The antibacterial peptide or salt thereof according to claim 3, wherein the antibacterial peptide consists of any one amino acid sequence selected from the group consisting of Trp-Leu-Val-Trp-Ile-Trp-Arg-Arg-Lys-Arg (SEQ ID NO: 2), Trp-Leu-Val-Trp-Ile-Trp-Arg-Arg-Arg (SEQ ID NO: 3), Trp-Leu-Val-Trp-Ile-Trp-Gln-Arg-Arg-Arg (SEQ ID NO: 4), Trp-Leu-Val-Trp-Ile-Trp-Arg-Arg-Gln-Arg (SEQ ID NO: 5), Trp-Leu-Val-Trp-Ile-Trp-Arg-Arg (SEQ ID NO: 6), Lys-Trp-Leu-Val-Trp-Ile-Trp-Arg-Arg-Arg (SEQ ID NO: 7) and Trp-Leu-Val-Trp-Ile-Trp-Arg-Arg-Lys-Arg (SEQ ID NO: 75).
5. The antibacterial peptide or salt thereof according to claim 1, wherein the antibacterial peptide consists of the amino acid sequence of Trp-Val-Val-Trp-Val-Val-Trp-Arg-Arg-Arg (SEQ ID NO: 8).
6. The antibacterial peptide or salt thereof according to claim 1, wherein B consists of the amino acid sequence of Trp-Ile-Trp-Val-Leu-Trp (SEQ ID NO: 9).
7. The antibacterial peptide or salt thereof according to claim 6, wherein the antibacterial peptide consists of any one amino acid sequence selected from the group consisting of Arg-Arg-Arg-Trp-Ile-Trp-Val-Leu-Trp-Lys (SEQ ID NO: 10), Lys-Lys-Lys-Trp-Ile-Trp-Val-Leu-Trp-Lys (SEQ ID NO: 11), Arg-Arg-Trp-Ile-Trp-Val-Leu-Trp-Arg (SEQ ID NO: 12), Asn (glycosylated)-Arg-Arg-Arg-Trp-Ile-Trp-Val-Leu-Trp-Lys (SEQ ID NO: 57), Arg-Arg-Arg-Trp-Ile-Trp-Val-Leu-Trp-Lys-Asn (SEQ ID NO: 79) and Arg-Arg-Arg-Trp-Ile-Trp-Val-Leu-Trp (SEQ ID NO: 84).
8. The antibacterial peptide or salt thereof according to claim 7, wherein Arg at the C-terminus of the antibacterial peptide consisting of the amino acid sequence of Arg-Arg-Trp-Ile-Trp-Val-Leu-Trp-Arg (SEQ ID NO: 12) is an L-form or a D-form.
9. The antibacterial peptide or salt thereof according to claim 1, wherein the antibacterial peptide consists of any one amino acid sequence selected from the group consisting of Trp-Leu-Val-Trp-Lys-Trp-Arg-Arg-Arg (SEQ ID NO: 14), Trp-Leu-Val-Trp-Ser-Trp-Arg-Arg-Arg (SEQ ID NO: 15), Trp-Val-Val-Trp-Val-Trp-Arg-Arg-Arg(SEQ ID NO: 16), Trp-Leu-Trp-Ile-Trp-Arg-Arg-Arg (SEQ ID NO: 17), Arg-Arg-Trp-Val-Trp-Val-Val-Trp-Lys (SEQ ID NO: 18), Arg-Arg-Arg-Trp-Val-Trp-Val-Val-Trp-Lys (SEQ ID NO: 19), Arg-Arg-Arg-Trp-Ile-Trp-Ile-Leu-Trp (SEQ ID NO: 20), Arg-Arg-Arg-Trp-Ile-Trp-Ile-Ile-Trp (SEQ ID NO: 21), Trp-Ile-Ile-Trp-Ile-Trp-Arg-Arg-Arg (SEQ ID NO: 22), Trp-Val-Leu-Trp-Val-Trp-Leu-Arg-Lys-Lys-Arg (SEQ ID NO: 67), Asp-Trp-Leu-Val-Trp-Ile-Trp-Arg-Arg Arg (SEQ ID NO: 56), Trp-Val-Val-Trp-Val-Trp-Lys-Lys-Lys (SEQ ID NO: 77), Trp-Val-Val-Trp-Val-Val-Trp-Lys-Lys-Lys (SEQ ID NO: 78), Trp-Ala-Ala-Trp-Val-Val-Trp-Arg-Arg-Arg (SEQ ID NO: 81), Trp-Val-Val-Trp-Ala-Ala-Trp-Arg-Arg-Arg (SEQ ID NO: 83), Arg-Arg-Arg-Gly-Trp-Val-Trp-Val-Val-Trp-Lys (SEQ ID NO: 98), Arg-Arg-Arg-Trp-Ile-Trp-Trp-Leu-Trp-Lys (SEQ ID NO: 40), Arg-Arg-Lys-Trp-Ile-Trp-Trp-Leu-Trp-Lys (SEQ ID NO: 41), Arg-Arg-Lys-Trp-Ile-Trp-Trp-Leu-Trp-Lys-Lys-Lys (SEQ ID NO: 42), Trp-Leu-Leu-Trp-Val-Leu-Trp-Arg-Lys-Lys-Arg (SEQ ID NO: 69), Trp-Val-Ala-Trp-Ala-Val-Trp-Arg-Arg-Arg (SEQ ID NO: 82), Arg-Arg-Arg-Trp-Ile-Trp-Ala-Ala-Trp-Lys (SEQ ID NO: 85), Arg-Arg-Arg-Trp-Val-Trp-Trp-Val-Trp-Trp (SEQ ID NO: 90), Arg-Arg-Arg-Trp-Val-Trp-Trp-Val-Val-Trp (SEQ ID NO: 91), Arg-Arg-Arg-Trp-Val-Trp-Val-Val-Trp-Trp(SEQ ID NO: 92), Arg-Arg-Arg-Trp-Val-Val-Trp-Val-Trp-Trp (SEQ ID NO: 94), Arg-Arg-Arg-Trp-Val-Val-Trp-Val-Val-Trp (SEQ ID NO: 95) and Arg-Arg-His-His-Trp-Val-Val-Trp-Val-Val-Trp-Lys (SEQ ID NO: 99).
10. An antibacterial peptide or a salt thereof, wherein the antibacterial peptide consists of any one amino acid sequence selected from the group consisting of Trp-Leu-Leu-Trp-Ile-Gly-Leu-Arg-Lys-Lys-Arg (SEQ ID NO: 24), Trp-Leu-Leu-Trp-Ile-Ala-Leu-Arg-Lys-Lys (SEQ ID NO: 25), Lys-Trp-Leu-Leu-Trp-Ile-Gly-Leu-Arg-Lys-Lys-Arg (SEQ ID NO: 26), Trp-Ala-Ala-Trp-Ile-Gly-Leu-Arg-Lys-Lys-Arg (SEQ ID NO: 27), Trp-Leu-Val-Trp-Ile-Gly-Leu-Arg-Lys-Lys-Arg (SEQ ID NO: 28), Trp-Leu-Ile-Trp-Ile-Gly-Leu-Arg-Lys-Lys-Arg (SEQ ID NO: 29), Trp-Leu-Phe-Trp-Ile-Gly-Leu-Arg-Lys-Lys-Arg (SEQ ID NO: 30), Trp-Leu-Tyr-Trp-Ile-Leu-Leu-Arg-Lys-Lys-Arg (SEQ ID NO: 31), Trp-Leu-Val-Trp-Ile-Tyr-Leu-Arg-Lys-Lys-Arg (SEQ ID NO: 32), Trp-Leu-Val-Trp-Val-Tyr-Leu-Arg-Lys-Lys-Arg (SEQ ID NO: 33), Trp-Leu-Val-Trp-Ile-Tyr-Val-Arg-Lys-Lys-Arg (SEQ ID NO: 34), Trp-Leu-Val-Trp-Ile-Tyr-Arg-Lys-Lys-Arg (SEQ ID NO: 35), Trp-Leu-Ile-Trp-Val-Tyr-Arg-Lys-Lys-Arg (SEQ ID NO: 36), Trp-Leu-Val-Trp-Ile-Tyr-Arg-Arg-Arg (SEQ ID NO: 37), Trp-Val-Val-Val-Val-Trp-Arg-Arg-Arg (SEQ ID NO: 38), Trp-Val-Val-His-Val-Val-Trp-Arg-Arg-Arg(SEQ ID NO: 39), Arg-Arg-Arg-His-Val-His-Val-Val-Trp-Lys (SEQ ID NO: 43), Trp-Leu-Leu-Trp-Ile-Gly-Leu-Arg-Lys-Lys-Arg-Tyr (SEQ ID NO: 65), Trp-Leu-Val-Tyr-Val-Trp-Leu-Arg-Lys-Lys-Arg (SEQ ID NO: 68), Ser-Trp-Leu-Leu-Trp-Ile-Gly-Leu-Arg-Lys-Lys-Arg (SEQ ID NO: 55), Trp-Val-Val-His-Val-Val-Trp-Arg-Arg-Arg-Asn (glycosylated) (SEQ ID NO: 59), Arg-Arg-Arg-Trp-Val-Val-Val-Val-Val-Trp (SEQ ID NO: 97), Trp-Leu-Leu-Trp-Ile-Ala-Leu-Arg-Lys-Lys-Arg (SEQ ID NO: 23), Trp-Leu-Leu-Trp-Ile-Gly-Leu-Arg-Lys-Lys (SEQ ID NO: 47), Leu-Leu-Trp-Ile-Ala-Leu-Arg-Lys-Lys-Arg (SEQ ID NO: 49), Leu-Leu-Trp-Ile-Ala-Leu-Lys-Lys-Lys-Lys (SEQ ID NO: 50), Leu-Leu-Trp-Ile-Gly-Leu-Arg-Lys-Lys-Arg (SEQ ID NO: 51), Tyr-Trp-Leu-Leu-Trp-Ile-Gly-Leu-Arg-Lys-Lys-Arg (SEQ ID NO: 64), Trp-Leu-Leu-Trp-Val-Tyr-Val-Arg-Lys-Lys-Arg (SEQ ID NO: 66), Trp-Leu-Trp-Val-Trp-Val-Arg-Lys-Lys-Arg (SEQ ID NO: 70), Val-Trp-Val-Trp-Val-Trp-Val-Arg-Lys-Lys-Arg (SEQ ID NO: 76), Trp-Val-Val-Trp-Val-Tyr-Val-Arg-Lys-Lys-Arg (SEQ ID NO: 71), Trp-Val-Val-Trp-Val-Val-Tyr-Arg-Lys-Lys-Arg (SEQ ID NO: 72), Trp-Leu-Ala-Trp-Leu-Tyr-Leu-Arg-Lys-Lys-Arg (SEQ ID NO: 73), Trp-Leu-Leu-Trp-Leu-Tyr-Ala-Arg-Lys-Lys-Arg (SEQ ID NO: 74), Trp-Ala-Ala-Trp-Ile-Gly-Leu-Arg-Arg-Arg (SEQ ID NO: 80), Trp-Ala-Ala-Trp-Ile-Gly-Leu-Arg-Lys-Lys-Lys (SEQ ID NO: 86), Trp-Ala-Ala-Trp-Ile-Gly-Leu-Arg-Arg-Lys-Lys (SEQ ID NO: 87), Trp-Ala-Ala-Trp-Ile-Gly-Leu-Arg-Lys-Arg-Lys (SEQ ID NO: 88), Trp-Ala-Ala-Trp-Ile-Gly-Leu-Arg-Arg (SEQ ID NO: 89), Arg-Arg-Arg-Trp-Val-Trp-Val-Val-Val-Trp (SEQ ID NO: 91) and Arg-Arg-Arg-Trp-Val-Val-Val-Val-Trp-Trp (SEQ ID NO: 96).
11. The antibacterial peptide or salt thereof according to claim 10, wherein a C.sub.3 to C.sub.10 fatty acid is further bound to the amine group of Lys at the N-terminus of the antibacterial peptide consisting of the amino acid sequence of Lys-Trp-Leu-Leu-Trp-Ile-Gly-Leu-Arg-Lys-Lys-Arg (SEQ ID NO: 26).
12. The antibacterial peptide or salt thereof according to claim 11, wherein the C.sub.3 to C.sub.10 fatty acid is caproic acid or capric acid.
13. The antibacterial peptide or salt thereof according to claim 10, wherein a C.sub.3 to C.sub.10 fatty acid is further bound to the amine group of Lys at the N-terminus of the antibacterial peptide consisting of the amino acid sequence of Trp-Leu-Leu-Trp-Ile-Ala-Leu-Arg-Lys-Lys-Arg (SEQ ID NO: 23), Trp-Leu-Leu-Trp-Ile-Ala-Leu-Arg-Lys-Lys (SEQ ID NO: 25).
14. The antibacterial peptide or salt thereof according to claim 13, wherein the C.sub.3 to C.sub.10 fatty acid is capric acid.
15. The antibacterial peptide or salt thereof according to claim 10, wherein Arg at the C-terminus of the antibacterial peptide consisting of the amino acid sequence of Trp-Leu-Leu-Trp-Ile-Ala-Leu-Arg-Lys-Lys-Arg (SEQ ID NO: 23) is an L-form or a D-form.
16. The antibacterial peptide or salt thereof according to claim 10, wherein Trp of the antibacterial peptide consisting of the amino acid sequence of Trp-Leu-Leu-Trp-Ile-Gly-Leu-Arg-Lys-Lys-Arg (SEQ ID NO: 24) may be substituted with C.sub.1-6 alkoxy or halogen, or nitrogen (N) in the indole ring of Trp may be modified with sulfur (S).
17. An antibiotic comprising the antibacterial peptide or salt thereof according to claim 1 as an active ingredient.
18. A cosmetic composition comprising the antibacterial peptide or salt thereof according to claim 1 as an active ingredient.
19. A food additive comprising the antibacterial peptide or salt thereof according to claim 1 as an active ingredient.
20. An antibacterial feed additive composition comprising the antibacterial peptide or salt thereof according to claim 1 as an active ingredient.
Description
BRIEF DESCRIPTION OF DRAWINGS
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BEST MODE FOR CARRYING OUT THE INVENTION
[0180] Hereinafter, the present invention will be described in more detail.
[0181] In one aspect of the present invention, there is provided an antibacterial peptide containing three Trps or a salt thereof. In this case, the three Trps may be separated by one or two amino acids. In this case, the amino acid present between Trps may be any one amino acid selected from the group consisting of Val, Leu, Ile, Gly, Ala, Ser, Phe, Tyr, Trp, Lys and His. In addition, the antibacterial peptide may further comprise at least one or more amino acids at the N-terminus and/or C-terminus. In this case, the amino acid bound to the N-terminus and/or C-terminus may be any one amino acid selected from the group consisting of Arg, Lys, Asn, Gln, Asp, Val, Leu, Ser, His, Gly and Tyr The antibacterial peptide may consist of 6 to 12 amino acids. In this case, it may be in a form in which —COOH at the C-terminus of the antibacterial peptide is modified with —CONH.sub.2. In addition, a fatty acid may be bound to amino acids constituting the peptide.
[0182] In one aspect of the present invention, there is provided an antibacterial peptide represented by Structural Formula I or Structural Formula II below or a salt thereof:
##STR00003##
##STR00004##
in which, [0183] N′ is the N-terminus of the antibacterial peptide, [0184] C′ is the C-terminus of the antibacterial peptide, [0185] the C-terminus is one in which a carboxy group or hydroxy (—OH) of the carboxy group is substituted with an amine group (-NH.sub.2), [0186] A consists of the amino acid sequence of (X.sub.1).sub.a-(X.sub.2).sub.b-(X.sub.3).sub.c-(X4).sub.d, [0187] wherein a, b, c and d are each independently 0 or 1, and [0188] X.sub.1 to X.sub.4 are each independently any one amino acid selected from the group consisting of Arg, Lys, Asn, Gin, Asp, Val, Leu, Ser, His, Gly and Tyr, and [0189] B consists of the amino acid sequence of Trp-(Z.sub.1).sub.e-(Z.sub.2).sub.f-Trp-(Z.sub.3).sub.g-(Z.sub.4).sub.h-Trp, [0190] wherein e, f, g and h are each independently 0 or 1, and [0191] Z.sub.1 to Z.sub.4 are each independently any one amino acid selected from the group consisting of Val, Leu, 11e, Gly, Ala, Ser, Phe, Tyr, Trp, Lys and His, and [0192] C consists of the amino acid sequence of (X.sub.5).sub.i-(X.sub.6).sub.j-(X.sub.7).sub.k-(X.sub.8).sub.1, [0193] wherein i, j, k and 1 are each independently 0 or 1, and [0194] X.sub.5 to X.sub.8 are each independently any one amino acid selected from the group consisting of Arg, Lys, Asn, Gin, Asp, Val, Leu, Ser, His, Gly and Tyr, and [0195] wherein Trp may be substituted with C.sub.1-6 alkoxy or halogen, or nitrogen (N) in the indole ring of Trp may be modified with sulfur (S), and [0196] when an amino acid at the N-terminus is Lys, a C.sub.3 to C.sub.10 fatty acid may be further bound to an amine group, [0197] Tyr may be substituted with halogen, and [0198] Asn may be glycosylated.
[0199] The above “alkoxy,” unless otherwise indicated, refers to a group having the Formula -O-alkyl, in which an alkyl group as defined above is attached to a parent compound through an oxygen atom. The alkyl portion of the alkoxy group may have 1 to 20 carbon atoms (i.e., C.sub.1-C.sub.20 alkoxy), 1 to 12 carbon atoms (i.e., C.sub.1-C.sub.12 alkoxy), or 1 to 6 carbon atoms (i.e., C.sub.1-C.sub.6 alkoxy). Examples of suitable alkoxy groups include methoxy (—O—CH.sub.3 or —OMe), ethoxy (-OCH.sub.2CH.sub.3 or -OEt), t-butoxy (—O—C(CH.sub.3).sub.3 or -O-tBu), and the like.
[0200] As used herein, the term “halogen,” unless otherwise stated, refers to F, Cl, Br or 1.
[0201] In the present invention, any one of e, f, g and h may be 0, and the remainder may be 1.
[0202] In addition, X.sub.1 to X.sub.8, Z.sub.1 to Z.sub.4 and Trp may be each independently an L-form or D-form amino acid.
[0203] In the present invention, said C′ of the antibacterial peptide may be one in which a hydroxy group (—OH) of a carboxy group is substituted with an amine group (—NH.sub.2).
[0204] The antibacterial peptide according to the present invention may have excellent antibacterial activity against Gram-positive bacteria. In addition, the antibacterial peptide may have excellent antibacterial activity against Gram-negative bacteria.
[0205] In the present invention, the “Gram-positive bacteria” is a type of prokaryotes, and refers to bacteria whose cell walls are stained purple by the Gram staining method. Since the cell wall of Gram-positive bacteria is composed of several layers of peptidoglycan, it appears purple without discoloration even if it is treated with ethanol after staining with a basic dye such as crystal violet. In the present invention, the Gram-positive bacteria may be Staphylococcus aureus, Streptococcus pneumoniae, Enterococcus faecium or Lactobacillus lactis, but is not limited thereto. In the present invention, the Gram-positive bacteria may be bacteria resistant to antibiotics, and may be Gram-positive multi-drug resistant bacteria that are resistant to two or more antibiotics.
[0206] In the present invention, the “Gram-negative bacteria” is a type of prokaryotic cells, and has an outer membrane composed of lipopolysaccharide, lipoprotein, and other complex high molecular substances, instead of having a relatively small amount of peptidoglycan in the cell wall compared to Gram-positive bacteria. After staining with a basic dye such as crystal violet, treatment with ethanol causes discoloration, and counterstaining with a red dye such as safranin results in a red color. The cell wall of Gram-negative bacteria is composed of very thin peptidoglycan and outer membrane compared to Gram-positive bacteria. Peptidoglycan is bound to the lipoprotein connected to the outer membrane and does not contain teichoic acid. The periplasm, a space having a thickness of about 15 nm, is present between the outer and inner membranes of Gram-negative bacteria, and contains a high concentration of protein and maintains a cytoplasm-like state.
[0207] In the present invention, the Gram-negative bacteria may be Acinetobacter baumannii, Escherichia coli, Klebsiella penumoniae, Salmonella spp., Pseudomonas aeruginosa, Haemophilus influenzae, Enterobacter spp. or Yersinia pestis, but is not limited thereto. In the present invention, the Gram-negative bacteria may be bacteria resistant to antibiotics, and may be Gram-negative multi-drug resistant bacteria that are resistant to two or more antibiotics.
[0208] The bacteria resistant to antibiotics may be Acinetobacter baumimnii, Pseudomonas aeruginosa, Enterococcus faecalis or Staphylococcus aureus, but is not limited thereto. The antibiotics may include antibiotics such as aminoglycoside class (aminoglycoside, gentamicin, neomycin, and the like), penicillin class (ampicillin and the like), sulfonamide class, beta-lactam class (beta-lactam, amoxicillin/clavulanic acid, and the like), chloramphenicol class, erythromycin class, florfenicol class, fosfomycin class, kanamycin class, lincomycin class, methicillin class, quinolone class, streptomycin class, tetracycline class, trimethoprim class, and vancomycin class, but are not limited thereto.
[0209] In one embodiment of the antibacterial peptide of the present invention, B may consist of the amino acid sequence of Trp-Leu-Val-Trp-Ile-Trp (SEQ ID NO: 1). In this case, the antibacterial peptide may be a peptide consisting of any one amino acid sequence selected from the group consisting of Trp-Leu-Val-Trp-Ile-Trp-Arg-Arg-Lys-Arg (SEQ ID NO: 2), Trp-Leu-Val-Trp-Ile-Trp-Arg-Arg-Arg (SEQ ID NO: 3), Trp-Leu-Val-Trp-Ile-Trp-Gin-Arg-Arg-Arg (SEQ ID NO: 4), Trp-Leu-Val-Trp-Ile-Trp-Arg-Arg-Gln-Arg (SEQ ID NO: 5), Trp-Leu-Val-Trp-Ile-Trp-Arg-Arg (SEQ ID NO: 6), Lys-Trp-Leu-Val-Trp-Ile-Trp-Arg-Arg-Arg (SEQ ID NO: 7) and Trp-Leu-Val-Trp-Ile-Trp-Arg-Arg-Lys-Arg (SEQ ID NO: 75).
[0210] In one embodiment of the antibacterial peptide of the present invention, the antibacterial peptide may consist of the amino acid sequence of Trp-Val-Val-Trp-Val-Val-Trp-Arg-Arg-Arg (SEQ ID NO: 8).
[0211] In one embodiment of the antibacterial peptide of the present invention, B may consist of the amino acid sequence of Trp-Ile-Trp-Val-Leu-Trp (SEQ ID NO: 9). In this case, the antibacterial peptide may consist of any one amino acid sequence selected from the group consisting of Arg-Arg-Arg-Trp-Ile-Trp-Val-Leu-Trp-Lys (SEQ ID NO: 10), [0212] Lys-Lys-Lys-Trp-Ile-Trp-Val-Leu-Trp-Lys (SEQ ID NO: 11), [0213] Arg-Arg-Trp-Ile-Trp-Val-Leu-Trp-Arg (D-form) (SEQ ID NO: 12), [0214] Arg-Arg-Trp-Ile-Trp-Val-Leu-Trp-Arg (L-form) (SEQ ID NO: 13), Asn [0215] (glycosylated)-Arg-Arg-Arg-Trp-Ile-Trp-Val-Leu-Trp-Lys (SEQ ID NO: 57), [0216] Arg-Arg-Arg-Trp-Ile-Trp-Val-Leu-Trp-Lys-Asn (SEQ ID NO: 79) and [0217] Arg-Arg-Arg-Trp-Ile-Trp-Val-Leu-Trp (SEQ ID NO: 84).
[0218] In one embodiment of the present invention, the antibacterial peptide may consist of any one amino acid sequence selected from the group consisting of [0219] Trp-Leu-Val-Trp-Lys-Trp-Arg-Arg-Arg (SEQ ID NO: 14), [0220] Trp-Leu-Val-Trp-Ser-Trp-Arg-Arg-Arg (SEQ ID NO: 15), [0221] Trp-Val-Val-Trp-Val-Trp-Arg-Arg-Arg (SEQ ID NO: 16), [0222] Trp-Leu-Trp-Ile-Trp-Arg-Arg-Arg (SEQ ID NO: 17), [0223] Arg-Arg-Trp-Val-Trp-Val-Val-Trp-Lys (SEQ ID NO: 18). [0224] Arg-Arg-Arg-Trp-Val-Trp-Val-Val-Trp-Lys (SEQ ID NO: 19), [0225] Arg-Arg-Arg-Trp-Ile-Trp-Ile-Leu-Trp (SEQ ID NO: 20), [0226] Arg-Arg-Arg-Trp-Ile-Trp-Ile-Ile-Trp (SEQ ID NO: 21), and [0227] Trp-Ile-Ile-Trp-Ile-Trp-Arg-Arg-Arg (SEQ ID NO: 22). [0228] Trp-Val-Leu-Trp-Val-Trp-Leu-Arg-Lys-Lys-Arg (SEQ ID NO: 67). [0229] Asp-Trp-Leu-Val-Trp-Ile-Trp-Arg-Arg-Arg (SEQ ID NO: 56), [0230] Trp-Val-Val-Trp-Val-Trp-Lys-Lys-Lys (SEQ ID NO: 77), [0231] Trp-Val-Val-Trp-Val-Val-Trp-Lys-Lys-Lys (SEQ ID NO: 78), [0232] Trp-Ala-Ala-Trp-Val-Val-Trp-Arg-Arg-Arg (SEQ ID NO: 81), [0233] Trp-Val-Val-Trp-Ala-Ala-Trp-Arg-Arg-Arg (SEQ ID NO: 83). [0234] Arg-Arg-Arg-Gly-Trp-Val-Trp-Val-Val-Trp-Lys (SEQ ID NO: 98) and [0235] Arg-Arg-Arg-Trp-Ile-Trp-Trp-Leu-Trp-Lys (SEQ ID NO: 40), [0236] Arg-Arg-Lys-Trp-Ile-Trp-Trp-Leu-Trp-Lys (SEQ ID NO: 41), [0237] Arg-Arg-Lys-Trp-Ile-Trp-Trp-Leu-Trp-Lys-Lys-Lys (SEQ ID NO: 42), [0238] Trp-Leu-Leu-Trp-Val-Leu-Trp-Arg-Lys-Lys-Arg (SEQ ID NO: 69), [0239] Trp-Val-Ala-Trp-Ala-Val-Trp-Arg-Arg-Arg (SEQ ID NO: 82), [0240] Arg-Arg-Arg-Trp-Ile-Trp-Ala-Ala-Trp-Lys (SEQ ID NO: 85), [0241] Arg-Arg-Arg-Trp-Val-Trp-Trp-Val-Trp-Trp (SEQ ID NO: 90). [0242] Arg-Arg-Arg-Trp-Val-Trp-Trp-Val-Val-Trp (SEQ ID NO: 91), [0243] Arg-Arg-Arg-Trp-Val-Trp-Val-Val-Trp-Trp (SEQ ID NO: 92), [0244] Arg-Arg-Arg-Trp-Val-Val-Trp-Val-Trp-Trp (SEQ ID NO: 94), [0245] Arg-Arg-Arg-Trp-Val-Val-Trp-Val-Val-Trp (SEQ ID NO: 95) and [0246] Arg-Arg-His-His-Trp-Val-Val-Trp-Val-Val-Trp-Lys (SEQ ID NO: 99).
[0247] In another aspect of the present invention, there is provided an antibacterial peptide consisting of any one amino acid sequence selected from the group consisting of [0248] Trp-Leu-Leu-Trp-Ile-Gly-Leu-Arg-Lys-Lys-Arg (SEQ ID NO: 24). [0249] Trp-Leu-Leu-Trp-Ile-Ala-Leu-Arg-Lys-Lys (SEQ ID NO: 25), [0250] Lys-Trp-Leu-Leu-Trp-Ile-Gly-Leu-Arg-Lys-Lys-Arg (SEQ ID NO: 48), [0251] Trp-Ala-Ala-Trp-Ile-Gly-Leu-Arg-Lys-Lys-Arg (SEQ ID NO: 27), [0252] Trp-Leu-Val-Trp-Ile-Gly-Leu-Arg-Lys-Lys-Arg (SEQ ID NO: 28), [0253] Trp-Leu-Ile-Trp-Ile-Gly-Leu-Arg-Lys-Lys-Arg (SEQ ID NO: 29), [0254] Trp-Leu-Phe-Trp-Ile-Gly-Leu-Arg-Lys-Lys-Arg (SEQ ID NO: 30), [0255] Trp-Leu-Tyr-Trp-Ile-Leu-Leu-Arg-Lys-Lys-Arg (SEQ ID NO: 31), [0256] Trp-Leu-Val-Trp-Ile-Tyr-Leu-Arg-Lys-Lys-Arg (SEQ ID NO: 32), [0257] Trp-Leu-Val-Trp-Val-Tyr-Leu-Arg-Lys-Lys-Arg (SEQ ID NO: 33), [0258] Trp-Leu-Val-Trp-Ile-Tyr-Val-Arg-Lys-Lys-Arg (SEQ ID NO: 34), [0259] Trp-Leu-Val-Trp-Ile-Tyr-Arg-Lys-Lys-Arg (SEQ ID NO: 35), [0260] Trp-Leu-Ile-Trp-Val-Tyr-Arg-Lys-Lvs-Arg (SEQ ID NO: 36), [0261] Trp-Leu-Val-Trp-Ile-Tyr-Arg-Arg-Arg (SEQ ID NO: 37), [0262] Trp-Val-Val-Val-Val-Trp-Arg-Arg-Arg (SEQ ID NO: 38). [0263] Trp-Val-Val-His-Val-Val-Trp-Arg-Arg-Arg (SEQ ID NO: 39), [0264] Arg-Arg-Arg-His-Val-His-Val-Val-Trp-Lys (SEQ ID NO: 43), [0265] Trp-Leu-Leu-Trp-Ile-Gly-Leu-Arg-Lys-Lys-Arg-Tyr (SEQ ID NO: 65), [0266] Trp-Leu-Val-Tyr-Val-Trp-Leu-Arg-Lys-Lys-Arg (SEQ ID NO: 68), [0267] Ser-Trp-Leu-Leu-Trp-Ile-Gly-Leu-Arg-Lys-Lys-Arg (SEQ ID NO: 55). [0268] Trp-Val-Val-His-Val-Val-Trp-Arg-Arg-Arg-Asn (glycosylated) (SEQ ID NO: 59), [0269] Arg-Arg-Arg-Trp-Val-Val-Val-Val-Val-Trp (SEQ ID NO: 97), [0270] Trp-Leu-Leu-Trp-Ile-Ala-Leu-Arg-Lys-Lys-Arg (SEQ ID NO: 23), Lys(caproic acid (C.sub.6) [0271] attached)-Trp-Leu-Leu-Trp-Ile-Gly-Leu-Arg-Lys-Lys-Arg (SEQ ID NO: 26), [0272] Trp-Leu-Leu-Trp-Ile-Gly-Leu-Arg-Lys-Lys (SEQ ID NO: 47), [0273] Leu-Leu-Trp-lle-Ala-Leu-Arg-Lys-Lys-Arg (SEQ ID NO: 49), [0274] Leu-Leu-Trp-Ile-Ala-Leu-Lys-Lys-Lys-Lys (SEQ ID NO: 50), [0275] Leu-Leu-Trp-Ile-Gly-Leu-Arg-Lys-Lys-Arg (SEQ ID NO: 51), [0276] Tyr-Trp-Leu-Leu-Trp-Ile-Gly-Leu-Arg-Lys-Lys-Arg (SEQ ID NO: 64), Trp Leu Leu Trp Val [0277] Tyr Val Arg Lys Lys Arg (SEQ ID NO: 66), Trp-Leu-Trp-Val-Trp-Val-Arg-Lys-Lys-Arg [0278] (SEQ ID NO: 70), Val-Trp-Val-Trp-Val-Trp-Val-Arg-Lys-Lys-Arg (SEQ ID NO: 76), [0279] Trp-Val-Val-Trp-Val-Tyr-Val-Arg-Lys-Lys-Arg (SEQ ID NO: 71), [0280] Trp-Val-Val-Trp-Val-Val-Tyr-Arg-Lys-Lys-Arg (SEQ ID NO: 72), [0281] Trp-Leu-Ala-Trp-Leu-Tyr-Leu-Arg-Lys-Lys-Arg (SEQ ID NO: 73), [0282] Trp-Leu-Leu-Trp-Leu-Tyr-Ala-Arg-Lys-Lys-Arg (SEQ ID NO: 74), [0283] Trp-Ala-Ala-Trp-Ile-Gly-Leu-Arg-Arg-Arg (SEQ ID NO: 80), [0284] Trp-Ala-Ala-Trp-Ile-Gly-Leu-Arg-Lys-Lys-Lys (SEQ ID NO: 86), [0285] Trp-Ala-Ala-Trp-Ile-Gly-Leu-Arg-Arg-Lys-Lys (SEQ ID NO: 87). [0286] Trp-Ala-Ala-Trp-Ile-Gly-Leu-Arg-Lys-Arg-Lys (SEQ ID NO: 88), [0287] Trp-Ala-Ala-Trp-Ile-Gly-Leu-Arg-Arg (SEQ ID NO: 89), [0288] Arg-Arg-Arg-Trp-Val-Trp-Val-Val-Val-Trp (SEQ ID NO: 93) and [0289] Arg-Arg-Arg-Trp-Val-Val-Val-Val-Trp-Trp (SEQ ID NO: 96).
[0290] In one embodiment of the present invention, the amino acid of the antibacterial peptide may be an L-form or a D-form. Specifically, the amino acid that may have an L-form in the antibacterial peptide is represented by an L-form, but the form of the amino acid represented by an L-form may include a D-form depending on the processing environment. Thus, the form of the amino acid is not limited by the indication.
[0291] In one embodiment of the present invention, each amino acid of the antibacterial peptide may be a modified derivative. Specifically, the tryptophan (Trp) may be methoxy-tryptophan (Wm) represented by Formula 1 below, may be benzothienyl-alanine (Ws) represented by Formula 2 below, and may be fluoro-tryptophan (Wf) represented by Formula 3 below.
##STR00005##
##STR00006##
##STR00007##
[0292] In addition, in one embodiment of the present invention, the tyrosine (Tyr) may be monoiodotyrosine represented by Formula 4 below.
##STR00008##
[0293] In addition, in one embodiment of the present invention, hydrogen, C.sub.1-10 alkyl or C.sub.1-C.sub.20 fatty acid may be bound to the NH.sub.3.sup.+ terminus of the lysine (Lys), and in one embodiment, it may be caproic acid (C.sub.6 fatty acid) or capric acid (C.sub.10 fatty acid), but is not limited thereto. On the other hand, in one embodiment of the present invention, the lysine of the antibacterial peptide may form multiple antigenic peptide conjugation (MAP conjugation). The multiple antigenic peptide (MAP) is an artificially branched peptide, and lysine moieties can be used as a scaffolding core to support the formation of 8 or less branches having variable or identical peptide sequences. In one embodiment of the present invention, the lysine of the antibacterial peptide formed a branch with the peptide containing tryptophan.
[0294] In addition, in one embodiment of the present invention, the asparagine (Asn) may be a glycosylated asparagine.
[0295] In one embodiment of the present invention, the antibacterial peptide consisting of the amino acid sequence of Lys-Trp-Leu-Leu-Trp-Ile-Gly-Leu-Arg-Lys-Lys-Arg may be one in which a C.sub.1 to C.sub.20 fatty acid is further bound to the amine group of Lys at the N-terminus. Specifically, it may be one in which a C.sub.3 to C.sub.10 fatty acid is further bound. The fatty acid may be caproic acid or capric acid. Specifically, it may be caproic acid, but is not limited thereto.
[0296] The antibacterial peptide consisting of the amino acid sequence of Trp-Leu-Leu-Trp-Ile-Ala-Leu-Arg-Lys-Lys-Arg-Trp-Leu-Leu-Trp-Ile-Ala-Leu-Arg-Lys-Lys may be one in which a C.sub.3 to C.sub.10 fatty acid is further bound to the amine group of Lys at the N-terminus. Specifically, it may be one in which a C.sub.3 to C.sub.10 fatty acid is further bound. The fatty acid may be caproic acid or capric acid. Specifically, it may be caproic acid, but is not limited thereto.
[0297] Arg at the C-terminus of the antibacterial peptide consisting of the amino acid sequence of Trp-Leu-Leu-Trp-Ile-Ala-Leu-Arg-Lys-Lys-Arg may be an L-form or a D-form.
[0298] Trp of the antibacterial peptide consisting of the amino acid sequence of Trp-Leu-Leu-Trp-Ile-Gly-Leu-Arg-Lys-Lys-Arg may be substituted with C.sub.1-6 alkoxy or halogen, or nitrogen (N) in the indole ring of Trp may be modified with sulfur (S).
[0299] The salt should have low toxicity to humans and should not have any negative effect on the biological activity and physicochemical properties of the parent compound. For example, the salt may be an acid addition salt formed by a pharmaceutically acceptable free acid.
[0300] The free acid may be an inorganic acid or an organic acid, wherein the inorganic acid may be hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, perchloric acid, bromic acid, and the like, and the organic acid may be acetic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, fumaric acid, maleic acid, malonic acid, phthalic acid, succinic acid, lactic acid, citric acid, gluconic acid, tartaric acid, salicylic acid, malic acid, oxalic acid, benzoic acid, embonic acid, aspartic acid, glutamic acid, and the like.
[0301] The acid addition salt can be prepared by a conventional method, for example, by dissolving the peptide in an excess aqueous acid solution, and precipitating the salt using a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile..
[0302] In addition, the salt may be an alkali metal salt (sodium salt, etc.) or an alkaline earth metal salt (potassium salt, etc.). The alkali metal salt or alkaline earth metal salt can be obtained, for example, by dissolving the peptide in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the undissolved compound salt, and then evaporating and drying the filtrate.
[0303] The antibacterial peptide of the present invention may exhibit excellent antibacterial activity compared to commercially available antibiotics. Specifically, in one embodiment of the present invention, when treated with carbapenem, an antibiotic known as an indicator of whether multi-drug resistant bacteria is determined, the number of strains having KPC (Klebsiella pneumoniae carbapenemase) and NDM (New Delhi metallo-beta-lactamase), which are resistance enzymes, was increased, but it was confirmed that the antibacterial peptide of the present invention exhibits excellent antibacterial activity against carbapenem-resistant Acinetobacter baumannii and Pseudomonas aeruginosa. In addition, in one embodiment of the present invention, the antibiotic tetracycline has insufficient sterilization ability and the target protein tends to be mutated, but it was confirmed that the antibacterial peptide of the present invention exhibits antibacterial activity against tetracycline-resistant Acinetobacter baumannii, and has excellent sterilization ability, and the tendency to mutate the target protein is reduced. In addition, in one embodiment of the present invention, vancomycin, an antibiotic against Gram-positive bacteria, has a problem in that resistant strains occur, but it was confirmed that the antibacterial peptide of the present invention exhibits antibacterial activity against vancomycin-resistant Enterococcus faecalis. In one embodiment of the present invention, bezlotoxumab, known as a novel antibody-based antibiotic, is used exclusively for preventing reinfection of Clostridium Difficile (C. Difficile), but the antibacterial peptide of the present invention has the advantage that the spectrum of pathogens, against which the antibacterial peptide of the present invention exhibits antibacterial activity, is wide.
[0304] In one embodiment of the present invention, it was confirmed that the antibacterial peptide of the present invention exhibits antibacterial activity against Gram-positive bacteria and Gram-negative bacteria, but daptomycin and gramicidin, which are commercially available antibiotics, exhibit antibacterial activity only against Gram-positive bacteria. In addition, protegrin, a peptide that partially satisfies the minimum growth inhibitory concentration for Gram-positive bacteria and Gram-negative bacteria, has a high cytotoxicity problem.
[0305] On the other hand, colistin, which is a multi-drug resistant Gram-negative bacteria antibiotic, causes fatal kidney damage, it is difficult to administer in combination with other drugs, and there are problems that resistant bacteria occur. Accordingly, antibiotics such as SPR206 and SPR741 have been developed. Specifically, the SPR206 increased the antibacterial activity of colistin by 3 to 4 times, and reduced renal toxicity by ⅓. However, there is a problem in that resistant bacteria are shared by colistin. The SPR741 eliminates the renal toxicity of colistin, but has no antibacterial activity by itself, so there is a problem that it must be administered in combination with antibiotics of Gram-positive bacteria. On the other hand, the antibacterial peptide of the present invention not only exhibits antibacterial activity against Gram-positive bacteria and Gram-negative bacteria, but also exhibits antibacterial activity against colistin-resistant bacteria, and also inhibits the generation of resistant strains.
[0306] The antibacterial peptide of the present invention may have low cytotoxicity to human-derived cells.
[0307] In another aspect of the present invention, there is provided an antibiotic comprising the antibacterial peptide as an active ingredient.
[0308] The antibacterial peptide of the present invention may be administered orally or parenterally during clinical administration, and may be used in the form of general pharmaceutical preparations. Formulations for oral administration may take various forms such as syrups, tablets, capsules, creams and lozenges. Parenteral administration may refer to administration via a route other than oral administration, such as rectal, intravenous, peritoneal, muscle, arterial, transdermal, nasal, inhalation, ocular, and subcutaneous administration. When the antibacterial peptide of the present invention is used as a pharmaceutical, it may further include one or more active ingredients exhibiting the same or similar function.
[0309] That is, the antibacterial peptide of the present invention may be administered in a variety of formulations for parenteral administration. When it is formulated, it is prepared using a commonly used diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, a surfactant, and the like. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. As the non-aqueous solvents and suspending agents, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. As the base of the suppositories, Witepsol, Macrogol, Tween 61, cacao butter, laurin, glycerogelatin, and the like may be used.
[0310] In addition, the antibacterial peptide of the present invention may be used by mixing with various pharmaceutically acceptable carriers such as physiological saline or organic solvents. In addition, carbohydrates such as glucose, sucrose or dextran, for increasing stability or absorbability, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers may be used as pharmaceuticals.
[0311] The effective dose of the antibacterial peptide of the present invention is 0.01 .Math.g/kg to 2 mg/kg, specifically 0.5 mg/kg to 1 mg/kg, and may be administered once to 3 times a day.
[0312] In the antibiotic of the present invention, the total effective amount of the novel peptide of the present invention may be administered to a patient in a single dose in the form of a bolus or by infusion for a relatively short period of time, and multiple doses may be administered by a fractionated treatment protocol in which they are administered over a long period of time. The effective dose of the patient is determined by considering various factors such as the age and health status of the patient as well as the route of administration and the frequency of treatments. Considering this point, one of ordinary skill in the art will be able to determine an appropriate effective dose according to the specific use of the novel peptide of the present invention as an antibiotic.
[0313] In another aspect of the present invention, there is provided an antibacterial cosmetic composition comprising the antibacterial peptide as an active ingredient.
[0314] The cosmetic composition of the present invention includes components commonly used in cosmetic compositions in addition to the antibacterial peptide, for example, conventional adjuvants such as antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances, and carriers.
[0315] In the cosmetic composition of the present invention, the antibacterial peptide of the present invention may be added in an amount of 0.1 to 50 % by weight, preferably 1 to 10% by weight, in a cosmetic composition usually contained therein.
[0316] The cosmetic composition of the present invention may be prepared in any formulation conventionally prepared in the art. It may be formulated as, for example, a solution, a suspension, an emulsion, a paste, a gel, a cream, a lotion, a powder, a soap, a surfactant-containing cleansing agent, an oil, a powder foundation, an emulsion foundation, a wax foundation and a spray, etc., but is not limited thereto. More specifically, it may be prepared in the form of a skin sotfner (skin toner), a nourishing lotion (milk lotion), a nourishing cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray, or a powder..
[0317] When the formulation of the present invention is a paste, a cream or a gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, and the like may be used as a carrier component. When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, a propellant such as chlorofluorohydrocarbon, propane/butane or dimethyl ether may be further included. When the formulation of the present invention is a solution or an emulsion, a solvent, a solubilizer or an emulsifier is used as a carrier component, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylglycol oil, glycerol fatty esters, fatty acid esters of polyethylene glycol or sorbitan. When the formulation of the present invention is a suspension, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, and microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tragacanth may be used as a carrier component. When the formulation of the present invention is a surfactant-containing cleansing agent, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide ether sulfate, alkylamidobetaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative or ethoxylated glycerol fatty acid ester and the like may be used as a carrier component.
[0318] In another aspect of the present invention, there is provided an antibacterial food additive comprising the antibacterial peptide as an active ingredient.
[0319] When the antibacterial peptide of the present invention is used as a food additive, the antibacterial peptide may be added as it is or used together with other food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined according to the purpose of its use In general, the peptide of the present invention is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, based on the raw material. However, in the case of long-term administration, the amount may be below the above range, and since there is no problem in terms of stability, the active ingredient may be used in an amount above the above range.
[0320] The type of the food is not particularly limited. Examples of foods to which the above substances can be added include meat, sausage, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages and vitamin complexes, and the like, and include all foods in a conventional sense.
[0321] In another aspect of the present invention, there is provided an antibacterial feed additive comprising the antibacterial peptide as an active ingredient.
[0322] The feed composition of the present invention replaces the existing antibiotics and inhibits the growth of harmful food pathogens, thereby improving the health status of the animal body, improving the weight gain and meat quality of livestock, and increasing milk production and immunity. The feed composition of the present invention may be prepared in the form of a fermented feed, a compound feed, a pellet form, a silage, and the like
[0323] The fermented feed may be prepared by fermenting organic matter by adding various microbial populations or enzymes other than the peptide of the present invention, and the compounded feed may be prepared by mixing several types of general feed with the peptide of the present invention. The feed in the form of pellets may be prepared by applying heat and pressure to the compounded feed, etc. in a pellet machine, and the silage may be prepared by fermenting green fodder with microorganisms. The wet fermented feed may be prepared by collecting and transporting organic matter such as food waste, mixing excipients for sterilization process and moisture control in a certain ratio, and then fermenting it at a temperature suitable for fermentation for more than 24 hours so that the moisture content is about 70%. The dry fermented feed may be prepared by applying an additional drying process to the wet fermented feed so that the moisture content is about 30% to 40%.
[0324] In another aspect of the present invention, there is provided an antibacterial biotic pesticide comprising the antibacterial peptide as an active ingredient.
[0325] In another aspect of the present invention, there is provided an preservative composition comprising the antibacterial peptide as an active ingredient.
[0326] The preservative composition includes a cosmetic preservative or a pharmaceutical preservative. The food preservatives, cosmetic preservatives and pharmaceutical preservatives are additives used to prevent deterioration, decay, discoloration and chemical changes of pharmaceuticals, and include a sterilizing agent and an antioxidant, and also include functional antibiotics that inhibit the proliferation of microorganisms such as bacteria, mold, yeast, and the like, thereby inhibiting the growth or sterilization of spoilage microorganisms in food and pharmaceuticals. Under ideal conditions for such a preservative composition, it should be non-toxic and should be effective even in a small amount.
[0327] In another aspect of the present invention, there is provided an antibacterial quasi-drug product composition comprising the antibacterial peptide as an active ingredient.
[0328] When the antibacterial peptide is used as a quasi-drug product additive, the antibacterial peptide may be added as it is or used together with other quasi-drug products or quasi-drug product ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined according to the purpose of its use. The quasi-drug product composition of the present invention may be a disinfectant cleaner, a shower foam, a gargrin, a wet tissue, a detergent soap, a handwash, a humidifier filler, a mask, an ointment, a patch or a filter filler, but is not limited thereto.
[0329] In another aspect of the present invention, there is provided an antibacterial method comprising administering a pharmaceutically effective amount of the antibacterial peptide to a subject. The subject may be a mammal other than a human, but is not limited thereto.
MODE FOR CARRYING OUT THE INVENTION
[0330] Hereinafter, the present invention will be described in detail by way of Examples. However, the following examples are only for illustrating the present invention, and the present invention is not limited to the following examples.
Example 1. Preparation of Antibacterial Peptides
[0331] The antibacterial peptides of the present invention were prepared by the preparation method shown in
[0332] Specifically, Rink-amide-MBHA resin was swelled with dichloromethane and then deprotected with piperidine. D,D-dimethylformaide, diisopropylcarbodiimide and 1-hydroxybenzotriazole solutions were used to connect Fmoc-protected amino acids while changing, and then cleaved with thioanisole and TFA to prepare the antibacterial peptide of the present invention.
[0333] The prepared antibacterial peptides were purified with acetonitrile using HPLC C18 colume.
Experimental Example 1. Analysis of Antibacterial Peptides
Experimental Example 1.1. Analysis of Solubility of Antibacterial Peptides
[0334] The solubility of the antibacterial peptides of the present invention was measured, and the results are shown in Table 1 below.
TABLE-US-00001 Component Solubility in solution (.Math.M) H.sub.2O PBS CAMHB KSH29 >2000 100 50 KSH30 >2000 25 50 KSH31 >2000 100 25 KSH32 >2000 25 50 KSH35 >2000 >200 25 KSH36 >2000 >200 100 KSH40 >2000 100 100 KSH42 >2000 100 100 KSH43 >2000 >200 >25 KSH44 >2000 100 100 KSH45 >2000 >200 50 KSH46 >2000 >200 200 Colistin >2000 >200 >200 Vancomycin >2000 >200 >200
[0335] The concentration of the peptide was 25 to 200 .Math.M, and was observed for 12 hours at room temperature.
[0336] In addition, the results obtained by analyzing the stability of KSH42 and KSH43 of the antibacterial peptides of the present invention for pronase are shown in
[0337] In addition, the results obtained by comparing the degree of artificial cell membrane leakage according to treatment with KSH29, KSH42 and KSH43 are shown in
Experimental Example 1.2. Measurement of Molecular Weight and Purity of Antibacterial Peptides
[0338] The purity and molecular weight of the antibacterial peptides of the present invention were measured, respectively, and the results are shown in Table 2 below and
TABLE-US-00002 Component Amino acid sequence (5′ to 3′) Molecular weight Purity Expected value Measured value KSH 1 WLLWIALRKKR 1481.9 1492.3 97.219 KSH 3 wllwialckkr 1481.9 1482.2 99.454 KSH 4 wllwialrkkr 1636.0 1636.3 99.231 KSH 5 wllwiglrkkr 1467.9 1467.2 95.276 KSH 6 wllwialrkk 1325.7 1325.0 99.503 KSH 7 wllwialrkk 1479.9 1479.7 99.173 KSH 8 wllwiglrkk 1311.7 1311.5 99.187 KSH 9 kwllwiglrkkr 1694.2 1693.9 97.418 KSH 10 kwllwiglrkkr 1750.3 1750.0 98.472 KSH 11 llwialrkkr 1295.7 1295.2 98.990 KSH 12 llwialkkkk 1239.6 1239.5 98.512 KSH 13 llwiglrkkr 1281.6 1281.3 98.373 KSH 15 w.sub.Mllwiglrkkr 1495.8 1495.4 99.340 KSH 16 w.sub.Sllwiglrkkr 1484.9 1484.2 98.220 KSH 18 w.sub.Fllwigirkkr 1485.9 1485.5 98.034 KSH 19 Swllwiglrkkr 1555.0 1555.1 99.001 KSH 20 waawiglrkkr 1383.7 1383.4 95.894 IKSH 5-1 Ywllwiglrkkr 1757.9 1757.1 98.325 IKSH 5-2 wllwiglrkkrY 1757.9 1757.1 98.169 KSH YL wlywillrkkr 1574.0 1573.0 99.077 KSH VY wlvwiylrkkr 1560.0 1559.4 99.071 KSH VY2 wlvwvylrkkr 1545.9 1546.3 98.687 KSH VY3 wlvwiyvrkkr 1545.9 1545.8 98.569 KSH VY4 wlvwiyrkkr 1446.8 1446.9 97.749 KSH 27 wliwyrkkr 1446.8 1445.7 98.415 KSH 28 wlvwiwrrkr 1497.9 1497.0 97.448 XSH 2 WLLWVYVRKKR 1545.9 1546.8 77.714 LSH 28 WLVWIWRRKR 1469.9 1470.0 96.827 LSH 1 WVLWVWLRKKR 1569.0 1570.5 95.680 LSH 2 WLVYVWLRKKR 1545.9 1547.3 98.361 LSH 3 WLLWVLWRKKR 1583.0 1583.9 95.542 LSH 5 WLWVWVRKKR 1455.8 1456.5 98.155 LSH 99 VWVWVWVRKKR 1540.9 1540.3 93.474 LSH 6 WVVWVYVRKKR 1517.9 1520.2 98.372 LSH 7 WVVWVVYRKKR 1517.9 1520.0 97.509 LSH 8 WLAWLYLRKKR 1531.9 1534.1 98.728 LSH 9 WLLWLYARKKR 1531.9 1534.0 97.372 KSH 29 wlvwiwrrr 1369.7 1369.7 98.784 KSH 30 wlvwiwqrrr 1497.8 1497.6 96.018 KSH 31 wlvwiwrrqr 1497.8 1497.3 98.882 KSH 32 wlvwiwrr 1213.5 1213.5 98.961 KSH 33 wlvwiyrrr 1346.6 1346.3 98.869 KSH 35 kwlvwiwrr 1497.8 1497.8 99.323 KSH 36 dwlvwiwrrr 1484.8 1483.9 96.340 KSH 37 wlvwkwrrr 1384.7 1384.1 96.120 KSH 39 wlvwswrrr 1343.6 1342.9 95.095 KSH 40 wvvwwvrrr 1341.6 1340.7 96.948 KSH 41 wlwiwrrr 1270.5 1269.5 99.478 KSH 42 wvvwvvwrrr 1440.8 1440.1 97.988 KSH 43 rrrwiwvlwk 1497.8 1497.2 98.707 KSH 44 wvvvvrrr 1254.5 1254.2 97.954 KSH 45 wvvhvvwrrr 1391.7 1390.8 99.616 KSH 46 kkkwiwvlwk 1413.8 1412.8 98.596 KSH 47 Nrrrwiwvlwk 1815.5 1816.1 93.431 KSH 48 rrrwiwvlwkN 1815.5 1816.5 99.225 KSH 49 wvvhvvwrrrN 1709.4 1710.0 98.865 KSH 50 rrrwiwwlwk 1584.9 1584.9 98.916 KSH 51 rrkwiwwlwk 1556.9 1557.1 98.814 KSH 52 rrkwiwwlwkkk 1813.3 1813.2 98.896 KSH 54 rrwiwvlwr 1369.7 1369.9 99.074 KSH 55 rrwiwvlwR 1369.7 1369.8 99.331 KSH 56 rrwvwvvwk 1313.6 1313.4 97.172 KSH 57 rrrwvwvvwk 1427.7 1427.6 98.509 KSH 58 rrrhvhvvwk 1371.7 1371.7 97.610 KSH 59 rrrwiwilw 1383.7 1382.8 98.520 KSH 60 rrrwiwiiw 1383.7 1383.4 96.978 KSH 61 wiiwiwrrr 1383.7 1383.2 98.758 KSH 62 Rrrwiwvlwk 1497.8 1498.2 98.804 KSH 63 rRrwiwvlwk 1497.8 1498.2 98.713 KSH 64 rrRwiwvlwk 1497.8 1498.3 98.759 KSH 66 wvvwvwkkk 1257.6 1257.6 97.7 KSH 67 wvvwvvwkkk 1356.7 1356.7 96.7 KSH 68 rrrwiwvlwkn 1611.9 1612.3 96.7 KSH 69 waawiglrrr 1283.5 1284.3 96.5 KSH 70 waawvvvwrrr 1384.6 1385.1 98.6 KSH 71 wvawavwrrr 1384.6 1385.1 97.7 KSH 72 wvvwaawrrr 1384.6 1385.2 95.2 KSH 73 rrrwiwvlw 1369.7 1369.9 96.7 KSH 74 rrrwiwaawk 1427.7 1428.2 97.7 KSH 75 waawiglrkkk 1355.7 1356.6 97.6 KSH 76 waawiglrrkk 1383.7 1384.1 97.4 KSH 77 waawiglrkrk 1383.7 1383.9 98.1 KSH 78 waawiglrr 1127.3 1127.9 95.3 KSH 79 rrrwvwwvww 1614.9 1614.8 99.0 KSH 80 rrrwvwwvvw 1527.8 1527.7 99.1 KSH 81 rrrwvwvvww 1527.8 1527.0 98.9 KSH 82 rrrwvwvvvw 1440.7 1440.9 98.2 KSH 83 rrrwvvwvww 1527.8 1527.3 99.3 KSH 84 rrrwvvwvvw 1440.7 1440.2 99.0 KSH 85 rrrwvvvvww 1440.7 1440.2 98.7 KSH 86 rrrwvvvvvw 1353.7 1353.7 96.3 KSH 90 rrrgwvwvvwk 1526.8 1526.6 96.3 KSH 91 rrhhwvvwvvwk 1843.2 1842.3 95.3 KSH V wlvwiglrkk r 1453.8 1454.1 100.00 KSH I wliwiglrkk r 1467.9 1468.2 96.73 KSH F wlfwiglrkk r 1501.9 1501.9 96.65
[0339] In the amino acid sequences of Table 2 above, an uppercase letter is an L-form amino acid and a lowercase letter is a D-form amino acid. k is lysine to which capric acid (C.sub.10 fatty acid) is bound, and k is lysine to which caproic acid (C.sub.6 fatty acid) is bound. In addition, Wm is methoxy-tryptophan (Wm) represented by Formula 1 below. Ws is benzothienyl-alanine represented by Formula 2 below, and Wf is fluoro-tryptophan represented by Formula 3 below.
##STR00009##
##STR00010##
##STR00011##
wherein Y is monoiodotyrosine represented by Formula 4 below.
##STR00012##
wherein N is glycosylated asparagine.
Experimental Example 2. Analysis of Minimum Growth Inhibitory Concentration (MIC)
[0340] The minimum growth inhibitory concentrations of the antibacterial peptides of the present invention weremeasured, and the results are shown in Table 3 below.
[0341] Specifically, the strains were grown in CAMHB until the midlog phase, and then diluted with PBS buffer to 1× 10.sup.6 cfu/ml to prepare the bacterial solution, and the antibacterial peptide prepared in Example 1above was diluted with CAMHB to 80 .Math.M, 40 .Math.M, 20 .Math.M, 10 .Math.M, 5 .Math.M, 2.5 .Math.M, 1.25 .Math.M and 0.63 .Math.M, and then the bacterial solution was treated with the same volume. It was cultured at 37° C. for 18 hours, and then the MIC was measured.
TABLE-US-00003 Component MRPA (P. aeruginosa) CCARM 2180 MRAB (A. baumannii) ATCC BAA 1605 MRSA (S. aureus) KCCM 40510 VRE (E. faecalis) ATCC 51575 KSH1 40 .Math.M 5 .Math.M 2.5 .Math.M 5 .Math.M KSH3 5 2.5 12.5 2.5 KSH4 >40 >40 5 5 KSH5 10 1.25 1.25 2.5 KSH6 20 2.5 1.25 2.5 KSH7 >40 >40 10 10 KSH8 40 5 2.5 5 KSH9 10 5 1.25 1.25 KSH10 >20 >20 5 5 KSH11 20 5 10 5 KSH12 >40 10 20 20 KSH13 >40 5 40 40 KSH15 10 1.25 1.25 2.5 KSH16 10 1.25 1.25 2.5 KSH18 5 1.25 1.25 2.5 KSH19 10 2.5 2.5 2.5 KSH20 >40 2.5 >40 >40 KSHY 20 1.25 10 2.5 KSH V 10 1.25 2.5 2.5 KSH I 10 1.25 2.5 2.5 KSH F 10 1.25 2.5 2.5 IKSH5-1 >40 5 2.5 2.5 IKSH5-2 10 1.25 1.25 2.5 KSH YL 5 1.25 1.25 2.5 KSH VY 5 1.25 1.25 1.25 KSH VY2 10 2.5 2.5 2.5 KSH VY3 10 2.5 2.5 2.5 KSH VY4 10 1.25 1.25 1.25 KSH27 5 0.63 1.25 1.25 KSH28 5 0.63 1.25 1.25 XSH2 20 1.25 25 5 LSH28 40 5 10 10 LSH1 20 2.5 2.5 2.5 LSH2 20 1.25 5 5 LSH3 20 1.25 2.5 2.5 LSH5 40 2.5 10 10 LSH99 >40 2.5 >40 10 LSH6 40 2.5 10 10 LSH7 >40 5 20 20 LSH8 >40 5 20 20 LSH9 40 5 10 10 KSH29 5 0.63 1.25 1.25 KSH30 >5 1.25 1.25 1.25 KSH31 >5 1.25 1.25 1.25 KSH32 >5 1.25 1.25 1.25 KSH33 5 0.63 0.63 0.63 KSH35 2.5 0.63 1.25 1.25 KSH36 >5 5 25 2.5 KSH37 40 2.5 5 5 KSH39 >40 2.5 10 10 KSH40 5 1.25 1.25 1.25 KSH41 20 2.5 2.5 1.25 KSH42 2.5 0.63 0.63 0.63 KSH43 1.25 0.63 0.63 0.63 KSH44 5 2.5 1.25 1.25 KSH45 2.5 1.25 1.25 1.25 KSH46 1.25 0.63 1.25 1.25 KSH47 10 1.25 2.5 2.5 KSH48 2.5 0.63 2.5 2.5 KSH49 10 2.5 1.25 1.25 KSH50 2.5 0.63 1.25 1.25 KSH51 5 1.25 1.25 1.25 KSH52 5 1.25 5 5 KSH54 2.5 0.63 0.63 0.63 KSH55 2.5 0.63 0.63 0.63 KSH56 5 1.25 1.25 1.25 KSH57 20 5 20 20 KSH58 10 5 10 10 KSH59 2.5 1.25 0.63 0.63 KSH60 5 1.25 0.63 0.63 KSH61 5 1.25 0.63 0.63 KSH62 2.5 0.63 1.25 1.25 KSH63 2.5 2.5 2.5 2.5 KSH64 2.5 2.5 2.5 2.5 KSH66 >10 1.25 1.25 0.63 KSH67 5 1.25 0.63 0.63 KSH68 2.5 0.63 1.25 1.25 KSH69 >10 2.5 >10 >10 KSH70 10 1.25 2.5 2.5 KSH71 >10 1.25 5 2.5 KSH72 10 2.5 2.5 5 KSH73 2.5 2.5 1.25 1.25 KSH74 >10 2.5 >10 >10 KSH75 >10 2.5 >10 >10 KSH76 >10 1.25~2.5 >10 >10 KSH77 >10 2.5 >10 >10 KSH78 >10 5 >10 >10 KSH79 2.5 10 1.25 1.25 KSH80 2.5 10 1.25 1.25 KSH81 5 10 1.25 1.25 KSH82 10 10 1.25 1.25 KSH83 5 10 1.25 1.25 KSH84 2.5 10 1.25 1.25 KSH85 10 10 1.25 1.25 KSH86 10 10 1.25 1.25 KSH90 5 5 1.25 1.25 KSH91 5 10 1.25 1.25 Melittin >40 1.25 1.25 1.25
[0342] The MIC values were determined using cation-adjusted Mueller Hinton broth (containing 10 mg/L Mg.sup.2+ and 50 mg/L Ca.sup.2+). The results are the average value of three independent experiments.
Experimental Example 3. Analysis of Antibacterial Activity in Vitro
[0343] The morphology of the multi-drug resistant strains (Acinetobacter baumannii and Staphylococcus aureus) according to treatment with KSH29 was photographed under an electron microscope, and the results are shown in
[0344] Specifically, MRAB and MRSA were treated with the antibacterial peptide prepared in Example 1at the same concentration as MIC for 0, 15, 30, and 60 minutes, and treated with 4% paraformaldehyde and 1% osmium tetraoxide solution, respectively, for 1 hour and fixed. Thereafter, it was freeze-dried after rapid freezing with liquid nitrogen. It was observed using Pt-coating Field Emission Scanning Electron Microscope (S-4700, EMAX System) at 10,000 magnification.
[0345] In addition, the results obtained by analyzing the degree of liposome leakage according to treatment with KSH29, KSH42 and KSH43 are shown in
[0346] The liposome was prepared in the following manner.
[0347] Specifically, 7 .Math.M DMPC (phosphatidylcholine) and 3 .Math.M DMPG (phosphatidylglycerol) were dissolved in methanol and then evaporated to dryness. Thereafter, Tris buffer containing 70 mM calcein was added, and vortexing and freezing-thawing were repeated. A liposome having a size of 100 .Math.m was formed using Avestin 50x Polycaronate membrane (diam = 0.75 nm. Pore diam = 100 nm) filter. The formed liposome was pore-separated using a Sephadex G50 column, and diluted to 200 .Math.M to prepare it.
[0348] In addition, the the degree of liposome leakage was analyzed in the following manner.
[0349] Specifically, the prepared liposome was diluted with the antibacterial peptide prepared in Example 1 above at 20 .Math.M, 2 .Math.M and 0.2 .Math.M (a ratio of lipid : peptide is 10 : 1, 100 : 1 and 1000 : 1, respectively). It was photographed at Flex conditions, excitation 490 nm, emission 520 nm for a total of 1,200 seconds, and the antibacterial peptide was added 30 seconds after the start of photographing.
[0350] In addition, the results obtained by analyzing the dynamic light scattering according to treatment with KSH29. KSH42 and KSH43 are shown in
[0351] The dynamic light scattering was analyzed in the following manner.
[0352] Specifically, the prepared liposome was diluted with the antibacterial peptide prepared in Example 1 above at 20 .Math.M (a ratio of lipid : peptide is 10 : 1). 5 .Math.l of the dilution was loaded onto AvidNano black cell, and a hydrophobic diameter was measured after setting as follows: solute: liposome, solvent: water, run: 10, acquistion: 10.
[0353] In addition, the results obtained by analyzing the degree of inducing membrane potential difference disturbance of multi-drug resistant Staphylococcus aureus (MRSA) according to treatment with KSH29, KSH42 and KSH43 are shown in
[0354] The degree of inducing membrane potential difference disturbance was analyzed in the following manner.
[0355] Specifically, 25 .Math.M gramicidin, 25 .Math.M colistin, and sample peptides at MIC 40x, MIC 20x, MIC 10x and MIC 5x were diluted in 5 mM HEPES (hydroxyethyl piperazine ethane sulfonic acid), 20 mM glucose and 100 mM KCl buffer to prepare the reagent. The strains cultured in CAMHB until the midlog phase were washed three times with 5 mM HEPES and 20 mM glucose buffer, and diluted with 5 mM HEPES, 20 mM glucose and 100 mM KCl buffer to an OD value of 0.1 to prepare the bacterial solution. diSC35 dye was added to the diluted bacterial solution to 2 .Math.M, incubated for 30 minutes, and then dispensed by 90 .Math.l and prepared. It was photographed at Flex conditions, excitation 620 nm, emission 670 nm for a total of 600 seconds, and 10 .Math.l of the reagent was added to the bacterial solution 120 seconds after the start of photographing.
[0356] In addition, the results obtained by analyzing the degree of inducing membrane potential difference disturbance of multi-drug resistant Enterococcus faecalis (MREF) according to treatment with KSH29, KSH42 and KSH43 are shown in
[0357] In addition, the results obtained by analyzing the degree of inducing membrane potential difference disturbance of multi-drug resistant Acinetobacter baumannii (MRAB) according to treatment with KSH29, KSH42 and KSH43 are shown in
[0358] In addition, the results obtained by analyzing the cell membrane permeability of E. coli according to treatment with KSH29, KSH42 and KSH43 and nitrocefin or ONPG (O-nitrophenyl-β-D-galactopyranoside) are shown in
[0359] The cell membrane permeability of E. coli was analyzed in the following manner.
[0360] Specifically, the strains cultured in CAMHB until the midlog phase were washed three times, and then diluted with PBS buffer to an OD value of 0.4 to prepare the bacterial solution. 10 mM ONPG (O-nitrophenyl-p-D-galactopyranoside) and 120 .Math.M nitrocefin were loaded, and then the antibacterial peptide prepared in Example 1 above was added to 4 times of a desired concentration, respectively, to prepare the reagent. The bacterial solution was treated with the reagent, and then the absorbance was measured at 420 nm for ONPG and 490 nm for nitrocefin at 37° C. at an interval of 1 minute for 1 hour.
[0361] In addition, the results obtained by analyzing the antibacterial effect of KSH29 for each treatment time against multi-drug resistant Pseudomonas aeruginosa (MRPA), multi-drug resistant Enterococcus faecalis (MREF), multi-drug resistant Acinetobacter baumannii (MRAB) and multi-drug resistant Staphylococcus aureus (MRSA) are shown in
[0362] The antibacterial effect for each treatment time was analyzed in the following manner.
[0363] Specifically, the strains were grown in CAMHB until the midlog phase, and then diluted with PBS buffer to 1 × 10.sup.6 cfu/ml to prepare the bacterial solution, and the antibacterial peptide prepared in Example 1 above was diluted to 8 times, 4 times and 2 times the MIC for each strain, and then the bacterial solution was treated with the same volume and cultured at 37° C. The mixture was recovered every 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 16 hours and 18 hours, and then diluted 1 / 10,000 or 1 / 100,000, dispensed in CAMHB agar, and cultured at 37° C., and then the number of colonies was measured.
[0364] In addition, the results obtained by analyzing the antibacterial effect of KSH42 and KSH43 for each treatment time against multi-drug resistant Acinetobacter baumannii (MRAB) and multi-drug resistant Staphylococcus aureus (MRSA) are shown in
[0365] In addition, the results obtained by measuring the minimum inhibitory concentrations of KSH29, KSH42 and KSH43 against 30 species of Acinetobacter baumannii (A. baumannii) are shown in Table 4 below.
[0366] The minimum inhibitory concentrations (MIC) against the 30 species of Acinetobacter baumannii were measured in the following manner.
[0367] Specifically, the strains were grown in CAMHB until the midlog phase, and then diluted with PBS buffer to 1 × 10.sup.6 cfu/ml to prepare the bacterial solution, and the antibacterial peptide prepared in Example 1 above was diluted with CAMHB to 80 .Math.M, 40 .Math.M, 20 .Math.M, 10 .Math.M, 5 .Math.M, 2.5 .Math.M, 1.25 .Math.M and 0.63 .Math.M, and then the bacterial solution was treated with the same volume and cultured at 37° C. for 18 hours, and then the MIC was confirmed.
TABLE-US-00004 Strain KSH29 KSH42 KSH43 Colistin 1 1.25 .Math.M 1.25 .Math.M 1.25 .Math.M 0.63 .Math.M 3 (COL.sup.R) 2.5 1.25 1.25 >40 6 (TGC.sup.I) 1.25 1.25 0.63 2.5 10 1.25 1.25 0.63 2.5 14 1.25 1.25 1.25 1.25 15 1.25 1.25 0.63 0.63 16 1.25 1.25 1.25 <0.31 19 1.25 1.25 0.63 <0.31 22 (TGC.sup.I) 1.25 1.25 0.63 1.25 39 1.25 1.25 0.63 <0.31 44 1.25 1.25 1.25 1.25 66 1.25 1.25 1.25 1.25 68 1.25 1.25 1.25 0.63 69 1.25 1.25 0.63 1.25 71 1.25 1.25 0.63 1.25 76 1.25 1.25 0.31 <0.31 80 0.63 1.25 0.63 0.63 82 1.25 1.25 0.63 0.63 86 0.63 1.25 0.63 0.63 89 1.25 1.25 1.25 2.5 102 (TGC.sup.I) 1.25 1.25 0.63 0.63 127 1.25 1.25 1.25 0.63 141 (TGC.sup.R) 1.25 1.25 0.63 <0.31 144 1.25 2.5 1.25 063 147 (TGC.sup.R) 1.25 2.5 1.25 5 152 (TGC.sup.R) 1.25 2.5 0.63 1.25 19606 1.25 1.25 0.63 1.25 10087 1.25 1.25 1.25 1.25 1605 0.63 0.63 0.63 0.63 40203 1.25 1.25 0.63 0.63 The strains 1 to 152, 10087 and 19606 were purified and obtained from Kyungpook National University College of Medicine. The strains are resistant to the antibiotics imipenem, meropenem, doripenem, cefotaxime, tobramycin, ciprofloxacin (except strain 22), gentamycin (except strain 22), ceftazidime (except strain 22) and tetracycline (except strain 22).
[0368] The strain 1605 was purchased from ATCC (American Type Culture Collection, USA). In addition, the strain 40203 was purchased from the Korean Culture Center of Microorganisms.
[0369] The COL.sup.R refers to resistance to colistin, the TGC.sup.R refers to resistance to tigecycline, and the TGC.sup.I refers to tigecycline intermediate strains.
[0370] In addition, the MIC50 and MIC90 of the peptides against 30 species of Acinetobacter baumannii are shown in Table 5 below.
TABLE-US-00005 Component MIC50 MIC90 KSH29 1.25 1.25 KSH42 1.25 1.25 KSH43 0.63 1.25
[0371] In addition, the results obtained by analyzing the degree of resistance acquisition induction of microorganisms according to treatment with KSH29, KSH42 and KSH43 are shown in
Experimental Example 4. In Vivo Antibacterial Activity in Larvae
[0372] The results obtained by analyzing the antibacterial activity against bacteria (A. baumannii, S. aureus and E. faecium) according to treatment with KSH37 (negative control). KSH42 and KSH43 as a survival rate of Galleria mellonella are shown in
[0373] Specifically, bacteria (A. baumannii, S. aureus and E. faecium) were grown in CAMHB until the midlog phase, and then washed with PBS buffer, and infected to 1 × 10.sup.6, 5 × 10.sup.5, 5 × 10.sup.7 cfu/ml, and treated with the KSH37 (negative control). KSH42 and KSH43 to be 5 .Math.g/larvae. Thereafter, the survival rate of the larvae was checked for 5 days and compared to measure the in vivo antibacterial activity.
Experimental Example 5. Analysis of Toxicity
[0374] The results obtained by analyzing the toxicity evaluation of the antibacterial peptides of the present invention are shown in
[0375] Specifically, 8% hRBCs (human red blood cells) were diluted with PBS. Each antibiotic was diluted with PBS to 200 .Math.M, 100 .Math.M, 50 .Math.M, 25 .Math.M, 12.5 .Math.M, 6.25 .Math.M, 3.13 .Math.M and 1.07 .Math.M, and the 8% hRBCs were treated with the dilutions, and the 8% hRBCs were treated with the antibacterial peptide prepared in Example 1 above in the same amount, and reacted at 37° C. for 1 hour. The positive control was treated with 1% Triton X100 instead of the antibacterial peptide of the present invention. For hemolysis of red blood cells, the soup was recovered by spinning down at 1,000x g, and the absorbance was measured at 540 nm. The absorbance of hRBCs reacted with 0.2% Triton X-100 was considered as 100%, and the absorbance of hRBCs reacted with PBS was considered as 0%, and the comparison was performed.
Experimental Example 6. In Vivo Antibacterial Activity in Mice
[0376] The results obtained by analyzing the antibacterial activity against bacteria (E. coli) according to treatment with KSH43 as a survival rate of mice (BALB/c, female. 7-weeks) are shown in
[0377] Specifically, the strains were grown in CAMHB until the log phase, and then washed with PBS buffer, and infected by subcutaneous injection into mice to 1 × 10.sup.6 cfu/mouse. After 1 hour, 24 hours, and 48 hours, KSH43 and PBS (negative control) were administered at a dose of 100 mg/kg. Thereafter, the survival rate of the mice was checked every 12 hours for 7 days and compared to measure the in vivo antibacterial activity.
[0378] As a result, all mice administered with KSH43 survived for 7 days, whereas in the case of mice administered with PBS, only about 10% of mice survived on day 3 (