WO2023048411A1 - Novel antimicrobial peptide and use thereof - Google Patents

Abstract

The present invention relates to a novel antimicrobial peptide and use thereof. The novel antimicrobial peptide of the present invention consisting of any one of the amino acid sequences of SEQ ID NOs: 6 to 8 has excellent antimicrobial activity against gram-positive bacteria, gram-negative bacteria and antibiotic-resistant bacteria and a cell regeneration effect, and thus can be effectively used in various fields such as antimicrobial compositions or the prevention and treatment of infectious diseases, cosmetic compositions, and preservative compositions.

Description

Novel antimicrobial peptides and uses thereof

The present invention relates to novel antimicrobial peptides and uses thereof.

It is known that most living organisms on Earth produce antimicrobial peptide (AMP) to prevent invasion from external microorganisms and protect themselves. Antimicrobial peptides are mainly positively charged basic peptides with a molecular weight of 2 to 6 KDa composed of 10 to 50 amino acids. It is a host defense factor. Antibacterial peptides are the only defense system for invertebrates without an immune system, such as insects and crustaceans, and for vertebrates with an immune system, they have an initial primary defense mechanism against attacks by external microorganisms and play an important role in the subsequent immune action. It is known to do (G. Wang, Pharmaceuticals (2014) 7:545-594).

The first antibiotic discovered was known as penicillin discovered by Alexander Fleming in 1928, but before that, in 1922, Fleming discovered lysozyme with antibacterial activity in human saliva. Later, in the 1940s, peptide antibiotics such as Gramicidin were developed and used for treatment. Since the 1980s, as science and technology developed, many peptides were discovered as science and technology for separation and structural analysis of peptides developed. In particular, what started to attract attention in the medical world was Dr. Jaslov of the National Institutes of Health in the United States, in 1987, an antibacterial peptide called magainin from an African frog was approved by the FDA for the treatment of diabetic food ulcers. It started to attract a lot of attention as it completed clinical trials and attempted to obtain new drug approval. Since then, the existence of various antibacterial peptides from various organisms has been revealed, such as the discovery of a peptide called buforin from the stomach of a Korean toad ( Bufo bufo gargarizans ) in 1996, and many studies on their characteristics and functions have been conducted. Antimicrobial peptides have been isolated from about 1,500 different organisms. However, when looking at the studies on many antimicrobial peptides discovered so far, they have general tendencies such as α-helix or β-sheet and basicity in terms of structure or activity, but have almost similarity in amino acid sequence. It is not visible. It is believed that this is because each organism has evolved its own peptide structure and sequence differently to suit the environment in which it lives. Various mechanism models have been proposed, and as an example, it is known that an antibacterial peptide having an alpha-helix structure acts on a bacterial cell membrane to destroy the cell membrane, thereby killing cells.

The advantages of these antibacterial peptides as raw materials for new medicines and cosmetics are summarized as follows.

First, strains resistant to antibiotics continue to emerge due to misuse of antibiotics, emerging as a serious threat to human health. Representative resistant bacteria include gram-positive methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococci (VRE), gram-negative bacteria Pseudomonas aeruginosa , and Acinetobacter baumani. ( Acinetobacter baumannii ), etc., and as new multidrug-resistant bacteria are continuously discovered, antibiotic-resistant bacteria survive regardless of body parts and organs, such as acute sepsis, respiratory infections, cystic fibrosis, and urinary tract infections, and treatment is difficult, causing high mortality. there is. However, antimicrobial peptides have a mechanism of action that physically destroys microbial cell membranes, and thus can prevent the emergence of antibiotic-resistant bacteria. Unlike general chemically synthesized peptides that stop the growth of bacteria by interfering with the cell wall or nucleic acid synthesis, antibacterial peptides can directly destroy cells by penetrating the cell membrane, thereby avoiding the adaptive mechanism of resistance to antibiotics.

Second, antimicrobial peptides are rapid, effective and have a broad antibacterial spectrum.

Third, antibacterial peptides selectively act only on pathogens, so they are effective in the human body. This is because, in the case of human eukaryotic cells, a large amount of neutrally charged cholesterol and the like are present in the cell membrane, which hinders the passage of antimicrobial peptides.

Fourth, since antimicrobial peptides do not undergo secondary modification such as glycosylation, they can be mass-produced using a genetic engineering process and thus have low production costs during development.

Fifth, most antibacterial peptides have cell regeneration, that is, wound healing activity, so they can be used for skin beauty along with antibacterial activity (Ana Gomes et. al., Molecules (2017) 22:1743-1761 ).

Among the natural world, research on antibacterial peptides for plants and terrestrial organisms is actively in progress, and has been isolated from various tissues from vertebrates to invertebrates so far. Research has been conducted to prepare an antibacterial peptide library based on physicochemical properties and to search for peptides with improved disadvantages therefrom, or to search for and improve new peptides in nature. In particular, the potency of antimicrobial peptides is greatly affected by various physical properties, such as length and electrical properties. In general, the amino acid length should be less than 50, the number of cationic amino acids should be high, and it should be hydrophobic and have an amphipathic structure. In particular, the distribution and hydrophobicity of cationic amino acids are important because the cell membrane surface of bacteria is negatively charged so that they can easily bind. In the case of the alpha helix structure, since the amino acid composition affects the number, structure, and characteristics of helix turns, a software program can be used as a means for visually expressing them. Using these programs, it is possible to analyze the sequence of cationic amino acids, where hydrophobic amino acids are concentrated at any part of the helix, and which are easy to bind to bacterial cell membranes. In addition, it was found that antibacterial activity of alpha-helical antimicrobial peptides increases when proline is present in the middle to serve as a structural hinge (Ruth Ann Veach. et al., J. Biol. Chem. ( 2004) 279(12):11425-11431). Therefore, it is necessary to improve peptides that increase antibacterial activity while reflecting these characteristics.

Meanwhile, Korean Patent No. 1734331 discloses 'a novel antibacterial active peptide isolated from Korean sea cucumber and its use', and Korean Patent No. 2039400 discloses 'a novel antibacterial peptide derived from mBjAMP1 peptide and its use'. However, the novel antimicrobial peptide of the present invention and its use have not been described.

The present invention was derived from the above needs, and some of the amino acid sequences of the novel antibacterial peptide (SEQ ID NO: 5) prepared by combining the amino acid sequences of the peptides of SEQ ID NO: 1 and SEQ ID NO: 2, which have known antibacterial activity A peptide (H12) of SEQ ID NO: 6 was prepared by substituting residues, and a peptide of SEQ ID NO: 7 ( H103) was prepared, and a peptide of SEQ ID NO: 8 (H123) was prepared by substituting some residues in the peptide of SEQ ID NO: 6. Then, as a result of confirming the antibacterial activity of the novel peptides of SEQ ID NOs: 6 to 8 prepared, it was confirmed that each peptide had significantly increased antibacterial activity against gram-positive bacteria, gram-negative bacteria and antibiotic-resistant bacteria compared to the parent peptide, the present invention has been completed.

In order to solve the above problems, the present invention provides an antimicrobial peptide consisting of any one of the amino acid sequences of SEQ ID NOs: 6 to 8.

In addition, the present invention provides a polynucleotide encoding the antimicrobial peptide.

In addition, the present invention provides an antibiotic containing the antimicrobial peptide as an active ingredient.

In addition, the present invention provides an antibacterial quasi-drug composition containing the antimicrobial peptide as an active ingredient.

In addition, the present invention provides an antibacterial cosmetic composition containing the antimicrobial peptide as an active ingredient.

In addition, the present invention provides an antibacterial food additive containing the antimicrobial peptide as an active ingredient.

In addition, the present invention provides an antibacterial feed additive containing the antimicrobial peptide as an active ingredient.

In addition, the present invention provides an antibacterial biological pesticide containing the antimicrobial peptide as an active ingredient.

In addition, the present invention provides an antiseptic composition containing the antimicrobial peptide as an active ingredient.

In addition, the present invention provides an antibacterial method in a subject comprising administering a pharmaceutically effective amount of the antimicrobial peptide to the subject.

Since the novel antimicrobial peptide of the present invention has excellent antibacterial activity against gram-positive bacteria, gram-negative bacteria and antibiotic-resistant bacteria, and has a cell regeneration effect, it is used in various fields such as antibacterial compositions, prevention and treatment of infectious diseases, cosmetic compositions, and preservative compositions. could be useful.

1 is a helical wheel diagram of an antimicrobial peptide (H10) consisting of the amino acid sequence of SEQ ID NO: 4.

Figure 2 is a helical wheel diagram of the antimicrobial peptide (H12) consisting of the amino acid sequence of SEQ ID NO: 6.

3 is a helical wheel diagram of the peptide (H103) consisting of the amino acid sequence of SEQ ID NO: 7.

4 is a helical wheel diagram of the peptide (H123) consisting of the amino acid sequence of SEQ ID NO: 8.

5 is a 3D secondary structure (JSmol 3D secondary structure) whose structural characteristics were identified using PHYRE II and JSmol structure visualization software for predicting the protein structure of the peptide (msi 78) consisting of the amino acid sequence of SEQ ID NO: 2.

6 is a 3D secondary structure of the peptide (H123) consisting of the amino acid sequence of SEQ ID NO: 8 whose structural characteristics were identified using PHYRE II and JSmol structure visualization software for protein structure prediction.

Figure 7 shows the result of scratch wound healing assay (scratch wound healing assay) for measuring the cell regeneration effect of the antibacterial peptide (H10) consisting of the amino acid sequence of SEQ ID NO: 4 (A), cell density (B) and average spacing (C) is the result shown. *, *** means that the results of the peptide treatment group compared to the untreated group are statistically significantly different, * is p <0.05, *** is p <0.001. scale bar=200 μm.

8 is a result of scratch wound healing analysis (A), cell density (B), and average spacing (C) for measuring the cell regeneration effect of the antibacterial peptide (H12) consisting of the amino acid sequence of SEQ ID NO: 6. *, **, *** mean that there is a statistically significant difference in the results of the peptide treatment group compared to the untreated group, * is p <0.05, ** is p <0.01, *** is p <0.001. scale bar=200 μm.

FIG. 9 shows results of scratch healing analysis (A), cell density (B), and average interval (C) for measuring the cell regeneration effect of the peptide (H123) having the amino acid sequence of SEQ ID NO: 8. **, *** means that there is a statistically significant difference in the results of the peptide treatment group compared to the untreated group, ** is p <0.01, *** is p <0.001. scale bar=200 μm.

In order to achieve the object of the present invention, the present invention provides an antimicrobial peptide consisting of any one of the amino acid sequences of SEQ ID NOs: 6 to 8.

As used herein, the term "antibacterial peptide" is a polymer composed of amino acids linked by amide bonds (or peptide bonds) and has growth inhibitory activity against microorganisms.

The novel antimicrobial peptide having the amino acid sequence of SEQ ID NO: 6 according to the present invention is prepared by combining the amino acid sequences of known antimicrobial peptides (SEQ ID NO: 1 and SEQ ID NO: 2), the amino acid sequence (RLLLRRLLRPK KF GK A FVKILK K ) was prepared by replacing the 11th, 12th, 15th, and 22nd amino acids (underlined) with alternative amino acids having the same characteristics as each. Specifically, SEQ ID NO: 5 is an antibacterial peptide consisting of the amino acid sequence of SEQ ID NO: 1 (RLLRRLLR) known to have antibacterial activity and the amino acid sequence of SEQ ID NO: 2 (GIGKFLKKA KKFGKAFVKILKK ) from the 10th to the 22nd (underlined) rear part It was prepared by joining the sequences, and was prepared by inserting Proline (P) to act as a structurally folding part (hinge) between the two sequences in order to double the effect of increasing antibacterial activity. A novel antibacterial peptide ( RLLRRLLRPK RW GK L FVKILK R , SEQ ID NO: 6) was prepared. In addition, SEQ ID NO: 4 was prepared by conjugating the antibacterial peptide consisting of the amino acid sequence of SEQ ID NO: 1 and the rear part sequence from the 4th to the 14th of the antimicrobial peptide known as LK peptide (SEQ ID NO: 3, LKKLLLKLLKKLLKL). SEQ ID NO: 4 was also prepared by inserting P to act as a structurally folding part (hinge) between the two sequences (see Tables 1 and 2).

In addition, the peptide consisting of the amino acid sequence of SEQ ID NO: 7 according to the present invention is L and lysine in the amino acid sequence of SEQ ID NO: 4 (RLLRRLLRPI LLKLLKKLLKL ) prepared by combining the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 3 known as antibacterial peptides The repetitive sequence (underlined) of (Lysine, K) was replaced with repetitive sequences of W and K to have similar sequence characteristics. In addition, isoleucine (I) at the 10th position of SEQ ID NO: 4 was placed at positions 14 and 17 of SEQ ID NO: 7 so that amino acids having the same characteristics were not omitted, and finally, two amino acids were omitted to obtain the sequence of SEQ ID NO: 4. The length of the peptide was shorter than that of the peptide. Specifically, in the 10th to 19th amino acids (underlined) of the peptide (RLLLRRLLRP WKKWIKWIKK ) consisting of the amino acid sequence of SEQ ID NO: 7, repeats of K are maintained for K rich characteristics, which is one of the characteristics of cationic antibacterial peptides, and L Instead, it was substituted with W, which has the characteristics of an amphiphilic cell-penetrating peptide. In addition, a peptide consisting of the amino acid sequence of SEQ ID NO: 7 ( RLLRRLLR P WKKWIKWIKK ) was prepared by inserting P to serve as a structurally folding portion (hinge) in the ninth sequence of the peptide consisting of the amino acid sequence of SEQ ID NO: 7 (Table 1 and Table 2).

In addition, the peptide consisting of the amino acid sequence of SEQ ID NO: 8 is prepared by combining the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2, known as antimicrobial peptides, in the amino acid sequence of SEQ ID NO: 6 ( R LL RR LL R PKRWGKLFVKILKR) , 5th and 8th amino acids (underlined) R were substituted with K to prepare a peptide consisting of the amino acids of SEQ ID NO: 8 ( K LL KK LL K PKRWGKLFVKILKR) (see Tables 1 and 2).

In the present invention, the amino acid of the antimicrobial peptide preferably has an L form, but it is not excluded from the present invention that it is substituted with a D- form.

The antimicrobial peptide may be prepared by a conventional peptide synthesis method known in the art. As a method for the synthesis, it is preferable to synthesize by a chemical synthesis method, specifically, a solution phase peptide synthesis method, a solid-phase peptide synthesis method, a fragment condensation method, and F-moc or T -Synthesis by BOC chemistry may be more preferable, but is not limited thereto. In addition, there is a method of biologically producing a polynucleotide encoding an antimicrobial peptide by expressing it in genetically engineered E. coli (Cheng KT et al., Molecules, (2018) 23(4):800-811), but is not limited thereto.

The antimicrobial peptide preferably has antibacterial activity against gram-positive bacteria, gram-negative bacteria or antibiotic-resistant bacteria, but is not limited thereto.

The gram-positive bacteria include all gram-positive bacteria known in the art including Staphylococcus, Enterococcus, Bacillus, Listeria, or Lactobacillus. It may be a positive bacteria, preferably a gram-positive bacteria of the genus Staphylococcus, Enterococcus or Bacillus, most preferably Staphylococcus aureus, Enterococcus faecium or Bacillus Cereus ( Bacillus cereus ) It may be, but is not limited thereto.

The gram-negative bacteria are Escherichia, Pseudomonas, Acinetobacter, Klebsiella, Salmonella, Leptospira or Rickettsia. The gram-negative bacteria included may be any gram-negative bacteria known in the art, preferably gram-negative bacteria of the genus Escherichia coli, the genus Pseudomonas, the genus Acinetobacter or the genus Klebsiella, most preferably Escherichia coli , Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), Acinetobacter baumani ( Acinetobacter baumannii ) and Klebsiella pneumoniae ( Klebsiella pneumoniae ) It may be any one or more selected from the group consisting of, but is not limited thereto.

In addition to the gram-positive bacteria and gram-negative bacteria, the antibacterial peptide of the present invention may have antibacterial activity against antibiotic-resistant bacteria. The antibiotics are not limited thereto, but are aminoglycoside-based (aminoglycoside, gentamicin, neomycin, etc.), penicillin-based (ampicillin, etc.), sulfonamide-based, beta-lactam-based (beta-lactam, amoxicillin/clamyl) Bulanic acid, etc.), chloramphenicol series, erythromycin series, florphenicol series, fosfomycin series, kanamycin series, lincomycin series, methicillin series, quinolone series, streptomycin series, tetracycline series, trimethoprim series and vancomycin class of antibiotics.

In one embodiment of the present invention, the antibiotic-resistant bacteria are vancomycin-resistant Enterococcus faecium , methicillin-resistant Staphylococcus aureus , ESBL (Extended-spectrum β) -lactamase) producing Escherichia coli ( Escherichia coli ), carbapenem (carbapenem) resistant E. coli, carbapenem-resistant Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), carbapenem-resistant Acinetobacter baumannii ( Acinetobacter baumannii ) and carbapenem-resistant Klebsi Ella pneumoniae ( Klebsiella pneumoniae ) It may be any one or more selected from the group consisting of, but is not limited thereto.

The antimicrobial peptide of the present invention is characterized in that it has low cytotoxicity to human-derived cells and has a cell regeneration effect.

In addition, the present invention provides a polynucleotide encoding the antimicrobial peptide.

In addition, the present invention provides an antibiotic containing the antimicrobial peptide as an active ingredient.

The antimicrobial peptide of the present invention is a peptide having an amino acid sequence of any one of SEQ ID NOs: 6 to 8, as described above. Since the antimicrobial peptide of the present invention exhibits strong antibacterial activity against gram-negative bacteria, gram-positive bacteria and antibiotic-resistant bacteria, the antibacterial peptide of the present invention can be usefully used as an active ingredient of antibacterial antibiotics.

The peptide of the present invention can be administered parenterally during clinical administration and can be used in the form of general pharmaceutical preparations. Parenteral administration may refer to administration through an administration route other than oral administration, such as rectal, intravenous, peritoneal, intramuscular, arterial, transdermal, nasal, inhalational, ocular and subcutaneous administration. When the antibacterial peptide of the present invention is used as a medicine, it may additionally contain one or more active ingredients exhibiting the same or similar functions.

The antibacterial peptide of the present invention can be prepared in various parenteral formulations. When formulated, it is prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Formulations for parenteral administration include sterilized aqueous solutions, water-insoluble agents, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for the suppository, Witepsol, Macrogol, Tween 61, cacao butter, liurine fat, glycerogeratin and the like may be used.

In addition, the antibacterial peptide of the present invention can be used in combination with various pharmaceutically acceptable carriers such as physiological saline or organic solvents, and to increase stability or absorption, carbohydrates such as glucose, sucrose or dextran, Antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers may be used as pharmaceuticals.

In the antibiotic of the present invention, the total effective amount of the novel peptide of the present invention can be administered to the patient in a single dose in the form of a bolus or by infusion for a relatively short period of time, and multiple administration It can be administered by a fractionated treatment protocol in which multiple doses are administered over a long period of time. Since the effective dose of the patient is determined considering various factors such as the patient's age and health condition as well as the drug administration route and number of treatments, considering this point, those of ordinary skill in the art can use the present invention It will be possible to determine an appropriate effective dosage according to the specific use of the novel peptide of <RTI ID=0.0>as an antibiotic.

In addition, the present invention provides an antibacterial quasi-drug composition containing the antimicrobial peptide as an active ingredient.

When the composition of the present invention is used as a quasi-drug additive, the peptide may be added as it is or used together with other quasi-drugs or quasi-drug ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of use.

The antibacterial quasi-drug of the present invention is not limited thereto, but is preferably a disinfectant cleaner, shower foam, gargreen, wet tissue, detergent soap, hand wash, humidifier filler, mask, ointment, patch, or filter filler.

In addition, the present invention provides an antibacterial cosmetic composition containing the antimicrobial peptide as an active ingredient.

Since the antimicrobial peptide has excellent antibacterial activity, low cytotoxicity to human-derived cells and has a cell regeneration effect, it can be used very usefully as an antibacterial cosmetic composition.

The cosmetic composition of the present invention includes components commonly used in cosmetic compositions in addition to the antimicrobial peptide, for example, antioxidants, stabilizers, solubilizers, vitamins, pigments and flavoring agents, and carriers.

In the cosmetic composition of the present invention, the 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 the cosmetic composition that is normally contained.

The cosmetic composition of the present invention can be prepared in any formulation conventionally prepared in the art, for example, a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing , It may be formulated as an oil, powder foundation, emulsion foundation, wax foundation and spray, but is not limited thereto. More specifically, it can be formulated into a formulation of softening lotion (skin), nourishing lotion (milk lotion), nourishing cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray or powder. .

When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc. are used as carrier components. It can be.

When the formulation of the present invention is a powder or 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, additional chlorofluorohydrocarbon, propane / May contain a propellant such as butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizing agent or emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butyl glycol oil, fatty acid esters of glycerol, polyethylene glycol or sorbitan.

When the formulation of the present invention is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystals Star cellulose, aluminum metahydroxide, bentonite, agar or tragacantha and the like may be used.

When the formulation of the present invention is surfactant-containing cleansing, as carrier components, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters and the like can be used.

In addition, the present invention provides an antibacterial food additive containing the antimicrobial peptide as an active ingredient.

Since the antimicrobial peptide has excellent antibacterial activity, low cytotoxicity to human-derived cells, and a cell regeneration effect, it can be very useful as an antibacterial food additive.

When the peptide of the present invention is used as a food additive, the 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 depending on the purpose of its use. Generally, 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 intake, the amount may be less than 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.

There is no particular limitation on the type of food. Examples of foods to which the substance can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, There are alcoholic beverages, vitamin complexes, and the like, and includes all foods in a conventional sense.

In addition, the present invention provides an antibacterial feed additive containing the antimicrobial peptide as an active ingredient.

The feed composition of the present invention replaces conventional antibiotics and suppresses the growth of harmful food pathogens to improve the health of animals, improve weight gain and quality of livestock, and increase milk production and immunity. The feed composition of the present invention may be prepared in the form of fermented feed, formulated feed, pellet form and silage.

The fermented feed can be prepared by fermenting organic matter by adding various microorganisms or enzymes other than the peptide of the present invention, and the formulated feed can be prepared by mixing various kinds of general feed with the peptide of the present invention. Feed in the form of pellets can be prepared by applying heat and pressure to the formulated feed, etc. in a pellet machine, and silage can be prepared by fermenting green feed with microorganisms. Wet fermented feed collects and transports organic matter such as food waste, mixes excipients for sterilization and moisture control in a certain ratio, and ferments for more than 24 hours at a temperature suitable for fermentation, so that the moisture content is about 70%. It can be made by adjusting. The fermented dry feed can be prepared by adjusting the wet fermented feed to have a moisture content of about 30% to 40% through an additional drying process.

In addition, the present invention provides an antibacterial biological pesticide containing the antimicrobial peptide as an active ingredient.

In addition, the present invention provides an antiseptic composition containing the antimicrobial peptide as an active ingredient.

The antimicrobial peptide of the present invention is a peptide having any one amino acid sequence of SEQ ID NOs: 6 to 8, and exhibits strong antibacterial activity against gram-negative bacteria, gram-positive bacteria and antibiotic-resistant bacteria, so the antibacterial peptide of the present invention is an antibacterial biological pesticide Or it can be usefully utilized as an active ingredient of an antiseptic composition.

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 change of medicines, and include bactericides and antioxidants, and inhibit the growth of microorganisms such as bacteria, molds, and yeasts, thereby inhibiting the growth of food products. And functional antibiotics such as inhibiting the growth of spoilage microorganisms or sterilizing action in pharmaceuticals are also included. As an ideal condition for such an antiseptic composition, it should not be toxic and should be effective even in a small amount.

In addition, the present invention provides an antibacterial method in a subject comprising administering a pharmaceutically effective amount of the antimicrobial peptide to the subject. The subject may be a mammal other than human, but is not limited thereto.

In the present invention, a pharmacologically effective amount means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is dependent on the type, severity, activity of the drug, and drug sensitivity, time of administration, route of administration and excretion rate, duration of treatment, factors including concomitantly used drugs, and other factors well known in the medical field. The antimicrobial peptide of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, sequentially or simultaneously with conventional therapeutic agents, and can be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.

The dosage of the antimicrobial peptide of the present invention may be administered in a variety of ranges depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of the disease, but the route of administration, obesity Since it may increase or decrease according to severity, gender, weight, age, etc., the dosage is not limited to the scope of the present invention in any way.

The amino acid sequences used herein are abbreviated according to the IUPAC-IUB nomenclature as follows: alanine A, arginine R, asparagine N, aspartic acid D, cysteine C, glutamic acid E, glutamine Q, glycine G, histidine H, Isoleucine I, Leucine L, Lysine K, Methionine M, Phenylalanine F, Proline P, Serine S, Threonine T, Tryptophan W, Tyrosine Y, Valine V. In addition, the amino acids may include both D-type and L-type amino acids. It may, but is not limited thereto.

Hereinafter, the present invention will be described in detail by examples. However, the following examples are only to illustrate the present invention, and the content of the present invention is not limited to the following examples.

Example 1. Peptide synthesis

In the present invention, the peptides in Table 1 were synthesized through a solid / solution phase using the Fmoc chemistry method, a peptide synthesis technology, and purified by high-performance liquid chromatography to a purity of 95% or more .

synthesized peptide information

sequence number	antibacterial peptide	number of amino acids	note
One	RLLRRLLR	8	Korea Patent No. 1341210
2	GIGKFLKKA KKFGKAFVKILKK	22	magainin, msi 78
3	LKKLLKLLKKLLKL	14	LK peptide
4	RLLRRLLRP ILLKLLKKLLKL	21	Novel derivative peptide (H10)
5	RLLRRLLRPK KF GK A FVKILK K	22	Novel derivative peptide (H11)
6	RLLRRLLRPK RW GK L FVKILK R	22	Novel derivative peptide (H12)
7	RLLRRLLRP WKKWIKWIKK	19	Novel derivative peptide (H103)
8	K LL KK LL K PKRWGKLFVKILKR	22	Novel derivative peptide (H123)

The peptide of SEQ ID NO: 4 (H10) was synthesized by combining the peptides of SEQ ID NO: 1 and SEQ ID NO: 3, and the peptide of SEQ ID NO: 5 (H11) was synthesized by combining the peptides of SEQ ID NO: 1 and SEQ ID NO: 2 (underlined) , The peptide of SEQ ID NO: 6 (H12) was synthesized by replacing the 11th, 12th, 15th, and 22nd amino acids (underlined) in the peptide of SEQ ID NO: 5 with alternative amino acids identical to each characteristic (non-polar or positively charged) . In addition, in the peptide of SEQ ID NO: 4, the repetitive sequences of L and K (underlined) were replaced with repetitive sequences of W and K to have similar sequence characteristics, and in the peptide of SEQ ID NO: 4, the 10th to 19th amino acids (underlined ) The peptide of SEQ ID NO: 7 (H103) was synthesized by maintaining the repetition of K in ) and replacing L with W having the characteristics of an amphipathic cell-penetrating peptide. In addition, the peptide of SEQ ID NO: 8 (H123) was synthesized by substituting K for the 1st, 4th, 5th and 8th amino acids R in the peptide of SEQ ID NO: 6.

All of the peptides in the table above have L-forms, but those substituted with D-forms are not excluded in the present invention.

Example 2. Measurement of antibacterial activity of novel derivative peptides

In order to compare the antibacterial activity of the synthesized novel derivative peptide, the antibacterial activity was measured against non-resistant or antibiotic-resistant strains of gram-negative and gram-positive bacteria, respectively. For the antibacterial activity, the minimal inhibitory concentration (MIC) of the peptide that does not divide the cells was measured in MH (Mueller Hinton) medium with sufficient nutrients. After obtaining the strains shown in Table 3 below, diluting with MH medium so that the number of bacteria is 2 × 10 ⁶ CFUs (colony-forming units) per ml, and dispensing 100 μl into a 96-well microtiter plate, 100 μl of a peptide solution diluted with MH medium was added to each well. The MIC was determined by measuring the absorbance at 620 nm after the plates were incubated at 37° C. for 16 hours.

The antibacterial peptide of SEQ ID NO: 1 has been reported to exhibit an MIC of 1 mg/ml, that is, 1,000 μg/ml, for both Gram-positive and Gram-negative bacteria (refer to Korean Patent Registration No. 1341210). As a result of the antibacterial activity analysis, it was found that the antibacterial peptides of SEQ ID NOs: 6 and 7, which are novel antimicrobial peptides, exhibit significantly increased antibacterial activity compared to the respective parent peptides (SEQ ID NOs: 5, H11 and SEQ ID NOs: 4, H10). In addition, in the case of SEQ ID NO: 8, a novel derivative antimicrobial peptide derived from the antimicrobial peptide of SEQ ID NO: 6, it was found to be improved than the antimicrobial peptide of SEQ ID NO: 6 except for Enterococcus faecium (Table 3) .

According to the report of the Korea Centers for Disease Control and Prevention, vancomycin-resistance E. faecium (VRE) shows a significant increase of 12-23% depending on the region for the last three years. It was confirmed that the antibacterial activity of the antibacterial peptides of SEQ ID NOs: 6 to 8 against VRE was superior to that of a non-susceptible strain.

In the case of antibacterial activity against methicillin-resistance Staphylococcus aureus (MRSA), which is famous for multi-resistant bacteria, the antibacterial peptide of SEQ ID NO: 6 showed a MIC value similar to that of non-resistant strains, but SEQ ID NO: 7 And the antibacterial peptide of SEQ ID NO: 8 was confirmed to have excellent antibacterial activity against MRSA compared to non-resistant strains.

For carbapenem-resistant Escherichia coli strains, the antibacterial peptides of SEQ ID NOs: 6 to 8 showed superior antibacterial activity compared to SEQ ID NOs: 4 and 5. Extended-spectrum β-lactamase (ESBL) is an enzyme propagated by a plasmid. The frequency of hospital infection by Gram-negative strains that produce ESBL is gradually increasing, and penicillin, cephalosporin, and kava It has been reported that it is difficult to treat infection due to multi-resistance to penem, etc. (Yoon Pil-hun et al. (2014) Korean J Nosocomial Infect Control 19(2):45-51). They also have carbapenem resistance, which is a last resort, and there is a report that showed a mortality rate of 12.9%. The novel antibacterial peptides of SEQ ID NOs: 6 to 8 showed antibacterial activity against these ESBL multi-resistant strains.

Pseudomonas aeruginosa is a major medical-related causative agent that causes skin infections, pneumonia, bedsores, sepsis, meningitis, and the like. This also shows multi-resistance to antibiotics such as carbapenems and amicoglycosides. The antibacterial peptides of SEQ ID NOs: 6 and 8 showed rather excellent antibacterial activity in carbapenem-resistant Pseudomonas aeruginosa compared to non-carbapenem-resistant strains. The antibacterial peptide of SEQ ID NO: 7 showed improved antibacterial activity compared to the parent peptide (H10), but no significant difference in antibacterial activity was observed between carbapenem-resistant or non-resistant strains.

Acinetobacter baumannii ( Acinetobacter baumannii ) Since 2007, the number of infections in hospitals worldwide has increased, and the multidrug resistance rate, including carbapenem-based antibiotics, has also begun to increase significantly. In 2010, at the University of Tokyo Hospital in Japan, 46 people were infected with Acinetobacter bacteria and 10 of them died. It was shocking. It is showing a rapid increase at 71.7% per year. Multidrug-resistant Acinetobacter baumani is a resistant bacteria that requires special attention in hospitals because it can cause infection in patients with immunosuppression, chronic lung disease, or long-term hospitalization. The antibacterial peptides of SEQ ID NOs: 6 to 8 showed superior antibacterial activity against carbapenem-resistant Acinetobacter baumani bacteria compared to non-resistant bacteria.

In the case of the antibacterial peptide of SEQ ID NO: 7, it showed strong antibacterial activity against carbapenem-resistant bacteria of Klebsiella pneumoniae called Klebsiella pneumoniae .

From the above results, the antimicrobial peptides (SEQ ID NOs: 6 to 8) of the present invention show excellent antibacterial activity against various antibiotic-resistant bacteria, including gram-positive and gram-negative bacteria, so that antibacterial compositions or infectious disease prevention and treatment and cosmetic compositions, It was predicted that it could be usefully used in various fields such as preservative compositions.

Example 3. Characterization of novel antimicrobial peptides

In order to analyze the excellent antibacterial activity of the novel antimicrobial peptides (SEQ ID NOs: 6 to 8), the structural characteristics were identified using the helical wheel diagrams technique.

Comparing the helical wheel structure of SEQ ID NO: 6 (FIG. 2) and the helical wheel structure of SEQ ID NO: 4 (FIG. 1), in SEQ ID NO: 4, the position of amino acid P serving as a hinge is not on the hydrophobicity face. However, SEQ ID NO: 6 was determined to have enhanced hydrophobicity in the hydrophobicity face. In addition, SEQ ID NO: 6, like the helical wheel structure of SEQ ID NO: 1 and the helical wheel structure of SEQ ID NO: 2, hydrophobic amino acids are regularly arranged at the bottom of the peptide, so it was determined that the basic characteristics that antibacterial peptides should have are well equipped. In addition, the peptide of SEQ ID NO: 6 has a mean hydrophobicity of 0.362, which is higher than 0.280 of SEQ ID NO: 5, supporting excellent antibacterial activity.

Similar to SEQ ID NO: 6, the helical wheel structure of the peptide of SEQ ID NO: 7 was determined to be more hydrophobic because the position of P was in the hydrophobic cross section (FIG. 3). In addition, the peptide of SEQ ID NO: 4 has 7 hydrophobic cross-sections, whereas the peptide of SEQ ID NO: 7 has 10 hydrophobic cross-sections, so it was judged to have excellent cell penetration ability and thus more excellent antibacterial activity. In addition, the hydrophobic moment (hydrophobic moment (uH)) is a measure of amphiphilicity, meaning that the larger the hydrophobic moment, the greater the amphiphilicity and the larger the area of the hydrophobic surface. The hydrophobic moment of the peptide was found to be 0.892.

In the helical wheel structure of the peptide of SEQ ID NO: 8 (FIG. 4), the position of amino acid P serving as a hinge was determined to have a more enhanced hydrophobic property in the hydrophobic face. In addition, it was confirmed that the hydrophobic moment of SEQ ID NO: 8 was 0.365.

To predict the structure of the peptide of SEQ ID NO: 8, structural characterization was performed using PHYRE II and JSmol structural visualization software. Comparing the 3D secondary structure of SEQ ID NO: 8 (FIG. 6) and the 3D secondary structure of SEQ ID NO: 2 (FIG. 5), SEQ ID NO: 8 has physical cell permeability due to the amino acid proline serving as a folding structure (hinge). was found to be stronger. Specifically, SEQ ID NO: 2 is msi 78, which is well known as an antibacterial peptide called magainin in the Protein Data Bank. There are 17 Aligned Residues, a confidence of 97.61%, and PDB info are accurately analyzed as antibiotics. It takes the form of a secondary structure where the helical wheel emerges clearly. On the other hand, SEQ ID NO: 8 is a novel antibacterial peptide, with 23 Aligned Residues, 14.18% confidence, PDB info predicted as antiviral, and structural features include a helical wheel, one of the characteristics of antibacterial peptides, as well as a proline sequence. The biggest structural difference is that cell permeability and antibacterial activity are increased by the introduction of a hinge. The similarity and homology of the protein sequence was also very low at 14.18%, indicating high potential as a novel antibacterial peptide.

Example 4. Measurement of hemolytic activity

In order to compare the cytotoxicity of the peptides prepared by the method of Example 1, the hemolytic activity of the synthesized peptides was measured.

Specifically, human erythrocytes were diluted with PBS (pH 7.0) to a concentration of 8%, and the peptide of SEQ ID NO: 2, 4 or 6 was treated at a concentration of 1, 5, 10, 20 or 100 μg / ml per well, respectively. , and reacted at 37° C. for 1 hour. Then, the amount of hemoglobin contained in the supernatant obtained by centrifugation at 1,000 xg was confirmed by measuring absorbance at a wavelength of 414 nm. As a control for the degree of cell destruction, the absorbance of the supernatant obtained after treatment with 0.2% Triton and reaction at 37 ° C. for 1 hour was measured. The hemolytic activity (hemolysis) of each peptide was calculated using the formula. All hemolytic activities (%) were expressed as mean ± standard deviation.

Hemolytic activity = (absorbance A-absorbance B)/(absorbance C-absorbance B)

(Absorbance A: absorbance of the reaction solution treated with each peptide measured at a wavelength of 414 nm, absorbance B: absorbance of the reaction solution treated with PBS measured at a wavelength of 414 nm, absorbance C: 0.2% Triton measured at a wavelength of 414 nm Absorbance of the reaction solution treated with.)

As a result of comparing the hemolytic activity (%) expressed as a percentage, the hemolytic activity of the peptide of SEQ ID NO: 2 was 0.5±0.4%, 1.2±0.4%, 1.2±0.4%, 1.8±0.7%, 2.9±1.0% and 9.2±2.9%. The hemolytic activity of the peptide of SEQ ID NO: 4 (H10) was 7.2±0.6%, 34.4±2.2%, 53.8±3.9%, 75.7±4.3% and 95.7±1.9% at concentrations of 1, 5, 10, 20 and 100 μg/ml, respectively. appeared in %. On the other hand, the hemolytic activity of the peptide of SEQ ID NO: 6 (H12) was -0.9±0.6%, 0±0.9%, 0.7±0.4%, 1.2±0.8% and 1.2±0.8% at concentrations of 1, 5, 10, 20 and 100 μg/ml, respectively. It was found to be 6±1.5%. In addition, the hemolytic activity of the peptide of SEQ ID NO: 8 was 0±0%, 0±0%, 0±0%, 0.2±0%, 2.3%±1.1 at concentrations of 1, 5, 10, 20, and 100 μg/ml, respectively. % (Table 4).

Based on the above results, it was confirmed that the hemolytic activity of the novel antimicrobial peptides (SEQ ID NOs: 6 and 8) of the present invention was less than 1% at low concentrations (1, 5 and 10 μg/ml), thereby making it the most stable compared to other antibacterial peptides. and showed no cytotoxicity to human erythrocytes.

Example 5. Cell regeneration effect measurement

In order to measure the cell regeneration efficacy of the antibacterial peptides of SEQ ID NOs: 4, 6 and 8, a scratch wound healing assay was performed. HaCaT cells were dispensed in a 12-well plate at 5×10 ⁴ cells/ml, cultured in DMEM medium containing 10% FBS for 24 hours to stabilize, and then replaced with DMEM medium excluding 10% FBS, followed by starvation for 24 hours. proceeded. When the cells in the well showed 100% confluency, the center of each well was regularly scratched using a 200 μl tip, and then the medium was removed and washed with PBS. Thereafter, the peptide was treated at a concentration of 1, 5, 10, 25 or 50 μg/ml, respectively, and incubated for 24 hours, observed using a microscope, and artificially created gaps between cells compared to wells not treated with the peptide ( The change in whether the gap) was filled was confirmed.

As a result, it was confirmed that the peptides of SEQ ID NOs: 4, 6 and 8 had no cell regeneration effect at a high concentration of 50 μg/ml. The peptide of SEQ ID NO: 4 had the best cell regeneration effect at a concentration of 10 μg/ml, whereas the peptide of SEQ ID NO: 6 had the best cell regeneration effect at a concentration of 5 to 25 μg/ml, and the peptide of SEQ ID NO: 8 It was confirmed that the cell regeneration effect was excellent at a concentration of 1 to 25 μg/ml (FIGS. 7 to 9).

Claims (14)

1. An antibacterial peptide consisting of any one of the amino acid sequences of SEQ ID NOs: 6 to 8.
2. The antimicrobial peptide according to claim 1, wherein the antimicrobial peptide has antibacterial activity against gram-positive bacteria, gram-negative bacteria, or antibiotic-resistant bacteria.
3. The method of claim 2, wherein the Gram-positive bacteria is at least one selected from the group consisting of Staphylococcus aureus , Enterococcus faecium and Bacillus cereus . antibacterial peptide.
4. The method of claim 2, wherein the gram-negative bacteria are selected from the group consisting of Escherichia coli , Pseudomonas aeruginosa , Acinetobacter baumannii and Klebsiella pneumoniae Antimicrobial peptide, characterized in that any one or more of the.
5. The method of claim 2, wherein the antibiotic-resistant bacteria are vancomycin-resistant Enterococcus faecium , methicillin-resistant Staphylococcus aureus , ESBL (Extended-spectrum β-lactamase) E. coli that produces ( Escherichia coli ), carbapenem (carbapenem) resistant Escherichia coli, carbapenem-resistant Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), carbapenem-resistant Acinetobacter baumannii ( Acinetobacter baumannii ) and carbapenem-resistant Klebsiella pneumoniae Ae ( Klebsiella pneumoniae ) Antimicrobial peptide, characterized in that any one or more selected from the group consisting of.
6. A polynucleotide encoding the antimicrobial peptide of claim 1.
7. An antibiotic containing the antimicrobial peptide of claim 1 as an active ingredient.
8. An antibacterial quasi-drug composition containing the antimicrobial peptide of claim 1 as an active ingredient.
9. An antibacterial cosmetic composition containing the antimicrobial peptide of claim 1 as an active ingredient.
10. An antibacterial food additive containing the antimicrobial peptide of claim 1 as an active ingredient.
11. An antibacterial feed additive containing the antimicrobial peptide of claim 1 as an active ingredient.
12. An antibacterial biological pesticide containing the antimicrobial peptide of claim 1 as an active ingredient.
13. An antiseptic composition containing the antimicrobial peptide of claim 1 as an active ingredient.
14. An antibacterial method in a subject comprising administering a pharmaceutically effective amount of the antimicrobial peptide of claim 1 to the subject.

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