KR20130104718A - Novel analogues of antibacterial peptide derived from styela clava and the use thereof - Google Patents

Abstract

PURPOSE: An antibacterial peptide is provided to be used as an antibacterial or antiseptic composition, an antibiotic pharmaceutical composition, a pharmaceutical composition for preventing and treating pathogenic bacterial infection, and an antibiotic food additive. CONSTITUTION: An antibacterial peptide is prepared by substituting 9th and 12th amino acids (isoleucine) from N-terminal in an amino acid sequence of sequence number 1 with lysine. The peptide has an amino acid sequence of sequence number 2 and has an antibacterial activity against gram-positive bacteria or gram-negative bacteria. The gram-negative bacteria are Escherichia coli or Proteus vulgaris. The gram-positive bacteria are Listeria monocytogenes or Staphylococcus epidermidis.

Description

Novel analogues of antibacterial peptide derived from Styela clava and the use

The present invention relates to an antimicrobial peptide and its use, and in particular, isoleucine (I) located at the 9th and 12th amino acids of an antimicrobial peptide having antimicrobial activity derived from midderm described in SEQ ID NO: 1. It relates to a novel antimicrobial peptide synthesized by replacing with lysine (Lysine, K) and a pharmaceutical composition containing the same as an active ingredient.

Antibiotics are substances derived from living organisms that inhibit or kill the growth of other microorganisms. A. Fleming first discovered penicillin in 1928, and more than 5,000 antibiotics were discovered after streptomycin was discovered in 1943, and over 50 antibiotics were developed in 400 clinical trials. It is used. Conventional antibiotics inhibit or kill the growth of microorganisms mainly by the following mechanisms. 1) Block the synthesis of cell wall (Cycloserine, Vancomycin, Bacitracin, Penicillin, Cephalosporin (porin), Monobactam, Carbapenem, Ampicillin), 2) Destroy cell structure (Polymyxin), 3) Inhibition of metabolism (Trimethroprim, Sulfonamide), 4) Inhibition of DNA or RNA synthesis (Actinomycin, Nalidixic acid, Ciprofloxacin, Novobiocin, Rifampin, Streptovaricin), 5) Inhibition of protein synthesis (Erythromycin, Chloramphenicol, Clindamycin, Lincomycin, Tetracycline, Streptinomycin, Streptomycin, Gentamicin, Kanamycin, Amikacin, Amikacin) Mupirocin, Puromycin).

Decreased physical activity caused by economic growth leads to a decrease in immunity, exposing the risk of various diseases from microbial infections such as bacteria, gom palm, and parasites (Clark GC et al. Journal) . of medical microbiology , 58, 977, 2009), although various drugs are currently used for disease prevention and treatment worldwide, misuse of drugs has increased the resistance of pathogens. In particular, the rise of vancomysin resistant enterococci (VREs), called meridinin resistant Staphylococcus aureus (MRSA) and multidrug-resistant pathogens (MDRP), called superbugs, is of global interest (Jabra-Rizk. Et al M. A, PloS). pathog , 6 2010, Woo. J. et al J. Food Prot ., 73, 2285, 2010).

This increase in antibiotic resistance was first discovered in Pneumococcus sp. In the 1970s and provided important clues to the mechanism of action of penicillin (Tomasz et al., Nature , 227, 138-140, 1970). Tolerant species stop growing in the presence of the usual concentration of antibiotics but do not die as a result. Resistance is due to the absence of the activity of bacterial autolytic enzymes such as autolysin when antibiotics inhibit cell wall synthase, which in the case of penicillin activates endogenous hydrolytic enzymes. By killing bacteria, but inhibits the activity of the enzyme will show a result of survival even during antibiotic treatment.

Tolerance to antibiotics is a distinct phenomenon from antibiotic resistance, first discovered in Pneumococcus sp. In the 1970s and providing an important clue to the mechanism of action of penicillin (Tomasz). et al., Nature, 227, 138-140, 1970). Tolerant species stop growing in the presence of the usual concentration of antibiotics but do not die as a result.

It is clinically very important for bacteria to be resistant to antibiotics, because the inability to eradicate resistant bacteria makes antibiotic treatment less effective in clinical infections (Handwerger and Tomasz, Rev. Infec . Dis ., 7, 368-386, 1985). In addition, resistance is regarded as a prerequisite for bacterial resistance to antibiotics, because strains survive even antibiotic treatment. These strains acquire new genetic elements that are resistant to antibiotics and continue to grow even in the presence of antibiotics. In fact, all bacteria that are resistant to antibiotics are known to be resistant to the antibiotics (Liu and Tomasz, J. Infect . Dis ., 152, 365-372, 1985), which can kill antibiotic resistant bacteria. There is a need for the development of new antibiotics.

In terms of the mechanism of action, resistance is largely divided into two, the first is the phenotypic resistance that occurs when the growth rate decreases in all bacteria (Tuomanen E., Revs . Infect . Dis ., 3, S279). -S291, 1986), the second is genetic resistance by mutations in certain bacteria. In both cases, the underlying phenomenon is the down regulation of the activity of the autolysin enzyme, which is transient in the case of external resistance to external stimuli, In the case of genetic resistance, mutations that cause change are permanent. Obviously, the simplest case of genetic resistance is due to a deficiency of the autolysine enzyme, for which there is no clinically found strain resistant to this deficiency of this autolytic enzyme for a variety of reasons. Tolerance found in is a process that modulates the activity of the autolysine enzyme (Tuomanen et al., J. infect. Dis ., 158, 36-43, 1988).

Bacteria become resistant to antibiotics: a) enzyme activation by enzymes, b) changes in target sites, c) antibiotic permeability of cell membranes, and d) antibiotics out of cells. There are various mechanisms such as efflux. Bacteria become resistant to antibiotics by working with one or several of these. Beta-lactamase is a typical example of enzyme inactivation. Beta-lactamase hydrolyzes beta-lactam rings to inactivate drugs that exert antimicrobial activity by this structure. However, the bacterium produces beta-lactamse and is not resistant to all beta-lactam antibiotics. For example, Staphylococcus aureus , which produces beta-lactamase, Although it is resistant to penicillin, ampicillin, piperacillin and the like, it is susceptible to methicillin, oxacillin, naphcillin and the like which are stable to this enzyme. This is because the range of the hydrolyzable substrate (antibiotic) varies depending on the type of beta-lactamase. Enzymes that inactivate aminoglycosides include aminoglycoside acetyltransferase, aminoglycoside phosphotransferase, and aminoglycoside adenyltransferases.

Some bacteria obtain resistance by changing the target protein to which the antibiotic binds or by overproducing the target protein. Gram-positive bacteria such as Staphylococcus aureus and Streptococcus pneumoniae often acquire resistance to beta-lactam antibiotics by this mechanism. The target of the beta-lactam family of antibiotics is the penicillin-binding protein (PBP). Staphylococcus aureus produces new PBPs (PBP2) that have low affinity with beta-lactam antibiotics, making them resistant to methicillin, a gene called mec. Streptococcus pneumoniae acquires resistance to beta-lactam antibiotics by conventional PBP denaturation. Pseudomonas Pseudomonas aeruginosa), while Gram-negative bacteria such as Enterobacter foil TB subgenus (Enterobacteriaceae) has a bacteria acquired resistance to beta-lactam antibiotics by such mechanism. Vancomycin-Resistant Enterococcus (VRE) is also an example of obtaining resistance to vancomycin by denaturation of the target substance. Vancomycin binds to the N-terminal D-Ala-D-Ala of the peptidoglycan precursor, preventing PBP from binding to this site. As a result, bacteria are unable to synthesize cell walls by peptidoglycan polymerization. However, the N-terminal of VRE is changed to D-Ala-D-Lac and the like, and the affinity with vancomycin is reduced to less than 1 / 1,000, thereby obtaining resistance to vancomycin. Mutations between DNA gyrase and topoisomerase IV, the target sites of quinolones, result in resistance to quinolones.

Porin is a protein in the outer membrane that exists only in Gram-negative bacteria. Porin accepts nutrients and releases metabolites. Most antibiotics enter this cell through this structure. The outer membrane of E. coli contains a variety of porins, but antibiotics mainly pass through OmpF and OmpC. Porin permeability of antibiotics depends on the charge, hydrophobicity and molecular weight of the antibiotic molecules. Negative charge molecules pass through porin slower than positive or positive charge molecules. Hydrophobic molecules are more hydrophilic, and higher molecular weight antibiotics do not penetrate popin better than lower molecular weight antibiotics. Because methicillin is hydrophobic, it does not penetrate porin well, and thus has no antimicrobial activity against Gram-negative bacteria. As described above, in Gram-negative bacillus, porin affects antibiotic susceptibility. If porin is lost or changed, resistance to antibiotics increases.

Some bacteria actively release antibiotics that enter the cell. This resistance mechanism can be found in bacteria that are resistant to tetracycline or macholide, which inhibits the production of ribosomes, and bacteria that are resistant to quinolone, which inhibits DNA gyrase activity. P. aeruginosa OprK protein in the extracellular membrane has a function of releasing toxic components in the cell to the outside of the cell, and the mutant expressing a large amount of this protein is released by ciprofloxacin, nalidixic acid, and tetracycline ) And chloramphenicol are resistant to many antibiotics. This mechanism of obtaining resistance to various antibiotics by active efflux is called a multidrug resistance pump (MDR). MDR pumps include Gram-positive bacteria such as S. aureus, E. coli and P. aeruginosa In addition to Gram-negative bacteria, fungi Candida albicans and protozoa Plasmodium falciparum It also has a back. As the MDR pump of S. aureus , NorA, QacA, QacB and the like are known. Strains that express large amounts of QacA protein are resistant to chlorhexidine, and strains that express large amounts of NorA are resistant to quinolone. EmrAB is known as the MDR pump of E. coli , and the high-expressing strain is resistant to nalidixic acid and thiolactomycin.

As described above, the development of a new antibiotic is necessary to remove bacteria that exhibit resistance to antibiotics, and the development of a new antibiotic that acts independently of the activity of the autolysine enzyme.

The mechanism by which antimicrobial peptides act on pathogens is very different from that of conventional antibiotics. They bind to the cell surface of microorganisms and then form pores in the cell membrane, disrupting the normal permeability of the cell membrane and causing the pathogen to die rapidly (Pharmacol. Rev. 55: 27-55, 2003). They are also known to be able to prevent sepsis caused by pathogenic endotoxins (Biochim. Biophys. Acta. 1562: 32-36, 2002) and act as signaling molecules that mediate acquired immune responses in higher animals. (J. Leukoc. Biol. 60: 415-422, 1996). Due to these characteristics, antimicrobial peptides are attracting attention as new antibiotics or new bioimmune activators that can control antibiotic resistant bacteria. Thus, peptide antibiotics have been tested in various organisms to find antibiotics that can replace conventional antibiotics. In practice, however, there are very few cases in which the product is being used in the market, because the stability of the peptide antibiotic when injected in vivo or the safety of the human body has been raised. In order to solve this difficulty, it is necessary to analyze the amino acid sequence and structure of peptides having antimicrobial or antifungal activity to find a structure that can effectively kill microorganisms without harming the human body. To this end, information on the amino acid sequence and structure of various antibiotic active peptides beyond those reported so far is required.

Insects, animals and marine organisms, on the other hand, can produce antimicrobial peptides on their own (Bevins et al., Ann. Rev. Biochem ., 59, 395-414, 1990). . The first is a cysteine-rich -sheet peptide, the second-a helical amphiphilic peptide molecule, and the third is a proline-rich peptide. Mayasaki et al., Int. J. Antimicrob.Agents , 9, 269-280, 1998). These antimicrobial peptides are known to play an important role in host defense and the innate immune system (Boman, HG, Cell, 65: 205, 1991; Boman, HG, Annu. Rev. Microbiol. , 13:61, 1995). In addition, the antimicrobial peptides have various structures depending on the amino acid sequence.

Styela clava is a marine organism belonging to the chromosome, the off-white animal, which has contributed to the increase of fishermen's income since full-scale farming began in the mid-1980s (Ministry of Agriculture, Statistical Yearbook, p.291. 1993). In addition, midderok is a marine organism found all over Korea, and especially 80% of Korea's consumption is produced in Masan, Gyeongsangnam-do. It is used all year round because of its unique taste and fragrant aroma, and it is the peak of production between April and July. Consumption of midderk is mainly used in foods such as steamed or miso stew, and others collected from April to May for sashimi. Studies on midderk have been studied in terms of sterol content (Jo YG. 1978. The sterol composition of Styela clava.Kor Fish Soc 11: 97-101), and seasonal changes in nutritional composition (Lee KH, Park CS, Hong BI, Jung BC). , Cho HS, Jea YG. 1995. Seasonal variations of nutrients in warty sea squirt (Styela clava) .J Korean Soc.Food Nutr 24: 268-273; Ahn SH. 2003. Extraction of glycosaminoglycans from Styela clava tunic.Biotechnol Bioproc Eng 18: 180-185), and most of the studies were conducted on constituents, and glucosaminoglycan was extracted from the skin as a functional ingredient (Lehrer RI. 2001. Clavanins and styelins, alpha-helical antimicrobial peptides from the hemocytes). of Styela clava.Adv Exp Med Biol 484: 71-76).

However, as described in the present invention, a novel peptide described as SEQ ID NO: 2 (CSP-4) prepared by substituting lysine for isoleucine located at amino acid sequences 9 and 12 of the antimicrobial peptide derived from the midderm tissue as described herein. Was not.

Thus, the present inventors have prepared by substituting isoleucine (Isoleucine, I) located in Nos. 9 and 12 with lysine (Lysine, K) from an antimicrobial peptide having an amino acid sequence described in SEQ ID NO: 1 previously known. New peptides having an amino acid sequence set forth as 2 (CSP-4) were prepared. The antimicrobial peptide of the present invention has a significantly reduced aggregation at pH 7.4 than the peptide having the amino acid sequence of SEQ ID NO: 1 (Clavaspirin), has a low cytotoxicity, and the antimicrobial activity is not limited by pH. In addition, the antimicrobial peptide having the SEQ ID NO: 2 (CSP-4) is a histidine (histidine) to the antimicrobial peptide (clavaspirin) described in SEQ ID NO: 1 known that the bacterial membrane action at pH 7.4 shows a faster action as compared to pH 5.5 ) It contains a lot of residues, and when it is lower than pH 6.04, it shows strong electrostatic interaction with the cell membrane due to increase of cation electrode, but it is not affected by histidine at pH 7.4. The antimicrobial peptides of the invention described in number 2 completed the invention by identifying faster cell membrane action.

It is an object of the present invention to provide an antimicrobial peptide in which isoleucine (I) located at the 9th and 12th amino acids from the N-terminus in the amino acid sequence of SEQ ID NO: 1 is substituted with lysine (Lysine, K).

Another object of the present invention is to provide an antimicrobial or antiseptic composition containing the peptide as an active ingredient.

Another object of the present invention to provide an antibiotic pharmaceutical composition containing the peptide as an active ingredient.

Another object of the present invention to provide a pharmaceutical composition for the prevention and treatment of pathogenic bacterial infections containing the peptide as an active ingredient.

Another object of the present invention is to provide an antibiotic food additive containing the peptide as an active ingredient.

In order to solve the above object, the present invention provides an antimicrobial peptide in which isoleucine (Isoleucine, I) located at the 9th and 12th amino acids from the N-terminus in the amino acid sequence of SEQ ID NO: 1 is substituted with lysine (Lysine, K). This provides a novel antimicrobial peptide having low cytotoxicity and antimicrobial activity that is not limited by pH.

In another aspect, the present invention is to provide an antimicrobial or antiseptic composition containing the peptide as an active ingredient.

The present invention also provides a pharmaceutical composition for antibiotics containing the peptide as an active ingredient.

The present invention also provides a pharmaceutical composition for the prevention and treatment of pathogenic bacterial infections containing the peptide as an active ingredient.

The present invention also provides an antibiotic food additive containing the peptide as an active ingredient.

In the known amino acid sequence of SEQ ID NO: 1 according to the present invention, the antimicrobial peptide in which isoleucine (Isoleucine, I) located at the 9th and 12th amino acids from the N-terminus with lysine (Lysine, K) is SEQ ID NO: 1 ( Agglomeration was significantly reduced at pH 7.4, peptides with amino acid sequences described as Clavaspirin), have low cytotoxicity, antimicrobial activity is not limited to pH, and bacterial membrane degradation activity at pH 7.4 compared to pH 5.5. As a result, the Clavaspirin peptide contains a lot of histidine residues, and when the pH is lower than 6.04, the cation electrode increases, resulting in strong electrostatic interaction to the cell membrane, but the histidine at pH 7.4. (Histidine) is not affected by the faster cell membrane degradation activity at pH 7.4.

1 is a graph showing the degree of scattering of light through agglomeration of the parent antimicrobial peptide (Clavaspirin) and the antimicrobial peptide (CSP-4) prepared in the present invention at pH 5.5 and pH 7.4.
2 is a graph showing the effect of the antimicrobial peptide (CSP-4) and the parent antimicrobial peptide (Clavaspirin) prepared in the present invention on the cell membrane at pH 5.5 and pH 7.4.
Figure 3 is a diagram showing the cell membrane degradation of the antimicrobial peptide (CSP-4) and the parent antimicrobial peptide prepared in the present invention (A) pH 5.5 and (B) pH 7.4, each of the top is a parent antibacterial peptide, the bottom is Peptides prepared in the present invention are shown.

Hereinafter, the present invention will be described in detail.

The present invention provides an antimicrobial peptide in which isoleucine (I) is replaced with lysine (Lysine, K) located at amino acids 9 and 12 from the N-terminus in the amino acid sequence of SEQ ID NO: 1.

The present invention also provides an antimicrobial peptide having the amino acid sequence of SEQ ID NO: 2.

The amino acid sequence of SEQ ID NO: 1 of the present invention can be prepared by conventional peptide synthesis methods known in the art as an antimicrobial peptide (Clavaspirin) derived from Styela clava, and the production method is not particularly limited.

NMR helix structure of Clavaspirin, a parent peptide having the amino acid sequence of the conventionally known SEQ ID NO: 1, shows the hydrophobicity of the peptide and isoleucine (Isoleucine, I) located side by side at No. 9 and No. 12 Strong hydrophobicity increases with) the aggregation of peptides by formation of coiled-coli structures at neutral pH due to hydrophobic interaction between peptides. Antibacterial peptide (Clavaspiein) described in SEQ ID NO: 1 shows strong activity against Gram-positive bacteria and Gram-negative bacteria in low pH environment, but low activity and strong cytotoxicity to normal cells in neutral pH environment.

Thus, in one embodiment of the present invention, according to the Merrifield liquid phase method (Merrifield, RB., J. Am. Chem. Soc., 85, 2149, 196), SEQ ID NO: 1 of the parent peptide Substituting two isoleucines (Isoleusine, I) located side by side of the amino acid sequence described in the following by a low hydrophobic lysine (Kys) to provide a CSP-4 peptide having an amino acid sequence interrupted by SEQ ID NO: 2 (see Table 1). ). Antimicrobial peptides having an amino acid sequence set forth in SEQ ID NO: 2 of the present invention can be prepared by conventional peptide synthesis methods known in the art, the production method is not particularly limited.

In addition to the lysine (Lysine, K) of the present invention, it is possible to substitute a positively charged amino acid Arginine (Arginine, R) or histidine (Hstidine, H), preferably lysine (Lysine, K).

The antimicrobial peptide (CSP-4) according to the present invention has a strong antimicrobial activity that is significantly reduced in aggregation and cytotoxicity as a result of contrast with the parent peptide Clavaspirin (Clavaspirin).

In the present invention, an antimicrobial peptide in which isoleucine (I) is replaced with lysine (Lysine, K) at positions 9 and 12 from the N-terminus in the amino acid sequence of SEQ ID NO: 1 It provides an antimicrobial peptide having activity.

The Gram-negative bacteria is, for example, but not limited to, The Genus Bordetella, The Genera Pseudomonas, The Genus Burkholderia, The Genera Aeromonas, Plesi The Genera Plesiomonas, The Genera Vibrio, The Genera Moraxella, The Genera Neisseria, The Genus Actinobacillus, and Manheimia Genera Mannheimia, The Genera Pasteurella, The Genera Haemophilus, The Genera Histophilus, The Genera Taylorella, The Genus Brucella ), The Genus Francisella, The Genus Escherichia, The Genus Shigella, The Genera Klebsiella, The Genera Enterobacter, Sheet The Genera Citrobacter, Ph.D. The Genera Proteus, The Genera Morganella, The Genera Edwardsiella, The Genus Salmonella or The Genus Yersinia. Preferably Escherichia is coli) or Proteus vulgaris (Proteus vulgaris).

The Gram-positive bacteria is, for example, but not limited to, The Genera Actinomyces, The Genera Arcanobacterium, The Genus Bacillus, The Genus Bacillus, The Genus Corynebacterium, The Genera Dermatophilus, The Genera Nocardia, The Genera Listeria, The Genera Erysipelothrix, The Genus Mycobacterium Or The Genus Rhodococcus, preferably Listeria monocytogenes ( Listeria) monocytogenes ) or Staphylococcus epidermidis ).

In one embodiment of the present invention, to determine whether the antimicrobial peptide CSP-4 exhibits antimicrobial activity, the minimum growth concentration (MIC) of various bacterial strains was measured. As a result, the antimicrobial peptide of the present invention is Gram-negative bacterium Escherichia coli. coli) and Proteus vulgaris (Proteus showed a high antibacterial activity, regardless of the pH with respect to vulgaris), a gram-positive Listeria monocytogenes presence (Listeria monocytogenes ) or Staphylococcus epidermidis also showed high antibacterial activity regardless of pH.

The present invention provides an antimicrobial peptide wherein the antimicrobial peptide (CSP-4) exhibits antimicrobial activity in acid, neutral and alkaline.

In one embodiment of the present invention, the parent peptide (Clavaspirin) described in SEQ ID NO: 1 is markedly inferior in antimicrobial activity at pH 6.2 or above pH 7.4. In addition, in one embodiment of the present invention, as a result of measuring the aggregation of the parent peptide using a fluorescence photometer, the aggregation phenomenon is increased at pH 7.4.

In one embodiment of the present invention, the antimicrobial peptide of the present invention had an antimicrobial activity of 8 to 16 times the parent peptide antimicrobial activity at pH 7.4, and showed a higher antimicrobial activity than the parent peptide at pH 4 to 10, preferably Is pH 6-8.5 and even more preferably pH 7.4. There is also no increase in aggregation at pH 7.4.

In one embodiment of the present invention, CSP-4 peptide, the antimicrobial peptide of the present invention, is treated with SYTOX-Green on E. coli cells cultured at pH 5.5 and pH 7.4 to confirm the action on the cell membrane. After reacting in a light incubator for 15 to 20 minutes, the peptide was treated at the minimum inhibitory concentration to measure the effect on the cell membrane. As a result, the antimicrobial peptide of the present invention (CSP-4) showed a strong effect on the cell membrane of the antimicrobial peptide of the present invention by increasing the strong fluorescence at pH 5.5 and pH 7.4 compared to the parent Clavaspirin peptide. Therefore, it was found that the CSP-4 peptide, the antimicrobial peptide of the present invention, was not affected by pH due to its strong action on the cell membrane at pH 5.5 and pH 7.4 (see FIG. 2). In addition, the pH conditions, pH 4.0, pH 6.2, pH 8.5, pH 9.8 measured in addition to the antimicrobial peptide (CSP-4) of the present invention was found to have a fast bacterial membrane degradation action in acidic, neutral, basic (Table 8). The antimicrobial peptide of the present invention exhibits rapid bacterial membrane degradation activity at pH 7.4, which contains a lot of histidine residues in the parent peptide and shows a strong electrostatic interaction with the cell membrane due to an increase in the cationic electrode when the pH is lower than 6.04. This is because the antimicrobial peptides are not affected by histidine at pH 7.4.

Therefore, the antimicrobial peptides according to the present invention exhibit antimicrobial activity in acidic, neutral and basic, preferably pH 4.0 to 10, more preferably pH 6 to 8.5, even more preferably pH 7.4.

The present invention provides an antimicrobial peptide in which the antimicrobial peptide (CSP-4) is significantly reduced in cytotoxicity.

In one embodiment of the present invention, in order to confirm the cytotoxicity of the peptide, normal human blood was used to measure erythrocyte hemolytic activity against the antimicrobial peptide. In addition, in one embodiment of the present invention, after treating the anti-bacterial peptide to the human keratinocyte line (HaCaT cell line) was confirmed the degree of cell survival. As a result, the antimicrobial peptide described in SEQ ID NO: 2 of the present invention had little hemolytic action than the antimicrobial peptide described in SEQ ID NO: 1, and confirmed that cell viability was maintained high even at high concentrations.

Therefore, the present invention provides an antimicrobial peptide in which isoleucine (Isoleucine, I) located at the 9th and 12th amino acids from the N-terminus in the amino acid sequence of SEQ ID NO: 1 is replaced with lysine (Lysine, K). The present invention provides an antimicrobial peptide having antimicrobial activity against both Gram-positive and Gram-negative bacteria, having no cytotoxicity, and having no antimicrobial activity affected by pH change.

The present invention can be used as an antimicrobial or antiseptic composition containing the antimicrobial peptide as an active ingredient.

In a specific embodiment of the present invention, the antimicrobial peptides according to the present invention are not cytotoxic and have antimicrobial activity against Gram-negative bacteria and Gram-positive bacteria regardless of pH, thus preventing any microbial growth. It can be usefully used as a bouillon composition. The use is not limited to a food preservative composition, it can be used as a preservative for inhibiting the growth of microorganisms in all substances requiring antimicrobial activity such as cosmetic preservatives, pharmaceutical preservatives.

The present invention provides an antibiotic pharmaceutical composition containing the antimicrobial peptide as an active ingredient.

The present invention also provides a pharmaceutical composition for the prevention and treatment of pathogenic bacterial infections containing the antimicrobial peptide as an active ingredient.

In a specific embodiment of the present invention, the antimicrobial peptides according to the present invention have antimicrobial activity in Gram-negative and Gram-positive bacteria irrespective of pH, have no cytotoxicity, and do not have agglomeration, thereby preventing antibiotic compositions or pathogenic bacterial infections. And it can be usefully used as a therapeutic pharmaceutical composition.

The composition comprising the antimicrobial peptide of the present invention preferably includes 0.1 to 50% by weight of the antimicrobial peptide, based on the total weight of the composition, but is not limited thereto.

The compositions of the present invention may further comprise suitable carriers, excipients and diluents conventionally used in the manufacture of medicaments.

The composition according to the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., oral preparations, suppositories and sterilized injection solutions according to a conventional method have. Examples of carriers, excipients and diluents that can be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, (sucrose), lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The composition of the present invention can be administered orally or parenterally, any parenteral administration method can be used, systemic or topical administration is possible, but systemic administration is more preferred, and intravenous administration is most preferred.

The preferred dosage of the composition of the present invention varies depending on the condition and the weight of the patient, the degree of disease, the type of drug, the route of administration and the period of time, but can be appropriately selected by those skilled in the art. However, for the desired effect, the effective dose of the antimicrobial peptide of the present invention is 1 to 2 mg / kg, preferably 0.5 to 1 mg / kg, and can be administered 1 to 3 times a day. The dose is not intended to limit the scope of the invention in any way.

The antibiotic containing the antimicrobial peptide of the present invention as an active ingredient may be administered to a patient in a single dose by bolus form or by infusion for a relatively short period of time, and may be administered in multiple doses. dose) may be administered by a fractionated treatment protocol with long term administration. Since the effective dose of the antimicrobial peptide of the present invention is determined in consideration of various factors such as the age and health condition of the patient as well as the route of administration and the number of treatments of the drug, it is common knowledge in the art Anyone with A can determine the appropriate effective dose.

The pharmaceutical compositions of the present invention may be prepared in unit dose form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporation into a multi-dose container. The formulation can be used in any form suitable for pharmaceutical preparations, including powders, granules, tablets, capsules, suspensions, emulsions, syrups, oral formulations such as aerosols, external preparations such as ointments, creams, suppositories, and sterile injectable solutions. It may further comprise a dispersant or stabilizer.

The present invention also provides an antibiotic food additive containing the peptide as an active ingredient.

In a specific embodiment of the present invention, the antimicrobial peptides according to the present invention have antimicrobial activity in Gram-negative and Gram-positive bacteria regardless of pH, have no cytotoxicity, and do not have agglomeration, and thus may be usefully used as food additives for antibiotics. . In addition, the antimicrobial peptide of the present invention may be usefully used as a food additive as well as a food additive.

There is no particular limitation on the kind of the food. Examples of the foods to which the above substances can be added include dairy products including dairy products such as drinks, meat, sausage, bread, biscuits, rice cakes, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, Beverages, alcoholic beverages, and vitamin complexes, all of which include health functional foods in a conventional sense.

The antimicrobial peptides of the present invention can be added as is to foods or used with other foods or food ingredients, and can be suitably used according to conventional methods. The amount of the active ingredient to be mixed can be suitably determined according to its use purpose (for prevention or improvement). In general, the amount of the antimicrobial peptide in the food may be added at 0.1 to 90 parts by weight of the total food weight. However, in the case of long-term intake intended for health and hygiene purposes or for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

The beverage composition of the present invention is not particularly limited to other ingredients except for containing the antimicrobial peptide as essential ingredients in the indicated ratios, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. Natural flavors (tau martin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavors (saccharin, aspartame, etc.) can be advantageously used as flavors other than those described above The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the composition of the present invention.

In addition to the above, the antimicrobial peptides of the present invention are various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, alginic acid and Salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. In addition, the antimicrobial peptides of the present invention may contain pulp for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components may be used independently or in combination. The proportion of such additives is not so critical but is generally selected in the range of 0.1 to about 20 parts by weight per 100 parts by weight of the antimicrobial peptide of the present invention.

Hereinafter, the present invention will be described in detail with reference to Examples, Experimental Examples and Preparation Examples.

However, the following Examples, Experimental Examples and Preparation Examples are merely illustrative of the present invention, and the present invention is not limited to the following Examples, Experimental Examples and Production Examples.

< Example  1> Of peptide  Synthesis and separation purification

The inventors have described Clavaspirin (amino acid sequence having the amino acid sequence set forth in SEQ ID NO: 1 of the parent peptide according to Merrifield, RB., J. Am. Chem. Soc., 85, 2149, 196). Lee et al., J. peptide Res. 58, 2001 / 445-456), replacing isoleucine (I) with lysine (K) at positions 9 and 12, the hydrophobic moieties. CSP-4 peptide (SEQ ID NO: 2) was synthesized (Table 1).

Specifically, the peptide of the peptide designed in the present invention is NH2 form of the carboxyl terminus was used as a starting material Rink Amide MBHA-Resin, the peptide of the OH carboxyl end is Fmoc-amino acid-Wang Resin as a starting material Used.

The extension of the peptide chain by the coupling of Fmoc-amino acids was carried out by DCC (N-hydroxybenzo triazole (HOBt) -dicyclo-hexycarbodiimide) method. After coupling the Fmoc-amino acid at the amino terminal of each peptide, remove the Fmoc group with NMP (20% piperidine / N-methyl pyrolidone) solution, wash it several times with NMP and DCM (dichoromethane), and then use nitrogen gas. Dried. A solution of trifluoroacetic acid (TFA) -phenol-thioanisole-H2O-triisopropylsilane (85: 5: 5: 2.5: 2.5, vol. / Vol.) Was added thereto and reacted for 2 to 3 hours to remove the protecting group and to separate the peptide from the resin. After the preparation, the peptide was precipitated with diethylether. The crude peptide obtained by the above method was purified by reverse phase (RP) -HPLC column (Delta Pak, C18300A, 15,19.0 mm ×) in an acetonitrile gradient containing 0.05% TFA. 30 cm, Waters, USA). After the synthetic peptide was hydrolyzed with 6 N HCl at 110 ° C, the residue was concentrated under reduced pressure, dissolved in 0.02 N HCl, and the amino acid composition was measured with an amino acid analyzer (Hitachi 8500 A). As a result of confirming the purity of the peptides prepared by the above method, the purity was over 95%, and the molecular weight was determined from the amino acid sequence using MALDI mass spectrometry (Hill, et al., Rapid Commun. Mass Spectrometry, 5: 395, 1991). As a result of comparing with the molecular weight obtained by calculation, it confirmed that the value corresponded.

Peptides  Sequence, molecular weight and retention time Peptides Amino acid sequence Molecular Weight Holding time Clavaspirin FLRFIGSVIHGIGHLVHHIGVAL-NH2 (SEQ ID NO: 1) 2492.01 35.2 CSP-4 FLRFIGSVKHGKGHLVHHIGVAL-NH2 (SEQ ID NO: 2) 2522.04 20.2

< Experimental Example  1> antibacterial Of peptide  Antimicrobial Activity Assay

In order to compare the antimicrobial activity of the peptides prepared by the method of <Example 1>, the present inventors measured the minimum growth concentration (MIC) value, which is the minimum concentration of the peptides in which cells are not cleaved.

Specifically, Escherichia coli , P vulgaris ( Proteus vulgaris ) as Gram-negative bacteria, L. Listeria monocytogenes, S. Epidermis ( Staphylococcus epidermidis ) was used, Escherichia coli ) (ATCC 25922), and Listeria monocytogenes (ATCC 19115) were sold from the "American Type Culture Collection" and Staphylococcus epidermidis) (KCTC 3096), Proteus vulgaris (Proteus vulgaris ) (KCTC 2433) was distributed from "Korean Collection for Type Cultures" and each strain was isolated from LB medium (1% bacto tryptone, 0.5% bacto yeast extract, 1% sodium chloride; Sigma, USA) pH 5.5, pH 6.2, Incubate at pH 8.5 and pH 9.8 until the mid-log phase, and then dilute to 1 x bacterium peptone medium (Difco, USA) at a cell concentration of 5 x 10 ⁵ cells / 100 μl of micro titrate plates ( Nunc, USA). The antimicrobial peptides and parent peptides of the present invention synthesized in Example 1 were diluted at pH 5.5, pH 6.2, pH 8.5, and pH 9.8 by 1 / 2-fold from 96 wells, respectively, and then added to the plate at 37 ° C. Were cultured for 12 hours, and the absorbance was measured at a wavelength of 620 nm using a micro titrate plate reader (Merck Elisa reader, Germany) to determine the MIC value of each strain, and the results are shown in Tables 2 to 5 below. It was.

Antibacterial against Escherichia coli (Gram negative bacteria) Of peptide  Antimicrobial activity
Peptide Growth Inhibitory Concentration (μM) Escherichia coli (gram negative) pH 4.0 pH 5.5 pH 6.2 pH 7.4 pH 8.5 pH 9.8 Clavaspirin 2 2 32 64 128 180 CSP-4 2 2 4 4 16 96

P . To vulgaris (gram negative bacterium)  Against antibacterial Of peptide  Antimicrobial activity
Peptide Growth Inhibitory Concentration (μM) P. Bvlgari (gram negative bacteria) pH 4.0 pH 5.5 pH 6.2 pH 7.4 pH 8.5 pH 9.8 Clavaspirin 8 8 32 64 80-90 85-100 CSP-4 4 4 8-10 8 24 83

L . To monocytogenes (gram negative bacteria)  Against antibacterial Of peptide  Antimicrobial activity
Peptide Growth Inhibitory Concentration (μM) L. Monocytogen (gram positive bacteria) pH 4.0 pH 5.5 pH 6.2 pH 7.4 pH 8.5 pH 9.8 Clavaspirin 16 16 64 64 120-130 150 CSP-4 4 4 4 4 16 80-90

S . To epidermis (gram negative bacteria)  Against antibacterial Of peptide  Antimicrobial activity
Peptide Growth Inhibitory Concentration (μM) S. epidermis pH 4.0 pH 5.5 pH 6.2 pH 7.4 pH 8.5 pH 9.8 Clavaspirin 4 4 32 32 60-70 128 CSP-4 4 4 4 4 8 60-65

As a result, as shown in Tables 2 to 5, the peptide (CSP-4) described in SEQ ID NO: 2 of the present invention had the same or lower MIC at pH 5.5 or less than the peptide (Clavaspirin) described in SEQ ID NO: 1, and pH 6.2, pH 7.4, pH 8.5, pH 9.8 showed significantly higher antimicrobial activity than the parent peptide (Clavaspirin). Therefore, the parent peptide (Clavaspirin) in Gram-positive bacteria and Gram-negative bacteria showed a strong activity at pH 5.5 or lower, but low activity in all strains above pH 6.2, whereas the antimicrobial peptide (CSP-4) of the present invention in most strains It was found to show high antimicrobial activity regardless of pH (Tables 2 to 5).

< Experimental Example  2> antibacterial Of peptide  Hemolytic Activity Assay

The present inventors measured the erythrocyte hemolytic activity of the peptides in order to compare the cytotoxicity of the peptides prepared by the method of <Example 1>.

First, dilute human erythrocytes with phosphate buffer (PBS, pH 7.0) to a concentration of 8% and continuously sequence the peptides described in SEQ ID NOS: 1 to 2 in Table 1 at concentrations from 12.5 μM / well to 1/2 It was diluted with and reacted at 37 ℃ for 1 hour. Then, the amount of hemoglobin contained in the supernatant by centrifugation at 1,000 g was investigated by measuring the absorbance at 414 nm wavelength. In order to investigate the degree of cell destruction, 1% Triton X-100 (sigma, USA) was added to human red blood cells to measure the absorbance of the supernatant. The cell destruction capacity of the 1% Triton X-100 was 100%, and the red blood cell destruction ability of the antimicrobial peptide (CSP-4) and the parent peptides (Clavaspirin) of the present invention was calculated according to [Formula 1] below. The results are shown in Table 6 below.

Equation 1

In the above formula, absorbance A is the absorbance of the peptide solution at 414 nm wavelength, absorbance B is the absorbance of PBS at 414 nm wavelength and absorbance C is the absorbance of 1% Triton X-100 at 414 nm wavelength.

Antibacterial Of peptide  Hemolytic Activity Measurement Peptides % Erythrocyte destructive capacity (each peptide concentration, μM) 200 100 50 25 12.5 Clavaspirin 66 62 42 22 7 CSP-4 10 One 0 0 0

As a result, as shown in Table 3, the parent peptide (Clavaspirin) described in SEQ ID NO: 1 had a hemolytic action of 66% at 200 µM, whereas the peptide (CSP-4) described in SEQ ID NO: 2 of the present invention had the same concentration. Hemolysis occurred in only 10% of cases.

Therefore, it can be seen that the antimicrobial peptide of the present invention hardly occurs cytotoxicity (Table 6).

< Experimental Example  3> antibacterial Of peptide  Cytotoxicity Analysis in Normal Cell Lines

The inventors of the present invention, using a human keratinocyte cell line (HaCaT cell line, Dr. NE. Fusenig, Heidelberg, Germany) to confirm the cytotoxicity of the peptides prepared by the method of <Example 1> Toxicity was measured.

Specifically, a human keratinocyte cell line (HaCaT cell line) cultured in DMEM medium containing 10% FBS (Fetal Bovine Serum) was dispensed in 96 well plates by 3 × 10 ³ and incubated for 24 hours. 5% CO ₂ for 24 hours by treating each peptide prepared in Example 1> by concentration And reacted in an incubator. After incubation, 20 μl of Thiozolyl Blue Tetrazolium Bromide (MTT) solution dissolved in phosphate buffered saline (PBS) at a concentration of 5 mg / ml was added to each well and reacted for 4 hours. The supernatant was removed, 200 μl of DMSO was added to melt the formed MTT crystal, and the results were confirmed at 560 nm. The results are shown in Table 7.

Antibacterial Peptides  Cytotoxicity measurement Peptides % HaCa T cell viability (each peptide concentration, μM) 200 100 50 25 12.5 Clavaspirin 16 66 100 100 100 CSP-4 88 100 100 100 100

As a result, as shown in Table 7, the parent peptide (Clavaspirin) described in SEQ ID NO: 1 in the human keratinocyte cell line (HaCaT cells) shows high toxicity, whereas the present invention described in SEQ ID NO: 2 of the present invention. Peptide (CSP-4) showed little cytotoxicity.

Thus, the antimicrobial peptide of the present invention was found to show little cytotoxicity (Table 7).

< Experimental Example  4> antibacterial Of peptide pH Agglomeration phenomenon

In order to compare the aggregation of the peptides prepared by the method of <Example 1> at pH 5.5 and pH 7.4, the inventors measured the values by treating the peptides with respective peptide concentrations. In order to confirm the morphological changes of the antimicrobial peptides in 10 mM sodium phosphate buffer at pH 5.5 and pH 7.4, the scattering of light at 90 ° C. was performed using the Perkin-Elmer LS55 fluorometer. As confirmed by the antimicrobial peptide (CSP-4), the peptide was reacted for 12 hours by treating different concentrations in pH 5.5 and pH 7.4 buffer, and the result was confirmed by 400 nm on a fluorescence spectrometer.

As a result, the parent peptide (Clavaspirin) described in SEQ ID NO: 1 as shown in Figure 1 confirmed the phenomenon of high light scattering occurs at pH 7.4, but the antimicrobial peptide of the present invention described in SEQ ID NO: 2 (CSP-4) Shows low light scattering at pH 5.5 and pH 7.4.

Therefore, it was found that the antimicrobial peptide (CSP-4) of the present invention was not significantly affected by the pH change (FIG. 1).

< Experimental Example  5> pH Antibacterial Of peptide Saitoks -Green( SYTOX - Green A) fluorescence light intensity analysis

The inventors of the peptide prepared by the method of <Example 1> at the time of pH 4.0, pH 5.5, pH 6.2, pH 7.4, pH 8.5, pH 9.8 SYTOX-Green fluorescence and brightness Measured.

Specifically, E. coli was incubated at 37 ° C. with LB broth at pH 5.5 and pH 7.4, and E. coli cells were adjusted to 2 × 10 ⁷ cells / ml in 10 mM sodium phosphate buffer. The E. coli cells were treated with 1 μM of SYTOX-Gree and reacted in a light incubator for 15 to 20 minutes, and then the Clavaspirin and CSP-4 peptides were minimized. Inhibition concentration (MIC) and the value of fluorescence light intensity increase from the SYTOX-Green (SYTOX-Green) according to the pH was determined by measuring the emission wavelength at 485nm and radiation wavelength 520nm, the results are shown in FIG.

As a result, as shown in FIG. 2, the peptide described in SEQ ID NO: 2 of the present invention (CSP-4) has a parent peptide of the present invention at pH 5.5 compared to the parent peptide (Clavaspirin) described in SEQ ID NO: 1. The fluorescence light intensity was about 2 times higher at 60 minutes, and the parent peptide showed no response at pH 7.4, but the antimicrobial peptide of the present invention showed very high fluorescence light intensity.

In addition, E. coli was incubated at 37 ° C. with LB broth at pH 4.0, pH 5.5, pH 6.2, pH 7.4, pH 8.5, and pH 9.8, and then E. coli cells were added at 2 × 10 ⁷ cells / ml in each pH of 10 mM sodium phosphate buffer. Fit. These E. coli cells were treated with 1 μM of SYTOX-Gree and reacted in a light incubator for 15 to 20 minutes, followed by parental peptide (Clavaspirin) and the antimicrobial peptide of the present invention (CSP-4). ) At a minimum inhibitory concentration (MIC) and record the fluorescence light intensity 30 minutes after beginning to measure the fluorescence light intensity increase from SYTOX-Green with pH at 485 nm and 520 nm. (Table 8). As a result, the antimicrobial peptide of the present invention showed high fluorescence intensity regardless of pH.

pH In accordance Of peptide  Fluorescent light intensity
Peptide Fluorescent light intensity (arbiratry uints) Escherichia coli pH 4.0 pH 5.5 pH 6.2 pH 7.4 pH 8.5 pH 9.8 MIC of
Clavaspirin 66 ± 5.21 ¹⁾ 65 ± 6.45 38 ± 4.23 12 ± 1.25 15 ± 3.48 10 ± 2.36 MIC of
CSP-4 72 ± 6.33 78 ± 5.46 129 ± 8.17 218 ± 8.55 115 ± 6.27 13 ± 1.92

¹⁾ Mean ± SD (n = 5)

Therefore, the antimicrobial peptide (CSP-4) of the present invention showed strong fluorescence intensity at different pHs as compared with the parent peptide Clavaspirin, and it was found that E. coli membrane destruction showed a stronger action than the parent peptide (FIG. 2). And Table 8).

< Experimental Example  6> artificially made giant Single membrane  parcel( GUVs Antibacterial The peptide  Each pH Structural phenomena represented by

The present inventors treated the peptides with artificial liposomes made artificially from the peptides prepared by the method of <Example 1> to measure structural phenomena appearing according to respective pH.

First, the giant unilamellar vesicles (GUVs) were made by diluting several phospholipids and stimulating them with electrical stimulation. Phosphatidyl ethanolamine-rhodamine (PE / PG / PE-Rhodamin, 69/30/1) was diluted. The diluted aqueous solution of phospholipid was loaded with 100 μl of diluted lipid on indium tin oxide (ITO) coated glass (25 × 35 × 1.1 mm Sigma-Aldrich, st, Louis, MO) and then phospholipid at 600 rpm for 5 minutes in spin coat. Spread out. Thereafter, the lipid and the diluted organic solvent are removed for at least 2 hours in a vacuum evacuation state. The combination of lipid-attached indium tin oxide (ITO) and non-attached indium tin oxide (ITO) in the middle of the electroformation chamber (25x25x1 mm) for the urea of dimethylsiloxane polyurea fiber (dimethylsiloxane) The chamber is made by inserting, and injected into the chamber at each pH with 5 mM HEPES buffer (pH 5.5, pH 7.4) added with 0.1 M sucrose. In addition, a 1.7 V electrode and 10 Hz frequency were injected into the indium tin oxide (ITO) glass using a generator (generator-Agilent 33220A, Agilent Technology, US), followed by reaction for 4 hours and 10 Hz. Convert the frequency and react for 10 minutes.

The liposome aqueous solution is removed from the electroforming chamber, and 5 mM HEPES buffer (pH 5.5, pH 7.4) with 0.1 M glucose is added to the liposome aqueous solution. This is to prevent large single-membrane liposomes (GUVs) from bursting by osmotic phenomena and to allow for the deposition of well on slide glass using the weight of external glucose (180.16 MW) and internal sucrose (342.30 MW). to be. Then, the phenomenon was photographed / analyzed using a digital CCD camera (DP71, Olympus) of a fluorescence phase contrast microscope (Ix71, Olympus, Tokyo, Japan).

As a result, as shown in FIG. 3, A (pH 5.5) and B (pH 7.4), respectively, at different pHs, the parent peptide (Clavaspirin) and the antimicrobial peptide (CSP-4) of the present invention, respectively, can be seen. The upper part of A (pH 5.5) is the parent peptide (Clavaspirin), and the lower part is the antimicrobial peptide (CSP-4) of the present invention, compared to the parent peptide. It was found that the top of B (pH 7.4) was the parent peptide (Clavaspirin), which hardly occurs even after a long time to act on the lipid membrane, but the antimicrobial peptide (CSP-4) of the present invention was very fast. It was confirmed to act by destroying the lipid membrane.

Therefore, the antimicrobial peptide of the present invention is hardly affected by the reaction of pH, and it can be seen that it acts on the lipid membrane in a short time and destroys the membrane (FIG. 3).

As a result, the antimicrobial peptides according to the present invention have little cytotoxicity, no agglomeration phenomenon, show strong antimicrobial activity regardless of pH, and lipids in a quick time regardless of pH, compared to the antimicrobial peptides set forth in SEQ ID NO: 1. It was confirmed that it acts on the membrane to destroy the membrane.

The following are some examples of formulations containing the antimicrobial peptides of the present invention, but the present invention is not limited thereto.

< Manufacturing example  1> Preparation of Pharmaceutical Formulations

<1-1> tablet (direct pressure)

After sifting 5.0 mg of the antibacterial peptide, 14.1 mg of lactose, 0.8 mg of crospovidone USNF, and 0.1 mg of magnesium stearate were mixed and pressed to prepare a tablet.

<1-2> tablets (wet granulation)

After sifting 5.0 mg of the antimicrobial peptide, 16.0 mg of lactose and 4.0 mg of starch were mixed. 0.3 mg of Polysorbate 80 was dissolved in pure water and then an appropriate amount of this solution was added and then atomized. After drying, the fine particles were sieved and mixed with 2.7 mg of colloidal silicon dioxide and 2.0 mg of magnesium stearate. The fine particles were pressed to prepare tablets.

<1-3> with powder Capsule

After sifting 5.0 mg of the antibacterial peptide, it was mixed with 14.8 mg of lactose, 10.0 mg of polyvinyl pyrrolidone, and 0.2 mg of magnesium stearate. The mixture was extruded through a hard No. 5 gelatin capsules.

<1-4> injection

Injectables were prepared by containing 100 mg of antimicrobial peptide, and other than 180 mg of mannitol, 26 mg of Na ₂ HPO ₄ 12H ₂ O and 2974 mg of distilled water.

< Manufacturing example  2> Manufacturing of food

<1-2> Preparation of antimicrobial food

Antibacterial Peptide 100mg

Vitamin mixture quantity

70 [mu] g of vitamin A acetate

Vitamin E 1.0 mg

0.13 mg vitamin B1

0.15 mg of vitamin B2

0.5 mg vitamin B6

0.2 [mu] g vitamin B12

10 mg vitamin C

Biotin 10 μg

Nicotinic acid amide 1.7 mg

50 ㎍ of folic acid

Calcium pantothenate 0.5 mg

Mineral mixture quantity

1.75 mg of ferrous sulfate

0.82 mg of zinc oxide

Magnesium carbonate 25.3 mg

15 mg of potassium phosphate monobasic

Secondary calcium phosphate 55 mg

Potassium citrate 90 mg

100 mg of calcium carbonate

24.8 mg of magnesium chloride

Although the composition ratio of the above-mentioned vitamin and mineral mixtures is mixed with a component suitable for a health food in a preferred embodiment, the composition ratio may be arbitrarily modified, and the above components are mixed according to a conventional antimicrobial food production method. Next, the granules may be prepared and used for preparing the antimicrobial food composition according to a conventional method.

<1-2> Preparation of antibacterial beverage

       Antibacterial Peptide 100mg

       Citric acid 100 mg

       100 mg of oligosaccharide

       Plum concentrate 2 mg

       100 mg taurine

       Purified water was added to 500 ml

After mixing the above components according to a conventional auxiliary beverage manufacturing method, and stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 1 L container, sealed sterilization and then refrigerated Used to prepare the healthy beverage composition of the invention.

Although the composition ratio is a mixture of the components suitable for the preferred beverage as a preferred embodiment, the blending ratio may be arbitrarily varied according to the regional and national preferences such as the demand level, the demanding country, and the intended use.

The present invention is not limited to the above-described embodiments, experimental examples, and preparation examples, which can bring about various modifications and alterations by those skilled in the art, and can be applied to the human body thinly according to its efficacy. And ointments, which may be used in the manufacture, which fall within the spirit and scope of the invention as defined in the appended claims.

According to the present invention, it becomes possible to produce antimicrobial peptides which have little cytotoxicity and are not affected by pH change and have excellent antimicrobial activity. Therefore, the antimicrobial peptide of the present invention can be usefully used in the field of prevention and treatment of pathogenic bacterial infections.

<110> Industry-Academic Cooperation Foundation, Chosun University <120> Novel analogues of antibacterial peptide derived from Styela          clava and the use <130> 12p-02-30 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 23 <212> PRT <213> Styela clava <400> 1 Phe Leu Arg Phe Ile Gly Ser Val Ile His Gly Ile Gly His Leu Val   1 5 10 15 His His Ile Gly Val Ala Leu              20 <210> 2 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> CSP-4 <400> 2 Phe Leu Arg Phe Ile Gly Ser Val Lys His Gly Lys Gly His Leu Val   1 5 10 15 His His Ile Gly Val Ala Leu              20

Claims (11)

An antibacterial peptide in which isoleucine (I) located at amino acids 9 and 12 from the N-terminus of the amino acid sequence of SEQ ID NO: 1 is substituted with lysine (Lysine, K).
According to claim 1, wherein the peptide is an antimicrobial peptide having an amino acid sequence of SEQ ID NO: 2.
According to claim 1, wherein the peptide is an antimicrobial peptide, characterized in that it has an antimicrobial activity against Gram-negative bacteria or Gram-positive bacteria.
4. The method according to claim 3, wherein the gram-negative bacteria are Escherichia coli and coli) or Proteus vulgaris (Proteus vulgaris), the Gram-positive bacteria are Listeria monocytogenes presence (Listeria monocytogenes ) or Staphylococcus epidermidis .
The antimicrobial peptide of claim 1, wherein the peptide exhibits antimicrobial activity in acid, neutral, and alkaline.
The antimicrobial peptide of claim 5 wherein the peptide exhibits antimicrobial activity at pH 6 to 8.5.
According to claim 1, wherein the peptide is an antimicrobial peptide, characterized in that the cytotoxicity is reduced.
Antimicrobial or antiseptic composition containing the peptide of claim 1 as an active ingredient.
An antibiotic pharmaceutical composition comprising a therapeutically effective amount of the peptide according to claim 1 as an active ingredient.
A pharmaceutical composition for the prevention and treatment of pathogenic bacterial infections containing a therapeutically effective amount of the peptide according to claim 1 as an active ingredient.
An antibiotic food additive containing the peptide of claim 1 as an active ingredient.

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