KR102032056B1 - Novel tetrapeptide and composition for anti-algae or anti-bacteria containing the same - Google Patents

Abstract

The present invention relates to a novel tetrapeptide and a composition for anti-red tide or anti-bacteria containing the same. The tetrapeptide of the present invention is excellent in anti-red tide effect or antibacterial activity that selectively inhibits only toxic or harmful red tide organisms except harmless or non-toxic red tide organisms. In addition, the present invention is derived from a decomposition product of Scomber japonicus waste liquid, and can provide a method for recycling fish waste liquid with economic feasibility, thereby capable of being usefully used in related industries.

Description

Novel tetrapeptide and composition for anti-algae or anti-bacteria containing the same

The present invention relates to a novel tetrapeptide and a composition for a bath or antimicrobial containing the same.

Red tides (Red tides or Harmful Algal Blooms) Plankton that lives in the oceans reproduces in large quantities, discoloring the water color to brown or red and destroying the ecosystem. There are more than 200 species of red tide creatures around the world, and about 40 species of red tide creatures appear off the coast of Korea. The damage caused by the red tide can be divided into two categories: firstly, large-scale death or damage of fish and shellfish, and second, damage to the tourism industry. Specifically, if the red tide is toxic and toxic, fish and fish eat poisonous red tide in the process of sucking water to catch food, and the poisoning of the toxin eventually occurs. When a red tide breeds in large quantities and consumes nutrients such as nitrogen and phosphorus, it dies and sinks to the sea floor. At this time, the bacteria break down them, and in this process, the bacteria use up all the oxygen and the bottom becomes anoxic. Shellfish that are highly motile will suffocate and die. Red tide also occurs frequently in late spring and summer, when the water temperature is high and not windy. This is the time when many tourists go swimming or watching the beach. When swimming, many European countries such as the United States, Spain, and Italy shut down the beach because they can drink water containing toxic red tide and die or become paralyzed. As a result, tourism damages are enormous. In Korea, red tide occurs in the wide coastal areas of the south coast. Red tide occurs in the east and west coasts.

Microbes are everywhere around us. In particular, telephones, mobile phones, flooring, refrigerators, air-conditioners, humidifiers, toys, wallpaper, and gas kits that we can easily encounter in everyday life can easily cause various kinds of bacteria and molds to produce odors, even the human body. Harmful bacteria can multiply and cause various diseases, and serious social problems such as food poisoning caused by Escherichia coli O-157 and Legionella bacteria that cause pneumonia occurring in baths. Therefore, a high functional antimicrobial material is required to prevent this. To date, many researches on these antimicrobial materials have been conducted, and recently, studies to separate new antimicrobial materials from natural products have been actively conducted.

An object of the present invention can provide a tetrapeptide comprising the amino acid sequence represented by SEQ ID NO: 1.

It is also an object of the present invention can provide a composition for inhibiting red tide containing the tetrapeptide.

It is also an object of the present invention can provide a pharmaceutical composition for antimicrobial containing the tetrapeptide.

In addition, an object of the present invention can provide an antimicrobial food composition comprising the tetrapeptide.

It is also an object of the present invention can provide an antimicrobial cosmetic composition comprising the tetrapeptide.

It is also an object of the present invention can provide an antimicrobial feed composition comprising the tetrapeptide.

It is also an object of the present invention can provide an antimicrobial biological pesticide composition comprising the tetrapeptide.

In order to achieve the above object, the present invention provides a tetrapeptide comprising the amino acid sequence represented by SEQ ID NO: 1.

In another aspect, the present invention provides a composition for inhibiting red tide containing the tetrapeptide.

The present invention also provides an antimicrobial pharmaceutical composition comprising the tetrapeptide.

The present invention also provides an antimicrobial food composition comprising the tetrapeptide.

The present invention also provides an antimicrobial cosmetic composition comprising the tetrapeptide.

The present invention also provides an antimicrobial feed composition comprising the tetrapeptide.

In another aspect, the present invention provides a biological pesticide composition for antimicrobial containing the tetrapeptide.

The tetrapeptide of the present invention is excellent in the tide effect that selectively inhibits only toxic or harmful red tide organisms except harmless or non-toxic red tide organisms. Moreover, it is excellent in the antimicrobial activity which suppresses gram negative bacteria and gram positive bacteria effectively. In addition, it is derived from the mackerel ( Scomber japonicas ) waste product decomposition products, and can be used in the relevant industries because it is possible to prepare a recycling method of the fish waste fluid and economical.

1 is a diagram showing the results of analyzing the RP-HPLC chromatogram of the tetrapeptide of the present invention.
Figure 2 is a diagram showing the results of analyzing the MS spectrum of the tetrapeptide of the present invention.
3 is a diagram showing the results of performing UPLC-ESI-MS / MS analysis for de novo sequencing for the analysis of the tetrapeptide of the present invention.
Figure 4 is a diagram showing the structure of the tetrapeptide of the present invention.
5 is a diagram confirming the morphological changes of H. triquetra, P. minimum and A. fundyense after treatment of the tetrapeptide of the present invention (C is the control group, T means the experimental group, A species H. triquetra , Class B P. minimum , Class C A. fundyense .)
6 is a diagram showing the results of confirming the degree of inhibition of S. costatum after treatment of the tetrapeptide of the present invention.
7 is a diagram confirming the inhibitory effect of a) E. coli and b) S. aureus after treating the tetrapeptide of the present invention.

An object of the present invention can provide a tetrapeptide comprising the amino acid sequence represented by SEQ ID NO: 1.

In the present invention, the term "tetrapeptide" is one of peptides classified in oligopeptides, and consists of only four amino acids linked by peptide bonds. Tetrapeptides have strong pharmacological activity and exhibit affinity and specificity for various receptors in protein-protein signal transduction. Linear or cyclic tetrapeptides are present and can be cyclized by peptide bonds or covalent bonds.

The tetra peptide may be mackerel (Scomber japonicas) is derived from the waste decomposition products, the mackerel waste liquid decomposition product is inoculated with Bacillus (Bacillus), Breda ratio Bacillus (Brevibacillus) and their mixed culture and cultured in the decomposition.

The term "culture" in the present invention means to grow microorganisms under environmental conditions that are appropriately artificially controlled.

The mixed strain and mackerel waste liquid can be grown in a conventional medium. In order to cultivate a specific microorganism, a nutrient substance required by the microorganism to be cultured, that is, the culture medium, may be added and mixed with a material for a special purpose. The medium may also be referred to as an incubator or a culture medium, and is a concept that includes all natural, synthetic, or selective media.

The medium used for the cultivation should meet the requirements of the particular strain in an appropriate manner while controlling the temperature, pH, etc. in a conventional medium containing a suitable carbon source, nitrogen source, amino acids, vitamins and the like. Carbon sources that can be used include mixed sugars of glucose and xylose as the main carbon source, and sugars and carbohydrates such as sucrose, lactose, fructose, maltose, starch and cellulose, soybean oil, sunflower oil, castor oil, coconut Oils such as oils and fats, fatty acids such as palmitic acid, stearic acid, linoleic acid, alcohols such as glycerol, ethanol, organic acids such as acetic acid. These materials can be used individually or as a mixture. Nitrogen sources that can be used include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, anmonium carbonate, and ammonium nitrate; Amino acids such as glutamic acid, methionine, glutamine and organic nitrogen sources such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or its degradation product, skim soy cake or its degradation product Can be. These nitrogen sources may be used alone or in combination. The medium may include, as personnel, monopotassium phosphate, dipotassium phosphate and corresponding sodium-containing salts. Personnel that may be used include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts. In addition, as the inorganic compound, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate and calcium carbonate may be used. Finally, in addition to the above substances, essential growth substances such as amino acids and vitamins can be used.

In addition, suitable precursors to the culture medium may be used. The raw materials described above may be added batchwise, fed-batch or continuous in a suitable manner to the culture in the culture process, but is not particularly limited thereto. Basic compounds such as sodium hydroxide, potassium hydroxide, ammonia or acid compounds such as phosphoric acid or sulfuric acid can be used in an appropriate manner to adjust the pH of the culture.

In one embodiment of the present invention, there are 11 complex microorganisms used for the biological decomposition of mackerel waste, Pknufermbacteria (KACC 91274P), a mixed strain described in KR 10-0822239, and Wormbacteria, a mixed strain described in KR 10-1058245. (KACC 91409P) was used. Specifically, the Pknufermbacteria is a total of seven bacteria and Bacillus subtilis (DQ219358), Bacillus licheniformis ( Bacillus licheniformis ) (AY468373), Bacillus coagulans (AF466695), Bacillus Bacillus circulans (Y13064), Bacillus anthracis (AY138279), Bacillus fusiformis (AY548950) and Brevibacillus agri (AY319301). Wormbacteria are four species, Bacillus licheniformis (EF113324), Bacillus cereus (DQ923487), Brevibacillus agri (AJ586388) and Brevibacillus parabrevis Brevibacillus parabrevis (AB215101) was used to inoculate the mackerel waste solution and incubated and digested. The effect of tide tide on harmful or toxic red algae microalgae was identified and the tetrapeptides of the invention were identified in the mackerel wastewater degradation products.

The tetrapeptide does not inhibit the activity of harmless or nontoxic red tide organisms, and may selectively inhibit the activity of toxic or harmful red tide organisms. Wherein the toxic or harmful red tide organisms hetero kapsa in (Heterocapsa), pro Centrum in (Prorocentrum), Alexandria Solarium in (Alexandrium), kokeulrodinium in (Cochlodinium), Jiro di nium in (Gyrodinium), Dino pi system in (Dinophysis ), pseudo-Chemnitz cyano in (Pssudo-nitzschia) and load nodi nium in (Gymnodinium) at least one member selected from the group consisting of: or, preferably, the hetero kapsa in (Heterocapsa), Centrum in (Prorocentrum) to Pro or Alexandrite Alexandrium , more preferably Heterocapsa triquetra , Prorocentrum minimum , Alexandrium catenella and Alexandrium fundyense , The tetrapeptide of the present invention is not limited thereto, as long as it can inhibit the wake trap.

The harmless sex or toxic red tide organisms skeletal Loreto Cinema in (Skeletonema), Quito-year-old Ross in (Chaetoceros), Tala's coming LA (Thalassiosira), yukam Pia in (Eucamphia), Akashi right in (Akashiwo), Serra tium in ( Ceratium ), the genus Eutreptiella and Heterosigma are one or more selected from the group consisting of, preferably, Skeletonema , and more preferably Skeletonema Costatum ( Skeletonema costatum ) (diatom), but is not limited thereto.

The tetrapeptide may have antimicrobial activity against Gram negative bacteria, Gram positive bacteria or antibiotic resistant bacteria. The gram-negative bacteria is an S. genus Escherichia (Escherichia) or Shigella in (Shigella), preferably not limited to Escherichia coli (Escherichia coli) and, to this. The Gram-positive bacteria is Staphylococcus , preferably Staphylococcus aureus , but is not limited thereto.

The antibiotic-resistant bacteria include Escherichia coli , Staphylococcus aureus , Streptococcus pyogenes , Pseudomonas aeruginosa , and Clostridium, which have antibiotic resistance. At least one selected from the group consisting of Clostridium difficile , Salmonella enteritidis and Salmonella typhimurium , but is not limited thereto.

In the present invention, the Staphylococcus aureus ( Staphylococcus aureus ) is the main antibiotic resistance bacteria, depending on antibiotic resistance and degree, multi-drug resistant Staphylococcus aureus (MDRSA), methicillin resistant yellow grapes can be classified into aureus (methicillin-resistant Staphylococcus aureus, MRSA ), vancomycin-resistant Staphylococcus aureus (vancomycin resistant Staphylococcus aureus, VRSA) and vancomycin intermediate resistant Staphylococcus aureus (vancomycin intermediate Staphylococcus aureus, VISA) .

In the present invention, the antibiotic may be divided into beta-lactam-based or non-beta-lactam-based beta-lactam, lactam-based antibiotics, penicillin-based antibiotics, cephalosporin-based antibiotics, carbapenem-based May contain antibiotics. In addition, non-beta-lactam antibiotics include aminoglycoside series, tetracycline series, macrolide series, quinolone series, rifampicin series, sulfonamides series, Polypeptide-based, Lincosamide-based, fluoroquinolone-based and Sephalosporin-based, and antibiotic-resistant bacteria of the present invention may include resistant bacteria caused by the antibiotic can do.

The range of tetrapeptides according to the present invention includes proteins having the amino acid sequence represented by SEQ ID NO: 1 isolated from the mackerel waste separation product and functional equivalents of the proteins. "Functional equivalent" means at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 70% of the amino acid sequence represented by SEQ ID NO: 1 as a result of the addition, substitution, or deletion of the amino acid; Refers to a protein having a sequence homology of 95% or more and exhibiting substantially homogeneous physiological activity with the protein represented by SEQ ID NO: 1. Tetrapeptides of the invention include not only proteins having their native amino acid sequence, but also amino acid sequence variants thereof are also included within the scope of the present invention.

A variant of a tetrapeptide means a protein in which the natural amino acid sequence of the tetrapeptide and one or more amino acid residues have different sequences by deletion, insertion, non-conservative or conservative substitution, or a combination thereof. Amino acid exchange in proteins and peptides that do not alter the activity of the molecule as a whole is known in the art (H. Neurode, R. L. Hill, The Proteins, Academic Press, New York, 1979). In addition, tetrapeptides or variants thereof can be extracted from nature or synthesized (Merrifleld, J. Amer. Chem. Soc. 85: 2149-2156, 1963) or by genetic recombination methods based on DNA sequences (Sambrook). et al, Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, 2nd edition, 1989).

The amino acid variation is made based on the relative similarity of amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, and the like. By analysis of the size, shape and type of amino acid side chain substituents, arginine, lysine and histidine are all positively charged residues; Alanine, glycine and serine have similar sizes; It can be seen that phenylalanine, tryptophan and tyrosine have a similar shape. Thus, based on these considerations, arginine, lysine and histidine; Alanine, glycine and serine; Phenylalanine, tryptophan and tyrosine are biologically equivalent functions.

In introducing mutations, the hydropathic index of amino acids can be considered. Each amino acid is assigned a hydrophobicity index depending on its hydrophobicity and charge: isoleucine (+4.5); Valine (+4.2); Leucine (+3.8); Phenylalanine (+2.8); Cysteine / cysteine (+2.5); Methionine (+1.9); Alanine (+1.8); Glycine (-0.4); Threonine (-0.7); Serine (-0.8); Tryptophan (-0.9); Tyrosine (-1.3); Proline (-1.6); Histidine (-3.2); Glutamate (-3.5); Glutamine (-3.5); Aspartate (-3.5); Asparagine (-3.5); Lysine (-3.9); And arginine (-4.5).

The hydrophobic amino acid index is very important in conferring the interactive biological function of proteins. It is known that substitution with amino acids having similar hydrophobicity indexes can retain similar biological activity. When introducing mutations with reference to the hydrophobicity index, substitutions are made between amino acids which exhibit a hydrophobicity index difference of preferably within ± 2, more preferably within ± 1, even more preferably within ± 0.5.

On the other hand, it is also well known that substitutions between amino acids having similar hydrophilicity values result in proteins with equivalent biological activity. As disclosed in US Pat. No. 4,554,101, the following hydrophilicity values are assigned to each amino acid residue: arginine (+3.0); Lysine (+3.0); Asphaltate (+ 3.0 ± 1); Glutamate (+ 3.0 ± 1); Serine (+0.3); Asparagine (+0.2); Glutamine (+0.2); Glycine (0); Threonine (-0.4); Proline (-0.5 ± 1); Alanine (-0.5); Histidine (-0.5); Cysteine (-1.0); Methionine (-1.3); Valine (-1.5); Leucine (-1.8); Isoleucine (-1.8); Tyrosine (-2.3); Phenylalanine (-2.5); Tryptophan (-3.4).

When introducing mutations with reference to hydrophilicity values, substitutions are made between amino acids which exhibit a hydrophilicity value difference of preferably within ± 2, more preferably within ± 1 and even more preferably within ± 0.5.

Amino acid exchange in proteins that do not alter the activity of the molecule as a whole is known in the art (H. Neurath, R. L. Hill, The Proteins, Academic Press, New York, 1979). The most commonly occurring exchanges are amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Exchange between Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, Asp / Gly.

Tetrapeptides according to the invention can be synthesized by solid phase methods using conventional peptide synthesizers.

Typically, the amino acids used are used in protected form at the N-terminus and in protected side chains. As the protecting group for protecting the N-terminus, 9-fluorenylmethyloxycarbonyl group (Fmoc) is generally used. As the protecting group for protecting the reactive side chain, triphenylmethyl, butyloxycarbonyl, t-butyl ester, pentamethylchroman-6-sulfonyl and the like are used.

The resin used for the solid phase reaction is applied differently depending on whether the C-terminal form of the polypeptide is carboxylic acid or amide form. When the C-terminus is a carboxylic acid, 2-chlorotrityl chloride (CTC), 4-benzyloxybenzyl alcohol (Wang), hydroxymethylphenoxyacetyl (HMPA) resin may be used. When the C-terminus is an amide, the terminal of the resin is made of an amine group. For example, trialkoxybenzhydrylamine (Rink amide) or methylbenzhydrylamine (MBHA) resin may be used.

In using all of the above resins, peptides are synthesized from the C-terminus to the N-terminus. Upon coupling, the carboxyl group of the amino acid is activated with an activator, and after coupling, the N-terminal protecting group is removed with piperidine and the next coupling is performed. Carboxyl activators include HATU (N-[(dimethylamino-)-1H-1,2,3-triazolo [4,5-b] pyridin-1-ylmethylene] -N-methylmethanaminium hexafluorophosphate N-oxide) and 1-hydroxy -7-azabenzotriazole (1-hydroxy-7-azabenzotriazole, HOAt) or HBTU (N-[(1H-benzotriazol-1-yl) (dimethylamino) methylene] -Nmethylmethanaminium hexafluorophosphate N-oxide) and 1-hydroxy DIEA (N, N-diisopropylethylamine) can be used in benzotriazole (HOBt), and N-N'-diisopropylcarbodiimide (DIC) and 1-hydroxy-7 as addition aid Azabenzotriazole (HOAt) or 1-hydroxybenzotriazole (HOBt) can be used together.

In the present invention, after four amino acids are coupled, the N-terminal amine group has an acyl activator having a desired acyl group, for example, acyl anhydride and DMAP (4- (N, N-dimethylamino) pyridine) together to form an amide bond at the N-terminus or carboxylic acid (Fatty acid) with N, N-diisopropylcarbodiimide (DIC), 1-hydroxy-7-azabenzotriazole ( HOAt) or 1-hydroxybenzotriazole (HOBt) may be used together to form an amide bond. The polypeptide analogue is then separated from the solid resin by the cleavage solution comprising trifluoroacetic acid (TFA) and the protecting group bound to its side chain is removed.

The tetrapeptide of the present invention can be produced by inserting a gene encoding the same into a vector.

As used herein, the term “vector” refers to a plasmid vector as a means for expressing a gene of interest in a host cell; Phagemid vector; Cosmid vector; And viral vectors such as bacteriophage vectors, adenovirus vectors, retroviral vectors and adeno-associated virus vectors, and the like.

According to a preferred embodiment of the invention, the gene encoding the tetrapeptide in the vector of the invention is operatively linked with the promoter.

In the present invention, the term "operably linked" means a functional binding between a gene expression control sequence (eg, a promoter, signal sequence, or array of transcriptional regulator binding sites) and another gene sequence, thereby The regulatory sequence will control transcription and / or translation of the other gene sequence.

The vector system of the present invention may be constructed through various methods known in the art, and specific methods thereof are disclosed in Sambrook et al. (2001), Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, This document is incorporated herein by reference.

Vectors of the present invention can typically be constructed as vectors for cloning or vectors for expression. In addition, the vector of the present invention can be constructed using prokaryotic or eukaryotic cells as hosts. When the vector of the present invention is an expression vector and the prokaryotic cell is a host, powerful promoters capable of promoting transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoters, recA promoters, SP6 promoters, trp promoters and T7 promoters, etc.), ribosome binding sites for initiation of translation, and transcription / detox termination sequences. When E. coli (eg, HB101, BL21, DH5α, etc.) is used as the host cell, the promoter and operator sites of the E. coli tryptophan biosynthetic pathway (Yanofsky, C. (1984), J. Bacteriol., 158: 1018-). 1024) and a phage λ left promoter (pLλ promoter, Herskowitz, I. and Hagen, D. (1980), Ann. Rev. Genet., 14: 399-445) can be used as regulatory sites.

On the other hand, vectors that can be used in the present invention are plasmids often used in the art (eg, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14). , pGEX series, pET series and pUC19, etc.), phagemids (eg pComb3X), phage or virus (eg SV40, etc.) can be engineered.

On the other hand, when the vector of the present invention is an expression vector and the eukaryotic cell is a host, a promoter derived from the mammalian cell genome (eg, metallothionine promoter) or a promoter derived from a mammalian virus (eg adeno) Late viral promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter and four promoters of HSV) can be used and generally have a polyadenylation sequence as a transcription termination sequence.

The vector of the present invention may be fused with other sequences as needed to facilitate purification of the protein, and the sequences to be fused include, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA). ), FLAG (IBI, USA) and 6x His (hexahistidine; Quiagen, USA) and the like can be used, but are not limited thereto. In addition, the expression vector of the present invention may include an antibiotic resistance gene commonly used in the art as a selectable label, for example, ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin Resistance genes to neomycin and tetracycline.

It is also an object of the present invention can provide a composition for inhibiting red tide containing the tetrapeptide.

In the present invention, the term "red tide suppression" is used in various terms, such as drops, red tide prevention, algae and anti algae, to inhibit the growth, kill or inhibit the movement of algae causing red tide, etc. It means preventing the algae growth and removing the phenomenon causing red tide. In addition, it also includes the meaning of killing or inhibiting growth of algae causing red tide.

For the purposes of the present invention, it does not inhibit harmless or non-toxic algae, and has the effect of selectively inhibiting only harmful or toxic algae.

It is also an object of the present invention can provide a pharmaceutical composition for antimicrobial containing the tetrapeptide.

The pharmaceutical composition of the present invention may further include an adjuvant in addition to the tetrapeptide. The adjuvant may be used without any limitation as long as it is known in the art, but may further include the Freund's complete adjuvant or incomplete adjuvant to increase its effect.

The pharmaceutical composition according to the present invention may be prepared in a form in which the active ingredient is incorporated into a pharmaceutically acceptable carrier. Here, pharmaceutically acceptable carriers include carriers, excipients and diluents commonly used in the pharmaceutical art. Pharmaceutically acceptable carriers that can be used in the pharmaceutical compositions of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, Calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The pharmaceutical compositions of the present invention may be used in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral formulations, external preparations, suppositories, or sterile injectable solutions, respectively, according to conventional methods. .

When formulated, it may be prepared using conventional diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, and the like. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations contain at least one excipient in the active ingredient, for example starch, calcium carbonate, sucrose, lactose, gelatin It can be prepared by mixing. In addition to simple excipients, lubricants such as magnesium stearate, talc can also be used. Liquid preparations for oral administration include suspensions, solvents, emulsions, and syrups.In addition to commonly used diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. Can be. Formulations for parenteral administration include sterile aqueous solutions, water-insoluble solvents, suspensions, emulsions, lyophilized formulations and suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The pharmaceutical composition according to the present invention can be administered to a subject by various routes. All modes of administration can be expected, for example by oral, intravenous, intramuscular, subcutaneous, intraperitoneal injection.

The dosage of the pharmaceutical composition according to the present invention is selected in consideration of the age, weight, sex, physical condition, etc. of the individual. It is apparent that the concentration of tetrapeptide included in the pharmaceutical composition can be variously selected according to the object, and preferably, the pharmaceutical composition is included in the pharmaceutical composition at a concentration of 0.01 to 5,000 μg / ml. If the concentration is less than 0.01 μg / ml, the pharmaceutical activity may not appear, and when the concentration exceeds 5,000 μg / ml, the human body may be toxic.

The tetrapeptide according to the present invention may exhibit an antimicrobial effect against an infectious disease caused by a Gram-negative bacterium, Gram-positive bacteria or an antibiotic endogenous strain, thereby exhibiting a preventive and therapeutic effect of the infectious disease. The infectious diseases include acne, atopic dermatitis, purulent purpura, inflammatory periodontal disease, gastric disease, food poisoning, sepsis, septic shock, endocarditis, enteritis, meningitis, osteomyelitis, spinal caries, pneumonia, osteomyelitis, gonococcal infection and meningococcal infection At least one selected from the disease consisting of, but is not limited thereto.

In addition, an object of the present invention can provide an antimicrobial food composition comprising the tetrapeptide.

In addition to the tetrapeptide as an active ingredient, the food composition of the present invention may contain various flavors, natural carbohydrates, and the like as additional ingredients, as in general food compositions.

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. The aforementioned flavoring agents can advantageously be used natural flavoring agents (tautin), stevia extracts (for example rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.). The food composition of the present invention may be formulated in the same manner as the pharmaceutical composition, used as a functional food, or added to various foods. Foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gums, candy, ice creams, alcoholic beverages, vitamin complexes and health supplements. There is this.

In addition to the extract as an active ingredient, the food composition may include 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 thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the food composition of the present invention may contain a fruit flesh for producing natural fruit juice and fruit juice beverage and vegetable beverage.

The functional food composition of the present invention may be prepared and processed in the form of tablets, capsules, powders, granules, liquids, pills and the like for the purpose of preventing or treating ischemic brain injury. In the present invention, the "health functional food composition" refers to a food manufactured and processed using raw materials or ingredients having functional properties useful for the human body according to Act No. 6767 of the Health Functional Food Act. Ingestion is intended to obtain useful effects for health purposes such as nutrient control or physiological action. The health functional food of the present invention may include a conventional food additive, and the suitability as a food additive, unless otherwise specified, in accordance with the General Regulations of the Food Additives and General Test Methods approved by the Food and Drug Administration, etc. Judging by the standards and standards. Examples of the items listed in the 'Food Additive Revolution' include chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; Natural additives such as dark blue pigment, licorice extract, crystalline cellulose, high color pigment and guar gum; Mixed preparations, such as a sodium L- glutamate preparation, an addition of an alkali, a preservative preparation, and a tar coloring agent, etc. are mentioned. For example, the health functional food in the form of tablets is a mixture of the active ingredient of the present invention with excipients, binders, disintegrants and other additives by granulating in a conventional manner, and then compression-molded with a lubricant or the like, or The mixture can be compression molded directly. In addition, the health functional food in the form of tablets may contain a mating agent or the like as necessary. Hard capsules among the health functional foods in the form of capsules may be prepared by filling a conventional hard capsule with a mixture of the active ingredient of the present invention mixed with additives such as excipients, and the soft capsule agent may include the active ingredient of the present invention as an additive such as an excipient. It can be prepared by mixing the mixture with a capsule base such as gelatin. The soft capsule agent may contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like, as necessary. The health functional food in the form of a cyclic form can be prepared by molding a mixture of the active ingredient and the excipient, the binder, the disintegrant, etc. of the present invention by a known method, and can be avoided with sucrose or other epidermis, if necessary, Alternatively, the surface may be coated with a material such as starch or talc. The health functional food in the form of granules may be prepared by granulating a mixture of an excipient, a binder, a disintegrant, and the like of the active ingredient of the present invention in a known manner, and may contain a flavoring agent, a coagulant, etc. as necessary. Can be.

It is also an object of the present invention can provide an antimicrobial cosmetic composition comprising the tetrapeptide.

The composition containing the tetrapeptide of the present invention can be used in various ways for the prevention or improvement of antibiotic resistant bacteria infectious skin diseases. Examples of products to which the present composition can be added include cosmetics such as various creams, lotions, skins, essences, shampoos, rinses, cleansing agents, face washes, soaps, treatments, packs, and essences.

The cosmetic of the present invention comprises a composition selected from the group consisting of water soluble vitamins, oil soluble vitamins, polymer peptides, polymer polysaccharides, sphingolipids and seaweed extracts.

The water-soluble vitamins may be any compound that can be incorporated into cosmetics, but preferably vitamin B1, vitamin B2, vitamin B6, pyridoxine, pyridoxine, vitamin B12, pantothenic acid, nicotinic acid, nicotinic acid amide, folic acid, vitamin C, vitamin H, and the like. Their salts (thiamine hydrochloride, sodium ascorbate salt, etc.) and derivatives (ascorbic acid-2-sodium phosphate salt, ascorbic acid-2-magnesium phosphate salt, etc.) may also be used in the water-soluble vitamins that can be used in the present invention. Included. The water-soluble vitamins can be obtained by conventional methods such as microbial transformation, purification from microorganism culture, enzyme or chemical synthesis.

The oil-soluble vitamin may be any compound that can be incorporated into cosmetics, but preferably vitamin A, carotene, vitamin D2, vitamin D3, vitamin E (d1-alpha tocopherol, d-alpha tocopherol, d-alpha tocopherol), and the like. And derivatives thereof (ascorbic palmitate, ascorbic stearate, ascorbic acid dipalmitate, dl-alpha tocopherol acetate, dl-alpha tocopherolvitamin E, DL-pantothenyl alcohol, D-pantothenyl alcohol, pantothenyl ethyl) Ethers, etc.) are also included in the oil-soluble vitamins used in the present invention. Oil-soluble vitamins can be obtained by conventional methods such as microbial transformation, purification of microorganism culture, enzyme or chemical synthesis.

The polymer peptide may be any compound as long as it can be incorporated into cosmetics. Preferably, collagen, hydrolyzed collagen, gelatin, elastin, hydrolyzed elastin, keratin, and the like can be given. Polymeric peptides can be purified and obtained by conventional methods such as purification from microbial cultures, enzymatic methods or chemical synthesis methods, or can be purified and used from natural products such as dermis and pig silk such as pigs and cattle.

The polymer polysaccharide may be any compound as long as it can be blended into cosmetics. Preferably, hydroxyethyl cellulose, xanthan gum, sodium hyaluronate, chondroitin sulfate or a salt thereof (sodium salt, etc.) may be mentioned. For example, chondroitin sulfate or its salt can be normally purified from a mammal or fish.

The sphingolipid may be any compound as long as it can be blended into cosmetics. Preferably, ceramide, phytosphingosine, sphingosaccharide lipid, etc. may be mentioned. Sphingo lipids can usually be purified from mammals, fish, shellfish, yeasts or plants by conventional methods or obtained by chemical synthesis.

The seaweed extract may be any compound as long as it can be formulated into cosmetics. Preferably, the seaweed extract may include brown algae extract, red algae extract, green algae extract, and the like, and calginane, arginic acid, sodium arginate purified from these seaweed extracts, Potassium arginate and the like are also included in the seaweed extract used in the present invention. Seaweed extract can be obtained by purification from seaweed by conventional methods.

In addition to the said essential component, you may mix | blend the cosmetics of this invention with the other components normally mix | blended with cosmetics as needed. Other ingredients that may be added include fats and oils, moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, fungicides, antioxidants, plant extracts, pH adjusters, alcohols, pigments, flavorings, Blood circulation accelerators, cooling agents, restriction agents, purified water and the like. Examples of the fat or oil component include ester fats, hydrocarbon fats, silicone fats, fluorine fats, animal fats, and vegetable fats and oils.

As ester fats and oils, glyceryl tri2-ethylhexanoate, cetyl 2-ethylhexanoate, isopropyl myristate, butyl mystinate, isopropyl palmitate, ethyl stearate, octyl palmitate, isocetyl isostearate, and stearic acid Butyl, ethyl linoleate, isopropyl linoleate, ethyl oleate, isocetyl acid, isocetyl acid, isostyl acid mystearyl, isostaryl palmitate, octylate acid octylate dodecyl, isostearic acid isetyl, diethyl sebacate, adipine Acid isopropyl, isoalkyl neopentane, tri (capryl, capric acid) glyceryl, tri2-ethylhexanoic acid trimethylolpropane, triisostearic acid trimethylolpropane, tetra2-ethylhexanoic acid pentaelistitol Cetyl caprylate, lauric acid decyl, hexyl laurate, decyl myristin, myristin acid myristyl, myritic acid cetyl, stearyl stearate, decyl oleate, lysinooleic acid Teyl, isostearyl laurate, isotridecyl myristin, isocetyl palmitate, octyl stearate, isocetyl stearate, isodecyl oleate, octyl dodecyl oleate, octyl dodecyl linoleate, isopropyl isopropyl acid, 2-ethylhexanoic acid cetostearyl, 2-ethylhexanoic acid stearyl, hexyl isostearate, ethylene glycol dioctanoate, ethylene glycol dioleate, propylene glycol dicapric acid, propylene glycol di (capryl, capric acid), Dicaprylic acid propylene glycol, dicapric acid neopentyl glycol, dioctanoate neopentyl glycol, tricaprylate glyceryl, trifundecyl glyceryl, triisopalmitate glyceryl, triisostearate, octyl neopentane Dodecyl, isostearyl octanoate, octyl isononanoate, hexyldecyl neodecanoate, octyldodecyl neodecanoate, isocetyl isostearate, isostearic isostearic acid , Isostearic acid octyldecyl, polyglycerol oleic acid ester, polyglycerin isostearic acid ester, triisocetyl citrate, triisoalkyl citrate, triisooctyl citrate, lauric lactate, myritic lactate, cetyl lactate, octyl decyl lactate, citric acid Triethyl, acetyl triethyl citrate, acetyl tributyl citrate, trioctyl citrate, diisostearyl malic acid, 2-ethylhexyl hydroxystearate, di2-ethylhexyl succinate, diisobutyl adipic acid, diisopropyl sebacic acid Dioctyl sebacinate, cholesteryl stearate, cholesteryl isostearate, cholesteryl hydroxystearate, cholesteryl oleate, oleic acid dihydrocholesteryl, physosterate isostearic acid, phosphate oleate Reel, 12-Steloylhydroxystearate isocetyl, 12-Steloylhydroxystearate stearyl, 12-ste Ester systems such as alloylhydroxystearic acid isostearyl; and the like.

Examples of the hydrocarbon-based oils and fats include hydrocarbon oils such as squalene, liquid paraffin, alpha-olefin oligomer, isoparaffin, ceresin, paraffin, liquid isoparaffin, polybutene, microcrystalline wax, and vaseline.

Silicone oils include polymethylsilicone, methylphenylsilicone, methylcyclopolysiloxane, octamethylpolysiloxane, decamethylpolysiloxane, dodecamethylcyclosiloxane, dimethylsiloxane and methylcetyloxysiloxane copolymer, dimethylsiloxane and methylsteoxysiloxane copolymer, alkyl Modified silicone oil, amino modified silicone oil and the like.

Perfluoro polyether etc. are mentioned as fluorine-based fats and oils.

Animal or vegetable oils include avocado oil, almond oil, olive oil, sesame oil, rice bran oil, soybean oil, soybean oil, corn oil, rapeseed oil, almond oil, palm kernel oil, palm oil, castor oil, sunflower oil, grape seed oil. , Cottonseed oil, Palm oil, Cucumber nut oil, Wheat germ oil, Rice germ oil, Shea butter, Walnut colostrum oil, Marker demia nut oil, Meadow home oil, Egg yolk oil, Uji, Horse oil, Mink oil, Orange rape oil, Jojoba oil And animal or plant fats and oils such as candeler wax, carnava wax, liquid lanolin and hardened castor oil.

Examples of the moisturizing agent include a water-soluble low molecular moisturizer, a fat-soluble molecular moisturizer, a water-soluble polymer, and a fat-soluble polymer.

Water-soluble low molecular humectants include serine, glutamine, sorbitol, mannitol, pyrrolidone-sodium carboxylate, glycerin, propylene glycol, 1,3-butylene glycol, ethylene glycol, polyethylene glycol B (polymerization degree n = 2 or more), polypropylene Glycol (polymerization degree n = 2 or more), polyglycerol B (polymerization degree n = 2 or more), lactic acid, lactic acid salt, etc. are mentioned.

Examples of the fat-soluble low molecular humectants include cholesterol and cholesterol esters.

Examples of the water-soluble polymer include carboxyvinyl polymer, polyasparaginate, tragacanth, xanthan gum, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, carboxymethyl cellulose, water soluble chitin, chitosan, and dextrin. Can be.

Examples of the fat-soluble polymers include polyvinylpyrrolidone and eicosene copolymers, polyvinylpyrrolidone and hexadecene copolymers, nitrocellulose, dextrin fatty acid esters, polymer silicones, and the like. Examples of the emollient include long-chain acyl glutamic acid cholesteryl esters, hydroxystearic acid cholesterol, 12-hydroxystearic acid, stearic acid, rosin acid, lanolin fatty acid cholesteryl esters, and the like.

As surfactant, nonionic surfactant, anionic surfactant, cationic surfactant, amphoteric surfactant, etc. are mentioned.

Nonionic surfactants include self-emulsifying glycerin monostearate, propylene glycol fatty acid esters, glycerin fatty acid esters, polyglycerol fatty acid esters, sorbitan fatty acid esters, POE (polyoxyethylene) sorbitan fatty acid esters, POE sorbitan fatty acid esters, POE Glycerin fatty acid ester, POE alkyl ether, POE fatty acid ester, POE hardened castor oil, POE castor oil, POE and POP (polyoxyethylene and polyoxypropylene) copolymer, POE and POP alkyl ether, polyether modified silicone, lauric acid Alkanolamide, alkylamine oxide, hydrogenated soybean phospholipid, etc. are mentioned.

As anionic surfactant, fatty acid soap, alpha-acyl sulfonate, alkyl sulfonate, alkyl allyl sulfonate, alkyl naphthalene sulfonate, alkyl sulfate, POE alkyl ether sulfate, alkylamide sulfate, alkyl phosphate, POE alkyl phosphorus industry, alkylamide Phosphates, alkyloylalkyltaurine salts, N-acylamino acid salts, POE alkyl ether carboxylate salts, alkyl sulfosuccinate salts, sodium alkyl sulfo acetates, acylated hydrolyzed collagen peptide salts, perfluoroalkyl phosphate esters, and the like. have.

As cationic surfactant, alkyl trimethylammonium chloride, stearyl trimethyl ammonium chloride, stearyl trimethyl ammonium chloride, cetostearyl trimethyl ammonium chloride, distearyl dimethyl ammonium chloride, stearyl dimethyl benzyl ammonium bromide, behenyl trimethyl ammonium chloride, chloride Benzalkonium, diethylaminoethylamide stearate, dimethylaminopropylamide stearate, lanolin derivatives, quaternary ammonium salts, and the like. Examples of amphoteric surfactants include carboxybetaine type, amidebetaine type, sulfobetaine type, hydroxysulfobetaine type, amide sulfobetaine type, phosphobetaine type, aminocarboxylate type, imidazoline derivative type and amideamine type. An amphoteric surfactant etc. are mentioned.

Organic and inorganic pigments include silicic acid, silicic anhydride, magnesium silicate, talc, sericite, mica, kaolin, bengala, clay, bentonite, titanium film mica, bismuth oxychloride, zirconium oxide, magnesium oxide, zinc oxide, titanium oxide, aluminum oxide Inorganic pigments such as calcium sulfate, barium sulfate, magnesium sulfate, calcium carbonate, magnesium carbonate, iron oxide, ultramarine blue, chromium oxide, chromium hydroxide, calamine, and composites thereof; Polyamide, polyester, polypropylene, polystyrene, polyurethane, vinyl resin, urea resin, phenol resin, fluorine resin, silicon resin, acrylic resin, melamine resin, epoxy resin, polycarbonate resin, divinylbenzene and styrene copolymer, Organic pigments such as silk powder, cellulose, CI pigment yellow, CI pigment orange, and composite pigments of these inorganic pigments and organic pigments;

As organic powder, Metal soaps, such as a calcium stearate; Alkyl phosphate metal salts such as sodium cetylinate, zinc lauryl acid and calcium laurate; Acylamino acid polyvalent metal salts such as N-lauroyl-beta-alanine calcium, N-lauroyl-beta-alanine zinc, and N-lauroylglycine calcium; Amide sulfonic acid polyvalent metal salts, such as N-lauroyl-taurine calcium and N-palmitoyl-taurine calcium; N-epsilon-lauroyl-L-lysine, N-epsilon-palmitolyzine, N-alpha-paratoylol nitin, N-alpha-lauroyl arginine, N-alpha-cured fatty acid acyl arginine -Acyl basic amino acid; N-acyl polypeptides, such as N-lauroyl glycyl glycine; Alpha-amino fatty acids such as alpha-aminocaprylic acid and alpha-aminolauric acid; Polyethylene, polypropylene, nylon, polymethyl methacrylate, polystyrene, divinylbenzene and styrene copolymer, ethylene tetrafluoride and the like.

Examples of the ultraviolet absorber include paraaminobenzoic acid, ethyl paraaminobenzoate, amyl paraaminobenzoic acid, octyl paraaminobenzoate, ethylene glycol salicylate, phenyl salicylate, octyl salicylate, benzyl salicylate, butylphenyl salicylate, homomentyl salicylic acid and benzyl cinnamic acid. , Paramethoxy cinnamic acid-2-ethoxyethyl, paramethoxy cinnamic acid octyl, diparamethoxy cinnamic acid mono-2-ethylhexaneglyceryl, paramethoxy cinnamic acid isopropyl, diisopropyl and diisopropyl cinnamic acid ester mixture, Urocanoic acid, ethyl urocanate, hydroxymethoxybenzophenone, hydroxymethoxybenzophenonesulfonic acid and salts thereof, dihydroxymethoxybenzophenone, dihydroxymethoxybenzophenonedisulfonic acid sodium salt, dihydroxybenzo Phenone, tetrahydroxybenzophenone, 4-tert-butyl-4'-methoxydibenzoylmethane, 2,4,6-trianilino-p- (carbo-2'-ethylhexyl-1'-oxy)- 1,3,5-triazine, 2- (2-hi And the like can be mentioned hydroxy-5-methylphenyl) benzotriazole.

As fungicides, hinokithiol, trichloric acid, trichlorohydroxydiphenyl ether, chlorhexidine gluconate, phenoxyethanol, resorcin, isopropylmethylphenol, azulene, salicylic acid, ginxitlionone, benzalkonium chloride, photosensitive Sodium No. 301, sodium mononitroguicol, undecylenic acid, and the like.

Examples of the antioxidant include butylhydroxyanisole, propyl gallic acid, and erythorbic acid.

Examples of the pH adjuster include citric acid, sodium citrate, malic acid, sodium malate, fmaric acid, sodium pmarate, succinic acid, sodium succinate, sodium hydroxide, sodium dihydrogen phosphate, and the like.

Examples of the alcohol include higher alcohols such as cetyl alcohol.

In addition, the compounding component which may be added other than this is not limited to this, Moreover, any of the above components can be mix | blended within the range which does not impair the objective and effect of this invention.

The cosmetic of the present invention may take the form of a solution, an emulsion, a viscous mixture, or the like.

The components included in the cosmetic composition of the present invention may include components conventionally used in the cosmetic composition as an active ingredient, for example, conventional auxiliaries and carriers such as stabilizers, solubilizers, vitamins, pigments and fragrances Include.

Skin whitening and skin anti-aging cosmetic composition of the present invention can be prepared in any formulation commonly prepared in the art, for example, emulsion, cream, lotion, pack, foundation, lotion, essence, hair cosmetics, etc. Can be.

Specifically, the cosmetic composition of the present invention skin lotion, skin softener, skin toner, milk lotion, astringent, lotion, moisturizing lotion, nutrition lotion, massage cream, nutrition cream, moisturizing cream, hand cream, foundation, essence, nutrition essence, Formulations of packs, soaps, cleansing foams, cleansing lotions, cleansing creams, hair lotions, hair tonics, hair essences, hair shampoos, hair rinses, hair treatments, body lotions and body cleansers.

When the formulation of the present invention is a paste, cream or gel, animal fibers, plant fibers, waxes, paraffins, starches, tracantes, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicas, talc or zinc oxides may be used as carrier components. Can be.

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used, in particular in the case of a spray, additionally chlorofluorohydrocarbon, propane Propellant such as butane or dimethyl ether.

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

When the formulation of the present invention is a suspension, liquid carrier diluents such as water, ethanol or propylene glycol, suspension agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol esters and polyoxyethylene sorbitan esters, microcrystalline Cellulose, aluminum metahydroxy, bentonite, agar or tracant and the like can be used.

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

It is also an object of the present invention can provide an antimicrobial feed composition comprising the tetrapeptide.

Feed composition of the present invention replaces the existing antibiotics and inhibit the growth of harmful food pathogenic bacteria, improve the health of the animal body, improve the weight gain and meat quality of the livestock, and expect the effect of increasing the yield and immunity Can be. Feed composition of the present invention can be prepared in the form of fermented feed, compound feed, pellet form and silage and the like. The fermented feed can be prepared by fermenting an organic material by adding various microorganisms or enzymes other than the peptide of the present invention, the compound feed can be prepared by mixing the peptides of the present invention with various kinds of general feed. Pellet-type feed may be prepared by applying heat and pressure to the blended feed, etc. in a pellet machine, silage can be prepared by fermenting the cheonye feed with the microorganism according to the present invention. The wet fermented feed collects and transports organic materials such as food waste, mixes excipients for sterilization and moisture control at a certain ratio, and then ferments at a temperature suitable for fermentation for 24 hours, so that the water content is about 70%. It can manufacture by adjusting. Fermented dry fertilizer can be prepared by adjusting the wet fermented feed to further contain about 30% to 40% of water through a drying process.

Feed composition of the present invention may further comprise a component added to the conventional feed. One example of the ingredients added to the feed may include grain powder, meat powder, legumes and the like. The grain powder in the above may be used at least one selected from rice flour, wheat flour, barley flour, and corn flour. The meat powder may be a meat powder powdered at least one selected from chicken, beef, pork, and ostrich meat. In the above beans can be used one or more selected from soybeans, kidney beans, peas, and black beans.

The feed composition of the present invention may be added to any one or more selected from nutrients and minerals to increase the nutritional properties of the feed in addition to cereal powder, meat powder, and soybean, which are added to the conventional feed mentioned above. It may include one or more selected from antifungal agents, antioxidants, anticoagulants, emulsifiers, and binders to prevent the degradation of.

It is also an object of the present invention can provide an antimicrobial biological pesticide composition comprising the tetrapeptide.

The term "biotic pesticide" of the present invention refers to a medicament for controlling pests and weeds by spraying or spinning in the same form as chemical pesticides using natural insects, natural enemies, antagonistic microorganisms, etc. Bio pesticides for pest control such as natural enemies, bio pesticides for pesticide killing fungal nuclei, bio pesticides for weed control, and the like.

The term "biological pesticide auxiliary composition" of the present invention is to perform the auxiliary role, such as to improve the activity of the pest pesticide biopesticides, pesticide biopesticides, weed control water pesticides or to expand the range It means composition to say

The composition may be coated on a medical device, medical material or medical implant. For example, the coating composition may coat tissues or organs derived from organisms such as skin and teeth, and various medical facilities, equipment, instruments, temporary or permanent prosthetic implants, lenses, valves, face makers, and surgical instruments. It is possible to coat a medical object such as a pin, an insertion conduit, a catheter, but the scope of the present invention is not limited thereto.

The present invention will be described in more detail with reference to the following examples. However, the following examples are only intended to embody the contents of the present invention, and the present invention is not limited thereto.

Example 1 Biological Degradation of Mackerel Waste Fluid Medium

In order to prepare the mackerel waste liquid medium for isolating the peptide of the present invention, the mackerel ( Scomber japonicus ) purchased on the market was chopped after washing with distilled water. Distilled water was put into chopped mackerel and sterilized by autoclaving at 121 ° C. for 15 minutes. After mashed mackerel treated with autoclave, only the liquid part was sieved and collected, and then the lipid part was removed. The lipid-free sample was sterilized again using distilled water to Chemical Oxygen Demand (COD) 49000 ± 10% (w / v) and used as a medium.

The complex microorganisms used for the biological decomposition of the mackerel waste liquid was 11 species, using a conventionally known strain. Specifically, the mixed strain "Pknufermbacteria" (KACC 91274P) described in KR 10-0822239 and the mixed strain "Wormbacteria" (KACC 91409P) described in KR 10-1058245 were used.

The Pknufermbacteria is a total of seven bacteria, Bacillus subtilis (DQ219358), Bacillus licheniformis ( Bacillus licheniformis ) (AY468373), Bacillus coagulans (AF466695), Bacillus circulan ( Bacillus circulans ) (Y13064), Bacillus anthracis (AY138279), Bacillus fusiformis (AY548950) and Brevibacillus agri (AY319301). Wormbacteria are four species, Bacillus licheniformis (EF113324), Bacillus cereus (DQ923487), Brevibacillus agri (AJ586388) and Brevibacillus parabrevis Brevibacillus parabrevis (AB215101). These complex strains were adapted by incubating for 12 hours at 45 ° C. in a mackerel waste medium which had no bacterial antagonist to each other and was inoculated in twice the diluted mackerel waste medium. The complex strain was inoculated in mackerel waste medium 10% and incubated at 45 rpm for 48 hours at 130 rpm.

Example 2 Culture of Algae

Algae used in the present invention is a harmful algae heterocapsa triquetra , paralytic shellfish poisoning (PSP), causing red tide, breeding in large quantities and anoxic phenomenon is a pro Centrum minimum (Prorocentrum minimum), Alexandria Solarium categories Nella (Alexandrium catenella) and Alexandria Solarium Fun dyense (Alexandrium fundyense), phytoplankton, diatoms, a skeleton Loreto Cinema course tatum (Skeletonema costatum) (diatom). the algae Korea It was distributed by the Library of Marine Samples Korea (LIMS) of the Institute of Marine Science and Technology, except that 4 species of harmful algae, except Skeletonema Costatumum, were cultured in modified Guillard's f / 2-si medium (filtered seawater). , skeletal retrograde nematic course of tatum is Guillard's f / 2 cells were cultured in medium (filtered water) 5 species of birds all 40μmol / m ² / s in light Group, Gwangam conditions were passaged 14:10 hours, and cultured at 20 ℃ condition maintenance of cell concentrations after counting the cells using a blood cell calculation board (hemocytometer) and Sedwick chamber.

Example 3 Harmful Red Tide Inhibition Effect of Mackerel Waste Supernatant and Extraction and Separation of Effective Substances

3-1. Confirmation of Harmful Red Tide Inhibition Effect of Mackerel Waste Supernatant

In order to confirm the algal inhibitory effect of the mackerel waste supernatant, the inhibitory effect of the mackerel waste supernatant cultured for 0, 12, 24, 36, 48, 72 hours with the complex strain of Example 1 was treated to the harmful alga of Example 2 It was confirmed. The results are shown in Table 1 below.

As shown in Table 1, it was confirmed that the harmful algae inhibitory effect appeared in the mackerel waste supernatant fermented for 48 hours.

HAB species Nutrient broth culture supernatant (as a control) IC ₅₀ (mg / ml) H. triquetra - 0.68 P. minimum - 0.56 A. fundyense - 0.54

3-2. Extraction and Separation of Active Substances in the Mackerel Waste Supernatant

Based on the above results, the effective substance of the mackerel waste liquid supernatant was low molecular weight, and it was estimated to be a peptide or a substance to which the peptide was bound. Therefore, in order to extract and separate the active material of the mackerel waste liquid supernatant, acetone precipitation was used, primary separation was separated by size exclusion chromatography (SEC), and secondary separation was performed by prep RP-HPLC ( 1). Subsequently, tertiary separation was performed once more by size exclusion chromatography (SEC), and only fractions with harmful algal inhibitory activity were subjected to UPLC-ESI-MSMS analysis.

Specifically, to separate the acetone precipitation method, the supernatant was collected by centrifuging the mackerel waste liquid medium incubated with the complex strain of Example 1 for 48 hours at 8000 rpm for 15 minutes. The culture supernatant and cold acetone (-20 ° C.) were mixed in a 1: 4 ratio and reacted at −20 ° C. for 1 hour. The supernatant was discarded by centrifugation at 13000 rpm for 10 minutes to collect the precipitate. After extraction, the collected pellets were dissolved in distilled water (pH 8.0), filtered, and separated by size exclusion chromatography (SEC) for primary separation. The resin used was Sephadex G-25 (GE Healthcare, USA) and 10 g of resin was filled in a glass column (49 cm x 2 cm). Mobile phase was sampled every 8 minutes with sterile distilled water (pH 8.0), flow rate (0.5 ml / min), the elution solution was used 0.2M NaOH.

The primary separation fraction with wake-up activity was lyophilized for 4 days, the fraction powder was dissolved in tertiary distilled water, filtered and loaded into prep-RP HPLC (Preparative-Reverse Phase High Performance Liquid Chromatography) for secondary separation. . HPLC system was carried out by xbridge prep C18 column (19 X 250 mm, 10um, Waters) at Korea Basic Science Institute, Seoul, Korea. Trifluoroacetic acid (trifluoroacetic acid), solvent B 0.1% trifluoroacetic acid / acetonitrile gradually changed the solvent B to 60-95% (linear gradient). As a result, as shown in FIG. 2, one fraction having antitanking activity was obtained after 30 minutes.

Afterwards, the secondary fraction with wake-up activity was lyophilized for 4 days, and the tertiary separation was carried out using SEC filled with the same resin, and the operational condition was 10% (v / v) ethanol / sterilized mobile phase. Distilled water (pH 8.0), flow rate was 0.05ml / min, aliquoted every 2 minutes, and the elution solution was 0.2M NaOH. Thereafter, only the tertiary fraction having the wake trough activity was lyophilized.

After tertiary separation, only fractions with harmful algal inhibitory activity were subjected to UPLC-ESI-MSMS analysis. As a result, as shown in FIG. 3, Mass was found to be 539.2524.

Example 4 Identification of Effective Substances of the Present Invention

Examples 2 and 3 identified the active substances causing the tide effect of the fractions.

Specifically, UPLC-ESI-MS / MS analysis for de novo sequencing was performed, which was performed by MaXis HD (Bruker Daltonics) connected with UPLC system in the Center for Research Facilities (Pukyong national university, Busan, Korea). , Bremen, Germnay). The UPLC column was UPLC C-18 (10 cm x 2.1 cm, 1.7 μm) and 0.1% formic acid was used as mobile phase A, 0.1% formic acid / acetonitrile as mobile phase B. The analysis gave a linear gradient of 5-95% of the mobile phase at a flow rate of 0.2 ml / min at 214 nm. MS scan analysis was in electrospray positive mode with a capillary voltage of 4500V, and a plate offset of -500V. Collision energy was fixed at 33.6 eV after analysis at 30-40 eV. Scan analysis was in the range of 50-3000 m / z. Nitrogen was used as the desolvation gas (8.0 l / min, 200 ° C). All raw data were converted and analyzed with Bruker Compass Data Analysis software (version 4.2, Bruker Daltonics, Bremen, Germnay). In order to derive or discriminate the ion formula, Bruker Compass DataAnalysis software was used to analyze the following parameters. Parameters were enzyme: none, missed cleavage: 1, mass tolerance: 2 ppm. De novo peptide sequencing of MS / MS data was performed using BioTools (version 3.2 Bruker Daltonics, Bremen, Germnay). In addition, ion formula, MS / MS data, and amino acid sequence were searched in the database [(APD: Antimicrobial Peptide Database, http://aps.unmc.edu/AP/main.php ), (Norine: A konwledgebase dedicated to non -ribosomal peptides, http://bioinfo.lifl.fr/norine/ ), (CAMP: Database that has been developed for advancement of the present understanding on antimicrobial peptides, http://www.camp.bicnirrh.res.in/ ), (BIOPEP: Database of biologically active peptide sequence, http://www.uwm.edu.pl/biochemia/index.php/pl/biopep ), (UniProt: Resource of protein sequence and functional information, http: // www.uniprot.org/), (METLIN: Includes 961,829 molecules ranging from lipids, steroids, plant & bacteria metabolites, small peptides, carbohydrates, exogenous drugs / metabolites, central carbon metabolites and toxicants, https://metlin.scripps.edu /) ]. The results are shown in FIG.

As shown in FIG. 3, a peptide was identified as a result of performing UPLC-ESI-MS / MS analysis for de novo sequencing. The peptide sequence was KMNF. In addition, de novo sequencing of 539.2524 peak among the peaks shown in FIG. 2 confirmed four possible sequences. Sequences that were difficult to identify due to technical limitations of mass spectrometry and electrospray ionization (ESI) were identified by de novo sequencing. In view of the peculiarities of the peptides having conventional tide activity and antimicrobial activity, the peptides are amphiphilic and often cations. Thus, one amphiphilic and cationic sequence was selected from a total of four possible sequences. As a result, KMNF was finally screened.

Example 5 Secondary Structure and Characterization of Peptides

In order to analyze the secondary structure of the peptide identified in the active substance of the present invention and to characterize it, the secondary structure analysis of the peptide was performed using PEP-FOLD ( http://bioserv.rpbs.univ-paris-diderot.fr/ PEPFOLD ) was used. ((Camproux, AC, et al., 1999, Protein engineering, 12 (12), 1063-1073), (Camproux, AC, et al., 2004, Journal of molecular biology, 339 (3), 591-605) , (Tuffery, P., et al., 2005, Journal of computational chemistry, 26 (5), 506-513), (Tuffery, P., et al., 2005, Structure, Function, and Bioinformatics, 61 (4) , 732-740), Maupetit, J., et al., 2007, Structure, Function, and Bioinformatics, 69 (2), 394-408, (Maupetit, J., et al., 2010, Journal of computational chemistry, 31 (4), 726-738). Peptide characterization was confirmed using the Expert Protein Analysis System (ExPASy) and ProtParam algorithm. The results are shown in Table 2 and FIG.

As shown in Table 2 and Figure 4, the peptide of the present invention was a novel tetrapeptide having a linear structure (linear structure), 4-mer, molecular weight is 538.66 g / mol.

Sequence NH-KMNF-COOH Molecular weight 538.66 g / mol pI pH 9.91 Net charge (pH 7) One Hydrophobic (%) 50

Example 6 Tetrapeptide Synthesis of the Invention

The synthesis of tetrapeptide was performed by AnyGen Co., Ltd. (Kwangju, Korea) as a solid phase method using Fmoc (9-fluorenyl-methoxycarbonyl) chemistry (using PTI SYMPHONY Auto synthesizer). Wang resin (wang resin) was used as the C-terminal and Fmoc-L-amino acids were coupled with O-Benzotriazole-N, N, N and HBTU (N'-tetramethyl-uronium-hexafluoro-phosphate). Amino acid side chains were protected with tert-butyl and tert-butyloxycarbonyl, trifluoroacetic acid, water, thioanisole and 1,2- Deprotection and cleavage were performed using a mixture of ethanediol (1,2-ethandithiol) (87.5, 5.0, 5.0, 2.5, v / v). Crude peptides were repeatedly washed with diethylether and dried in vacuo and purified by RP-HPLC, Shimadzu 5-μm Shimpak ODS C18 column (20Х250 mm). Purified peptides were identified by analytical RP-HPLC, Shimpak ODS C18 column (4.6Х250 mm). Molecular masses of the synthesized peptides were determined using a matrix-assisted laser desorption ionization MALDI-mass spectrometer (Shimadzu Axima Assurance).

Example 7 Selective Track Activity Analysis of the Peptides of the Invention

<7-1> Activity inhibitory effect of harmful microalgae according to the peptide treatment of the present invention

In order to confirm that the active substance obtained from the decomposition product of the mackerel waste liquid exhibits the effect of tide activity, After incubating the harmful algae H. triquetra, P. minimum, A. fundyense and A. catenella , algae in an exponential growth phase were dispensed into 24-well plates. 0.01-3 mg peptides of the invention were placed in a 24-well culture plate dispensed with algae and adjusted to a total volume of 1 ml. Peptides were dissolved in the same medium as algae culture, 24-well culture plates were incubated under the same conditions as algae culture, and changes of algae cells were observed under a microscope. The half maximal lethal concentration (LC ₅₀ ) was derived from the effective rate of algae cells, which was calculated by the following equation: Effective rate (%) = (Nc-Nt) / NcХ100. Nc means the total algal cell number in the control group and Nt means the total algal cell number in the treatment group. Affected algae were calculated as defined by no movement and morphological changes in algal cells (color change, shelling, swelling, bursting, cytoplasmic contraction, etc.). The results are shown in Table 3 below.

As a result, it was confirmed that the peptide of the present invention effectively inhibits harmful microalgae.

Algae LC ₅₀  (mg / ml) H. triquetra 0.85 P. minimum 1.24 A. fundyense 0.69 A. catenella 0.83

<7-2> Confirmation of Morphological Changes of Harmful Microalgae by Peptide Treatment of the Present Invention

In order to confirm the morphological changes of the harmful microalgae according to the peptide treatment of the present invention, the morphological changes of the representative microalgae H. triquetra, P. minimum, and A. fundyense toxic and causing red algae were identified. The throughput was the peptide of LC ₅₀ concentration of each algae, and the peptide was dissolved in the same medium as the algae culture, and the algae of the logarithmic phase were placed in a divided 24-well plate and adjusted to a total volume of 1 ml. After treatment, the cells were cultured under the same conditions as the algae culture, and 5 hours later, the change of the algae cells was observed under a microscope. 5, C means the control group, T means the experimental group, A species is H. triquetra, B species P. minimum, C species A. fundyense .

As shown in FIG. 5, it was confirmed that when the peptide was treated, changes in morphological changes and movements of harmful microalgae occurred. Specifically, it was confirmed that the algae movement immediately disappeared when the peptide was treated. In H. triquetra , defecation was observed, and P. minimum was observed to condense cytoplasm and cloud the cell membrane. In A. fundyense, the cytoplasm was eluted out of the cell, and shelling and cytoplasmic condensation were observed. Additional 5 days of affected birds There was no change in morphology or movement. That is, when the peptide of the present invention is treated, it was confirmed that the anti-red tide effect against harmful microalgae appeared in a short time.

<7-3> Non-inhibitory effect on the harmless microalgae according to the peptide treatment of the present invention

In order to confirm the non-inhibitory effect on the harmless microalgae according to the peptide treatment of the present invention, S. costatum was treated with the peptide of the present invention. Specifically, 20 ml of S. costatum in an exponential growth phase was dispensed into flasks, and peptides of 2.5 and 3 mg / ml concentrations were added to the total volume of 50 ml. Peptides were dissolved in diatom medium and analyzed under the same conditions as diatom culture. Chlorophyll a (Chl a) concentrations were measured for 24 h. Chl a extraction and concentration measurement were measured for absorbance after acetone extraction. Calculation of Chl a was calculated using the following formula: Chl a (mg / l) [(A ₆₆₅ -A ₇₅₀ ) Х13.7- (A ₆₄₉ -A ₇₅₀ ) Х5.76] ХV ₂ / V ₁ . A ₆₆₅ is the absorption of Chl a, A ₆₄₉ is the absorption of Chl a, A ₇₅₀ is the turbidity correction, V ₁ is the algal culture volume, V ₂ is the volume of the supernatant. The results are shown in FIG.

As shown in FIG. 6, the change in Chla a of the control and experimental groups for 24 hours was not affected when the peptide was treated with a concentration of 2.5 mg / ml. On the other hand, it was confirmed that the same concentration was applied to the four harmful algae H. triquetra, P. minimum, A. fundyense and A. catenella to show a 100% killing effect. In addition, the peptide is a concentration corresponding to the concentration of the LC ₉₀ or more (all birds used in the experiment LC90 = <2.5mg / ml) of the harmful algae, the treatment of the peptides invented at this concentration does not affect harmless algae It was confirmed that only harmful algae can be treated. In addition, since S.costatum used in the experiment is a standard marine ecotoxicity organism, the developed peptides are considered to have very low marine ecotoxicity. In addition, S. costatum is an algae that is used to feed mollusks and crustacean larvae, suggesting that peptide treatments can be effective in combating harmful algae without affecting the farm environment.

Example 8 Antimicrobial Activity Analysis of the Peptides of the Invention

The antimicrobial activity analysis of the peptide of the present invention used two gram-negative, one gram-positive. Escherichia coli (KCCM11234), Shigella flexneri (KCCM409483) and Staphylococcus aureus (KCCM12256) were purchased from the Korean Culture Center of Microorganisms (KCCM). As culture medium, E. coli was Luria-Bertani broth (pH 7.0), S. aureus was Brain-heart infusion broth (pH 7.4), and S. flexneri was Nutrient broth medium (pH 7.5). The antimicrobial effect on the bacteria was analyzed by the agar diffusion test. The antimicrobial effect of the peptide of the present invention was measured at a concentration of 1 mg / ml. The results are shown in Figure 7 and Table 4.

As shown in Figure 7 and Table 4, the peptide of the present invention was confirmed to inhibit the formation of colonies of E. coli and S. aureus .

Microorganisms Antimicrobial activity (1 mg / ml) F32-1 (KMNF) Gram Negative E. coli KCCM11234 + S. flexneri KCCM409483 + Positive S. aureus KCCM12256 + Clear zone diameter (cm) /-: Negative effects / +: <0.5 / ++: 0.5 ~ 1.0 / +++:> 1.0

<110> inje university industry-academic cooperation foundation <120> Novel tetrapeptide and composition for anti-algae or          anti-bacteria containing the same <130> PN1709-327 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> tetrapeptide <400> 1 Lys Met Asn Phe   One

Claims (16)

A tetrapeptide consisting of an amino acid sequence represented by SEQ ID NO: 1. The method of claim 1,
The tetrapeptide is characterized in that derived from the mackerel ( Scomber japonicas ) waste product degradation product, tetrapeptide. The method of claim 2,
The mackerel waste liquid degradation products Bacillus (Bacillus), Breda ratio Bacillus (Brevibacillus) and tetra-peptide, characterized in that the decomposition thereof in mixed culture. The method of claim 1,
The tetrapeptide is characterized in that it does not inhibit the activity of harmless or non-toxic red tide organisms, and selectively inhibits the activity of toxic or harmful red tide organisms. The method of claim 4, wherein
The toxic or harmful red tide organisms hetero kapsa in (Heterocapsa), Centrum in (Prorocentrum) to Pro, Alexandria Solarium in (Alexandrium), kokeulrodinium in (Cochlodinium), Jiro di nium in (Gyrodinium), Dino pi system in (Dinophysis) , Pseudo -nitzschia ( Pssudo-nitzschia ) and Gymnodinium ( Gymnodinium ), characterized in that at least one selected from the group consisting of, tetrapeptide. The method of claim 4, wherein
The harmless sex or toxic red tide organisms skeletal Loreto Cinema in (Skeletonema), Quito-year-old Ross in (Chaetoceros), Tala's coming LA (Thalassiosira), yukam Pia in (Eucamphia), Akashi right in (Akashiwo), Serra tium in ( Ceratium ), Eutreptiella genus ( Eutreptiella ) and Heterosigma genus ( Heterosigma ), characterized in that at least one selected from the group consisting of a tetrapeptide. The method of claim 1,
The tetrapeptide is characterized in that it has an antimicrobial activity against gram negative bacteria, gram positive bacteria or antibiotic resistant bacteria, tetrapeptide. The method of claim 7, wherein
The Gram-negative bacterium is characterized in that the genus Escherichia or Shigella, Tetrapeptide . The method of claim 7, wherein
The Gram-positive bacteria is Staphylococcus , characterized in that, tetrapeptide. The method of claim 7, wherein
The antibiotic-resistant bacteria include Escherichia coli , Staphylococcus aureus , Streptococcus pyogenes , Pseudomonas aeruginosa , and Clostridium, which have antibiotic resistance. A tetrapeptide, characterized in that at least one selected from the group consisting of Clostridium difficile , Salmonella enteritidis and Salmonella typhimurium . Red tide suppression composition comprising the tetrapeptide of claim 1. Claim 1 antimicrobial pharmaceutical composition comprising a tetrapeptide. Antimicrobial food composition comprising a tetrapeptide of claim 1. Antimicrobial cosmetic composition comprising a tetrapeptide of claim 1. Feed composition for antimicrobial comprising the tetrapeptide of claim 1. Antimicrobial biological pesticide composition comprising the tetrapeptide of claim 1.