KR102334571B1 - Antimicrobial peptide, Gryllotalpasin 1 derived from Gryllotalpa orientalis and uses thereof - Google Patents

Abstract

The present invention relates to a novel antibacterial or antifungal peptide derived from a puppies, and uses thereof, and more particularly, to grilotafasin-2 derived from puppies and an antibacterial or antifungal composition comprising the same. Grillotapasin-2 according to the present invention has excellent antibacterial and antifungal effects and has no cytotoxicity, so it can be used in various fields such as food, medicine, and cosmetics.

Description

Antimicrobial peptide, Gryllotalpasin 1 derived from Gryllotalpa orientalis and uses thereof

The present invention relates to a novel antibacterial or antifungal peptide derived from a tandoori dog and uses thereof, and more particularly, to a grillotapasin-1 derived from a tandoori dog and an antibacterial or antifungal composition comprising the same.

After penicillin, many antibiotics have been developed and used to combat bacteria that invaded from outside the living body. Although many new antibiotics are being developed to treat diseases caused by these antibiotic-resistant bacteria, most antibiotics have a mechanism of action similar to that of existing antibiotics, and remain in the body without being decomposed after antibiotic action, causing continuous antibiotic resistance problems. In addition, existing antibiotics not only induce resistance, but also cause other diseases by destroying normal bacterial flora through non-specific antibacterial action. In particular, Clostridium difficile infection (CDI), which is caused by the destruction of normal intestinal flora due to taking antibiotics, is emerging as a side effect of representative antibiotics. becomes this Therefore, there is an urgent need to develop a new paradigm of infection treatment to replace the existing antibiotics, and antibacterial peptides are attracting attention as a way to solve the existing problems.

Antibacterial peptide is a small-sized peptide composed of 10-50 amino acids that all living things have as one of the innate defense systems. It acts as a primary defense material when an organism is infected with a pathogen. The antibacterial peptide has a positive charge (+2~+9) as a whole, and contains about 30% of hydrophobic amino acid residues. Due to these characteristics, the antibacterial peptide forms an amphipathic α-helix or β-sheet structure after contact with a negatively charged bacterial cell membrane and intercalates into the cell membrane to increase the potential of the cell membrane. It kills bacteria by destroying the cell membrane by altering it or puncturing the cell membrane itself. In addition, some antibacterial peptides show antibacterial activity by paralyzing cell functions by entering cells and attacking intracellular targets. Therefore, antibacterial peptides are significantly less likely to induce resistance than conventional antibiotics that target specific receptors. In addition, antibacterial peptides have characteristics such as a rapid mechanism of action, action on existing antibiotic-resistant bacteria, and anti-endotoxicity, so they are suitable for development as a novel treatment for infection.

On the other hand, insects have evolved in the direction of securing their own special physiological control mechanisms and substances in order to adapt to and survive in the living environment. Insect Biochem. Mol. Biol. 41, 747-769].

While continuing research on insect-derived antibacterial peptides, the present inventors completed the present invention by confirming that grillotapasin-1, a novel peptide derived from land dogs, has excellent antibacterial and antifungal effects.

Accordingly, it is an object of the present invention to provide novel antibacterial/antifungal peptides and uses thereof.

In order to achieve the above object, the present invention provides an antibacterial or antifungal peptide represented by the amino acid sequence of SEQ ID NO: 1.

In addition, the present invention provides an antibacterial or antifungal composition comprising the peptide as an active ingredient.

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

In addition, the present invention provides an antibacterial or antifungal food preservative comprising the peptide as an active ingredient.

In addition, the present invention provides an antibacterial or antifungal cosmetic additive comprising the peptide as an active ingredient.

In addition, the present invention provides an antibacterial or antifungal pharmaceutical additive comprising the peptide as an active ingredient.

In addition, the present invention provides a feed additive comprising the peptide as an active ingredient.

Grillotapasin-1, which is a peptide newly isolated from puppies according to the present invention, has excellent antibacterial and antifungal effects and has no cytotoxicity, so it can be used in various fields such as food, pharmaceuticals, and cosmetics.

1 is a diagram showing the antibacterial and antifungal effects of gryllotafacin-1 on gram-positive bacteria, gram-negative bacteria, antibiotic-resistant bacteria and fungi according to the present invention.
2 is a diagram showing the results of confirming the toxicity of gryllotafacin-1 according to the present invention on normal cells, human keratinocytes or mouse fibroblasts.

Hereinafter, the present invention will be described in detail.

The present invention provides an antibacterial or antifungal peptide represented by the amino acid sequence of SEQ ID NO: 1. SEQ ID NO: 1 is represented as follows.

LKLKKVKKIL

As used herein, the term “peptide” refers to a linear molecule formed by combining amino acid residues with each other by peptide bonds. The peptide may be prepared according to a chemical synthesis method known in the art, and preferably according to a solid-phase synthesis technique, but is not limited thereto. The antibacterial or antifungal peptide according to the present invention may be one isolated from a puppies.

In the present invention, "ground dog ( Gryllotalpa orientalis )” is an insect of the locust family, also known as groundworm or groundworm. Adults have a body length of 30-35 mm, a yellowish-brown to blackish-brown color, and are covered with wool-like hairs all over their body. The forewings are small and the hind wings are large, and when not flying, it is long and slender and folded on the back. Females have a vein in the middle of the forewing, and males have a vein. Both male and female have about 10 pronounced protrusions on the simaek.

The peptide according to the present invention may include an amino acid in which an amino acid is added or deleted in the amino acid sequence consisting of SEQ ID NO: 1 as long as antibacterial activity is not reduced.

In an embodiment of the present invention, a novel peptide was isolated from a puppies, and it was named grillotapasin-1, and it was confirmed that the peptide had excellent antibacterial activity against gram-negative and gram-positive bacteria and antifungal activity against fungi. .

As used herein, the term “Gram-negative bacteria” refers to bacteria that are stained red when stained by the Gram staining method. Most of the Gram-negative bacteria have a thin cell wall (about 10 nm) and contain a large amount of lipopolysaccharide (LPS) on the outside. am. The gram-negative bacteria include, but are not limited to, for example, Escherichia genus, Pseudomonas genus, Salmonella genus, Leptospira genus, Rickettsia, and the like. In an embodiment of the present invention, Escherichia coli was used as a representative strain of Gram-negative bacteria.

As used herein, the term “Gram-positive bacteria” refers to bacteria whose cell wall is stained purple when stained by the Gram staining method, and the cell wall is composed of several layers of peptidoglycan. The Gram-positive bacteria include, for example, Staphylococcus, Listeria, Corynebacterium, Lactobacillus, Bacillus, Propionibacterium. ), but is not limited thereto. In one embodiment of the present invention, as a representative strain of Gram-positive bacteria, Staphylococcus aureus and Propionbacterium acnes ( Propionibacterium ) acnes ) was used.

The gram-negative bacteria and gram-positive bacteria may be antibiotic-resistant bacteria, but is not limited thereto.

As used herein, the term “fungi” refers to a group of microorganisms including molds, yeasts, and mushrooms, and belongs to eukaryotes having a nuclear membrane. In one embodiment of the present invention, Candida albicans was used as a representative strain of the fungus.

In one aspect, the present invention provides an antibacterial or antifungal composition comprising the peptide as an active ingredient.

In the present invention, the antibacterial or antifungal composition is a composition having an activity of inhibiting the growth of microorganisms such as bacteria or fungi, and may include any form used in various fields requiring an antibacterial or antifungal effect, for example, It may be in the form of pharmaceuticals, quasi-drugs, food additives, or feed additives. Specifically, for purposes such as antibiotics or antifouling agents in medicine, for antiseptic or antibacterial purposes in food, antibacterial, sterilization, and disinfection in agriculture, for anti-dandruff control, athlete's foot prevention, armpits in cosmetics and household products It may be used for products directly related to microorganisms such as collection suppression, anti-acne, etc., or used for antiseptic, antibacterial or sterilization purposes, such as cleaning agents or dishwashing detergents, but is not limited to these purposes.

Accordingly, the antibacterial or antifungal composition of the present invention may be prepared in the form of a pharmaceutical composition, such as an antibiotic, a food preservative, a cosmetic additive, a pharmaceutical additive, or a feed additive.

In addition, the antibacterial or antifungal composition of the present invention may contain one or more known active ingredients having an antibacterial or antifungal effect together with the peptide.

The antibacterial or antifungal pharmaceutical composition of the present invention can be prepared by including one or more pharmaceutically acceptable carriers in addition to the active ingredients described above for administration. The pharmaceutically acceptable carrier may be used in a mixture of saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components, and if necessary, antioxidants, buffers , and other conventional additives such as a bacteriostatic agent may be added. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to form an injectable formulation such as an aqueous solution, suspension, emulsion, etc., pills, capsules, granules or tablets. Furthermore, it can be preferably formulated according to each disease or component by an appropriate method of distribution of sugar or by using a method disclosed in Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA.

The antibacterial or antifungal pharmaceutical composition of the present invention may be administered orally or parenterally according to a desired method, and the dosage may be an individual's weight, age, sex, health status, diet, administration time, administration method, excretion rate. And the range varies depending on the severity of the disease. The daily dose of the peptide is 2 to 10 mg/kg, but may be increased or decreased according to clinical results, and it is preferable to divide and administer once to several times a day.

In another aspect, the present invention provides an antibiotic comprising the peptide as an active ingredient.

The antibiotic means an agent that is provided to an individual in the form of a drug to kill bacteria, and may be used in combination with a bactericide and an antibacterial agent.

In another aspect, the present invention provides an antibacterial or antifungal food preservative comprising the peptide as an active ingredient.

The antibacterial or antifungal food preservative of the present invention may be prepared by including at least one of known food preservatives. Food preservatives include, but are not limited to, dehydroacetic acid, potassium sorbate, calcium sorbate, sodium benzoate, potassium benzoate, calcium benzoate, methyl paraoxybenzoate, propyl paraoxybenzoate, sodium propionate, calcium propionate, and the like.

The antibacterial or antifungal food preservative of the present invention preferably contains 0.01 to 1% by weight of the peptide based on the total weight of the food preservative, but is not limited thereto.

In another aspect, the present invention provides an antibacterial or antifungal cosmetic additive or pharmaceutical additive comprising the peptide as an active ingredient.

The cosmetic additive or pharmaceutical additive for antibacterial or antifungal of the present invention means to be used for the preservation of cosmetics or pharmaceuticals.

The cosmetic additive may be prepared in the form of general emulsified formulations and solubilized formulations. Cosmetics with emulsified formulations include nutritional lotions, creams, and essences, and solubilized cosmetics include softened lotions. In addition, the cosmetic additive of the present invention can be prepared in the form of an adjuvant that can be applied topically or systemically commonly used in the field of dermatology by containing a dermatologically acceptable medium or base in addition to the peptide. In addition, suitable cosmetic formulations include, for example, a solution to which the peptide is added, a gel, a solid or kneaded anhydrous product, an emulsion obtained by dispersing an oil phase in an aqueous phase, a suspension, a microemulsion, a microcapsule, a microgranule or an ionic ( liposome), in the form of a non-ionic vesicular dispersant, cream, skin, lotion, powder, ointment, spray or in the form of a concealer stick. It may also be prepared in the form of a foam or an aerosol composition further containing a compressed propellant. In addition, as an excipient used in cosmetic additives, arbutin, kojic acid, rucinol, vitamin C and vitamin C derivatives may be additionally contained, and retinol for the purpose of improving wrinkles (retinol) and its derivatives, indol acetic acid and its derivatives, adenosin, tocoperol and its derivatives, kynitine, and the like may be additionally contained. In addition, the excipient may include ingredients commonly used in cosmetic additives, and for example, include conventional adjuvants and carriers such as stabilizers, solubilizers, vitamins, pigments and fragrances.

The antibacterial or antifungal cosmetic additive of the present invention preferably contains 0.01 to 1% by weight of the peptide based on the total weight of the cosmetic additive, but is not limited thereto.

In another aspect, the present invention provides a feed additive comprising the peptide as an active ingredient.

The feed additive of the present invention is provided as a feed by being appropriately mixed with feed raw materials. As the feed raw materials, grain oil, crude steel, vegetable oilseeds, animal feed raw materials, other feed raw materials, refined products, etc. are used, but are not limited thereto. . The feed may further include conventional additives that can increase the preservation of the feed in addition to the peptide of the present invention.

Other non-pathogenic microorganisms may be additionally added to the feed additive of the present invention. Microorganisms that can be added include Bacillus subtilis, such as Bacillus subtilis, capable of producing proteolytic enzymes, lipolytic enzymes and sugar converting enzymes, Lactobacillus having physiological activity and organic matter decomposition ability under anaerobic conditions such as the stomach of cattle Strain (Lactobacillus sp.), filamentous fungi such as Aspergillus oryzae, and Saccharomyces cerevisiae that increase the body weight of livestock, increase milk production, and increase the digestibility and absorption rate of feed ) can be selected from the group consisting of yeast such as

The feed additive of the present invention may include a binder, an emulsifier, a preservative, etc. added to prevent quality deterioration, and an amino acid agent, vitamin agent, enzyme agent, probiotic agent, flavor agent, non-protein nitrogen compound added to feed to increase efficacy , a silicate agent, a buffering agent, a colorant, an extractant, an oligosaccharide, etc. may be included, and in addition, a feed mixture agent and the like may be additionally included.

Hereinafter, the content of the present invention will be described in more detail through Examples and Experimental Examples. The following Examples and Experimental Examples only illustrate the present invention, and the content of the present invention is not limited by the following Examples and Experimental Examples.

Example 1. Antibacterial or antifungal derived from tang dog peptide Selection

The following experiment was performed to select peptides exhibiting antibacterial or antifungal activity from land dogs.

First, total RNA was isolated by rapidly freezing the tandoori with liquid nitrogen, and the genetic information on the tandoori transcript was confirmed using RNA-seq analysis. Using previously known antibacterial peptide properties, the entirety of the translated amino acid sequence from each gene was filtered, and peptides expected to be new antibacterial or antifungal peptides were selected.

The selected amino acid sequence was LKLKKVKKIL and consisted of 10 amino acids, which was named Grylotapasin 1 (Table 1). In subsequent experiments, gryllotafasin-1 of SEQ ID NO: 1 was synthesized, separated and purified.

peptide amino acid sequence Grillotaphacin-1 LKLKKVKKIL-NH ₂

Experimental example 1. Measurement of antibacterial and antifungal activity against pathogenic microorganisms

In order to confirm the antibacterial and antifungal activity of grillotapasin-1 selected in Example 1, a radial diffusion assay (RDA) was performed. Grillotapasin-1 was stored at -20°C until use, and was used by dissolving in sterile tertiary distilled water.

1-1. Determination of antibacterial activity

First, among bacteria, Escherichia coli , a representative strain of Gram-negative bacteria, Staphylococcus aureus , a representative strain of Gram-positive bacteria, and Propionibacterium acnes ), an antibiotic-resistant bacterium, MRSA (Methicillin resistant Staphylococcus aureus) was used. Each of the strains was placed in a 3% (w/v) TSB (tryptic soy broth) medium, and then cultured at 37° C. and 200 rpm for 18 hours. Again, the second culture was performed under the same conditions for 2 hours and 30 minutes, and absorbance was measured at 600 nm to become 4 × 10 ⁶ CFU (colony forming units). Then, 100 mM sodium phosphate/citrate buffer (9 mM sodium phosphate, 1 mM sodium citrate, pH 7.4) and 1% (w/v) type I electroendosmosis) agarose, 0.03 % TSB. The cultured strain (4×10 ⁶ CFU) was put on an underlay agar, mixed, and then poured onto a square plate. When the underlay agar was hardened, a hole with a diameter of 3 mm was made, and grillotapasin-1 diluted by concentration was added to each hole by 5 μl.

After incubation at 37°C for 3 hours to spread the peptide, 10 ml of sterilized overlay agar (6% TSB, 1% agarose) was poured and incubated at 37°C again to determine whether a clear zone was formed. Confirmed. The diameter measurement results are shown in FIG. 1 .

As shown in FIG. 1 , it was confirmed that the grilotafasin-1 peptide derived from the terrestrial dog had antibacterial activity against all of Gram-negative bacteria, Gram-positive bacteria and antibiotic-resistant bacteria, which increased in a concentration-dependent manner.

1-2. Determination of antifungal activity

Pathogenic fungi Candida albicans ( Candida albicans ) was used. After putting the strain in YPD medium (1% yeast extract, 2% peptone, 2% dextrose), incubate overnight at 30°C and 200 rpm conditions, inoculate it in a new medium, and incubate for 3 hours again to become a log phase did. Then, a sterile underlay consisting of 100 mM sodium phosphate/citrate buffer (9 mM sodium phosphate, 1 mM sodium citrate, pH 7.4) and 1% (w/v) type I (low electroendosmosis) agarose, 0.03% TSB. Cultured bacteria (4×10 ⁶ CFU/ml) was added to the agar, mixed, and poured onto a square plate. When the underlay agar was hardened, a hole with a diameter of 3 mm was made, and grillotapasin-1 diluted by concentration was added to each hole by 5 μl.

After incubation at 37° C. for 3 hours to spread the peptide, 10 ml of sterilized overlay agar (6% TSB, 1% agarose) was poured and incubated at 37°C again to check whether a clear zone was formed. The diameter measurement results are shown in FIG. 1 .

As shown in FIG. 1 , it was confirmed that the grilotapasin-1 peptide derived from terrestrial puppies had antifungal activity against the pathogenic fungus Candida albicans, which increased in a concentration-dependent manner.

Through the above experiment, it can be seen that grillotapasin-1 according to the present invention can be utilized as an antibacterial and antifungal agent against harmful strains.

Experimental example 2. Measurement of Cytotoxicity to Normal Cells

An MTS assay was performed to examine the cytotoxicity of grillotapasin-1 selected in Example 1. Cells were processed using HacaT (human keratinocyte), a human keratinocyte, and NIH3T3 (mouse fibroblast), a mouse fibroblast. Grillotapasin-1 was stored at -20°C until use, and was washed in sterile tertiary distilled water. It was melted and used.

^{Specifically, 100 μl of 2 × 10 4} cells/well cells were aliquoted into a 96-well culture vessel and then treated with grillotapasin-1 in a concentration-dependent manner, and then cultured for 24 hours. Thereafter, 10 μl CellTiter 96 AQueous One Solution Reagent (Promega, USA) was added and reacted for 3 to 4 hours to confirm color change and measure absorbance (590 mm). The results are shown in FIG. 2 .

As shown in FIG. 2 , it was confirmed that the grilotafasin-1 peptide derived from land dog had no cytotoxicity up to a concentration of up to 200 ug/ml.

These results indicate that grillotapasin-1 according to the present invention exhibits antibacterial and antifungal activity in the absence of cytotoxicity, suggesting that it can be used as a safe antibiotic, food preservative, cosmetic preservative, pharmaceutical, and the like.

Overall, grillotapasin-1, a peptide that the present inventors have newly isolated from puppies, has excellent antibacterial and antifungal effects and has no cytotoxicity, so it can be used in various fields such as food, pharmaceuticals, and cosmetics.

<110> REPUBLIC OF KOREA (MANAGEMENT : RURAL DEVELOPMENT ADMINISTRATION) <120> REPUBLIC OF KOREA (MANAGEMENT : RURAL DEVELOPMENT ADMINISTRATION) <130> 1-74 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Gryllotalpasin 1 <400> 1 Leu Lys Leu Lys Lys Val Lys Lys Ile Leu 1 5 10

Claims (10)

An antibacterial or antifungal composition comprising an antibacterial or antifungal peptide represented by the amino acid sequence of SEQ ID NO: 1 as an active ingredient. The composition according to claim 1, wherein the peptide is isolated from a land dog. The composition of claim 1, wherein the peptide exhibits antibacterial activity against Gram-negative bacteria and Gram-positive bacteria. The composition of claim 1, wherein the peptide exhibits antifungal activity against Candida albicans. delete An antibiotic comprising an antibacterial or antifungal peptide represented by the amino acid sequence of SEQ ID NO: 1 as an active ingredient. An antibacterial or antifungal food preservative comprising an antibacterial or antifungal peptide represented by the amino acid sequence of SEQ ID NO: 1 as an active ingredient. An antibacterial or antifungal cosmetic additive comprising an antibacterial or antifungal peptide represented by the amino acid sequence of SEQ ID NO: 1 as an active ingredient. An antibacterial or antifungal pharmaceutical additive comprising an antibacterial or antifungal peptide represented by the amino acid sequence of SEQ ID NO: 1 as an active ingredient. A feed additive comprising an antibacterial or antifungal peptide represented by the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

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