KR20210061495A - 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 Gryllotalpa orientalis, and uses thereof, and more particularly, to gryllotafacin-2 derived from Gryllotalpa orientalis 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, thereby being used in various fields such as food, medicine, and cosmetics.

Description

[Antimicrobial peptide, Gryllotalpasin 1 derived from Gryllotalpa orientalis and uses thereof] {Antimicrobial peptide, Gryllotalpasin 1 derived from Gryllotalpa orientalis and uses thereof}

The present invention relates to novel antibacterial or antifungal peptides derived from pups and their use, and more particularly, to grillotafacin-1 derived from pups and antibacterial or antifungal compositions comprising the same.

After penicillin, many antibiotics have been developed and used to combat bacteria invading from the outside of the body, but antibiotic-resistant bacteria are rapidly increasing due to the misuse of antibiotics in recent years. Many new antibiotics have been developed to treat diseases caused by these antibiotic-resistant bacteria, but most of the antibiotics have a mechanism of action similar to that of conventional antibiotics, and remain in the body without being degraded after the antibiotic action, causing persistent antibiotic resistance problems. In addition, conventional antibiotics not only induce resistance, but also cause other diseases by destroying the normal microflora through non-specific antibacterial action. In particular, Clostridium difficile infection (CDI), which occurs as a result of destruction of normal intestinal flora caused by taking antibiotics, is emerging as a side effect of representative antibiotics, and broad-spectrum antibiotics used for burn treatment also inhibit normal skin flora and cause fungal infection. Becomes. Therefore, there is an urgent need to develop a new paradigm infection treatment that will replace the existing antibiotics, and antibacterial peptides are attracting attention as a way to solve the existing problems.

Antimicrobial peptides are small-sized peptides composed of 10-50 amino acids possessed by all organisms as one of the innate defense systems, and act as a primary defense material when organisms are infected with pathogens. Antimicrobial peptides are generally positively charged (+2~+9) and contain about 30% of hydrophobic amino acid residues. Due to this characteristic, the antimicrobial peptide forms an amphipathic α-helix or β-fold structure after contacting the negatively charged bacterial cell membrane, which is inserted into the cell membrane to prevent the potential of the cell membrane. It kills bacteria by changing it or destroying the cell membrane by making a hole in the cell membrane itself. In addition, some antimicrobial peptides show antibacterial activity by entering the cell and then attacking the intracellular target to paralyze the cell function. Therefore, antimicrobial peptides are significantly less likely to induce resistance compared to conventional antibiotics targeting specific receptors. In addition, antimicrobial peptides are suitable for development as new infection treatments because they have a rapid mechanism of action, an action against existing antibiotic-resistant bacteria, and anti-endotoxicity.

On the other hand, insects have evolved in the direction of securing their own special physiological regulation mechanisms and substances in order to adapt to the living environment and survive. Recently, research on the development of new drug candidates using the physiologically active substances of insects is actively progressing [ Insect Biochem. Mol. Biol. 41, 747-769].

The present inventors completed the present invention by confirming that grillotafacin-1, a novel peptide derived from dog breeds, has excellent antibacterial and antifungal effects while continuing research on insect-derived antibacterial peptides.

Accordingly, it is an object of the present invention to provide a novel antibacterial/antifungal peptide and use 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.

Grillotafacin-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, medicine, and cosmetics.

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

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. The SEQ ID NO: 1 is represented as follows.

LKLKKVKKIL

In the present invention, the term “peptide” refers to a linear molecule formed by bonding of amino acid residues to each other by peptide bonds. The peptide may be prepared according to a chemical synthesis method known in the art, and preferably may be prepared according to a solid phase synthesis technique, but is not limited thereto. Antimicrobial or antifungal peptides according to the present invention may be isolated from pups.

In the present invention, " Gryllotalpa orientalis )” is an insect of the family Locust Order, and is also called a land dog and a land dog. The body length of an adult insect is 30-35 mm, the body color is yellow-brown to blackish brown, and the whole body is covered with hairs like ridges, and the forelimbs are strong and broad to be suitable for digging like a mole. The fore wing is small and the hind wing is large, and when it is not flying, it is folded long and thin on the back. The female has a longitudinal vein in the center of the fore wing, and the male has a vein. Both male and female have about 10 pronunciation protrusions in the vein.

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

In one embodiment of the present invention, a novel peptide was isolated from pups and named as grillotafacin-1, and it was confirmed that the peptide has excellent antibacterial activity against Gram-negative bacteria and Gram-positive bacteria and antifungal activity against fungi. .

In the present invention, the term "gram-negative bacteria" is a bacterium that dyes red when stained by the Gram staining method, and most of the cell walls of Gram-negative bacteria are thin (about 10 nm) and contain a large amount of lipopolysaccharide (LPS) on the outside. to be. Examples of the Gram-negative bacteria include, but are not limited to, Escherichia, Pseudomonas, Salmonella, Leptospira, Rickettsia, and the like. In one embodiment of the present invention, Escherichia coli was used as a representative strain of Gram-negative bacteria.

In the present invention, the term "gram-positive bacteria" is a bacterium whose cell wall is stained purple when stained by the Gram stain method, and the cell wall is composed of several layers of peptidoglycan. The Gram-positive bacteria include, for example, Staphylococcus, Listeria, Corynebacterium, Lactobacillus, Bacillus, and Propionbacterium. ), etc. In one embodiment of the present invention, as representative strains of Gram-positive bacteria, Staphylococcus aureus and Propionibacterium acnes (Propionibacterium acnes ) was used.

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

In the present invention, the term "fungi" refers to a group of microorganisms including fungi, yeast, 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 fungi.

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

In the present invention, the composition for antibacterial or antifungal is a composition having an activity of inhibiting the growth of microorganisms such as bacteria or fungi, and may include all forms 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, in medicine, for the same purpose as antibiotics and antifouling agents, in food, for antibacterial or antibacterial purposes, in agriculture, for antibacterial, sterilizing, and disinfecting purposes, and in cosmetics and household products, for dandruff control, athlete's foot, armpits It can be used for products directly related to microorganisms, such as for suppressing collection and anti-acne, or for antiseptic, antibacterial, or sterilizing purposes, such as cleaning detergents or dishwashing detergents, but is not limited to this purpose.

Accordingly, the antibacterial or antifungal composition of the present invention may be prepared in the form of a pharmaceutical composition, such as antibiotics, food preservatives, cosmetic additives, pharmaceutical additives, or feed additives.

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 may be prepared by including one or more pharmaceutically acceptable carriers in addition to the above-described active ingredients for administration. Pharmaceutically acceptable carriers can be used by mixing saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and at least one of these components, and antioxidants, buffer solutions, if necessary. , Other conventional additives such as bacteriostatic agents may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to prepare injectable formulations such as aqueous solutions, suspensions, emulsions, etc., pills, capsules, granules, or tablets. Furthermore, it can be preferably formulated according to each disease or component by an appropriate method of pre-sale of sugar or by using a method disclosed in Remington's Pharmaceutical Science (latest edition), Mack Publishing Company, Easton PA.

The antibacterial or antifungal pharmaceutical composition of the present invention can be administered orally or parenterally according to a desired method, and the dosage is the 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, and the like. The daily dose of the peptide may be 2 to 10 mg/kg, but may be added or subtracted according to clinical results, and it is preferable to administer it in divided doses from once to several times a day.

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

The antibiotic is provided in the form of a drug to an individual and means a preparation capable of killing bacteria, and may be used in combination with a fungicide 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 one or more of known food preservatives. As food preservatives, dehydroacetic acid, potassium sorbate, calcium sorbate, sodium benzoate, potassium benzoate, calcium benzoate, methyl paraoxybenzoate, propyl paraoxybenzoate, sodium propionate, calcium propionate, and the like are used, but are not limited thereto.

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

The cosmetic additive may be prepared in the form of a general emulsifying formulation and a solubilizing formulation. Cosmetics in emulsified form include nutrient lotion, cream, and essence, and cosmetic products in solubilized form include softening lotion. In addition, the cosmetic additive of the present invention may be prepared in the form of an adjuvant that can be applied topically or systemically 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 anhydrous product, an emulsion obtained by dispersing an oil phase in an aqueous phase, a suspension, a microemulsion, a microcapsule, a microgranular sphere or an ionic form ( Liposome), nonionic vesicle dispersant, cream, skin, lotion, powder, ointment, spray or conceal stick. It can also be prepared in the form of a foam (foam) or in the form of an aerosol composition further containing a compressed propellant. In addition, as excipients used in cosmetic additives, arbutin, kojic acid, rucinol, vitamin C and vitamin C derivatives may additionally be contained. Retinol is used for the purpose of improving wrinkles. (retinol) and its derivatives, indol acetic acid and its derivatives, adenosine, tocoperol and its derivatives, kinitine, and the like may further be contained. In addition, the excipients may include ingredients commonly used in cosmetic additives, and include conventional adjuvants and carriers such as stabilizers, solubilizers, vitamins, pigments and flavors.

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 appropriately mixed with feed ingredients to be provided as feed, and as the feed ingredients, grain oil, crude steel, vegetable oil meal, animal feed ingredients, other feed ingredients, refined products, etc. are used, but are not limited thereto. . In addition to the peptides of the present invention, the feed may further include conventional additives that can increase the preservability of feed.

Other non-pathogenic microorganisms may be additionally added to the feed additive of the present invention. Microorganisms that can be added include Bacillus subtilis, which can produce proteases, lipolytic enzymes, and sugar converting enzymes, and Lactobacillus, which has physiological activity and ability to degrade organic matter under anaerobic conditions such as the stomach of cattle. Strain (Lactobacillus sp.), filamentous fungi such as Aspergillus oryzae and Saccharomyces cerevisiae showing the effect of increasing the weight of livestock, increasing milk production and increasing the digestion and absorption rate of feed. ) May be selected from the group consisting of yeast.

The feed additive of the present invention may contain binders, emulsifiers, preservatives, etc. added to prevent quality deterioration, and amino acids, vitamins, enzymes, probiotics, flavoring agents, non-protein nitrogen compounds added to feed to increase utility , A silicate, a buffer, a colorant, an extractant, an oligosaccharide, etc. may be included, and in addition, a feed mixture 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 are merely illustrative of the present invention, and the contents of the present invention are not limited by the following examples and experimental examples.

Example 1. Antibacterial or antifungal derived from puppies Peptide Selection

In order to select peptides exhibiting antibacterial or antifungal activity from pups, the following experiment was performed.

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

The selected amino acid sequence was LKLKKVKKIL, which consisted of 10 amino acids, and was named as Gryllotapasin 1 (Table 1). In the subsequent experiment, grillotafacin-1 of SEQ ID NO: 1 was synthesized, separated and purified.

Peptide Amino acid sequence Grillotafacin-1 LKLKKVKKIL-NH ₂

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

In order to confirm the antibacterial and antifungal activities of grillotafacin-1 selected in Example 1, a radial diffusion assay (RDA) was performed. Grillotafacin-1 was stored at -20°C until use, and dissolved in sterilized tertiary distilled water for use.

1-1. Measurement of antibacterial activity

First, among bacteria, Escherichia coli , a representative strain of Gram-negative bacteria, Staphylococcus aureus , and Propionibacterium , a representative strain of Gram-positive bacteria. acnes ), an antibiotic resistant MRSA (Methicillin resistant Staphylococcus aureus) was used. Each of the above strains was added to 3% (w/v) TSB (tryptic soy broth) medium, followed by incubation at 37° C. and 200 rpm for 18 hours. Again, the secondary culture was performed for 2 hours and 30 minutes under the same conditions, and the absorbance was measured at 600 nm to obtain 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 sterilized under The strain (4×10 ⁶ CFU) cultured in the underlay agar was added, mixed, and then poured into a square plate. When the underlay agar was hardened, a hole having a diameter of 3 mm was made, and 5 µl of grillotafacin-1 diluted by concentration was added to the hole.

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

As shown in FIG. 1, it was confirmed that the grilotafacin-1 peptide derived from pups has antibacterial activity against all of gram-negative bacteria, gram-positive bacteria, and antibiotic-resistant bacteria, and this was confirmed to increase in a concentration-dependent manner.

1-2. Measurement of antifungal activity

The pathogenic fungus 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, and inoculate it in a new medium and incubate again for 3 hours to achieve logarithmic phase. I 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. Bacteria (4×10 ⁶ CFU/ml) cultured in the agar were added, mixed, and poured into a square plate. When the underlay agar was hardened, a hole having a diameter of 3 mm was made, and 5 µl of grillotafacin-1 diluted by concentration was added to the hole.

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 again at 37° C. to confirm the formation of a clear zone. The diameter measurement results are shown in FIG. 1.

As shown in FIG. 1, it was confirmed that the grilotafacin-1 peptide derived from the pug has antifungal activity against the pathogenic fungus Candida albicans, which is increased in a concentration-dependent manner.

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

Experimental example 2. Measurement of cytotoxicity to normal cells

In order to investigate the cytotoxicity of the grillotafacin-1 selected in Example 1, an MTS assay was performed. Cells were processed using human keratinocytes, HacaT (human keratinocyte) and mouse fibroblasts, NIH3T3 (mouse fibroblast), and grillotafacin-1 was stored at -20°C until use, and in sterilized tertiary distilled water. It was melted and used.

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

As shown in Fig. 2, it was confirmed that the grillotafacin-1 peptide derived from pups was not cytotoxic up to a concentration of up to 200 ug/ml.

These results suggest that grillotafacin-1 according to the present invention exhibits antibacterial and antifungal activity in a state of non-cytotoxicity, and thus it can be used as safe antibiotics, food preservatives, cosmetic preservatives, pharmaceuticals, and the like.

Overall, grillotafacin-1, a peptide newly isolated from pups by the present inventors, has excellent antibacterial and antifungal effects and has no cytotoxicity, so it can be used in various fields such as food, medicine, 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)

Antibacterial or antifungal peptide represented by the amino acid sequence of SEQ ID NO: 1. The peptide according to claim 1, wherein the peptide is isolated from pups. The peptide according to claim 1, wherein the peptide exhibits antimicrobial activity against Gram-negative bacteria and Gram-positive bacteria. The peptide according to claim 1, wherein the peptide exhibits antifungal activity against Candida albicans. An antibacterial or antifungal composition comprising the peptide of any one of claims 1 to 4 as an active ingredient. An antibiotic comprising the peptide of any one of claims 1 to 4 as an active ingredient. An antibacterial or antifungal food preservative comprising the peptide of any one of claims 1 to 4 as an active ingredient. An antibacterial or antifungal cosmetic additive comprising the peptide of any one of claims 1 to 4 as an active ingredient. An antibacterial or antifungal pharmaceutical additive comprising the peptide of any one of claims 1 to 4 as an active ingredient. A feed additive comprising the peptide of any one of claims 1 to 4 as an active ingredient.

Images