KR102540598B1 - Antibacterial peptide derived from Crassostrea gigas and its use - Google Patents

Abstract

The present invention is an antibacterial peptide derivative derived from Pacific oysters and an antibacterial pharmaceutical composition containing the same as an active ingredient, a pharmaceutical composition for preventing or treating high blood pressure, an antibacterial food additive, an antibacterial feed additive, an antibacterial cosmetic composition or hygiene products it's about
The antimicrobial peptide derived from Pacific oyster of the present invention is designed with cgRPL29, a peptide derived from the gills of Pacific oyster, as a parent, and is designed to be commercialized by reducing the number of amino acids. Since the designed antimicrobial peptide has excellent antibacterial activity despite the reduced number of amino acids, high membrane permeability, and low hemolytic activity, an antibacterial pharmaceutical composition, a pharmaceutical composition for preventing or treating food poisoning, an antibacterial food additive, It can be usefully utilized in the manufacture of antibacterial feed additives, antibacterial cosmetic compositions, or sanitary products.

Description

Antibacterial peptide derived from Pacific oyster and its use {Antibacterial peptide derived from Crassostrea gigas and its use}

The present invention is an antibacterial peptide derived from Pacific oyster ( Crassostrea gigas ) and an antibacterial pharmaceutical composition containing it as an active ingredient, a pharmaceutical composition for preventing or treating food poisoning, an antibacterial food additive, an antibacterial feed additive, an antibacterial cosmetic composition or hygiene It's about supplies.

cgRPL-29 is an antibacterial peptide composed of 54 amino acids with a molecular weight of about 6.4 kDa purified from the gills of Pacific oysters ( Crassostrea gigas ). However, it is an antibacterial peptide that does not show activity against the yeast Candida albicans . In order to overcome the limitations of the activity of cgRPL-29 and its length of 54 amino acids, it is urgent to develop new antimicrobial peptides, such as designing fragmented derivatives with increased antimicrobial activity.

Recent studies have reported that oysters, mussels, abalone, etc. play an important role in antibacterial activity peptides present in the gills or mantle as one of the effective defense mechanisms against external infectious agents. These results suggest that gills and mantle, which play a primary defense against the external environment, are organs that play an important role in the immune mechanism of shellfish, and are important targets for research on the search and purification of antibacterial active substances that perform the function of innate immunity. However, efforts to develop novel antimicrobial peptides with this as the main target are still lacking. Prior art literature related to the present invention is registered in Korea, which discloses the contents of prior research by the present inventors. Patent No. 10-1834884 (Patent Document 1) and Jung-Kil Seo et al ., Fish & Shellfish Immunology 67 (2017) 675-683 (Non-Patent Document 1). The prior art documents relate to antibacterial peptides derived from Pacific oysters, and relate to peptides having antibacterial activity against Escherichia coli or Bacillus subtilis. These prior art documents do not disclose antibacterial peptides derived from Pacific oysters that are composed of 15 or less amino acids, can be commercialized, and exhibit stronger antibacterial activity against more diverse strains.

KR 10-1834884 B

Jung-Kil Seo et al., Fish & Shellfish Immunology 67 (2017) 675-683, "Antimicrobial effect of the 60S ribosomal protein L29 (cgRPL29), purified from the gill of pacific oyster, Crassostrea gigas"

The present invention is derived from Pacific oyster ( Crassostrea gigas ) and uses cgRPL-29, a peptide with excellent antibacterial activity, to design and provide an antibacterial peptide with excellent antibacterial activity while reducing the number of amino acids to the extent that commercialization is possible. .

In addition, the present invention is to provide an antibacterial pharmaceutical composition containing the antimicrobial peptide as an active ingredient.

In addition, the present invention is to provide a pharmaceutical composition for preventing or treating food poisoning containing the antimicrobial peptide as an active ingredient.

The present invention is to provide an antibacterial food additive containing the antimicrobial peptide as an active ingredient.

The present invention is to provide an antibacterial feed additive containing the antimicrobial peptide as an active ingredient.

The present invention is to provide an antibacterial cosmetic composition containing the antimicrobial peptide as an active ingredient.

The present invention is to provide sanitary products containing the antibacterial peptide as an active ingredient.

The present invention is to provide an antibacterial peptide consisting of any one amino acid sequence selected from SEQ ID NOs: 2 to 4, derived from Pacific oyster ( Crassostrea gigas ).

The antimicrobial peptide may have an α-helix structure at the C-terminus.

The antimicrobial peptide may be one in which the C-terminus is amidated.

The antimicrobial peptide may have antibacterial activity against at least one selected from the group consisting of gram-positive bacteria, gram-negative bacteria, and yeast.

The antimicrobial peptide is Bacillus subtilis , Staphylococcus epidermidis , Staphylococcus mutans , Propionibacterium acnes ), Escherichia coli ( E. coli D31), E. coli ML35p, Shigella flexneri , Pseudomonas aeruginosa , Vibrio parahaemolyticus and Candida albicans selected from Any one or more of them may have antibacterial activity.

In addition, the present invention is to provide an antibacterial pharmaceutical composition comprising the antimicrobial peptide as an active ingredient.

In addition, the present invention is to provide a pharmaceutical composition for preventing or treating food poisoning comprising the antimicrobial peptide as an active ingredient.

In addition, the present invention is to provide an antibacterial food additive comprising the antimicrobial peptide as an active ingredient.

In addition, the present invention is to provide an antibacterial feed additive comprising the antimicrobial peptide as an active ingredient.

In addition, the present invention is to provide an antibacterial cosmetic composition comprising the antimicrobial peptide as an active ingredient.

In addition, the present invention is to provide a sanitary product containing the antimicrobial peptide as an active ingredient.

The antimicrobial peptide derived from Pacific oyster of the present invention is designed with cgRPL29, a peptide derived from the gills of Pacific oyster, as a parent, and is designed to be commercialized by reducing the number of amino acids. Since the designed antimicrobial peptide has excellent antibacterial activity despite the reduced number of amino acids, high membrane permeability, and low hemolytic activity, an antibacterial pharmaceutical composition, a pharmaceutical composition for preventing or treating food poisoning, an antibacterial food additive, It can be usefully utilized in the manufacture of antibacterial feed additives, antibacterial cosmetic compositions, or sanitary products.

1 is a result of confirming homology by aligning the amino acid sequences of cgRPL29, an antibacterial peptide derived from Pacific oysters, and ribosomal protein L29 of other organisms.
Figure 2 shows the amino acid sequence of cgRPL29, an antimicrobial peptide derived from Pacific oyster, and the location of a motif part (fragment) for the design of the antimicrobial peptide according to the present invention. In addition, FIG. 2 predicts the secondary structure of the peptide cgRPL29, indicating that an α-helix structure is formed. In the figure, 'H' represents an α-helix structure, 'E' represents a β-sheet structure, 'T' represents a turn structure, and 'C' represents a random coil structure.
Figure 3A shows the amino acid sequence and primary structure of the antimicrobial peptides according to the present invention, Figure 3B shows the secondary structure prediction results and helical wheel diagram of each antibacterial peptide according to the present invention.
Figure 4 shows the antibacterial activity of the antimicrobial peptides according to the present invention against various strains.
Figure 5 shows the antibacterial peptides according to the present invention to the endometrium permeability of Escherichia coli.
Figure 6 shows the results of measuring the MBC (Minimum Bactericidal Concentration) of E. coli of the antimicrobial peptides according to the present invention.
7 shows the results of a killing kinetics study on E. coli in 1×MBC and 5×MBC of antimicrobial peptides according to the present invention.

Hereinafter, the present invention will be described in detail.

The present inventors have completed the present invention as a result of intensive research efforts to develop an antimicrobial peptide derived from Pacific oyster ( Crassostrea gigas ) and having excellent antibacterial activity and commercially available.

Specifically, the antimicrobial peptide according to the present invention is derived from a peptide (cgRPL-29) extracted and purified from the gills of Pacific oyster ( Crassostrea gigas ), and this cgRPL-29 is a peptide with very excellent antibacterial activity. However, since the cgRPL-29 consists of a total of 54 amino acids, commercialization is difficult. Accordingly, the present inventors have developed an antibacterial peptide that is derived from cgRPL-29, a peptide derived from the Pacific oyster, and can be commercialized while maintaining excellent antibacterial activity.

To design the antimicrobial peptide, the present inventors first predicted the secondary structure of cgRPL-29 using a secondary structure prediction program. The predicted secondary structure of cgRPL-29 was a mixture of random coil structure, α-helix structure, turn structure, and β-sheet structure. Based on the predicted secondary structure, the part representing the amphiphilic α-helix structure, which is known as the most ideal structure for preferentially exhibiting antibacterial activity, is the motif region for the design of the peptide derivative of the present invention ) or parent peptide.

As a result of measuring the antibacterial activity of the selected fragment (Comparative Example; SEQ ID NO: 1: KFLKNLKFSKKH), the antibacterial activity was weak, and based on the fragment, amidation addition, amino acid addition, substitution or removal, and/or D-type amino acid substitution By designing a new antibacterial peptide using a method or the like, the antibacterial activity was remarkably increased.

The antimicrobial peptide according to the present invention is composed of any one amino acid sequence selected from SEQ ID NOs: 2 to 4.

The antimicrobial peptide may have an α-helix structure at its C-terminus.

In addition, the antimicrobial peptide may be amidated at the C-terminus. When the C-terminus of the antimicrobial peptide is amidated, it is preferable because the effect of improving resistance to proteases and improving positive net-charge is improved.

The antimicrobial peptide of the present invention designed and prepared as described above may have antibacterial activity against at least one selected from the group consisting of bacteria such as gram-positive bacteria and gram-negative bacteria and fungi such as yeast and mold.

Specifically, the antimicrobial peptide is Bacillus subtilis ( Bacillus subtilis ), Staphylococcus epidermidis ( Staphylococcus epidermidis ), Staphylococcus mutans ( Staphylococcus mutans ), Propionibacterium acnes ( Propionibacterium acnes ), Escherichia coli ( E. coli D31), Escherichia coli ( E. coli ML35p), Shigella flexneri ( Shigella flexneri ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), Vibrio parahaemolyticus ( Vibrio parahaemolyticus ) and Candida albicans ( Candida albicans ) It may exhibit antibacterial activity to any one or more selected from among.

The antimicrobial peptide according to the present invention has excellent antibacterial activity and a short amino acid sequence, so it can be commercialized. In addition, the antimicrobial peptide according to the present invention has strong inner membrane permeability. That is, the antibacterial peptide according to the present invention is highly likely to exhibit antibacterial activity by directly penetrating the bacterial inner membrane. In addition, the antibacterial peptide according to the present invention does not show hemolytic activity, so there is no problem of cytotoxicity.

In addition, as a second preferred embodiment, the present invention provides an antibacterial pharmaceutical composition comprising the antimicrobial peptide as an active ingredient.

The method of administering the pharmaceutical composition is not particularly limited, but may be injected into an artery or vein, subcutaneously, rectally, nasally, or any other parenteral route, more preferably intraarterially or intravenously. It is preferably administered intravenously, orally, or directly injected into muscle cells.

The dosage level selected from the composition will also depend on the activity of the compound, the route of administration, the severity of the condition being treated and the condition and previous medical history of the patient being treated. However, it is within the knowledge of the art to start with a dose of the compound at a level lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved, and the preferred dosage is age, sex and age. , body type, and weight can be determined. The composition may be further processed, preferably milled or ground into smaller particles, prior to formulation into pharmaceutically acceptable pharmaceutical preparations. The composition will also vary depending on the condition and the patient being treated, but this can be determined non-exclusively. For a desired effect, the effective dose of the antimicrobial peptide of the present invention is 1 to 2 mg/kg, preferably 0.5 to 1 mg/kg, and may be administered 1 to 3 times a day. The dosage is not intended to limit the scope of the present invention in any way.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulation using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by those skilled in the art. or it may be prepared by incorporating into a multi-dose container. At this time, the formulation can be used in any form suitable for pharmaceutical preparations, including oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups and aerosols, external preparations such as ointments and creams, suppositories and sterile injection solutions, etc. , a dispersing agent or a stabilizing agent may be additionally included.

In addition, the antimicrobial peptide exhibits antibacterial activity Bacillus subtilis , Staphylococcus epidermidis , Staphylococcus mutans , Propionibacterium acnes , Escherichia coli ( E. coli D31), Escherichia coli ( E. coli ML35p), Shigella flexneri ( Shigella flexneri ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), Vibrio parahaemolyticus ( Vibrio parahaemolyticus ) or Candida albicans ( Candida albicans ) Since it is possible to prevent or treat diseases such as food poisoning, candidiasis, typhoid fever, and cholera, which are diseases typically caused by, including the antimicrobial peptide as an active ingredient, prevention or treatment of infectious diseases caused by the bacteria It can be used as a pharmaceutical composition for

As a third preferred embodiment, the present invention provides a pharmaceutical composition for preventing or treating food poisoning comprising the antimicrobial peptide as an active ingredient.

As a fourth preferred embodiment, the present invention provides an antimicrobial food additive comprising the antimicrobial peptide as an active ingredient.

As a preferred fifth embodiment, the present invention provides an antibacterial feed additive comprising the antimicrobial peptide as an active ingredient.

In a specific embodiment of the present invention, the antimicrobial peptide according to the present invention has excellent antibacterial activity against gram-negative and gram-positive bacteria as well as fungi, and has no cytotoxicity, so it can be usefully used as an antibacterial food additive or feed additive.

There is no particular limitation on the type of food. Examples of foods to which the above substances can be added include drinks, meat, sausages, bread, biscuits, rice cakes, chocolates, candies, snacks, pizza, ramen, other noodles, gums, dairy products including ice creams, various soups, beverages, There are alcoholic beverages, vitamin complexes, etc., and includes all health functional foods in a conventional sense. The antibacterial peptide of the present invention can be added to food as it is or used together with other foods or food ingredients, and can be appropriately used according to conventional methods. The mixing amount of the active ingredient can be suitably determined depending on the purpose of its use (for prevention or improvement). In general, the amount of the antimicrobial peptide in the food may be added to 0.1 to 20% by weight of the total weight of the food. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range. .

In addition, as a preferred sixth embodiment, the present invention provides an antibacterial cosmetic composition comprising the antimicrobial peptide as an active ingredient.

At this time, the cosmetic composition may be in the form of a solution, powder, emulsion, lotion, spray, ointment, aerosol, cream or foam. In addition, in the cosmetic composition of the present invention, a carrier acceptable in cosmetic formulations may be included. Here, "acceptable carrier in cosmetic preparation" means a compound or composition that is already known and used, or a compound or composition to be developed in the future, which can be included in a cosmetic preparation, and has toxicity, instability, or irritation that is higher than the degree to which the human body can adapt when in contact with the skin. say nothing

The carrier may be included in the cosmetic composition of the present invention in an amount of about 1% to about 99.99% by weight, preferably about 90% to about 99.99% by weight, based on the total weight of the composition. Alcohols, oils, surfactants, fatty acids, silicone oils, wetting agents, humectants, viscosity modifiers, emulsions, stabilizers, UV scattering agents, UV absorbers, coloring agents, perfumes, and the like may be exemplified as the carrier. Compounds or compositions that can be used as the alcohol, oil, surfactant, fatty acid, silicone oil, wetting agent, humectant, viscosity modifier, emulsion, stabilizer, UV scattering agent, UV absorber, coloring agent, fragrance, etc. are already known in the art. Therefore, those skilled in the art can select and use an appropriate corresponding material or composition.

As a specific example of the present invention, the cosmetic composition according to the present invention may include glycerin, butylene glycol, propylene glycol, polyoxyethylene hydrogenated castor oil, ethanol, triethanolamine, etc. in addition to the peptides, preservatives, fragrances, coloring agents, A trace amount of purified water or the like may be included as needed.

The skin in the present invention is a concept that includes not only the face, but also the scalp and the whole body, and as a cosmetic composition that can be applied to the scalp, there are shampoo, rinse, treatment, hair growth agent, etc., body cleanser that can be applied to the whole body, etc. It can be manufactured in various forms for use.

As a seventh preferred embodiment, the present invention provides sanitary products containing the antimicrobial peptide as an active ingredient.

The sanitary products include wet wipes, hand sanitizers, mouth fresheners, mouth disinfectants, toothpaste additives, and the like.

Hereinafter, the present invention will be described in detail with reference to preferred embodiments so that those skilled in the art can easily practice the present invention. However, the present invention may be embodied in many different forms and is not limited to the embodiments described herein.

<Example>

<Materials and methods>

reagents and materials

Tryptic soy broth (TSB) and sabouraud dextrose broth (SDB) for antimicrobial activity measurement were purchased from Difco Bacto BBL and agarose type I (Low EEO Agar) from Sigma (St. Louis, MO, USA), respectively. did In addition, o-nitrophenyl-β-D-galactopyranoside (ONPG) used to measure membrane permeability was purchased from Sigma (St. Louis, MO, USA) and used. HPLC water and acetonitrile (CH ₃ CN) used in the purification process of the peptide compound were purchased from Tedia (Ohio, USA), and all other reagents were of special grade.

Design of antimicrobial peptides

For the design of the derivative of cgRPL29, the secondary structure was predicted using the Ganier program of EMBOSS GUI, a secondary structure prediction program. As a result, based on the predicted secondary structure of cgRPL29, a fragment showing the α-helix structure in the C-terminal direction was selected and used as a motif for derivative design. Each of the designed derivatives was composed of 11 or 12 amino acids, respectively, and was designed using methods such as amidation at the C-terminus and/or addition, substitution, and removal of amino acids at specific positions. In addition, in order to add stability to peptidase, derivatives in which all amino acids of the selected derivatives were substituted with D-forms were also designed.

Synthesis and purification of antimicrobial peptides

The designed peptide derivative was synthesized and purified with a purity of 95% or more and supplied from Peptron Co., Ltd. (Daejeon). The peptide derivatives were synthesized by the F-moc solid phase method using a peptide synthesizer ASP48S (Peptron, Daejeon Korea) and purified by RP-HPLC using a Shiseido Capcell-Pak C18 column (250 mm × 4.6 mm, 10 μm). . Purification conditions were carried out at a flow rate of 1 mL/min at 220 nm under a concentration gradient of 3-40% with a solvent system of H ₂ O-CH ₃ CN containing 0.1% (v/v) trifluoro acetic acid. The molecular weight of the purified peptide derivatives was measured using liquid chromatography/mass spectrometry (LC/MS; HP1100 series; Agilent, Santa Clara, CA, USA). The purified peptide derivatives were dissolved in 0.01% acetic acid at a concentration of 1000 ug/mL and stored at -70 °C for use in all subsequent experiments.

Antibacterial activity measurement method and strain used

In order to measure the antibacterial activity of the peptide derivatives, 4 types of Gram-positive bacteria ( Bacillus subtilis, Staphylococcus epidermidis , Staphylococcus mutans , Propionibacterium Acne ( Propionibacterium acnes )), 5 Gram-negative bacteria ( E. coli D31, E. coli ML35p), Shigella flexneri ( Shigella flexneri ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), Vibrio parahae Molyticus ( Vibrio parahaemolyticus )) and one yeast ( Candida albicans ) were used. As a method for measuring antibacterial activity, an ultrasensitive radial diffusion assay (URDA) method using two layers of media containing different concentrations was used. The strains used in URDA were pre-cultured for 18 hours at each medium and culture temperature, and then the bacterial concentration was measured at 84 using a color meter (Product No.52-1210, BioMerieux, Inc., USA). It was adjusted to be %T (≒ 1 x 10 ⁸ CFU/mL). Then, 0.5 mL of the bacterial solution diluted at each concentration was added to an underlay gel containing 9.5 mL of 0.03% TSB, 1% Type I agarose, and 10 mM phosphate buffer (PB) (pH 6.5), mixed well, and spread flat on a plate. poured and hardened After drilling a well with a diameter of 2.5 mm using a punch on a hard plate, each derivative was introduced at a concentration of 1000 ug/mL to 31.3 ug/mL in a volume of 5 μL. In all experiments, it was confirmed that there was no antibacterial effect by 0.01% acetic acid used as a solvent for the peptide derivative. When the peptide derivative permeates the medium, it is first cultured for 3 hours, then an overlay gel containing 10 mL of 6% TSB, 1% Type I agarose and 10 mM phosphate buffer (pH 6.5) is poured thereon, solidified, and the same temperature Secondary culture for 18 hours. The next day, the size (diameter) of the clearing zone formed around the well was measured to confirm the antibacterial activity. After removing the well diameter (2.5 mm) from the measured clear zone, the minimum effective concentration (ug/ml, MEC) was calculated as the slope value of the X-axis of the graph for log10 of the concentration of the peptide derivative used for measurement. . As a material for comparison of antibacterial activity, an antibacterial peptide derived from mast cells of American hybrid sea bass, piscidin 1, was used.

Measurement of cytoplasmic membrane permeabilization of bacteria

Bacterial endomembrane permeability was measured by measuring the activity of β-galactosidase present in the cytoplasm of E. coli ML35p using O-nitro-phenyl-β-D-galactopyranoside (ONPG), a nonmembrane-permeative chromogenic substrate. E. coli ML35p in the cultured mid-logarithmic phase was washed with 10 mM sodium phosphate buffer (pH7.4) and then dissolved in the same buffer containing 1.5 mM ONPG. Then, after adding 40 μg/mL of the peptide derivative to be measured, the degree of hydrolysis of ONPG to O-nitrophenol by β-galactosidase of E. coli ML35p leaked out was measured at 405 nm. As a comparative material for transmembrane activity, piscidin 1, an antibacterial peptide derived from mast cells of American hybrid sea bass, was used.

Measurement of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC)

The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the peptide derivatives were measured according to the method of Patrzykatet et al. (Patrzykat, A., Gallant, JW, Seo, JK, Pytyck, J., Douglas, S, E. Novel antimicrobial peptides derived from flatfish genes. Antimicrob Agents Chemother. 2003, 47(8), 2464-2470). The measured concentrations of each of the derivatives were prepared by serial dilution using 0.01% HAc from 100 ug/mL to 3.13 ug/mL, and the measurements were performed in 96-well polypropylene microtiter plates (Costar; Corning Incorporated, Corning, NY). E. coli ML35p used for the measurement was cultured for 16 hours in TSB medium to mid-logarithmic phase, and then finally diluted using TSB medium at a bacterial concentration of 10 ⁶ CFU/mL. In order to measure the minimum inhibitory concentration, after adding 90 uL of the final diluted bacterial solution to each well of a 96-well plate, 10 uL of each peptide derivative solution containing × 10 concentration of the target concentration was added to each well, , and incubated at 37 ° C. for 16 hours. As negative and positive controls for the measurement results, a state without the addition of the derivative and a state with the addition of the antibacterial peptide piscidin 1 were used. The minimum inhibitory concentration was defined as the concentration of the derivative added to the wells in which there was no bacterial growth after confirming the growth of the bacteria visually.

In addition, in order to confirm the form of antibacterial activity of the derivatives, bacterial solutions were taken from each well corresponding to the minimum inhibitory concentration of each derivative, spread on a TSA plate, and incubated at 37 ° C for 16 hours. ; MBC) was measured to confirm whether the antibacterial activity of the derivatives was bacteriostatic or bactericidal.

Killing kinetic study

For the killing kinetic study of the derivatives for which the bactericidal process was confirmed, the time-dependent killing rate was confirmed for 60 minutes at the concentrations of 1×MBC and 5×MBC for each derivative determined by MBC measurement. To this end, after adding the derivatives to an e-tube containing 10 ⁶ CFU/mL of E. coli ML35p, culturing at 37 ° C for a predetermined incubation time (5, 10, 20, 30, 40, 50, 60 minutes after the addition of the derivative) ), 10 uL of the reaction solution was taken from each measurement tube, spread on a TSA plate, and the number of colonies formed after culturing at 37 ° C for 16 hours was confirmed. As a control, a state without the addition of the derivative and a state with the addition of the antibacterial peptide piscidin 1 were used. The killing % of the derivatives was expressed as a relative ratio to the number of colonies generated in the condition without the addition of the derivatives.

<Test Example>

Test Example 1: Designed peptide derivatives of cgRPL29

1-1: cgRPL29

cgRPL-29 was purified from the gills of Pacific oysters ( Crassostrea gigas ), and is an antibacterial peptide composed of 54 amino acids and having a molecular weight of 6484.6 Da. cgRPL-29 showed almost identical sequence similarity to ribosomal protein L29, a protein derived from oyster gills (FIG. 1).

Figure 1 shows the sequence alignment (alignment) of the amino acid sequence of cgRPL-29 and ribosomal protein L29 present in other organisms. Conserved sequences in FIG. 1 are shown as black areas.

1-2: Secondary structure prediction of cgRPL29

From the antimicrobial peptide cgRPL-29, the secondary structure of cgRPL-29 was predicted using a secondary structure prediction program in order to design a more fragmented peptide derivative with improved antibacterial activity (FIG. 2). For this purpose, the secondary structure prediction of cgRPL29 was performed using the Ganier program of EMBOSS GUI.

As a result, cgRPL29 was predicted to have a mixed structure in which α-helix, turn, sheet, and random structures were mixed (Fig. 2). Based on these results, the C-terminus representing the amphiphilic α-helix structure, known as the most ideal structure for exhibiting antibacterial activity, was selected as the motif region for derivative design.

1-3: Primary structure of the designed peptide derivative

The structural characteristic of the selected C-terminal part (motif region) is an amphiphilic α-helix structure (FIG. 3B). This means that the C-terminal part of cgRPL-29 is the optimal region for derivative design, and based on the motif region, amidation addition at the C-terminus, amino acid addition, substitution or removal, and/or D-type amino acid substitution method was used to design three derivatives (Fig. 3A and Fig. 3B). Examples of each of the designed peptide derivatives are shown in Table 1 below. Each derivative consists of 11 or 12 amino acid residues. The synthesized derivatives were confirmed through molecular weight measurement.

sequence number peptide Sequence Count SEQ ID NO: 1 comparative example KFLKNLKFSKKH-NH ₂ 12 SEQ ID NO: 2 Example 1 KFLKLLKKLK-NH ₂ 11 SEQ ID NO: 3 Example 2 KFLKLLKKLLKH-NH ₂ 12 SEQ ID NO: 4 Example 3 D-KFLKLLKKLK-NH ₂ 11

In Table 1, the comparative example (SEQ ID NO: 1) is a "motif region" at the C-terminus of cgRPL29 for the design of the antimicrobial peptide derivative of the present invention, and corresponds to a comparative example of the peptide derivative of the present invention.

In addition, Example 1 (SEQ ID NO: 2), Example 2 (SEQ ID NO: 3), and Example 3 (SEQ ID NO: 4) are amidation addition, amino acid addition, substitution or removal, and / or D- It is a peptide derivative designed using the amino acid substitution method. In addition, Example 3 has the same amino acid sequence as Example 1, and is a peptide derivative in which all amino acids of Example 1 are substituted with D-type amino acids.

Test Example 2: Antibacterial activity of cgRPL-29 peptide derivatives

The antibacterial activity of the peptide derivatives and the antibacterial peptide piscidin 1 (an antibacterial peptide derived from American hybrid sea bass) used as a control against the above 9 bacteria and 1 yeast ( C. albican s) was tested using the URDA method and measured (Fig. 4 and Table 2).

As a result of the antibacterial activity measurement, the antimicrobial peptide corresponding to the comparative example showed weak antibacterial activity against all strains used for the measurement, whereas the three peptide derivatives of the present invention (Example 1, Example 2, and Example 3) showed strong antibacterial activity. In particular, the peptide derivatives of the present invention exhibited antibacterial activity similar to that of piscidin 1 used as a control, and in particular, showed stronger activity than piscidin 1 against C. albicans . Interestingly, the parent peptide cgRPL-29 showed no antibacterial activity against C. albicans , but the peptide derivatives of the present invention showed strong antibacterial activity against C. albicans . In addition, the peptide derivatives of the present invention exhibited strong antibacterial activity against S. epidermidis, P. acnes, C. albicans and S. mutans known to cause acne and tooth decay.

These results indicate that the peptide derivatives of the present invention can be used as candidates for the development of new peptide antibiotics because the peptide length is fragmented and the antibacterial activity is improved compared to the parent peptide, cgRPL29.

Microbes Gram Minimal Effectives Concentration (μg/mL) [μM] ⁿ comparative example Example 1 Example 2 Example 3 Piscidin1 B. subtilis KCTC1021 + 16.8 6.7 1.0 4.4 7.5 S. epidermidis KCTC1917 + >100 5.9 1.2 6.9 NT S.mutans KCCM40105 + 3.0 4.3 0.7 3.2 3.2 P. acnes KCTC11946 + >100 8.8 1.7 2.6 NT E. coli D31 - 18.2 10.8 4.9 9.0 3.8 E. coli ML35p - 1.8 6.2 1.7 3.5 2.3 S. flexneri KCTC2009 - 3.2 8.7 2.4 2.6 8.0 P. aeruginosa KCTC2004 - >100 8.5 4.2 3.0 8.0 V. parahaemolyticus KCCM42264 - 90.5 4.6 0.7 3.0 3.0 C. albicans KCTC7965 Yeast >100 9.7 6.0 9.4 >62.5

Test Example 3: Inner membrane permeability of bacteria

In order to confirm the bacterial inner membrane permeability of the peptide derivatives of the present invention and the antibacterial peptide piscidin 1 used as a control, the inner membrane permeability to E. coli ML35p was confirmed (FIG. 5).

As a result, the peptide derivatives according to Examples 1 to 3 of the present invention showed bacterial endomembrane permeability similar to that of piscidin 1, which is known to have strong endomembrane permeability, whereas the peptides of Comparative Examples showed very weak endomembrane permeability.

These results mean that, unlike the peptides of the Comparative Examples, the peptide derivatives of Examples 1 to 3 have a mechanism of action that exhibits antibacterial activity by recognizing and attacking the bacterial membrane as a direct target site.

Test Example 4: Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) measurement

MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) measurements were performed to determine whether the mechanism of action for the antibacterial activity of the peptide derivatives of the present invention was bacteriostatic or bactericidal.

As a result of the measurement, the peptide derivatives of the present invention showed a low MIC similar to that of piscidin 1 against E. coli ML35p (Table 3). On the other hand, the antimicrobial peptide of Comparative Example showed a high MIC of >100 ug/mL.

peptide Minimum Inhibitory Concentration (MIC, μg/mL) comparative example > 100 Example 1 3.13 Example 2 6.25 Example 3 25

In order to confirm the antimicrobial form of the peptide derivatives of the present invention, an MBC measurement experiment was performed based on the obtained MIC value, and as a result, it was confirmed that the peptide derivatives of the present invention and piscidin 1 showed the same MBC value as the MIC value ( Figure 6 and Table 4).

peptide Minimum Bactericidal Concentration (MBC, μg/mL) comparative example > 100 Example 1 3.13 Example 2 6.25 Example 3 25

These results mean that the peptide derivatives of the present invention have the same MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) and exhibit antibacterial activity through a bactericidal mechanism.

Test Example 5: Killing kinetic study of peptide derivatives

In order to confirm the killing rate over time of the peptide derivatives of the present invention, of which bactericidal activity was confirmed, a killing kinetic study was performed after reacting with E. coli ML35p at concentrations of 1xMBC and 5xMBC for 60 minutes (FIG. 7). ). As a result, the peptide derivatives according to the examples of the present invention were found to kill E. coli ML35p within 5 minutes at the concentrations of 1xMBC and 5xMBC, except for 1xMBC (3.13 μg/mL) of Example 1.

These results mean that the peptide derivatives of the present invention kill bacteria very quickly (within 5 minutes) after coming into contact with bacteria. Therefore, the peptide derivatives of the present invention are considered to have an action mechanism to sterilize bacteria in a very rapid time by directly attacking the bacterial membrane and penetrating the inner membrane to lyse the bacteria.

<result>

In order to develop a new antibacterial peptide derivative with fragmentation in length and improved antibacterial activity from the parent antibacterial peptide (cgRPL-29) whose primary structure was purified from the gills of Pacific oysters, the present inventors developed a peptide based on cgRPL-29. Derivatives were designed and synthesized by solid phase synthesis (FIG. 3). The peptide derivatives of the present invention were designed by selecting a site located in the direction of the C-terminus of cgRPL-29 as a motif region, substituting, adding or removing amino acids, and amidating the C-terminus. In addition, in order to add stability to peptidase, all amino acids of the designed derivative were substituted with D-forms.

As a result of measuring the antibacterial activity of the peptide derivatives of the present invention designed as described above (Example 1, Example 2, Example 3), strong antibacterial activity was shown against various strains including acne and tooth decay causing bacteria ( Fig. 4, Table 2). In addition, as a result of studying the mechanism of action of the derivatives, it was found that the peptide derivatives of the present invention exhibit antibacterial activity by quickly killing bacteria by penetrating or inhibiting the membrane after acting on the bacterial membrane (FIGS. 5 to 7). From the experimental results of this study, it is believed that the peptide derivatives of the present invention can be usefully used as candidates for developing antibiotic substitutes that can replace existing antibiotics.

Formulation examples for the antibacterial peptide derivatives of the present invention are exemplified below.

<Formulation Example 1> Preparation of pharmaceutical formulation

Formulation Example 1-1: Preparation of powder

20 mg of the antimicrobial peptide of the present invention

lactose 50 mg

Talc 10 mg

A powder was prepared by mixing the above components and filling in airtight bags.

Formulation Example 1-2: Manufacture of tablets

Antibacterial peptide of the present invention 10 mg

Corn Starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above ingredients, tablets were prepared by tableting according to a conventional tablet manufacturing method.

Formulation Example 1-3: Preparation of capsules

Antibacterial peptide of the present invention 10 mg

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium stearate 0.2 mg

Capsules were prepared by mixing the above ingredients and filling them into gelatin capsules according to a conventional capsule preparation method.

Formulation Example 1-4: Preparation of liquid formulation

20 mg of the antimicrobial peptide of the present invention

Isomerized sugar 10g

5g mannitol

Appropriate amount of purified water

According to the usual method for preparing liquids, each component is added to purified water to dissolve, lemon flavor is added in an appropriate amount, the above components are mixed, purified water is added to adjust the total volume to 100ml, and then the liquid is filled in a brown bottle and sterilized. manufactured.

Formulation Example 1-5: Preparation of injection

10 μg/mL of the antimicrobial peptide of the present invention

dilute hydrochloric acid BP until pH 7.6

Sodium Chloride BP for Injection up to 1 mL

Dissolve the peptide derivative of the present invention in an appropriate volume of sodium chloride BP for injection, adjust the pH of the resulting solution to pH 7.6 using dilute hydrochloric acid BP, adjust the volume using sodium chloride BP for injection, and mix thoroughly did The solution was filled into a 5 ml Type I ampoule made of transparent glass, sealed under an upper grid of air by dissolving the glass, and autoclaved sterilized at 120° C. for 15 minutes or longer to prepare an injection.

<Formulation Example 2> Manufacturing of cosmetics

Formulation Example 2-1: Softening lotion (skin)

In order to prepare an antibacterial softening lotion containing the peptide derivative of the present invention, it can be prepared according to a conventional manufacturing method in the field of cosmetics by mixing as shown in Table 5 below.

Ingredients Preparation Example 2-1
(weight%) Antimicrobial peptides of the present invention 0.01~30 1,3-butylene glycol 3.0 glycerin 5.0 Polyoxyethylene (60) hydrogenated castor oil 0.2 ethanol 8.0 citric acid 0.02 sodium citrate 0.06 antiseptic a very small amount Spices a very small amount Purified water to 100

Formulation Example 2-2: Nutrition lotion (lotion)

In order to prepare an antibacterial nutrient lotion containing the peptide derivative of the present invention, it can be prepared according to a conventional manufacturing method in the field of cosmetics by mixing as shown in Table 6 below.

Ingredients Production Example 2-2
(weight%) Antimicrobial peptides of the present invention 0.01~30 1,3-butylene glycol 3.0 glycerin 5.0 squalane 10.0 Polyoxyethylene sorbitan monooleate 2.0 Yuchangmok Oil 0.1~30 Polyoxyethylene (60) hydrogenated castor oil 0.2 ethanol 8.0 citric acid 0.02 sodium citrate 0.06 antiseptic a very small amount Spices a very small amount Purified water to 100

Formulation Example 2-3: Face wash (cleansing foam)

In order to prepare an antibacterial nutrient lotion containing the peptide derivative of the present invention, it can be prepared according to a conventional manufacturing method in the field of cosmetics by mixing as shown in Table 7 below.

Ingredients Manufacturing example 2-3
(weight%) Antimicrobial peptides of the present invention 0.01~30 Sodium N-acylglutamate 20.0 glycerin 10.0 PEG-400 15.0 propylene glycol 10.0 POE (15) oleyl alcohol ether 3.0 Laurine Derivatives 2.0 Methylparaben 0.2 EDTA-4Na 0.03 Spices a very small amount Purified water to 100

Formulation Example 2-4: Nutrition Cream

In order to prepare an antibacterial nutrient lotion containing the peptide derivative of the present invention, it can be prepared according to a conventional manufacturing method in the field of cosmetics by mixing as shown in Table 8 below.

Ingredients Manufacturing example 2-4
(weight%) Antimicrobial peptides of the present invention 0.01~30 vaseline 7.0 liquid paraffin 10.0 beeswax 2.0 Polysorbate 60 2.5 Sorbitan Sesquioleate 1.5 squalane 3.0 propylene glycol 6.0 glycerin 4.0 triethanolamine 0.5 xanthan gum 0.5 cotophenylacetate 0.1 flavorings, preservatives a very small amount Purified water to 100

Although the preferred embodiments of the present invention have been described above, the present invention is not limited thereto, and various modifications and implementations are possible within the scope of the technical spirit of the present invention, which also fall within the scope of the appended claims. It is natural.

<110> Industry-Academic Cooperation Foundation, Kunsan National University <120> Antibacterial peptide derived from Crassostrea gigas and its use <130> HPC-9907 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 12 <212> PRT 213 <Crossostrea gigas> <400> 1 Lys Phe Leu Lys Asn Leu Lys Phe Ser Lys Lys His 1 5 10 <210> 2 <211> 11 <212> PRT <213> artificial sequence <220> <223> Antibacterial peptide analogue derived from Crassostrea gigas <400> 2 Lys Phe Leu Lys Leu Leu Lys Lys Leu Leu Lys 1 5 10 <210> 3 <211> 12 <212> PRT <213> artificial sequence <220> <223> Antibacterial peptide analogue derived from Crassostrea gigas <400> 3 Lys Phe Leu Lys Leu Leu Lys Lys Leu Leu Lys His 1 5 10 <210> 4 <211> 11 <212> PRT <213> artificial sequence <220> <223> Antibacterial peptide analogue derived from Crassostrea gigas - D isomer of all amino acid residue <400> 4 Lys Phe Leu Lys Leu Leu Lys Lys Leu Leu Lys 1 5 10

Claims (12)

An antibacterial peptide consisting of any one amino acid sequence selected from SEQ ID NOs: 2 to 4 derived from Pacific oyster ( Crassostrea gigas ). According to claim 1,
The antimicrobial peptide is an antimicrobial peptide, characterized in that the C-terminus is an α-helix (α-helix) structure. According to claim 1,
The antimicrobial peptide is an antimicrobial peptide, characterized in that the C-terminus is amidated (amidation). According to claim 1,
The antimicrobial peptide is characterized in that it has an antibacterial activity against any one or more selected from the group consisting of gram-positive bacteria, gram-negative bacteria and yeast. According to claim 1,
The antibacterial peptide is Bacillus subtilis , Staphylococcus epidermidis , Staphylococcus mutans , Staphylococcus mutans, Propionibacterium acnes , E. coli D31), Escherichia coli ( E. coli ML35p), Shigella flexneri ( Shigella flexneri ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), Vibrio parahaemolyticus ( Vibrio parahaemolyticus ) and Candida albicans An antimicrobial peptide characterized in that it has antibacterial activity in any one or more. Pacific oyster ( Crassostrea gigas ) Antibacterial pharmaceutical composition comprising an antimicrobial peptide consisting of any one amino acid sequence selected from SEQ ID NOs: 2 to 4 as an active ingredient. According to claim 6,
The antibacterial peptide is Bacillus subtilis , Staphylococcus epidermidis , Staphylococcus mutans , Staphylococcus mutans, Propionibacterium acnes , E. coli D31), Escherichia coli ( E. coli ML35p), Shigella flexneri ( Shigella flexneri ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), Vibrio parahaemolyticus ( Vibrio parahaemolyticus ) and Candida albicans Antibacterial pharmaceutical composition characterized in that it has an antibacterial activity in any one or more. Pacific oyster ( Crassostrea gigas ) A pharmaceutical composition for preventing or treating food poisoning comprising an antimicrobial peptide consisting of any one amino acid sequence selected from SEQ ID NOs: 2 to 4 derived from as an active ingredient. Pacific oyster ( Crassostrea gigas ) An antibacterial food additive comprising as an active ingredient an antibacterial peptide consisting of any one amino acid sequence selected from SEQ ID NOs: 2 to 4 derived from. Pacific oyster ( Crassostrea gigas ) An antibacterial feed additive comprising as an active ingredient an antibacterial peptide consisting of any one amino acid sequence selected from SEQ ID NOs: 2 to 4 derived from. Pacific oyster ( Crassostrea gigas ) Antibacterial cosmetic composition comprising as an active ingredient an antibacterial peptide consisting of any one amino acid sequence selected from SEQ ID NOs: 2 to 4 derived from. Sanitary products containing an antibacterial peptide consisting of any one amino acid sequence selected from SEQ ID NOs: 2 to 4 derived from Pacific oyster ( Crassostrea gigas ) as an active ingredient.

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