KR102311729B1 - Anti-Microbial Peptide Plantaricin, Isolated from Lactobacillus Plantarum and Uses thereof - Google Patents

Abstract

The present invention relates to the antibacterial peptide planthalysin isolated from Lactobacillus plantanum and uses thereof.
The five novel plantaricin peptides isolated from the Lactobacillus plantarum strain of the present invention exhibit strong antibacterial activity against pathogenic bacteria and can be applied to various bacterial infections, and the composition of the present invention can be used in medicine, food or It can be usefully used for antibacterial purposes in various fields such as feed additive composition.

Description

Antimicrobial Peptide Plantaricin, Isolated from Lactobacillus Plantarum and Uses thereof

The present invention relates to a novel antibacterial peptide plantaricin isolated from lactic acid bacteria and uses thereof, and more particularly to an antibacterial peptide plantarisin isolated from Lactobacillus plantanum and uses thereof.

Some of the substances that play an important role in the process for maintaining homeostasis of living organisms are physiologically active substances derived from various living organisms. Until now, many studies have been conducted on numerous physiologically active substances, and among them, research on antibacterial and antifungal substances is a very important area in life science and medicine.

It is known that all living things produce antibacterial and antifungal substances for survival, and many studies are being made on microorganisms that produce antibacterial and antifungal substances. Studies to develop antibacterial agents have been attempted for a long time.

All living things are known to produce antibacterial substances for survival. Antibacterial peptides isolated from various living organisms are known to act in a variety of ways, ranging from bacteria, fungi and viruses, and play an important role in host defense and innate immune system. is known to do Organisms produce antimicrobial peptides on their own (Bevins et al., Ann. Rev. Biochem., 59, 395-414, 1990), forming small peptides of approximately 10 to 40 amino acids, and are largely divided into three can be divided into groups of dogs. The first is a cysteine-rich β-sheet peptide molecule, the second is an α-helix structure amphiphilic peptide molecule, and the third is a proline-rich peptide molecule.

These antibacterial peptides have various structures depending on the amino acid sequence, and the most common of these structures is a structure that does not have a cysteine residue and forms an amphiphilic alpha helix like cecropin, an antibacterial peptide found in insects. . As such, many studies have been made on the antimicrobial activity of peptides isolated from living organisms, and many studies have been attempted to develop antibacterial agents using peptides isolated from living organisms.

On the other hand, kimchi is rich in various fermentation products and ingredients such as dietary ingredients, vitamins, carotene, lactic acid, lactic acid bacteria, acetylcholine, dextran, acetate, etc., so that it enhances immunity, antioxidant, antibacterial, and antibacterial properties in the human body. It is known as a functional food having a function of preventing the production of cholesterol, carcinogens and mutants. In particular, dietary fiber rich in vegetables prevents constipation, and abundant lactic acid bacteria have a probiotic effect to improve intestinal flora by inhibiting the growth of harmful microorganisms in the intestine.

There are major strains belonging to the genus Lactobacillus, Leuconostoc, Pediococcus, Weissella, etc. in kimchi, and these strains play an important role in kimchi fermentation. is known

As described above, the Lactobacillus plantarum strain, a type of lactic acid bacteria contained in kimchi, is a lactic acid bacillus that performs homozygous or heterozygous fermentation, and is commonly found in the intestinal tract of animals, including humans, and in the fermentation process of dairy products or vegetables. The microorganisms of the genus Lactobacillus are known to produce various proteinaceous antibacterial substances, and by maintaining the intestinal pH at an acidic level, it suppresses the reproduction of harmful bacteria such as E. coli and Clostridium, and relieves diarrhea and constipation. It is known to play a role in vitamin synthesis, anti-cancer action, and lowering serum cholesterol [Michael and Philippe, Probiotics and prebiotics: Effects on diarrhea, The journal of nutrition, Volume 137, March 2007, pages 803S-811S ; Roberfroid, Prebiotics and probiotics: Are they functional foods?, American journal of clinical nutrition, Volume 71, June 2000, pages 1682S-1687S].

However, despite these research results, research results related to the discovery of Lactobacillus strains with high antibacterial activity that can be used industrially are insignificant. It is difficult to find.

Under this background, the present inventors made diligent efforts to discover novel antibacterial peptides isolated from lactic acid bacteria and having excellent antibacterial activity. It was confirmed that the taricin peptide has excellent antibacterial activity and can be usefully used for antibacterial purposes, and the present invention was completed.

Korean Patent Registration Publication No. 10-1772870 Korean Patent Registration No. 10-1757623 Korean Patent Registration Publication No. 10-1750468 Korean Patent Registration Publication No. 10-1658377

Accordingly, the main object of the present invention is to provide five kinds of plantaricin antibacterial peptides, which are novel antibacterial peptides isolated from Lactobacillus plantarum strains.

Another object of the present invention is to provide an antibacterial composition using the antibacterial peptide of planaricin.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a plantaricin peptide represented by any one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 5.

The term "Plantaricin peptide" of the present invention is a novel antibacterial peptide newly isolated by the present inventors from " Lactobacillus plantarum strain" isolated and identified from kimchi by the present inventors (antimicrobial peptides, AMPs), "Plantaricin", "Plantaricin" or "pln" can be used interchangeably.

In addition, the term "plantarisin peptide 1" of the present invention is represented by the amino acid sequence of SEQ ID NO: 1, "plantarisin peptide 2" is represented by the amino acid sequence of SEQ ID NO: 2, and "plantarisin peptide 3" is It is represented by the amino acid sequence of SEQ ID NO: 3, "Plantalysin peptide 4" is represented by the amino acid sequence of SEQ ID NO: 4, and "Plantalysin peptide 5" is represented by the amino acid sequence of SEQ ID NO: 5.

As used herein, the term “peptide” refers to a polymer composed of amino acids linked by amide bonds (or peptide bonds). For the purpose of the present invention, it refers to a peptide that exhibits a high antibacterial effect against harmful bacteria.

The peptide of the present invention is a plantarlysin peptide represented by SEQ ID NOs: 1 to 5.

The present inventors investigated and analyzed the antibacterial effect of lactic acid bacteria for several years for the development of a new antibacterial agent. The antibacterial activity of five new antibacterial peptides, Plantaricin peptide, was verified through genome and transcriptome analysis, and the present invention was completed.

In the present invention, the plantaricin peptide is characterized in that it is derived from a Lactobacillus plantarum strain isolated from kimchi.

In addition, the planaricin peptide of the present invention is characterized in that it exhibits antibacterial activity against pathogenic bacteria, and the pathogenic bacteria may include, but are not limited to, Salmonella Typhimurium.

The plan thalysin peptide of the present invention may be additionally linked to a cell internalization sequence or a protein transformation domain.

According to a recent study report, a transportan capable of transporting various cellular internalization sequences, for example, the TAT transactivation domain of HIV virus, antennapedia, etc. across the plasma membrane, is known. In addition, polyarginine is known as an attractive tool for transporting peptides into cells by much more efficient transport of peptides and proteins across the plasma membrane.

Accordingly, the planthalysin peptide of the present invention may include a cell internalization transporter or sequence. The cellular internalization sequence may be any internalization sequence known or newly discovered in the art, or a conservative variant thereof. Non-limiting examples of cell internalization transporters and sequences include polyarginine (eg R9), antennapedia sequence, TAT, HIVTat, penetratin, Antp-3A (Antp mutant), Buforin II, transportan, MAP (model amphiphilic peptide), KFGF, Ku70, prion, pVEC, Pep-1, SynB1, Pep-7, HN-1, BGSC (bis-guanidinium-spermidine-cholesterol) and BGTC (bis-gu anidinium-threne-cholesterol).

In addition, the planthalysin peptides of the present invention may also contain a targeting sequence, a capping motif, a tag, a labeled residue, an additional amino acid sequence designed for a specific purpose to increase the half-life or stability of the peptide. can In addition, the peptides of the present invention may be linked to coupling partners such as effectors, drugs, prodrugs, toxins, peptides, and delivery molecules.

In addition, the planthalysin peptide of the present invention may be prepared in the form of a pharmaceutically acceptable salt. Specifically, salts can be formed by addition of acids, for example, inorganic acids (e.g. hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid, etc.), organic carboxylic acids (e.g. acetic acid, trifluoroacetic acid, etc.) acetic acid, propionic acid, maleic acid, succinic acid, malic acid, citric acid, tartaric acid, salicylic acid), and acidic sugars (glucuronic acid, galacturonic acid, gluconic acid, ascorbic acid), acidic polysaccharides such as hyaluronic acid, chondroitin sulfate, arginic acid), organic sulfonic acids including sulfonic acid sugar esters such as chondroitin sulfate (eg, methane, sulfonic acid, p-toluene sulfonic acid), etc. may be added to form salts.

The planthalysin peptide of the present invention can be obtained by various methods well known in the art. Specifically, it can be prepared by chemical synthesis or genetic engineering method using a protein expression system, such as a method for synthesizing in vitro, and cell-free protein synthesis, and a conventional chemical synthesis method related to a method for producing a peptide is known in the art. It is well known.

In addition, the plantaricin peptide of the present invention can be produced through a genetic engineering method using the amino acid sequence represented by SEQ ID NO: 1 to SEQ ID NO: 5 or a nucleotide sequence encoding the same, an expression vector, and a host cell.

In the present invention, the term "expression vector" refers to a recombinant vector capable of expressing a target peptide in an appropriate host cell, and refers to a gene construct including essential regulatory elements operatively linked to express a gene insert. The expression vector of the present invention includes expression control elements such as a promoter, an operator, and an initiation codon as elements generally possessed by a suitable expression vector. The start codon and stop codon are generally considered part of the nucleotide sequence encoding the polypeptide, and must be functional in the subject when the genetic construct is administered and must be in-frame with the coding sequence. The promoter of the vector may be constitutive or inducible.

In addition, it may include a signal sequence for the release of the fusion polypeptide in order to facilitate the separation of the protein from the cell culture medium. A specific initiation signal may also be required for efficient translation of the inserted nucleic acid sequence. These signals include the ATG initiation codon and adjacent sequences. In some cases, an exogenous translational control signal, which may include an ATG initiation codon, must be provided. These exogenous translational control signals and initiation codons can be from a variety of natural and synthetic sources. Expression efficiency can be increased upon introduction of appropriate transcriptional or translational enhancing factors.

As the expression vector, any conventional expression vector may be used. For example, plasmid DNA, phage DNA, and the like can be used. Specific examples of the plasmid DNA include commercial plasmids such as pUC18 and pIDTSAMRT-AMP. Other examples of plasmids that can be used in the present invention include E. coli-derived plasmids (pYG601BR322, pBR325, pUC118 and pUC119), Bacillus subtilis-derived plasmids (pUB110 and pTP5) and yeast-derived plasmids (YEp13, YEp24 and YCp50). Specific examples of phage DNA include λ-phages (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11 and λZAP). In addition, retroviruses, animal viruses such as adenoviruses or vaccinia viruses, insect viruses such as baculoviruses can also be used. Since these expression vectors show different protein expression levels and modifications depending on the host cell, it is sufficient to select and use the most suitable host cell for the purpose.

In addition, as used herein, the term "transformation" refers to generating genetically stable inheritance so that the nucleotide fragment moves into the genome of a host organism to express a desired peptide.

Any transformation method may be used for the transformation method of the present invention, and it can be easily performed according to a conventional method in the art. In general, transformation methods include CaCl ₂ precipitation method, CaCl ₂ method using DMSO (dimethyl sulfoxide) as a reducing material, Hanahan method, electroporation, calcium phosphate precipitation method, protoplast fusion method, stirring using silicon carbide fiber method, agrobacterium mediated transformation method, transformation method using PEG, dextran sulfate, lipofectamine, and drying/inhibition mediated transformation method.

The host is not particularly limited as long as it allows expression of the peptide of the present invention. Specific examples of hosts that can be used in the present invention include E. coli (E. Escherichia (Escherichia) in strains, Lactobacillus Planta Room (Lactobacillus plantarum), such as Lactobacillus bacteria (Lactobacillus) in strains of Bacillus subtilis (Bacillus (Bacillus) spp, Pseudomonas footage is as Bacillus subtilis), such as coli) ( Pseudomonas putida ) such as Pseudomonas ( Pseudomonas ) genus strain, Saccharomyces cerevisiae ( Saccharomyces ) cerevisiae ) or Schizosaccharomyces pombe (Schizosaccharomyces pombe), such as yeast, animal cells, and insect cells.

According to another aspect of the present invention, the present invention provides an antibacterial composition comprising the plantaricin peptide of the present invention represented by any one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 5 as an active ingredient.

Since the plantaricin peptide of the present invention exhibits excellent antibacterial activity by directly affecting harmful microorganisms, it can be usefully used as an antibacterial pharmaceutical composition.

The pharmaceutical composition specifically has antibacterial activity against Gram-positive bacteria, Gram-negative bacteria and antibiotic-resistant strains. Specifically, there is antibacterial activity against any one or more selected from the group consisting of Salmonella sp. strain, Streptococcus sp. strain, Bacillus sp. strain, and Escherichia sp. strain, but is not limited thereto. However, it is not limited to the pathogenic bacteria listed above, considering that the action of the plantaricin peptide of the present invention to be attached to the microbial membrane and cause the collapse of the microbial membrane can act to inhibit the growth of almost all pathogenic bacteria.

The Salmonella sp. strain may include Salmonella gallinarum , and Salmonella Typhimurium , and the Bacillus sp. strain may include Bacillus cereus (B. cereus), and the S. cherry teeth sp is not intended to be, and the S. cherry Escherichia coli (E. coli), as the Streptococcus spp, but include a Streptococcus aureus (Staphylococcus aureus), limited.

The novel plantaricin 1 to plantarisin 5 peptides of the present invention may be administered parenterally during clinical administration and may be used in the form of general pharmaceutical preparations.

That is, the novel peptide of the present invention can actually be administered in various parenteral formulations, and when formulated, it is prepared using a diluent or excipient such as a commonly used filler, extender, binder, wetting agent, disintegrant, surfactant, etc. do. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, liulinji, glycerogelatin, and the like can be used.

In addition, the novel plantaricin peptide of the present invention can be used by mixing with various pharmaceutically acceptable carriers such as physiological saline or organic solvents, and in order to increase stability or absorption, such as glucose, sucrose or dextran Hydrocarbons, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers may be used as pharmaceuticals.

The effective dose of the novel plantaricin peptide of the present invention is 0.01 to 10 mg/kg, preferably 0.1 to 1 mg/kg, and may be administered 1 to 3 times a day.

The total effective amount of the novel plantaricin peptide of the present invention in the pharmaceutical composition of the present invention may be administered to a patient in a single dose by bolus form or infusion for a relatively short period of time. and may be administered by a fractionated treatment protocol in which multiple doses are administered for a long period of time. Since the concentration is determined by considering various factors such as the age and health status of the patient as well as the administration route and number of treatments, the effective dosage of the patient is determined. It will be possible to determine an appropriate effective dosage according to the specific use of the novel plantaricin peptide as a pharmaceutical composition.

In addition, since the plantaricin peptide of the present invention exhibits excellent antibacterial activity by directly affecting harmful microorganisms, it can be usefully used as an antibacterial cosmetic composition.

The cosmetic composition of the present invention includes ingredients commonly used in cosmetic compositions in addition to the novel plantaricin peptide as an active ingredient, for example, conventional auxiliary agents such as antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances; and carriers.

The cosmetic composition of the present invention may be prepared in any formulation conventionally prepared in the art, for example, solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing , oil, powder foundation, emulsion foundation, wax foundation, spray, etc., but is not limited thereto. More specifically, it may be prepared in the form of a flexible lotion (skin), a nourishing lotion (milk lotion), a nourishing cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray, or a powder.

When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide as a carrier component. can be used

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, additional chlorofluorohydrocarbon, propane /may contain propellants such as butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizer or emulsifier is used as a carrier component, for example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylglycol oil, glycerol fatty esters, fatty acid esters of polyethylene glycol or sorbitan.

When the formulation of the present invention is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystals Adult cellulose, aluminum metahydroxide, bentonite, agar or tragacanth may be used.

When the formulation of the present invention is a surfactant-containing cleansing agent, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide as carrier components Ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative or ethoxylated glycerol fatty acid ester and the like can be used.

In addition, since the plantaricin peptide of the present invention exhibits excellent antibacterial activity through direct effect on harmful microorganisms, it can be usefully used as an antibacterial composition for food preservation or feed preservation.

Food or feed preservatives, cosmetic preservatives, and pharmaceutical preservatives are additives used to prevent deterioration, spoilage, discoloration and chemical changes of food or pharmaceuticals. Functional antibacterial agents such as inhibiting the growth of spoilage microorganisms or sterilizing in food, feed, cosmetics, and pharmaceutical products are also included. As an ideal condition for preservatives in foods or feeds, cosmetics, and pharmaceuticals, it should not be toxic and should be effective even in a small amount. Pesticides to eradicate pests and pests in crops also suppress the growth of harmful microorganisms and humans can safely consume crops sprayed with pesticides only when there is no harm to the human body. The five novel plantaricin peptides of the present invention exhibit excellent antibacterial activity through direct effect on harmful microorganisms, and thus can be widely used as preservatives for food or feed, cosmetic preservatives, and pharmaceutical preservatives.

In another aspect, the present invention is for treating bacterial infection diseases of animals other than humans, comprising the step of killing pathogenic bacteria by administering the plantaricin peptide or a composition comprising the same to an individual in need of treatment. provide a way

Although the treatment method according to the present invention is a method of treating animals other than humans, it does not mean that the treatment method is ineffective in humans. In addition, in consideration of having a disease caused by a bacterial or fungal infection whose symptoms can be improved by administration of the therapeutic composition according to the present invention in the case of a human, it can be sufficiently used in human treatment.

In the present invention, the term "animals other than humans" refers to horses, sheep, pigs, goats, camels, except for humans, who have diseases caused by bacterial or fungal infection whose symptoms can be improved by administration of the therapeutic composition according to the present invention. , antelope, means animals such as dogs. By administering the therapeutic composition according to the present invention to animals other than humans, it is possible to effectively prevent and treat diseases caused by bacterial and fungal infections.

In the present invention, the term "administration" means introducing a predetermined substance to an animal by any suitable method, and the administration route of the therapeutic composition according to the present invention is oral or through any general route as long as it can reach the target tissue. It may be administered parenterally. In addition, the therapeutic composition according to the present invention may be administered by any device capable of moving the active ingredient to the target cell.

The preferred dosage of the therapeutic composition according to the present invention varies depending on the condition and weight of the animal to be treated, the degree of disease, the drug form, the route of administration, and the duration, but may be appropriately selected by those skilled in the art. However, for a desirable effect, the antimicrobial composition of the present invention is preferably administered at 0.01 to 10 mg/kg per day, preferably at 0.1 to 5 mg/kg. Administration may be administered once a day, or may be administered in several divided doses.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides five new plantaricin peptides isolated from Lactobacillus plantarum strain derived from kimchi, and an antibacterial composition using the same as an active ingredient.

(b) Five novel plantaricin peptides isolated from the Lactobacillus plantarum strain of the present invention exhibit strong antibacterial activity against pathogenic bacteria, and can be applied to various bacterial infections.

(c) In addition, the composition of the present invention can be usefully used for antibacterial purposes in various fields such as pharmaceutical, food or feed additive composition.

1 is a table summarizing the results of genome analysis of Lactobacillus plantarum strains derived from kimchi.
2 is a table summarizing the transcript analysis results of Lactobacillus plantarum strains derived from kimchi.
3 is a diagram showing the characteristics of the plan thalysin 1 peptide summarized.
4 is a schematic diagram showing the arrangement of adjacent gene sequences of the plantaricin 1 gene.
5 is a diagram showing the characteristics of the plan thalysin 2 peptide in summary.
6 is a schematic diagram showing the arrangement of adjacent gene sequences of the plantaricin 2 gene.
7 is a diagram showing the characteristics of the plan thalysin 3 peptide summarized.
8 is a diagram showing a comparison of the antibacterial activity of the synthetic plantaricin 3 peptide and the plantarisin 3 peptide cloned and expressed in E. coli.
9 is a diagram showing the measurement of antibacterial activity against four pathogenic strains of B. cereus, E. coli, S. aureus strains, and C. albicans strains of synthetic plantaricin 3 peptides.
10 is a schematic diagram showing the arrangement of adjacent gene sequences of the plantaricin 3 gene.
11 is a diagram showing the characteristics of the plan thalysin 4 peptide summarized.
12 is a schematic diagram showing the arrangement of adjacent gene sequences of the plantarlysin 4 gene.
13 is a diagram showing the characteristics of the plan thalysin 5 peptide in summary.
14 is a diagram showing the measurement of antibacterial activity of the plantaricin 5 peptide against four pathogenic strains of B. cereus, E. coli, S. aureus strains, and C. albicans strains.
15 is a diagram showing the antibacterial activity of the plantaricin 5 peptide expressed by cloning the plantaricin 5 gene into E. coli.
Fig. 16 is a schematic diagram showing the arrangement of adjacent gene sequences of the plantaricin 5 gene.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and therefore, the scope of the present invention is not to be construed as being limited by these examples.

Example

Throughout this specification, "%" used to indicate the concentration of a specific substance is (weight/weight)%, solid/liquid (weight/volume)%, and (weight/volume)%, and Liquid/liquid is (vol/vol)%.

Example 1. Isolation and identification of kimchi-derived strains with excellent antibacterial activity

1-1. strain isolation

In order to isolate new strains with excellent antibacterial activity from kimchi, well-ripened kimchi was randomly collected from a region near Gyeongnam, diluted 10-fold, and spread on MRS solid medium to secure growing strains.

First, among the strains grown on MRS solid medium, about 1,000 strains with excellent growth ability were first selected, and each selected strain was liquid-cultured in 5ml MRS medium.

For the analysis of antibacterial activity against 1,000 selected strains, after punching in LB plate medium with a 7mm cork borer, highly pathogenic Salmonella Typhimurium χ 3339 strain and pathogenic E. coli (E. coli ATCC 11775) It was plated on LB plate. Each strain culture solution isolated from kimchi was added at a concentration of 50 or 100 μl to the LB plate medium on which the highly pathogenic pathogen was smeared, and then cultured at 37° C. for 16 hours and observed at intervals of 1 hour to 2 hours.

As a result, a total of 12 strains were selected, and strain 6 having the best antibacterial activity was selected from among the 12 strains, and 16S rDNA sequence analysis was performed.

1-2. Strain identification

For 16S rDNA gene sequencing, the chromosomal DNA of the strain was extracted using a DNA isolation kit (GenElute Bacterial Genomic DNA Kit, Sigmaaldrich, USA) according to the manufacturer's protocol, and sequencing primer 785F (5'-GGATTAGATACCCTGGTA) -3') and 907R (5'-CCGTCAATTCMTTTRAGTTT-3') and PCR primers 27F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-TACGGYTACCTTGTTACGACTT-3') were used to generate the 16S rDNA gene fragment of the isolated strain. After amplification by polymerase chain reaction (PCR), the nucleotide sequence was analyzed. The phylogenetic relationship was confirmed by comparing the result with the nucleotide sequence of the NCBI GenBank database (http://ncbi.nlm.nih.gov).

As a result, the strain isolated in Example 1-1 was analyzed as a new strain having at least 99% homology with Lactobacillus plantarum.

Example 2. Analysis of omics (genomic and transcriptome)

2-1. genomic analysis

a) Preparation of genomic DNA isolation and sequencing from lactic acid bacteria samples

Genomic DNA was isolated from Lactobacillus plantarum isolated and identified in Example 1 using Bacterial Genomic DNA Extraction Kit Ver.3.0 (TaKaRa Korea, Seoul, Republic of Korea), and this DNA was subjected to DNA-Seq (DNA sequencing) was used for

b) DNA sequencing

After the isolated DNA is isolated (fragmentation) using the Covaris S2 ultra sonicator system, a library consisting of a 350 bp DNA fragment is constructed using the Truseq Nano DNA sample prep kit (Illumina, Inc., San Diego, CA). did. This final product was confirmed using the 2100 BioAnalyzer device, and sequence decoding was performed using the Illumina HiSeq platform (Illumina, Inc., San Diego, CA).

c) Bioinformatics analysis to secure genome information

The sequence reads obtained were obtained by filtering only high-quality reads using the sickle (v1.33) program and the BWA (ver. 0.7.12.) program, and these were obtained by filtering the Lactobacillus plantarum strain. It was used for mapping and genome assembly. After mapping, local realignment was performed using the GATK program (ver. 3.1-1), and the polymers (duplicates) were subjected to the Picard program (ver. 1.98) (http://picard.sourceforge.net). was used for mapping. Mutations in the sequence were called through the GATK program (ver 3.1-1), and annotation on the mutations was performed by the SNPEff (v.4.1) program.

For the identification of antibacterial peptides (AMPs), the similarity is obtained by comparing the sequence with the antibacterial peptides (AMPs) previously obtained from the CAMP (http://www.camp.bicnirrh.res.in/campHelp.php) database. More than 50% of the peptides (peptides) were classified as potential antibacterial peptides (AMPs), and were subjected to synthesis of artificial peptides and gene cloning.

As a result of the genome analysis, as shown in FIG. 1 , four well-known antibacterial peptides (AMPs) were searched. These were plantaricin N, plantaricin E/F complex, lysozyme, MazF, and bacteriophage holin.

In addition, as a result of the homology analysis, it was highly likely that the seven newly discovered peptides were antibacterial peptides (AMPs).

In particular, among the 7 types of peptides, 5 new peptides were predicted to exhibit high antibacterial activity, and they were named as plantaricin 1 to plantaricin 5, respectively.

2-2. Transcriptome analysis

a) Securing mRNA from Total RNA

mRNA was isolated from total RNA using an RNA-Seq sample preparation kit (Illumina, Inc., San Diego, CA), and this mRNA was used for RNA-Seq (RNA sequencing).

b) RNA sequencing

The isolated mRNA was subjected to an isolation (fragmentation) step, and was synthesized as a single-stranded cDNA through random hexamer priming, and a second strand was synthesized using this as a template. In addition, after sequentially undergoing end repair for making a blunt-end, A-tailing for attaching an adapter, and adapter ligation, PCR (Polymerase Chain Reaction) Amplification was performed using the instrument, and the final product was confirmed using the 2100 BioAnalyzer instrument. The created library was quantified using the KAPA library quantification kit, and the cluster generation was sequenced using a Hiseq 2500 instrument.

c) Bioinformatics analysis to secure transcriptome information

In the present invention, in order to remove a low-quality sequence, first, reads containing 10% or more of the bases represented by N in the sequence information or 40% or more of bases less than Q20 were removed, and the average quality Leads below this Q20 were also removed. The entire filtering process was performed with an internally created program.

For the reference genome used for sequence alignment and analysis, information provided by Ensembl (Flicek et al., 2013) was used, and version 72 was used. The filtered sequence was aligned to the genome sequence using the STAR 2.3.0e (Dobin et al., 2013) tool, and Ensembl 72 version of the genetic information was used in the sequence alignment process.

Expression level measurement was calculated using Cufflinks v2.1.1 (Trapnell et al., 2010) tool, and for this, Ensembl 72 version of genetic information was used, and for non-coding gene regions, mask option was used. was excluded from expression level measurement. In addition, multi-read-correction and frag-bias-correct options were additionally used to increase measurement accuracy, and other options were used as default values.

For specific expression gene analysis, the number of reads for each gene was calculated using the HTSeq-count v0.5.4p3 (Anders et al., 2015) program, and the intersection-non empty rule and paired end ( Calculations were made taking the paired-end sequence into account. Specific expression gene analysis was performed using the TCC (Sun et al., 2013) program using the calculated read number of each gene. For the TCC option, the iDEGES/edgeR method considering repetition was used, and the specific expression gene selection was based on the Q-value corrected for errors occurring during the multiple-testing process, and less than 0.05 was used as the reference value.

As a result of the transcriptome analysis, it was found that the expression of all of the novel antibacterial peptides (AMPs) including plantaricin 1 to 5 discovered in Example 2-1 was induced ( FIG. 2 ).

Example 3. Peptide Synthesis

Peptides of plantaricin 1 to plantaricin 5 were synthesized at the request of Cosmojintech Co., Ltd.

The synthesized antibacterial peptides were dissolved in water at a concentration of 10 mg/ml and stored at -20°C. To dissolve the dry powder, high-purity purified water ((Ultrapure grade H ₂ O) was used, and the resuspended sample was used to measure antibacterial activity.

Example 4. Antimicrobial Activity Assay

4-1. Securing samples such as pathogens

Pathogenic microorganisms used in the antibacterial activity test were subjected to antibacterial tests on Salmonella pathogens known to be highly pathogenic in actual livestock control sites. As a high-risk pathogen, a Salmonella Typhimurium (ST, KCTC12401) strain was selected.

4-2. Antibacterial activity assay test process

For the analysis of antimicrobial activity, a minimal inhibitory concentration (MIC) analysis was performed using a microtiter plate method. Analytical samples were used after preparing a stock, diluted to an appropriate concentration, and the selected strain was cultured overnight (O/N) in a 100 mL Erlenmeyer flask, centrifuged, and the supernatant was taken with a 0.25 μm syringe filter. ) after filtration, and used for antibacterial activity analysis.

For MIC analysis, strains cultured overnight (O/N) were used in the experiment after dilution to ^{10 6 CFU/ml.}

The concentrations of peptides used for MIC analysis were 0, 25, 50, 100, 250, 500, and 1,000 μg/ml concentrations. After the reaction solution was added to a microtiter plate, the reaction properties were observed while culturing O/N at 37°C, and the absorbance of each reaction solution was measured.

Significance test of experimental results was performed using ANOVA (one-way analysis of variance) and Duncan method in SPSS statistical program (IBM, Armonk, NY). In the experimental results, the bars in each graph mean the mean and standard error of the results obtained through at least three repeated experiments, and different characters indicate significant differences (p<0.05) between comparison subjects.

4-3. Plantaricin 1

Plantaricin 1 (Plantaricin 1; pln 1) is composed of 152 amino acids, it was found to be highly homologous to the gramminstin Pp3 (Gramminstin Pp3) gene (see FIG. 3). The region of homology was characterized by 50% hydrophobicity, +2 net charge, and a molecular weight of 2.161 kDa.

The secondary structure of the peptide was predicted to construct an alpha-helix structure. As a result of analyzing the antimicrobial activity by the synthesized peptide, the MIC ₅₀ (50% minimal inhibitory concentration) was measured to be 5.5 mg/ml, indicating excellent antibacterial activity (see FIG. 3 ).

Neighboring genes of plantaricin 1 are cspLA (cold shock-like protein), Yjjp (inner membrane protein), and LytR (transcriptional regulator), etc. (See Fig. 4).

4-4. Plantaricin 2

Plantaricin 2 (Plantaricin 2; pln 2) consists of a total of 58 amino acids, and showed homology to the indolicidin gene (see FIG. 5). In the process of genome analysis, the plantaricin 2 gene has a region homologous to the indolicidin gene at the C-terminus. Therefore, the present plantaricin 2 peptide was predicted to be highly likely to perform the function of antibacterial peptides (AMPs) after the signal peptide was cleaved. The homology region had 15% hydrophobicity, 0 net charge, and a molecular weight of 1.641 kDa, and an alpha-helix structure was predicted.

As a result of the antibacterial activity analysis, MIC ₅₀ (50% minimal inhibitory concentration) was measured to be 4.8 mg/ml, indicating excellent antibacterial activity.

This value is the second strongest antibacterial activity value among the five types of measured plntaricin peptide, and the plantaricin 2 peptide exhibited the most excellent antibacterial activity. A hypothetical protein was present in the neighboring gene of plantaricin 2, and it was found that ICDP (integrase core domain protein) and PinR_1 (putative DNA invertase from lambdoid prophage Ra) were located (see FIG. 6 ).

4-5. Plantaricin 3

Plantaricin 3 (Plantaricin 3; pln 3) is composed of a total of 46 amino acids, and showed homology to the bacteriopexin A (bactofencin A) gene. In addition, it was shown to have high homology with the L. allivarius cp400 peptide (see FIG. 7 ).

In particular, there was a well-known 'GG site' as the previously known 'ABC transporter signal sequence cutting site'. However, the signal sequence sequence was shown to be completely different from the bacteriopexin A (bactofencin A) gene.

These results can also be confirmed in the lower panel of FIG. 7 . As a result of genomic analysis, since this plantaricin 3 peptide was predicted to exhibit antibacterial activity after expression, the gene was cloned into the E. coli DH5α strain as well as peptide synthesis to further analyze the antimicrobial activity against the supernatant of the E. coli strain. did. For the antibacterial activity of synthetic peptides, in addition to the Salmonella Typhimurium (ST, KCTC12401) strain, 4 strains known for high pathogenicity acquired from the Korea Research Institute of Bioscience and Biotechnology were additionally tested.

As a result, as shown in FIG. 8, the antibacterial activity of the plantaricin 3 synthetic peptide against the Salmonella _{Typhimurium strain was MIC 50} A value of 5.2 mg/ml was measured to indicate excellent antibacterial activity (Fig. 8, lower left figure), and it was confirmed that the antibacterial activity against B. cereus, E. coli, and S. aureus strains was also excellent (Fig. 9 and Table 1). Reference).

[Table 1]

In addition, the antibacterial activity of plantaricin 3 protein cloned and expressed in E. coli was MIC _{50 against B. creus strain.} The value was 730ug peptide/ml, indicating that there was excellent activity (FIG. 8 right).

The neighboring genes of plantarisin 3 are lagD_2 (lactococcin G-processing and transport ATP-binding protein L) and hypothetical protein, and it is estimated that lagD_2 is partially related to the transport process of the ABC transporter (FIG. 10). .

4-6. Plantaricin 4

Plantaricin 4 (Plantaricin 4; pln 4) is composed of a total of 65 amino acids, it was found to be homologous to the hymenochirin-5B gene (Fig. 11 middle panel). Gly amino acids, which are specific cleavage sites for the signal sequence of the ABC transporter, were found to be present at amino acids 27 and 28 in the plan thalysin 4 peptide.

The synthesized peptide was artificially synthesized for amino acids 38 to 62 of the peptide sequence of Gene_01599 showing homology to the hymenochirin-5B peptide. The hydrophobicity of these amino acids was 64%, and it was predicted to have a +1 net charge, a molecular weight of 2.6263 kDa and an alpha-helix structure.

The antibacterial activity value (MIC ₅₀ ) was found to be relatively low among the five plantarlysin AMPs measured at 5.7 mg/ml.

However, the present plantaricin 4 peptide acts as an antibacterial peptide (AMPs) secreted due to the sequence consistent with the typical recognition sequence of the ABC transporter signal sequence and the hymenochirin-5B peptide at the C-terminus. was predicted to be highly probable.

Neighboring genes are Abetr (anaerobi benzoate catabolism transcriptional regulator) and pglH (; alNAc-alpha-(1->4)-GalNAc-alpha-(1->3)- diNAcBac-PP-undecaprenol alpha-1,4-N -acetyl-D-galactosaminyltransferase) genes, and these genes were estimated to have a low direct relationship to the expression of the present plantaricin 4 peptide (FIG. 12).

4-7. Plantaricin 5

Plantarisin 5 (Plantaricin 5; pln 5) is composed of a total of 54 amino acids and showed homology to the M-zodatoxin-Lt2a gene. This gene did not show a specific tendency for the signal sequence (see FIG. 13). In the region showing homology, the hydrophobicity was 28%, and the ratio of positive amino acids was very high with a +10 net charge. The molecular weight was 2.5851 kDa, and it was predicted to have an alpha-helix structure.

As a result, as shown in FIG. 13, the antibacterial activity of the plantaricin 3 synthetic peptide against the Salmonella _{Typhimurium strain was 4.4mg/ml MIC 50} value, which is the strongest antibacterial activity value among the five plantarlysin peptides measured. . In addition, it was confirmed that the antibacterial activity against B. cereus, E. coli, and S. aureus strains was also excellent (see FIG. 14 and Table 2).

[Table 2]

In addition, as a result of cloning the DNA sequence containing this gene into E. coli DH5α, it was estimated that the plantaricin 5 gene was highly likely to be expressed and secreted because it exhibited strong antibacterial activity (FIG. 15).

Neighboring genes of the plantaricin 5 peptide were a site-specific tyrosine recombinase (XerC) gene and a hypothetical protein ( FIG. 16 ). In particular, since there was no gene expressed in the upstream or downstream region of the plantaricin 5 peptide, it is presumed that there is a possibility that this part is related to the expression or functionality of the plantarisin 5 peptide. .

As described above in detail a specific part of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and it is clear that the scope of the present invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> National Institute of Biological Resources <120> Anti-Microbial Peptide Plantaricin, Isolated from Lactobacillus Plantarum and Uses thereof <130> PN190099AN <160> 5 <170> KoPatentIn 3.0 <210> 1 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Anti-Microbial Peptide Plantaricin 1 <400> 1 Val Leu Gly Ser Leu Ile Gly Ser Val Gly Ile Gly Val Leu Ser Ser 1 5 10 15 Leu Ala Ala Arg Tyr Lys 20 <210> 2 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Anti-Microbial Peptide Plantaricin 2 <400> 2 Ile Tyr Pro Glu Lys Gln Pro Glu Glu Pro Val Arg Arg 1 5 10 <210> 3 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Anti-Microbial Peptide Plantaricin 3 <400> 3 Lys Lys Ser Arg Arg Cys Gln Val Tyr Asn Asn Gly Met Pro Thr Gly 1 5 10 15 Met Tyr Thr Ser Cys 20 <210> 4 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> Anti-Microbial Peptide Plantaricin 4 <400> 4 Pro Ile Val Arg Glu Pro Phe Lys Ala Met Ala Val Gly Ile Ile Leu 1 5 10 15 Ala Val Met Ser Gly Leu Leu Val Thr 20 25 <210> 5 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Anti-Microbial Peptide Plantaricin 5 <400> 5 Lys Ala Lys Lys Arg Phe Leu Arg Asn Arg Leu Ser Gln Gln Ala Arg 1 5 10 15 Lys Ala Arg Thr Lys 20

Claims (6)

A plantaricin peptide represented by any one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 5.
According to claim 1,
The peptide is a plan taricin (Plantaricin) peptide, characterized in that it exhibits antibacterial activity.
3. The method of claim 2,
The antibacterial activity is Salmonella sp. strain, Bacillus sp. strain, Escherichia sp. strain, and Streptococcus sp. strain Plantaricin, characterized in that the antibacterial activity against one or more pathogens selected from the group consisting of (Plantaricin) peptide.
4. The method of claim 3,
The Salmonella sp. strain is Salmonella gallinarum (Salmonella gallinarum) or Salmonella Typhimurium (Salmonella Typhimurium), the Bacillus sp. strain is Bacillus cereus (B. cereus), and the Escherichia sp. strain is Escherichia coli (E. coli), and the Streptococcus sp. strain is Streptococcus aureus ( Staphylococcus aureus ) Plant taricin (Plantaricin) peptide, characterized in that the.
5. The method according to any one of claims 1 to 4,
The peptide additionally comprises a cell internalization sequence Plant lysin (Plantaricin) peptide.
An antibacterial composition comprising, as an active ingredient, a plantaricin peptide represented by any one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 5.

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