KR101502773B1 - A novel bifunctional peptide and its use with antimicrobial activity and anti inflammatory action from Bacillus strain - Google Patents

Abstract

The present invention relates to antibacterial and anti-inflammatory bifunctional peptides produced by Bacillus strains isolated from Korean traditional fermented food, Kimchi, and their uses. The bifunctional peptide according to the present invention is obtained by isolating the Bacillus genus CS32 (strain No. KCTC 18250P) from kimchi, which is stable at 90 ° C or lower and at pH 5.0-12.0, and is resistant to methicillin-resistant Staphylococcus aureus (MRSA), vancomycin- (MDR) bacteria including VRSA and vancomycin resistant enterococci (VRE), while inhibiting LPS-induced iNOS and COX-2 gene and protein expression and cytokine production , The peptide is very likely to be effectively used as an antibiotic or an anti-inflammatory agent.

Description

A novel bifunctional peptide and its use with antimicrobial activity and anti-inflammatory action from Bacillus strain,

The present invention relates to antibacterial and anti-inflammatory bifunctional peptides produced by a Bacillus strain isolated from kimchi, a traditional Korean fermented food, and its use.

Antimicrobial peptides (AMPs) are secreted by various microorganisms including bacteria and protect themselves from other microorganisms [Ref. 1]. These peptides are composed of polypeptides of 10-40 residues and affect the active component of the original immune response.

In addition, these antimicrobial peptides have been shown to be capable of killing gram-negative and gram-positive bacteria including mycobacteria, mycobacteria, protozoa, viruses, fungi and cancer cells [Reference 2]. Bacillus sp. Has been widely used in the fermentation industry due to its ability to produce resistant spores with Gram-positive bacteria with aerobic rod-like shape, as well as to produce antimicrobial substances as well as a number of extracellular enzymes [ 12].

Inflammation is a complex biological defense response against pathogens, damaged cells in vascular tissues, and various stimuli [13]. Inflammatory cells activated in the inflammatory process respond to increased amounts of nitric oxide (NO) and interleukin IL- 1β, IL-6, and tumor necrosis factor (TNF) -α [Reference: 15]. In the inflammatory reaction, the production of nitrogen monoxide as the main product is controlled by nitric oxide synthase (iNOS). iNOS is usually not present in cells, but when activated, it generates a large amount of nitrogen monoxide for a long time, and the generated nitrogen monoxide promotes inflammation and exacerbates inflammation. During inflammatory reactions, macrophages overexpress iNOS, COX-2 and cytokines and inflammatory mediators related to inflammation such as TNF- [alpha], IL-1 [beta], IL-6 and play an important role in the immune response [Reference: 15-16].

For the purpose of developing a microbial peptide having therapeutic use, the present inventors isolated the antimicrobial peptide producing strain from Kimchi, which is a Korean traditional food, and named it as Bacillus sp. CS32. The characteristics of the produced antimicrobial peptide, The anti-inflammatory activity was evaluated.

Document 1: Kim HK, Cheon BS, Kim YH, Kim SY, Kim HP. Effects of naturally occurring flavonoids on nitric oxide production in the macrophage cell line RAW 264.7 and their structure-activity relationships. Biochem Pharmacol. 1999; 58: 759-65.

The present inventors have made intensive studies to search for functional materials from Kimchi, a traditional Korean fermented food. As a result, the antimicrobial peptide produced by the Bacillus strain isolated from a traditional food exhibits a positive function and can be used as an effective antibiotic and anti-inflammatory agent And the present invention has been completed.

Accordingly, an object of the present invention is to provide a novel Bacillus strain isolated from Kimchi and a novel peptide produced by the strain, which simultaneously exhibits antimicrobial activity and anti-inflammatory action against multidrug-resistant bacteria and uses thereof.

It is to be understood that both the foregoing general description and the following detailed description of the present invention are exemplary and explanatory and are intended to provide further explanation of the invention as claimed. .

The bi-functional peptide according to the present invention was isolated from Korean traditional fermented food Kimchi with Bacillus sp. CS32 (strain accession number KCTC 18250P). The peptides produced by this bacillus are stable at 90 ° C or lower and at pH 5.0-12.0 and are stable in the presence of multidrug agents including methicillin resistant Staphylococcus aureus (MRSA), vancomycin resistant Staphylococcus aureus (VRSA) and vancomycin resistant enterococci (VRE) (MDR) bacteria and exhibit an anti-inflammatory action that inhibits the production of iNOS and COX-2 gene and protein, and cytokine induced by LPS. Therefore, the peptide is effectively used as an antibiotic or an anti-inflammatory agent The possibility of being used is very high.

Figure 1 shows the phylogenetic tree of Bacillus sp. CS32.
2 is a graph showing the column chromatography elution profile of Bacillus genus CS32.
Figure 3 shows Tricine SDS-PAGE and MALDI-TOF graphs for molecular weight confirmation.
4 is a graph showing the effect of CSP32 on cell viability.
Figure 5 is a graph and photographs showing the effect of LPS induced nitric oxide and inflammation related proteins in Raw 264.7 macrophages.
Figure 6 is a graph showing the effect of CSP32 on the inhibition of the production of TNF-a, IL-l [beta], and IL-6 cytokines in Raw 264.7 macrophage cells induced by LPS.
FIG. 7 is a photograph showing the effect of CSP32 on the expression of LPS-induced iNOS, COX-2, TNF-α, IL-1β, and IL-6 mRNA genes.

The present invention relates to antimicrobial and anti-inflammatory peptides produced by Bacillus genus CS32 (strain accession number KCTC 18250P) isolated from kimchi, a traditional Korean fermented food.

This peptide has a molecular weight of 5697.9 Da and has the following amino acid sequence as a result of N-terminal base sequence analysis (A-P-L-E-I-X-X-I-F-H-D-N). Stable at temperatures below 90 ° C and at pH 5.0-12.0 with temperature stability and pH stability and multidrug resistant (including methicillin resistant Staphylococcus aureus (MRSA), vancomycin resistant Staphylococcus aureus (VRSA) and vancomycin resistant enterococci (VRE) MDR) bacteria, and exhibits an anti-inflammatory action that inhibits the production of iNOS and COX-2 gene and protein induced by LPS and the production of inflammation-related cytokines. .

In a further aspect, the present invention provides an antibiotic or an anti-inflammatory agent comprising the peptide as an active ingredient.

In another further aspect, the present invention provides a method for producing Bacillus subtilis CS32, comprising culturing the Bacillus genus CS32 in a glucose-beef extract-peptone medium (1% glucose, 0.5% beef extract and 0.5% peptone) for 60 hours;

After culturing, cells are removed by centrifugation at 6000 rpm at 4 DEG C, and the effective peptide is precipitated at 4 DEG C with 60% saturation of ammonium sulfate in the culture supernatant;

The precipitate was collected by centrifugation at 6,000 rpm for 60 minutes at 4 ° C, suspended in 10 mM Tris-HCl buffer (pH 8.0), dialyzed and concentrated using a 1 kDa cut-off membrane (Millipore)

Extracting the crude extracts on a Sepharose CL-6B column followed by Sephadex G50 column, separating and eluting with the buffer; And

And collecting and concentrating the active fractions. The method for producing a bifunctional peptide exhibiting anti-microbial activity and anti-inflammatory action according to claim 1 is provided from the strain of Bacillus subtilis.

Hereinafter, the present invention will be described more specifically.

For the purpose of developing a microbial peptide having therapeutic use, the present inventors isolated a strain producing an antimicrobial peptide from Kimchi, a traditional Korean food, and named Bacillus genus CS32 (strain deposit number KCTC 18250P). The antimicrobial and anti-inflammatory peptide (CSP32) having a molecular weight of 5697.9 Da produced by this strain was purified and characterized. The amino acid sequence at the N-terminus of CSP32 was APLEIXXIFHDN (SEQ ID NO: 1). CSP32 was stable against a wide range of pH (5-10) and temperature (<90 ° C) as well as various chemical agents, lipase, protease K, α-chymotrypsin and trypsin. CSP32 exhibited antimicrobial activity against multidrug resistant (MDR) bacteria such as methicillin-resistant Staphylococcus aureus, vancomycin-resistant Staphylococcus aureus, and vancomycin-resistant enterococci. The minimum inhibitory concentration ranged from 0.156 to 80 mu g / ml. In addition, CSP32 was activated by LPS in the macrophage Raw 264.7, and showed the effect of inhibiting the production of increased nitrogen monoxide concentration-dependently. In addition, the expression of inducible nitric oxide (iNOS) and cyclooxygenase-2 (COX-2) induced by LPS in both inflammation-related protein and mRNA gene levels was inhibited in a concentration-dependent manner.

The release of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and IL-1β and mRNA expression levels of these cytokines were also decreased in a concentration-dependent manner by CSP32, This implies that Raw 264.7 cells affected the expression of proteins and genes of iNOS, COX-2, TNF-α, IL-6 and IL-1β.

CSP32 with both functional activity has therapeutic potential in the treatment of inflammation related diseases and MDR pathogens.

The subject of application of such antibiotics and antiinflammatory agents is clear and can be applied in accordance with the conventional methods known in the art.

The pharmaceutical composition according to the present invention may also contain pharmaceutically acceptable binders such as polyvinylpyrrolidone and hydroxypropylcellulose, disintegrants such as carboxymethylcellulose calcium, starch glycolate sodium, diluents such as corn starch (Such as lactose, soybean oil, crystalline cellulose, mannitol), a lubricant (e.g., magnesium stearate, talc), a sweetening agent (e.g., white sugar, fructose, sorbitol, aspartame), a stabilizer (carboxymethyl cellulose sodium, alpha or beta cyclodextrin, (Eg, ethyl vanillin, Masking Flavor, Menthol Flavono, herb flavor), and then mixed with a tablet, a capsule, a softener, etc. by mixing with a preservative (eg, methyl parahydroxybenzoate, propylparabenzoyl peroxide, sodium benzoate) Capsules, liquids, ointments or injections. The pharmaceutical composition of the present invention may be preferably administered in the form of an oral preparation or a parenteral preparation.

The dosage of the pharmaceutical composition of the present invention is appropriately set in accordance with the form of the pharmaceutical composition, the method of administration, the purpose of use, and the age, weight, and symptom of the individual to which the pharmaceutical composition is applied. For example, 1 mg to 2000 mg / kg per day for an adult. Of course, since the dosage varies depending on various conditions, the amount may be less than the above range, or may be more than the above range.

<Effective amount>

The dosage of the peptide as an active ingredient in the pharmaceutical composition of the present invention may be 1 to 2,000 mg per kg of body weight per day depending on the patient's age, sex, symptom, method of administration, or prevention purpose. have. Dosage levels for patients exhibiting the specific symptoms may vary depending on the patient's body weight, age, sex, health condition, diet, time of administration, method of administration, excretion rate, severity of disease, and the like.

<Examples>

Hereinafter, the present invention will be described more specifically by the following representative examples, but the present invention is not limited in any way by these embodiments.

Among the materials used in the following examples, Sepharose CL-6B and Sephadex G-50 were obtained from Pharmacia (Uppsala, Sweden). Trypsin, proteinase K, alpha -chimotrypsin, and lipolytic enzyme Sigma (St. Louis, USA). Lipopolysaccharide (LPS), dimethylsulfoxide (DMSO), Griess reagent and 3- (4,5-dimethylthiazol-2-2,5-diphenyltetrazolinium bromide (MTT) were also purchased from Sigma Dulbecco's modified Eagle's medium (DMEM), fetal calf serum (FBS), and penicillin-streptomycin solutions were purchased from Invitrogen (Grand Island, NY, USA). Rabbit anti-mouse iNOS polyclonal antibodies were purchased from Santa Cruz Biotech Inc. Mouse anti-mouse COX-2 was obtained from Cayman Co. HRP-conjugated donkey anti-rabbit Ig-G and anti-mouse IgG were purchased from Cell signaling. Phosphatase conjugated affinipure donkey anti-mouse IgG was purchased from Jackson Immuno-research laboratories INC. Mouse TNF-α, IL-6, IL-1B and ELISA kits were purchased from BD Biosciences (San Diego, CA, Chemicals and reagents used analytical grade.

Example 1 Screening and Culture of Bacillus sp. CS32

Korean traditional food samples were collected in various regions of Jeollanam - do. After the food samples were suspended and diluted in distilled water, Bacillus (MRS, Muller Hinton).

To confirm the antimicrobial activity of the isolated Bacillus strains, the paper disk diffusion method was used and the activity was confirmed by the diameter of the hypocotyl. In order to identify this strain, Bergey's ' Manual of systemic bacteriology ', 16s rRNA sequence analysis, was performed [Reference: 17]. The antimicrobial peptide production of this strain was optimized by using several carbon and nitrogen sources. The effect of various carbon sources on the production of antimicrobial peptides was determined using media containing 1% and 1% yeast extracts of glucose, mannitol, starch, lactose, fructose, sorbitol, sucrose or maltose. The culture was carried out in a 250 ml Erlenmeyer flask with 50 ml of medium and continuously shaking at 37 ° C and 180 rpm. The effect of nitrogen source on peptide production was then determined using media containing 1% each of beef extract, malt, tryptone, yeast extract, oatmeal, soybean oil, peptone and 1% glucose (optimal carbon source).

Analysis of the morphological and biochemical characteristics revealed that the strain CS32 was a Bacillus strain (data not shown). Followed by 16S rRNA sequencing to compare closely related sequences of Bacillus species. As a result of computer-assisted RNA search for the bacterial database, the 16S rRNA sequence of CS32 showed 99.392% homology with Bacillus licheniformis (see Table 1). The phylogenetic tree based on sequence alignment is shown in Fig. Figure 1 is the phylogenetic tree of the Bacillus genus CS32.

designation Strain Accession number Similarity Total nucleic acid difference One Bacillus licheniformis ATCC 14580 (T) CP000002 99.392 9/1480 2 Bacillus sonorensis NRRL B-23154 (T) AF302118 99.362 9/1410 3 Bacillus aerius 24K (T) AJ831843 99.323 10/1478 4 Bacillus subtilis subsp. spizizenii NRRL B-23049 (T) AF074970 98.652 19/1409 5 Bacillus mojavensis IFO 15718 (T) AB021191 98.648 20/1479 6 Brevibacterium halotolerans LMG 21660 (T) AJ620368 98.642 20/1473 7 Bacillus atrophaeus JCM 9070 (T) AB021181 98.642 20/1473 8 Bacillus subtilis subsp. subtilis NCIB 3610 (T) ABQL01000001 98.58 21/1479 9 Bacillus vallismortis DSM 11031 (T) AB021198 98.513 22/1479 10 Bacillus amyloliquefaciens subsp. amyloliquefaciens DSM 7 (T) FN597644 98.377 24/1479 11 Bacillus amyloliquefaciens subsp. plantarum FZB42 (T) CP000560 98.242 26/1479 12 Bacillus siamensis PD-A10 (T) GQ281299 98.174 27/1479 13 Bacillus methylotrophicus CBMB205 (T) EU194897 98.125 27/1440 14 Bacillus altitudinis 41KF2b (T) AJ831842 96.685 49/1478 15 Bacillus stratosphericus 41KF2a (T) AJ831841 96.685 49/1478

Example 2: Purification of antimicrobial and anti-inflammatory peptides produced by Bacillus CS32

The antimicrobial peptide (CSP32) production medium from Bacillus genus CS32 was optimized by using various carbon sources and nitrogen sources. Glucose and peptone and beef extracts have been found to be optimal carbon and nitrogen sources for maximum production of effective peptides. Finally, the production of CSP32 was carried out in GPB medium (1% glucose, 0.5% peptone and 0.5% beef extract) and the peptide production was from 12 hours after incubation and reached maximum production at 60 hours. The optimum conditions for production of CSP32, Bacillus sp. CS32 was cultured in GPB medium at 37 DEG C for 60 hours. Cells were removed by centrifugation (6000 rpm, 30 min, 4 ° C) and the peptides contained in the supernatant were precipitated overnight at 4 ° C with 60% saturation of ammonium sulphate. The precipitate was collected by centrifugation (6,000 rpm, 60 min, 4 ° C), suspended in 10 mM Tris-HCl buffer (pH 8.0) and concentrated using a 1 kDa cut-off membrane (Millipore) and dialyzed. CSP32 was purified by sequential chromatography on ammonium sulfate precipitation on Sepharose CL-6B and Sephadex G50 (see FIG. 2). 2 is a graph showing the column chromatography elution profile of the crude extract obtained after treatment with ammonium sulfate in the culture of Bacillus genus CS32. (a) Gel permeation chromatography on a Sepharose CL-6B column (2.2 cm x 116 cm) eluting with a flow rate of 5 ml / min (b) Sephadex G-50 column (1.5 cm x 70 cm) Gel permeation chromatography. Protein elutes at a flow rate of 1 ml / min.

Example 3: Analysis of molecular weight and N-terminal amino acid sequence of an effective peptide

The molecular weight of the purified peptide was confirmed by tricine SDS-PAGE. For in situ detection of the inhibitory activity, the gel was washed with 50 mM Tris-HCl buffer (pH 7.9) containing 2.5% Triton X-100 and overlaid on a soft agar containing the indicated strain (10 ⁶ colony forming units) &Lt; / RTI &gt; The molecular weight of the peptides was confirmed by MALDI-TOF-MASS. N-terminal amino acid sequencing was performed with a procise 492 amino sequencer (Applied Biosystems, Foster City, Calif.) According to the automated Edman degradation method.

The purified peptides were identified by Tricine SDS-PAGE and in situ inhibitory activity (see Figure 3a). MALDI-TOF MASS analysis confirmed that CSP32 had a molecular weight of 5,697.9 Da (see FIG. 3B). Fig. 3 is a graph showing electrophoresis and MALDI-TOF peaks with confirmed molecular weights, which is the result of trisin SDS-PAGE and staining of the active band of CSP32. (a) Tricine SDS-PAGE: Lane 1, a protein size marker with a corresponding value as shown on the left, kDa; Lane 2, purified CSP32; Lane 3, Confirmation of antimicrobial activity of protein bands (b) MALDI-TOF-MASS.

The first 12 amino acid residues at the N-terminus of CSP32 were determined to be APLEIXXIFHDN. This sequence is compared with the related antimicrobial peptides and is shown in Table 2.

Antimicrobial peptide N-terminal amino acid sequence Homology (%) Remarks The inventive peptide A P L E I X X I F H D N 100 SEQ ID NO: 1 Bacillus Lee Kenny Formis 26L-10 / 3RA I S L E I C X I F H D N 75 SEQ ID NO: 2 Bacillus subtilis subspecific JM4-A X X K E I X W I F H D N 58.33 SEQ ID NO: 3 Bacillus subtilis subpepper JM4-B X X K E I X H I F H D N 58.33 SEQ ID NO: 4

Example 4: Minimum Inhibitory Concentration of CSP32 against Pathogenic Microorganisms

The MIC (minimum inhibitory concentration) value was determined using Muller Hinton Broth [Refs. 17-18]. Pathogenic strains such as methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Staphylococcus aureus (VRSA) and vancomycin-resistant enterococci (VRE) Respectively. Bacitracin and vancomycin, antibiotics, were used as controls.

As shown in Table 3, CSP32 showed good antimicrobial activity against Gram-positive bacteria and no activity against Gram-negative bacteria, but CSP32 was found to be particularly effective against Micrococcus luteus when compared with control strains bacitracin and vancomycin, , MRSA, VRSA and VRE.

Test microorganism MIC CSP32 Bacitracin Vancomycin Alcaligenes faecalis ATCC 1004 > 80 > 80 > 80 Salmonella typhimurium KCTC 1925 > 80 > 80 > 80 Escherichia coli KCTC 1923 > 80 > 80 > 80 Pseudomonas aeruginosa KCTC 1637 > 80 > 80 > 80 Micrococcus luteus ATCC 9341 0.156 > 80 0.156 Mycobacterium smegmatis ATCC 9341 > 80 > 80 0.3125 Enterococcus faecalis ATCC 29212 5 5 2.5 Bacillus subtilis ATCC 6633 > 80 40 > 80 Listeria monocytogenes KCTC 3569 10 0.3125 0.156 Staphylococcus aureus KCTC 1928 5 40 0.3125 MRSA 639E 2.5 40 0.3125 MRSA 4-5 5 1.25 0.3125 MRSA 5-3 2.5 1.25 0.3125 MRSA U4 40 1.25 0.3125 MRSA S3 10 1.25 0.3125 MRSA P3 40 1.25 0.625 MSRA S1 40 1.25 0.625 VRSA 40 > 80 > 80 VRE 2 10 0.3125 > 80 VRE 3 > 80 20 > 80 VRE 4 10 5 > 80 VRE 5 10 1.25 > 80 VRE 6 > 80 > 80 > 80 VRE 82 80 40 > 80 VRE 89 > 80 40 > 80

Example 5 Stability of Antibacterial Peptides Produced by Bacillus CS32

CSP32 activity for thermal stability was assessed by measuring residual activities. After heat treatment (30 ~ 121 ℃ for 10 ~ 60 min), the samples were tested for residual antimicrobial activity against the indicated strain Micrococcus luteus ATCC9341.

The stability of pH to antimicrobial activity was measured by varying the pH level. First, the samples were adjusted to pH with citrate and Na ₂ HPO ₄ (pH 3.0-7.0), Tris (pH 7.0-9.0), NaHCO _3, NaOH (pH 10.0-11.0) buffer. The pH adjusted samples were mixed with the peptides and reacted at room temperature for 1 hour. The samples were again adjusted to pH 7.0 and tested for their antimicrobial activity.

Next, to confirm the stability against protease, each protease was treated with an antimicrobial peptide to a final concentration of 1 mg / ml; (50 mM Tris-HCl, pH 7.5), protease K (50 mM Tris-HCl, pH 7.5), a-chymotrypsin , pH 8.0). Each enzyme and the peptide reaction solution were reacted at room temperature for 1 hour, and the enzymes were inactivated by boiling at 100 ° C for 2 minutes. In order to confirm the stability against the chemical, each chemical (test concentration shown in Table 7) was added to CSP32, the samples were reacted at room temperature for 1 hour, and the residual antimicrobial activity against the test microorganisms was examined.

The test results are shown in Tables 4 and 5, respectively. From these results, it was confirmed that the activity of CSP32 was stable at 30 ° C or higher, 90 ° C or lower and pH 5.0-12.0 without any substantial decrease. However, the activity decreased sharply above 121 ℃. CSP32 peptides were stable against lipase, proteinase K, a-chymotrypsin and trypsin in the stability effect on protein hydrolytic enzymes determined by paper disk diffusion method (see Table 6).

CSP32 was also stable against a variety of chemicals and organic solvents (see Table 7).

Temperature time Residual activity (%) None 100 30 ℃ 10 minutes 100 30 minutes 100 60 minutes 100 50 ℃ 10 minutes 100 30 minutes 98 60 minutes 98 70 ℃ 10 minutes 98 30 minutes 98 60 minutes 98 90 ° C 10 minutes 98 30 minutes 95.9 60 minutes 95.9 121 ° C 105 kPa / 15 min 65.3

pH Residual activity (%) None 100 pH 2 61.5 pH 3 63 pH 4 89.8 pH 5 95.9 pH 6 100 pH 7 100 pH 8 100 pH 9 100 pH 10 98 pH 11 91.8 pH 12 91.8

Protease Residual activity (%) None 100 Lipase 98.3 Proteinase K 95.7 alpha-chymotrypsin 94.3 trypsin 100

process density Residual activity (%) None
Acetone
Chloroform
Ethanol
Methanol
Ethyl acetate
EDTA
Trichloroacetic acid
Triton X-100
Tween 20
Tween 80
10% (v / v)
10% (v / v)
10% (v / v)
10% (v / v)
10% (v / v)
10 mM / ml
100 mg / ml
1% (v / v)
10% (v / v)
10% (v / v) 100
103.6
103.8
105.9
101.56
101.8
101.8
101.3
91.6
109.6
108.3

Example 6: Cell culture and cell viability measurement

The rat macrophage cell line, Raw264.7, was cultured in DMEM medium containing 10% FBS and 5% CO _{2 at} 37 ° C. The toxicity of the antimicrobial peptide to cells was confirmed by the MTT method [Reference: 19]. Cells (10 ⁶ cells / ml) were dispensed into 96-well plates and treated with antimicrobial peptides (10, 50, 100 μg / ml) for 24 hours. 37 ℃ by the addition of 20μl of MTT solution per Well, 5% CO ₂ After 3 hours of reaction, the absorbance at 540 nm was measured using an ELISA reader, and the cell survival rate of the control group was expressed as a percentage. The results are shown in Fig. 4 is a graph showing the effect of CSP32 on cell viability. As can be seen from Fig. 4, CSP32 did not show cytotoxicity against Raw 264.7 cells in the range of 5-100 μg / ml. Therefore, the experiment was performed using CSP32 ELISA, western blot, and RT-PCR at a concentration of 100 μg / ml or less.

Example 7: Measurement of Nitric Oxide Production and Intracellular Inflammation-Related Protein Expression

The concentration of nitric oxide (NO) was determined by measuring the nitrate concentration in the culture medium using Griess reagent [Reference: 19]. Raw 264.7 cells (10 ⁶ cells / ml) were inoculated on a 6-well plate and the cells were treated with 1 μg / ml of LPS and treated with peptides at 10, 50 and 100 μg / Respectively. The supernatant of the culture was mixed with the same amount of Griess reagent (1% sulfanilamide in 5% phosphoric acid and 0.1% naphthyl ethylenediamine dihydrochloride), reacted for 10 minutes at room temperature and then absorbed at 540 nm with an ELISA reader And the production rate of nitrogen monoxide (NO) was confirmed.

The amount of nitrogen monoxide produced to confirm the effect of CSP32 on the production of nitrogen monoxide (NO) was measured using a Griess reagent. As a result, the change in the production amount of nitrogen monoxide treated with CSP32 after treatment with LPS in Raw 264.7 cells is shown in FIG. As shown in FIG. 5, the production of nitrogen monoxide was significantly increased in the LPS-treated cells compared to the untreated cells, and in the cells treated with CSP32 (10, 50, 100 μg / ml) It was confirmed that it was inhibited.

After confirming the production rate of nitrogen monoxide, the inflammatory effect of CSP32 was measured by measuring the amount of iNOS and COX-2 protein associated with inflammation. The cells were treated with 1 μg / ml of LPS and treated with CSP32 (10, 50, 100 μg / ml) for 1 hour. The cells were cultured for 24 hours, After washing with phosphate buffered saline (PBS), proteins were extracted using lysis buffer. This protein was quantitated by bradford assay and then separated by 10% SDS-PAGE and transferred to PVDF membrane. This membrane was reacted with primary and secondary antibodies against iNOS and C0X-2, and the expression pattern of inflammatory proteins was confirmed.

As shown in FIG. 5, when CSP32 was treated, the expression level of iNOS and COX-2 protein increased by LPS was significantly decreased in a concentration-dependent manner.

Example 8: Measurement of cytokine production amount

The inhibitory effect of CSP32 on the production of TNF-α, IL-1β and IL-6 in cell culture medium was determined by ELISA. Raw 264.7 cells (10 ⁶ cells / ml) were inoculated on a 6-well plate and the cells were treated with 1 μg / ml of LPS and treated with peptides at 10, 50 and 100 μg / Respectively. Then, the culture medium was taken to analyze the amounts of TNF-α, IL-1β and IL-6 produced. The results are shown in Fig. 6 is a graph showing the effect of CSP32 on the inhibition of TNF-α, IL-1β, and IL-6 production in Raw 264.7 macrophage cells activated by LPS. Macrophages play an important role in early inflammatory responses by secreting inflammatory mediators such as TNF-α, IL-1β and IL-6. LPS significantly increased TNF- [alpha], IL-1 [beta] and IL-6 production. As shown in Fig. 6, cytokine release induced by LPS was significantly inhibited by CSP32 in a concentration-dependent manner. These results indicate that CSP32 inhibited the production of inflammatory cytokines such as TNF-α, IL-1β and IL-6 in macrophages, suggesting that CSP32 is involved in anti-inflammatory actions.

Example 9: Analysis of intracellular inflammation-related mRNA expression by RT-PCR

To investigate the anti-inflammatory effects of CSP32, mRNA expression patterns of iNOS, COX-2, TNF-α, IL-Iβ and IL-6 were analyzed by RT-PCR. After culturing with macrophages, the cells were treated with CSP32, washed with phosphate buffered saline (PBS) 24 hours later, and RNA was isolated using Trizol. After mixing with 1 μg of RNA and PCR primers, RT-PCR was performed. Primer sequences are listed in Table 8.

primer Primer sequence Forward Reverse iNOS 5`-CCCTTCCGAAGTTTCTGGCAGCAGC-3` (5) 5`-GGCTGTCAGAGCCTCGTGGCTTTGG-3` (6) COX-2 5`-CACTACATCCTGACCCACTT-3` (7) 5`-ATGCTCCTGCTTGAGTATGT-3` (8) TNF-a 5`-TCTCATCAGTTCTATGGCCC-3` (9) 5`-GGGAGTAGACAAGGTACAAC-3` (10) IL-1? 5`-TGGACGGACCCCAAAAGATG-3` (11) 5`-AGAAGGTGCTCATGTCCTCA-3` (12) IL-6 5`-GTTCTCTGGGAAATCGTGGA-3` (13) 5`-TGTACTCCAGGTAGCTATGG-3` (14) GAPDH 5`-CACTCACGGCAAATTCAACGGCAC-3` (15) 5`-GACTCCACGACATACTCAGCAC-3` (16)

RT-PCR conditions were as follows: reaction was carried out at 42 ° C for 1 hour to prepare cDNA, and reaction was carried out at 94 ° C for 5 minutes to inactivate the reverse transcriptase. Thereafter, denaturation at 94 ° C for 30 seconds, annealing at 58 ° C for 30 seconds, and primer extension at 72 ° C for 40 seconds were repeated 30 times. The PCR products were separated on 1% agarose gel and bands were visualized by bromide ethidium staining.

Expression of iNOS, COX-2 and the cytokines TNF-α, IL-1β, and IL-6 mRNA was examined by RT-PCR in order to determine whether the effect of CSP32 on the production of nitric oxide was related to the gene level of iNOS and COX- -PCR. The results are shown in Fig. FIG. 7 is an electrophoresis image showing the effect of CSP32 on LPS-activated iNOS, COX-2, TNF-α, IL-1β, and IL-6 mRNA gene expression. GAPDH was used as a control for RT-PCR.

Cells treated with LPS with CSP32 significantly suppressed iNOS and COX-2 mRNA expression in a concentration-dependent manner. In addition, TNF-α, IL-1β, and IL-6 mRNA levels were increased when LPS was treated. In cells using CSP32, the expression of these cytokines decreased in a concentration-dependent manner (see FIG. 7).

Example 10: Pharmaceutical formulation

Manufacture of tablets

50 mg of the peptide of Example 2 (CSP32)

Lactose 80 mg

Starch 17 mg

Magnesium stearate 3 mg

Crystalline cellulose 10 mg

One embodiment of the present invention is the use of the peptide as an example of an effective ingredient of the tablet as described above. Tablets may be prepared by conventional methods and one preferred embodiment is in the form of tablets or soft capsules with conventional enteric coatings (e. G., Hydroxypropylmethylcellulose phthalate), sugar coating or coatings.

Based on the results of the above experiments, morphological, biochemical and 16S rRNA gene sequence analysis showed that the strain was identified as a Bacillus subtilis strain (CS32). This strain exhibited the highest 16S rRNA homology (99.392%) with that of Bacillus licheniformis as shown in Table 1. The antimicrobial peptide (called CSP32) produced by this strain in the optimal medium was homogeneously purified and the molecular weight of 5697.9 Da was confirmed. The first 12 amino acid residues at the N-terminus of CSP32 were APLEIXXIFHDN. As a result of the NCBI Blast search, the sequence of CSP32 showed the highest homology with the produced bacteriocin-like compound (Rikenin) from Bacillus licheniformis [Reference: 20]. In addition, this sequence also showed significant identity with the antimicrobial peptides produced by Bacillus subtilis JM4 with the shared sequence EIxxIFHDN (where x is an unidentified sequence) [Ref. 8]. Analysis of these results revealed that CSP32 is a novel type of antimicrobial peptide when compared with N - terminal amino acid sequence and molecular weight. CSP32 was stable at pH below 90 ° C and pH 5.0-12.0 and pH and temperature stability of CSP32 was comparable to that of antibiotic peptides from lichenin and Bacillus subtilis strain JM4 [ 8, 20]. CSP32 was stable against all tested proteases [Refs. 18, 20]. Although the proteolytic enzyme concentration was necessary to inactivate the antimicrobial activity, some of the products produced by Bacillus spp. Microbial agents are cyclic peptides that contain uncommon amino acids, which are more resistant to hydrolytic enzymes. The antimicrobial activity of CSP32 was evaluated for various gram positive and gram negative bacteria. CSP32 showed an effective antimicrobial activity against Gram-positive microorganisms, which was comparable to that of Bacillus sp. JM4 [Reference: 8] and the antimicrobial peptides of Bacillus licheniformis A89 [ Furthermore, the antimicrobial activity of CSP32 against multidrug-resistant bacteria such as MRSA, VRSA, and VRE showed better efficacy compared to basitracin and vancomycin, but it had a weaker antibacterial effect than BCP61 [ Reference: 18].

We also found that CSP32 significantly inhibited the production of proinflammatory mediators of nitric oxide (NO), iNOS and COX-2 protein in Raw 264.7 cells activated by LPS. These results indicate that the inhibitory effect of CSP32 is induced by inhibition of iNOS and COX-2 protein expression, and that RT-PCR analysis correlates with their protein levels at the mRNA level of iNOS and COX-2.

Thus, the inhibitory effect of CSP32 on iNOS and COX-2 gene expression seems to be one of the mechanisms involved in the anti-inflammatory action of the active peptides. CSP32 inhibited the expression of genes involved in inflammation, and we evaluated proinflammatory cytokine production in TNF-a, IL-l [beta] and IL-6. These cytokines are mainly produced by activated macrophages. Suppression of cytokine production is an important mechanism for evaluating inflammatory processes [Refs. 13-15]. Since antimicrobial peptides have been shown to inhibit proinflammatory mediators, we further investigated their efficacy against LPS-induced cytokine release by ELISA and RT-PCR. After 24 hours of incubation with LPS (1 ug / mL) and CSP32, significant inhibition was observed in Raw 264.7 cells as assessed by cytokine production (Fig. 6) and mRNA expression (Fig. 7).

These results indicate that CSP32 is a potent inhibitor of iNOS and COX-2 gene and protein expression, cytokine production, in Raw 264.7 activated by LPS, consistent with previous reports [Refs. 13-15].

In conclusion, CSP32 is a peptide with potent antimicrobial and anti-inflammatory activity purified from Bacillus strains isolated from Korean traditional foods. These results indicate that CSP32 can be a suitable candidate to be developed as a bifunctional peptide that can be used as a therapeutic agent in the treatment of multi-drug resistant pathogens as well as inflammation related diseases.

[Reference literature]

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[2] Rana M, Chatterjee S, Kochhar S, Pereira B. Antimicrobial peptides: a new dawn for regulating fertility and reproductive tract infections. J Endocrinol Reprod. 2006; 2: 88-95.

[3] Bizani D, Brandelli A. Characterization of a bacteriocin produced by a newly isolated Bacillus sp. Strain 8 A. J Appl Microbiol. 2002; 93: 512-9.

[4] Cherife, Ouzari H, Daffonchio D, Cherif H, Ben Slam K, Hassen A, et al. Thuricin 7: a novel bacteriocin produced by Bacillus thuringiensis BMG1.7, a new strain isolated from soil. Lett Appl Microbiol. 2001; 32: 243-7.

[5] Ahern M, Verschueren S, van Sinderen D. Isolation and characterization of a novel bacteriocin produced by Bacillus thuringiensis strain B439. FEMS Microbiol Lett. 2003; 220: 127-31.

[6] Dischinger J, Josten M, Szekat C, Sahl HG, Bierbaum G. Production of the novel two-peptide lantibiotic lichenicidin by Bacillus licheniformis DSM 13. PLoS One. 2009; 4: e6788.

[7] Oscar JC, Lasa I, Pisabarro AG. Detection and characterization of Bacillus cereus 7, a new bacteriocin produced by Bacillus cereus with a broad spectrum of activity. FEMS Microbiol Lett. 1999; 178: 337-41.

[8] Wu S, Jia S, Sun D, Chen M, Chen X, Zhong J, et al. Purification and characterization of two novel antimicrobial peptides Subpeptin JM4-A and Subpeptin JM4-B produced by Bacillus subtilis JM4. Curr Microbiol. 2005; 51: 292-6.

[9] Lee HJ, Joo YJ, Park CS, Kim SH, Hwang IK, Ahn JS, et al. Purification and characterization of a bacteriocin produced by Lactococcus lactis subsp. lactis H-559 isolated from kimchi. J Biosci Bioeng. 1999; 88: 153-9.

[10] Kayalvizhi N, Gunasekaran P. Production and characterization of a low-molecular-weight bacteriocin from Bacillus licheniformis MKU3. Lett Appl Microbiol. 2008; 47: 600-7.

[11] Khalil R, Elbahloul Y, Djadouni F, Omar S. Isolation and partial characterization of a bacteriocin produced by a newly isolated Bacillus megaterium 19 strain. Pak J Nutr. 2009; 8: 242-50.

[12] Rosenfeld Y, Shai Y. Lipopolysaccharide (endotoxin) -host defense antibacterial peptides interactions: Role in bacterial resistance and prevention of sepsis. Biochimica et Biophysica Acta (BBA) - Biomembranes. 2006; 1758: 1513-22.

[13] Sarkar D, Saha P, Gamre S, Bhattacharjee S, Hariharan C, Ganguly S, et al. Anti-inflammatory effect of allylpyrocatechol in LPS-induced macrophages is mediated by suppression of iNOS and COX-2 via the NF-kappaB pathway. Int Immunopharmacol. 2008; 8: 1264-71.

[14] Yoon WJ, Heo SJ, Han SC, Lee HJ, Kang GJ, Kang HK, et al. Anti-inflammatory effect of sargachromanol G isolated from Sargassum siliquastrum in RAW 264.7 cells. Arch Pharm Res. 2012; 35: 1421-30.

[15] Yu HY, Kim KS, Lee YC, Moon HI, Lee JH. Oleifolioside A, a New Active Compound, Attenuates LPS-Stimulated iNOS and COX-2 Expression through the Downregulation of NF-kappaB and MAPK Activities in RAW 264.7 Macrophages. Evid Based Complement Alternat Med. 2012;

[16] Yoon WJ, Kim SS, Oh TH, Lee NH, Hyun CG. Cryptomeria japonica essential oil inhibits the growth of drug-resistant skin pathogens and LPS-induced nitric oxide and pro-inflammatory cytokine production. Pol J Microbiol. 2009; 58: 61-8.

[17] Cho SS, Choi YH, Simkhada JR, Mander P, Park J, Yoo JC. A newly isolated Streptomyces sp. CS392 producing three antimicrobial compounds. Bioprocess Biosyst Eng. 2012; 35: 247-54.

[18] Choi YH, Cho SS, Simkhada JR, Yoo JC. A novel thermotolerant and acidotolerant peptide produced by a Bacillus strain is a newly isolated from a fermented food (kimchi) that shows activity against multidrug-resistant bacteria. Int J Antimicrob Agents. 2012; 40: 80-3.

[19] Kim HK, Cheon BS, Kim YH, Kim SY, Kim HP. Effects of naturally occurring flavonoids on nitric oxide production in the macrophage cell line RAW 264.7 and their structure-activity relationships. Biochem Pharmacol. 1999; 58: 759-65.

[20] Pattnaik P, Kaushik JK, Grover S, Batish VK. Purification and characterization of a bacteriocin-like compound (Lichenin) produced anaerobically by Bacillus licheniformis isolated from water buffalo. J Appl Microbiol. 2001; 91: 636-45.

[21] Mendo S, Faustino NA, Sarmento AC, Amado F, Moir AJ. Purification and characterization of a new peptide antibiotic produced by a thermotolerant Bacillus licheniformis strain. Biotechnol Lett. 2004; 26: 115-9.

Korea Biotechnology Research Institute KCTC18250P 20130717

<110> Chosun University Industry-Academic Cooperation Foundation <120> A novel bifunctional peptide and its use with antimicrobial          activity and anti-inflammatory action from Bacillus strain <130> pkr-453 <160> 16 <170> Kopatentin 1.71 <210> 1 <211> 12 <212> PRT <213> Streptomyces <400> 1 Ala Pro Leu Glu Ile Xaa Xaa Ile Phe His Asp Asn   1 5 10 <210> 2 <211> 12 <212> PRT <213> Bacillus licheniformis <400> 2 Ile Ser Leu Glu Ile Cys Xaa Ile Phe His Asp Asn   1 5 10 <210> 3 <211> 12 <212> PRT <213> Bacillus subtilis <400> 3 Xaa Xaa Lys Glu Ile Xaa Trp Ile Phe His Asp Asn   1 5 10 <210> 4 <211> 12 <212> PRT <213> Bacillus subtilis <400> 4 Xaa Xaa Lys Glu Ile Xaa His Ile Phe His Asp Asn   1 5 10 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 cccttccgaa gtttctggca gcagc 25 <210> 6 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 ggctgtcaga gcctcgtggc tttgg 25 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 cactacatcc tgacccactt 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 atgctcctgc ttgagtatgt 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 tctcatcagt tctatggccc 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 gggagtagac aaggtacaac 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 tggacggacc ccaaaagatg 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 agaaggtgct catgtcctca 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 gttctctggg aaatcgtgga 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 tgtactccag gtagctatgg 20 <210> 15 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 cactcacggc aaattcaacg gcac 24 <210> 16 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 gactccacga catactcagc ac 22

Claims (5)

delete delete delete delete Culturing the Bacillus genus CS32 strain in a glucose-beef extract-peptone medium containing 1% glucose, 0.5% beef extract and 0.5% peptone for 60 hours;
Cells were removed by centrifugation at 6000 rpm for 30 min at 4 &lt; 0 &gt; C, and the peptide in the supernatant was precipitated overnight at 4 &lt; 0 &gt; C with 60% saturation of ammonium sulphate;
The resulting precipitate was collected by centrifugation at 6,000 rpm for 60 minutes at 4 째 C, suspended in 10 mM Tris-HCl buffer (pH 8.0), and dialyzed using a 1 kDa cut-off membrane;
Adding the crude extracts obtained in the above step to Sepharose CL-6B column followed by Sephadex G50 column, separating and eluting with the buffer; And
Collecting and concentrating the active fractions,
The Bacillus genus CS32 strain is deposited with the strain Accession No. KCTC 18250P and exhibits 99.392% 16S rRNA homology with that of Bacillus licheniformis ,
The resulting peptide is APLEIXXIFHDN (SEQ ID NO: 1), the first 12 amino acid residues from the N-terminus, the molecular weight is 5697.9 Da, is stable at 90 ° C or lower and at pH 5.0 to 12.0 and is resistant to methicillin resistant Staphylococcus aureus (MDR) bacteria including Staphylococcus aureus (VRSA) and vancomycin resistant enterococci (VRE), as well as LPS-induced iNOS and COX-2 gene and protein expression and cytokine production And exhibiting an anti-microbial activity and an anti-inflammatory action from the strain of Bacillus subtilis.

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