KR101944713B1 - novel antibacterial composition - Google Patents

Abstract

The present invention provides an antibacterial composition derived from a strain of Bacillus sp. 2-4 (KCCM11107P) having high antibacterial activity against various pathogenic bacteria causing fish diseases.

Description

A novel antibacterial composition

The present invention relates to a novel antibacterial composition, and more particularly to a novel antibacterial composition having a high antibacterial activity against various pathogenic bacteria.

Research on natural materials that have physiological activity from natural materials has mainly been focused on land resources, but the new sources have been depleted through many years of research, leading to a decline in economic efficiency. As a result, developed countries have been intensively studying natural products derived from marine microorganisms. As a result, about 5,500 new materials have been developed in marine life. They are being developed competitively with pharmaceuticals, health supplements, and cosmetics. Approximately 40 substances have been patented for pharmaceuticals, and 5 of them are under development. Currently, oceanologists are keen to find new marine microorganisms and are keen to develop microorganisms that produce natural materials with new marine-derived structures. The development of marine-derived microorganisms' natural products is global, and recent global pharmaceutical companies have moved to shift from chemistry-based antibiotics to biologically-derived materials and to strengthen microbial market entry strategies using their metabolites have. Particularly, the development of next-generation anti-bacterial active materials derived from marine microorganisms and feed additives containing them will be an opportunity to secure leading technologies in the fisheries technology field. Seems to be.

On the other hand, the climate change due to global warming has greatly increased fish diseases. Fish diseases are caused by the destruction of balance between environment, pathogen, and avian disease, and the disease-causing mortality rate is rapidly increasing. For example, in the case of flounder, the damage caused by bacterial diseases such as Vibrio anguillarum, Vibrio harvevi, Edwardsiella tarda, and Streptococcus iniae is increasing. As a simple numerical value, the catching amount is about 122,000 per year. In the field survey of Jeju flounder farms, it is estimated that the economic damage is estimated to be 10 billion won per year based on the production of flounder, total bacterial infection rate and mortality rate in 2009 Damage is occurring. Therefore, there is a need for alternative substances, not antibiotics, to promote the prevention and growth of bacterial diseases in flounder. Antibiotics have advantages such as ease of use and efficacy of treatment. Such as the generation of resistant bacteria and the residual of antimicrobial substances in the body. Therefore, many researches have been focused on the development of probiotics for the prevention of fish diseases and maintenance of fish health. In particular, we have searched for non-pathogenic bacteria producing antimicrobial, antifungal, and antiviral substances and applied them to farms. Although the products of microbial probiotics currently being used for feed additives are being developed steadily, It is not attracting much attention in the market. In this connection, Korean Patent Registration No. 1544049 discloses a composition for controlling and treating squid chick disease of flounder.

However, the prior art has problems such as the generation of resistant bacteria and the residual of the antibacterial substance in the body including the antibacterial agent.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made to solve the above-mentioned problems, and it is an object of the present invention to provide a novel antibacterial composition having high antibacterial activity against various pathogenic bacteria including fish microbes. However, these problems are exemplary and do not limit the scope of the present invention.

According to one aspect of the present invention, there is provided a method for producing an antimicrobial agent having anti-bacterial activity against a bacterium selected from the group consisting of Staphylococcus aureus, Streptococcus mutans, Streptococcus iniae, and Streptococcus parauberis comprising basilomycin D having the following structural formula A bacterial composition is provided:

According to another aspect of the present invention, there is provided a bacterium having antibacterial activity against a bacterium selected from the group consisting of Staphylococcus aureus, Streptococcus mutans, Streptococcus iniae, and Streptococcus parauberis , Feed additives are provided:

According to another aspect of the present invention, there is provided a composition for preventing tooth decay comprising, as an active ingredient, basilomycin D having the following structural formula:

According to another aspect of the present invention there is provided a method for the treatment of bacteria selected from the group consisting of Staphylococcus aureus, Streptococcus mutans, Streptococcus iniae, and Streptococcus parauberis comprising the step of administering basilomycin D, There is provided a method for preventing and treating infectious diseases caused by:

According to one embodiment of the present invention as described above, the effect of producing an antibacterial composition having high antibacterial activity against various pathogenic bacteria including pathogenic bacteria causing fish diseases can be realized. Of course, the scope of the present invention is not limited by these effects.

1 is a diagram showing the structure of basilomycin D having antibacterial activity against various pathogenic bacteria purified according to an embodiment of the present invention.
FIG. 2 is a photograph showing the antibacterial activity against fish microbes for the activity test of the organic solvent extract according to one embodiment of the present invention. FIG.
Figure 3 is a graphical representation of Bacillus sp. And the production of a substance having an antimicrobial activity derived therefrom is confirmed by MS analysis.
Figure 4 is a graphical representation of Bacillus sp. And the production of a substance having an antimicrobial activity derived therefrom is confirmed by MS analysis.
Figure 5 is a graphical representation of Bacillus sp. And the production of a substance having an antimicrobial activity derived therefrom is confirmed by MS analysis.
FIG. 6 is a diagram showing the structures of bacillomycin D and surfactin having antimicrobial activity according to an embodiment of the present invention.
FIG. 7 is a graph showing the production of basilomycin D and surfactin by an LC-MS chromatogram according to culture conditions in a mass culture process according to an embodiment of the present invention.
FIG. 8 is a graph showing the HPLC analysis of the production of basilomycin D and surfactant by culture conditions in a mass culture process according to an embodiment of the present invention.
Figure 9 is a graphical representation of Bacillus sp. This is a photograph showing the process of culture concentration and solvent extraction.
10 is a graph showing the results of primary purification using LC analysis for identification of active compounds according to one embodiment of the present invention.
11 is a graph showing the results of secondary purification using PDA and MS analysis for identification of active compounds according to an embodiment of the present invention.
12 is a photograph showing the antimicrobial activity of surfactin, C14 vicormomycin D and C15 basilomycin D isolated and purified according to an embodiment of the present invention.

Definition of Terms:

As used herein, the term " bacillomycin " is an antifungal substance separated by acidic precipitation and ethanol extraction in the culture medium of Bacillus subtilis , a colorless microcrystal with a molecular weight of about 1,000 and is a weakly acidic, non-ribosomal family of cyclic polypeptides . Basilomycin has the structure of structure 1 below:

(Structural formula 1)

As used herein, the term " bacillomycin " means a basilomycin having 14 carbon atoms in the lipophilic < RTI ID = 0.0 > &Quot; bacillomycin " is known to belong to basilomycin D2 and D3 as a basilomycin having 15 carbon atoms in the lipophilic β-amino acid.

As used herein, the term " surfactin " is a very potent surfactant commonly used as an antibiotic. It is a bacterial cyclic lipopeptide and an antifungal agent produced by the gram-positive bacillus subtilis . The surfactants have been found to exhibit effective properties such as antimicrobial, antiviral, antifungal, antimyco plasma and hemolytic activity

DETAILED DESCRIPTION OF THE INVENTION [

According to one aspect of the present invention, there is provided a method for producing an antimicrobial agent having anti-bacterial activity against a bacterium selected from the group consisting of Staphylococcus aureus, Streptococcus mutans, Streptococcus iniae, and Streptococcus parauberis comprising basilomycin D having the following structural formula A bacterial composition is provided:

According to another aspect of the present invention, there is provided a bacterium having antibacterial activity against a bacterium selected from the group consisting of Staphylococcus aureus, Streptococcus mutans, Streptococcus iniae, and Streptococcus parauberis , Feed additives are provided:

According to another aspect of the present invention, there is provided a composition for preventing tooth decay comprising, as an active ingredient, basilomycin D having the following structural formula:

According to another aspect of the present invention there is provided a method for the treatment of bacteria selected from the group consisting of Staphylococcus aureus, Streptococcus mutans, Streptococcus iniae, and Streptococcus parauberis comprising the step of administering basilomycin D, There is provided a method for preventing and treating infectious diseases caused by:

Previously, antimicrobial substances of basilomycins have been reported, but all of them have been known as antifungal substances. However, the present inventors have found that when isolating and identifying an antimicrobial substance from a Bacillus sp. 2-4 (KCCM11107P) strain known as a probiotic strain having antibacterial activity (Patent No. 10-1186150), C14-benzylmycin D (3- -9 - ((R) -1-hydroxyethyl) -6- (2-amino-2-oxoethyl) -19- (4-hydroxybenzyl) - (hydroxymethyl) -1,4,7,10,14,17,20,23-octaoxo-12-undecylhexacosahydro-1H-pyrrolo [1,2- b] [1,4,7,10,13,16, 19,22] octaazacyclopentacosin-3-yl) propanoic acid) has antibacterial activity against some bacteria rather than the known antifungal activity (FIG. 1). Thus, the present inventors have completed the present invention by confirming the strong antibacterial activity against the specific bacterium of C14 vicormomycin D.

Hereinafter, the present invention will be described in more detail by way of examples. It should be understood, however, that the invention is not limited to the disclosed embodiments, but may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, Is provided to fully inform the user.

Example 1: Preparation of active fractions using an organic solvent

According to one embodiment of the present invention, Bacillus sp. 2-4 (KCCM11107P) strain was cultured in BHI medium at 30 ° C for 48 hours for sequential partition chromatography using an organic solvent, and then, using ethyl acetate as an organic solvent, Extraction was proceeded. The culture was centrifuged at 15,000 rpm for 15 minutes to separate the cells and culture supernatant. The supernatant and ethyl acetate were mixed at a ratio of 1: 3, and the mixture was added to a separating funnel and mixed and reacted for 1 hour. After 10 minutes, the material is divided into two layers. The lower layer is polar (water layer) and the upper layer is nonpolar (organic solvent layer). The material to be extracted is moved to a nonpolar state so that the lower layer is discarded and the upper layer and the collecting process are repeated twice to obtain an extract .

Example 2: Detection of antimicrobial activity of solvent fractions and tracing of active substance

In order to investigate the antimicrobial activity spectrum of the solvent extract obtained in Example 1, six bacterial pathogenic bacteria ( V. harvevi, S. iniae, E. tarda, S. parauberis, V. anguillarum, S. aureus ) Were investigated. Each pathogen was cultivated using marin broth, BHI broth, and Nutrient broth medium, and 100 μl of 1.0 x 10 ⁸ cfu / ml bacteria were plated on each solid medium. Thereafter, a 0.8 cm paper disk was placed on a plate, centrifuged, and about 50 μl of the culture extract was dropped. The size of the formed transparent ring was measured while culturing at 25-30 ° C for 24-48 hours.

As a result, it was confirmed that the organic solvent extract showed antibacterial activity against all the six pathogenic microorganisms used in the activity test (FIG. 2).

Example 3: Structure identification of active compounds

In order to purify the antimicrobial substance produced by the strain Bacillus sp. 2-4 (KCCM11107P) according to an embodiment of the present invention, the bacterial culture was centrifuged at 15,000 rpm for 15 minutes to culture the bacterial cells and the culture supernatant Respectively. Then, the supernatant and ethyl acetate were mixed at a ratio of 1: 3, placed in a separating funnel, and mixed and reacted for 1 hour. After the precipitate was removed, the material was extracted using a vacuum concentrator and 10 ml of ethyl acetate was added to recover. Thereafter, Bacillus sp. Secondary metabolite analysis to identify antimicrobial substances produced in the medium was carried out using LC-MS / MS in Kyunggi Bio Center, which has a mass spectrometer. The analytical equipment and conditions are shown in Table 1 Respectively.

As a result, MS analysis of secondary metabolites having antimicrobial activity produced by Bacillus sp. 2-4 (KCCM11107P) produced various secondary metabolites such as basilomycin D and Surfactin A1, B1, B2 and C1 (Figs. 3 to 5). In particular, basilomycin and surfactant are known to have various antiviral and antifungal activities and thus can be used as an active indicator component showing antimicrobial activity in the future (FIG. 6).

Analysis equipment LC system: UPLC system (Thermo scientific)
MS: LTQ-Orbitrap XL mass spectrometer (Thermo) Analysis condition Column: Acquity UPLC BEH C18 (2.1 x 150 mm, 1.7 um, Waters)
Elution solvent: A; DW (0.1% formic acid)
B; Acetonitrile (0.1% formic acid)
Elution rate: 400 uL / min
Injection vol: 2uL
Gradient: 5-70 B%, 0-20 min
70-100 B%, 20-24 min
100 B%, 24-27 min
Scan range: m / z 115-1,500
PDA scan: 200-600nm

Example 4: Development of QC assay for active compounds

In order to develop the QC method for active compounds, we observed changes in the production of antimicrobials during the scale-up of the mass culture process. First, each culture supernatant was extracted with ethyl acetate solvent in six kinds of samples (industrial medium, TSB, 30L Jar, conditional 2KL Jar), and analyzed by LC-MS chromatogram and HPLC analysis.

As a result, it was confirmed that the indicator substances, basilomycin and surfactant, were stably produced (Fig. 7). In Sample 6, a new peak different from the conventional one was found, (Fig. 8).

Example 5: Purification of Active Substance and Establishment of Analytical Method of Active Compound

According to one embodiment of the present invention, the active substance in the culture medium of the final mass culture process system was purified to measure the amount of the active substance. The culture broth was extracted with ethyl acetate twice at a ratio of 1: 3 and concentrated. The antimicrobial activity of the extracted active substance was measured by paper disk method. For the analysis of the antimicrobial spectrum of the active compound, purification was carried out by using a first Prep-LC-MS and a second auto purification system (FIG. 9).

As a result, it was confirmed through LC-MS analysis that surfactant A1, B1, B2, and C including C14 basilomycin D and C15 basilomycin D as target components were produced (FIGS. 10 and 11, Tables 2 and 3) . In the fourth purification step, C14 basilomycin D and C15 basilomycin D components were separated by 10 mg, respectively. In the case of surfactant, A, B1, B2, and C1 were not easily separated and 600 mg was collected (Table 4). The antimicrobial activity of nine bacterial strains was analyzed by using the separated purified basilomycin and serotactin. As a result, the serotactin was found to have antimicrobial activity against S. iniae and S. parauberis , C14 basilomycin D to S. aureus, S. mutans, S. iniae, S. parauberis and C. albicans (Fig. 12). Bacillus sp. (KCCM11107P) is shown in the following Table 2, and the purified amount of the active ingredient of the strain Bacillus sp. 2-4 (KCCM11107P) is shown in Table 3 below, Respectively. In particular, although the structure of C14 basilomycin D and C15 basilomycin D is very similar, C15 basilomycin D has only the antifungal activity against the fungus C. albicans , While in the case of C14 < RTI ID = 0.0 > basilomycin < / RTI & gt ; aureus, S. mutans, S. iniae, and S. parauberis. The fact that the antimicrobial substance of the basilomycin has antibacterial activity against bacteria is the first in the present invention It has been clarified.

RT (min) m / z
([M + H] < + >) [ Calculated
Formula △ ppm Expect compound Remarks 8.03 163.1323 unknown S6 New 9.76
(PDA) 883.2599 C39 H43 O18 N6 -3.334 Bacillibactin Schedule of new indicators 10.49 520.1785 unknown S6 New 14.36 1031.5377 C48 H75 O15 N10 -2.644 C14 Bacillomycin D Target 15.25 1045.5537 C49 H77 O15 N10 -2.609 C15 Bacillomycin D Target 23.77 994.6407 C50 H88 O13 N7 -2.727 [Val7] -
Surfactin C13ai Target 24.13 1008.6561 C51 H90 O13 N7 -2.748 Surfactin A1 Target 24.64 1022.6718 C52 H92 O13 N7 -2.945 Surfactin B1 Target 24.78 1022.6732 C52 H92 O13 N7 -1.567 Surfactin B2 Target 25.11 1036.6870 C53 H94 O13 N7 -2.568 Surfactin C1 Target

F2
1 ^st 2 ^nd 3 ^rd 4 ^th Total
Fraction. No mg Fraction. No mg Fraction. No mg Fraction. No mg Unknown
(m / z 520) 3
4
5
6 1.64
1.48
0.63
0.44 2
3
4 0.84
0.26
0.45 3.96
1.33
0.45 Bacillomycin D2
(m / z 1031) 9
10
11
12 0.07
0.85
2.52
0.47 29
30
31 0.68
2.5
3.92 38
39
40
41 0.77
1.26
4.14
1.26 31
32
33
34 One
0.51
1.47
0.82 2.52
10.04 Bacillomycin
D1, D3
(m / z 1045) 16
17
18
19 0.25
1.82
3.47
One 37
38
39
40 1.56
2.28
2.28
4.23 45
46
47
48
49
50 2.58
1.94
1.87
2.96
3.59
2.85 38
39
40
41 0.25
1.08
1.76
4.01 13.65
10.47
5.23 Bacillomycin D
(m / z 1059) 24
26 0.51
0.2 45
46
47
48 0.78
0.9
0.31
0.48 57
58
59
61
62
63 0.21
0.61
0.46
0.78
0.46
0.23 49
50
51
52
53 0.02
0.46
0.16
0.18
0.11 1.4
1.67
0.79

RT (min) m / z
([M + H] < ⁺ >) [ Expect compound Refined amount Unknown component 520.1785 unknown 1.33 mg Target 1 1031.5377 C14 Bacillomycin D 10.04 mg Target 2 1045.5537 C15 Bacillomycin D 10.47 mg
Target 3
1008.6561 Surfactin A1 600 mg


1022.6718 Surfactin B1 1022.6732 Surfactin B2 1036.6870 Surfactin C1

In conclusion, since the active compound derived from Bacillus sp. 2-4 (KCCM 11107P) purified according to an embodiment of the present invention has a high antibacterial activity against various pathogenic bacteria including pathogenic bacterium causing fish diseases It can be utilized as an active indicator component showing antimicrobial activity in the future.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. Accordingly, the true scope of the present invention should be determined by the technical idea of the appended claims.

Claims (4)

An antimicrobial composition having antibacterial activity against a bacterium selected from the group consisting of Staphylococcus aureus, Streptococcus mutans, Streptococcus iniae, and Streptococcus parauberis , which comprises as an active ingredient a basilomycin D having the following structural formula:

A feed additive having antibacterial activity against a bacterium selected from the group consisting of Staphylococcus aureus, Streptococcus mutans, Streptococcus iniae, and Streptococcus parauberis , which comprises as an active ingredient,

. A composition for preventing tooth decay comprising, as an active ingredient, barylomycin D having the following structural formula:

. A method for preventing and treating infectious diseases caused by a bacterium selected from the group consisting of Staphylococcus aureus, Streptococcus mutans, Streptococcus iniae, and Streptococcus parauberis , comprising the step of administering basilomycin D having the following structural formula to an animal other than human:

.

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