KR101229335B1 - Bacillus licheniformis nb109 having antimicrobial activities - Google Patents

Abstract

PURPOSE: A novel Bacillus licheniformis NB 109(KCTC 12256BP) with antibacterial activity is provided to ensure a broad antibacterial spectrum against various bacteria and to be used in agrochemicals, medicines, and cosmetic products. CONSTITUTION: A Bacillus licheniformis NB 109(KCTC 12256BP) has cytotoxicity against cancer cells. The strain has 16S rRNA sequence of sequence number 1. The strain has an antibacterial activity against Bacillus sp., Staphylococcus sp., Pseudomonas sp., Escherichia coli, Candida sp., Proteus sp., Erwinia sp., or Ralstonia sp. The strain has cancer cell-specific cytotoxicity.

Description

Bacillus Licheniformis NB109 Having Antimicrobial Activities

The present invention relates to a novel Bacillus licheniformis NB109 strain having antibacterial activity.

Biopesticides are generally pesticides derived from animals, plants and microorganisms. The definition of the scope is not clearly defined in Korea, but it is a product produced without artificial synthesis or manipulation of organisms or substances derived from them. It is defined. Biopesticides are divided into natural pesticides and biochemical pesticides, and among them, microbial pesticides refer to pesticides using living microorganisms such as bacteria, fungi, protozoa and viruses used for crop protection. The mechanism of action of microbial pesticides is that antibiotics produced by microorganisms inhibit the growth of pathogens; Necrosis parasites, competition to prevent pathogens from using plant nutrients or soil nutrients, and inducing resistance to increase plant immunity.

Microbial pesticides using this method is safe for soil, plants, insects, and human beings, and there is no residual worry and no harm, so it can be used regardless of the growth time. In addition, there is no resistance and stress to crops, crop growth is promoted, and soil pest control is beneficial in terms of long-term control.

Up to now, Korea has no practical concept of biopesticides, and soil microbial agents have been mainly used for fertilizer addition. However, by June 2000, the registration criteria and methods of microbial pesticides were announced, providing institutional support for the entry of microbial pesticides into products that are recognized for their bactericidal and pesticidal effects. We also recognize the need for environmentally friendly biopesticides and announce a plan to reduce or replace 30% of chemical pesticides currently in use by 2005 (December 2000). Has entered the era of However, in Korea, it is in the early stages of introduction of biopesticides, mainly microbial pesticides. Pest control using insect pathogenic microorganisms has been limitedly conducted by some researchers, and recently, prototypes showing the possibility of effectively controlling some plant diseases have been proposed, and overall, due to the lack of related experts and the inferior competitiveness of the technology. As a result, there are still some challenges to overcome. However, research and development are actively being conducted not only in government-funded research institutes but also in companies and universities, and the number of applications for microbial patents for biopesticides is increasing.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have isolated and identified bacteria having antimicrobial activity from fermentation compost in order to discover bacteria having biotechnological usefulness. As a result, a bacterium having antimicrobial activity was isolated and identified from the fermented compost sample, and the strain was Bacillus licheniformis ( Bacillus). licheniformis ) strain, and the present invention was completed by identifying that it has antibacterial and anticancer activity.

Accordingly, an object of the present invention is a novel Bacillus licheniformis ( Bacillus) licheniformis ) NB109 (KCTC 12256BP) strain to provide.

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

According to one aspect of the present invention, the present invention provides Bacillus licheniformis NB109 (KCTC 12256BP) strain.

The present inventors have isolated and identified a bacterium having antimicrobial activity from fermented compost to find a bacterium having biotechnological utility. Richinformis ( Bacillus licheniformis ) strain was identified as having antibacterial and anticancer activity.

According to the invention, one to determine the base sequence of 16S rRNA gene of the isolated microorganism phylogenetic analysis was identified as a novel microorganism belonging to the genus Bacillus (Bacillus) strain. It was named “ Bacillus licheniformis NB109 strain” and was deposited on August 2, 2012 to the Korean Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology, and was given accession number KTCT 12256BP.

According to a preferred embodiment of the present invention, the Bacillus licheniformis ( Bacillus licheniformis ) NB109 (KCTC 12256BP) strain has the 16S rRNA gene sequence of SEQ ID NO: 1.

The strain of the present invention exhibits antimicrobial activity against various microorganisms. The strains of the present invention exhibit a wide range of antimicrobial activity against various microorganisms, in particular bacteria (Figs. 10-13), have no cytotoxicity against normal cells (Fig. 14), acute oral toxicity (Table 10-13), and acute dermal toxicity ( Table 14-17) did not show acute fish toxicity (Table 18-20).

To as evidenced in one embodiment of the invention, the strain of the present invention is Bacillus (Bacillus) in bacteria, Staphylococcus aureus (Staphylococcus) bacteria belonging to the genus Pseudomonas (Pseudomonas) in bacteria, E. coli (Escherichia coli), has a Candida (Candida) spp, Proteus (Proteus) in bacteria, El beanie O (Erwinia) and bacteria belonging to the genus LAL antimicrobial activity against bacteria selected from Stony O (Ralstonia) bacteria belonging to the genus group consisting of.

Preferably, the strain of the present invention Bacillus subtilis ( Bacillus subtilis ), Bacillus megaterium megaterium ), Staphylococcus aureus ), Escherichia coli ), Pseudomonas aeruginosa ), Proteus vulgaris ), erwinia carotovora), bacterial blight (Erwinia chrisanthemi ), Ralstonia solanacearum , Pseudomonas fluorescens ), Pseudomonas syringae ), Erwinia antimicrobial activity against tracheiphila or Candida albicans .

According to a preferred embodiment of the present invention, the strain of the present invention is cytotoxic to cancer cells.

As demonstrated in one embodiment of the present invention below, the strain of the present invention does not have cytotoxicity in normal cells but specifically shows cytotoxicity in cancer cells (FIG. 14). Therefore, the strain of the present invention or the culture of the strain can be used for the preparation of various forms of anticancer composition.

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

(Iii) The present invention is a novel Bacillus richenium ( Bacillus ) having antibacterial activity licheniformis ) NB109 (KCTC 12256BP) strain.

(Ii) The strains of the present invention show a broad spectrum of antimicrobial activity against various bacteria and are safe substances that do not cause any toxicity or irritation in animal and fish stability tests. It is useful for sterilization, antibacterial and antiseptic purposes.

1 is a graph showing the results of fatty acid analysis of B. licheniformis NB109.
2 is a result of measuring the effect of the carbon source on the 24-hour growth of B. licheniformis NB109.
A, mannitol; B, fructose; C, glucose; D, D-sorbitol; E, cellobiose; F, xylose; G, galactose; H, maltose; I, liquid starch; J, arabinose; K, lactose; L, raffinose.
Figure 3 is the result of measuring the effect of the carbon source on the 48 hours growth of B. licheniformis NB109.
A, mannitol; B, fructose; C, glucose; D, D-sorbitol; E, cellobiose; F, xylose; G, galactose; H, maltose; I, liquid starch; J, arabinose; K, lactose; L, raffinose.
Figure 4 is the result of measuring the effect of nitrogen source on the 24-hour growth of B. licheniformis NB109.
A, yeast extract; B, malt extract; C, beef extract; D, NaNO ₃ ; E, NH ₄ NO ₃ ; F, L-asparagine; G, peptone; H, (NH ₄ ) HPO ₄ ; I, NH ₄ Cl.
5 is a result of measuring the effect of nitrogen source on the 48 hours growth of B. licheniformis NB109.
A, yeast extract; B, malt extract; C, beef extract; D, NaNO ₃ ; E, NH ₄ NO ₃ ; F, L-asparagine; G, peptone; H, (NH ₄ ) HPO ₄ ; I, NH ₄ Cl.
Figure 6 is the result of measuring the effect of inorganic salts on the 48 hours growth of B. licheniformis NB109.
A, MgCl ₂ ; B, AgNO ₃ ; C, HgCl ₂ ; D, ZnSO ₄ 7H ₂ O; E, MgSO ₄ 7H ₂ O; F, CaCl _{2 ..}
7 shows the results of measuring the effect of temperature on the 48-hour growth of B. licheniformis NB109.
(Circle), 30 degreeC; , 42 ° C .; □, 55 ° C .; ■, 65 ° C.
8 shows the results of measuring the effect of pH on the 24-hour growth of B. licheniformis NB109.
Figure 9 is the result of measuring the effect of the culture shaking speed on the 24 hours growth of B. licheniformis NB109.
10 B. licheniformis by disk diffusion method The minimum inhibitory concentrations for Gram-positive bacteria, Gram-negative bacteria and fungi of NB109 strains were measured.
11 is a result of measuring the concentration of phytopathogenic bacteria of B. licheniformis NB109 strain by the microdilution method.
12 is an electron micrograph of C. albicans .
A: B. licheniformis Untreated NB109 culture filtrate; B: B. licheniformis NB109 culture filtrate treatment group.
13 is Ralstonia An electron micrograph of solacearum .
A: B. licheniformis Untreated NB109 culture filtrate; B: chemical pesticide tebuconazole treatment group; C: B. licheniformis NB109 culture filtrate treatment group.
14 B. licheniformis Toxicity of NB109 culture filtrate to normal and cancer cell lines was measured.
Figure 15. B. licheniformis in kidney cancer-related cell lineage (K-562) It is the result of measuring cytotoxic effect (Facscan method) of NB109 culture filtrate.
16 B. licheniformis This is a result of measuring the effect of carbon source on 24-hour growth in industrial culture of NB109.
A, wheat flour; B, corn starch; C, sucrose; D, maltose; E, fructose; F, glucose.
17 B. licheniformis The result of measuring the effect of nitrogen source on 24 hours growth in the industrial culture of NB109.
A, rice bran; B, Corn steep liquor (CSL); C, soy flour; D, casein; E, yeast extract; F, molasses.
18 B. licheniformis The result of measuring the effect of nitrogen source on 36 hours growth in industrial culture of NB109.
A, rice bran; B, Corn steep liquor (CSL); C, soy flour; D, casein; E, yeast extract; F, molasses.
19 B. licheniformis The result of measuring the effect on the growth according to the concentration of yeast extract for 36 hours in the industrial culture of NB109.
20 B. licheniformis The results show the proper culture conditions for 24 hours in the industrial culture of NB109.
○, cell growth; ●, DO; □, pH.
FIG. 21 shows the results of antimicrobial activity against pepper blight and diarrhea on culture filtrates in which B. licheniformis NB109 strains were cultured for 24 hours in an industrial culture. A, Phytophthora capsici ; B, Pythium ultimum .
22 shows pathogenic contaminant microbial test results.
A: Escherichia coli ; B: Salmonella sp . ; C: Staphylococcus aureus ; D: Bacillus cereus ; E: Listeria monocytogenes

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Example 1 Screening of Microorganisms Having Antimicrobial Activity

The fermentation compost using ground manure, humus soil, and food waste from Jangseong, Jeollanam-do, was crushed and diluted with sterile water. After incubating for 2 days at ℃ temperature, single cells were inoculated in a new NA medium using platinum, followed by incubation at 45 ℃ for 2 days to secure single strains. Was designated as NB109. The selected microorganisms were mixed at a ratio of 1: 1 and 50% glycerin (glycerin) cultured in shaken culture in NB (Nutrient Broth, Difco, USA) medium and stored at -70 ° C to be used as microorganisms to identify microorganisms and to produce products. . Potato dextrose agar (2.0% dextrose, 0.4% potato starch, 1.5% bactoagar, pH = 6.0 ± 0.4) for the isolation and selection of antagonistic strains that can control crop pathogenic fungi Substituted cultures were performed to isolate strains showing antifungal activity. In the antibacterial activity test, phytophthora capsici was used among the phytopathogenic fungi, a pathogen which is very common in domestic pepper cultivation.

Example  2: Identification of Microorganisms Having Antimicrobial Activity

The 16S rRNA gene is a region that has a large diversity according to differentiation between species and genus, and contains information that can distinguish bacteria at the species level. These 16S rRNA gene sequencing changes are used as the most important information for identifying the softness of bacteria. . Purely isolated strains were inoculated in NA medium and incubated at 45 ° C. for 2 days, and then single cells were reinoculated in NB medium and shaken at 45 ° C. for 2 days. 16S rRNA gene PCR (Polymerase Chain Reaction) was amplified using the extracted DNA as a template using the modified benzyl chloride method. Primers used for 16S rRNA gene amplification were 27F (5'-AGAATTTGATCCTGGCTCAG-3 ') and 1492R (5'-GGTTACCTTGTTACGACTT-3'), PCR conditions were 94 ℃ 30 seconds, 58 ℃ after 2 minutes of reaction at 95 ℃ The reaction was carried out 30 times in a sequence of 30 seconds, 72 ° C. and 45 seconds, followed by 5 minutes of reaction at 72 ° C., and then left at 4 ° C. 16S rRNA gene PCR amplification products were purified using PCR Product Purification Kit (Qiagen, USA). 16S rRNA gene PCR purified product was analyzed by nucleotide sequence using Genetic Analyzer 310A (Applied Biosystems, USA). The base sequence was searched for homology in the database of DDBJ / NCBI / Genebank and Ribosomal Database Project II (RDP II).

Homology of 16S rRNA gene sequence (SEQ ID NO: 1) of the root antagonist microorganism NB109 from the databases of DDBJ / NCBI / Genebank and Ribosomal Database Project II (RDP II) showed a strain containing the species of the genus Bacillus Bacillus , a strain belonging to a chemical group licheniformis DSM13 ^T (X68416) was found to have a flexible relationship of 99.57%. Thus, the rhizosphere antagonist NB109 Bacillus identified as licheniformis .

Example  3: fatty acid composition analysis

In the case of higher organisms, since the cell fatty acid composition is simple and has the same components, it is difficult to use as a classification index, but in the case of bacteria, the cell fatty acid composition is used as a useful information in terms of chemical taxonomy. Purely isolated strains were inoculated in NA medium and incubated at 45 ° C. for 2 days, and then single cells were reinoculated with TSA (Trypticase Soy Broth, Difco, USA) and shaken at 45 ° C. for 2 days. Extraction and purification of cell fatty acids was performed by Ikemoto & Miyagawa method to extract the cell fatty acid methyl ester (50%) by the method of Ikemoto & Miyagawa. Colony fatty acids were analyzed by Gas Chromatograph (Gas Chromatograph, 6890N, Agilent Technologies Inc., USA) using a Microbial Identification System (MIDI: Microbial ID, Inc., Newark, Del., USA).

B. licheniformis Analysis of cell fatty acid composition of NB109 showed C _{15 : 0} anteiso (54.74%), C _{15 : 0} iso (19.30%) as major fatty acids, C _{14 : 0} iso, C _{16 : 0} iso, and C _{17 : 0} iso And various molecular fatty acids such as C _{17 : 0} anteiso (Table 1). Also B. licheniformis NB109-B1 is B. licheniformis It was confirmed that the cell fatty acid composition was similar to the main cell fatty acid composition of the DSM13 ^T (X68416) standard strain (FIG. 1).

Fatty Acid Composition of B. licheniformis NB109  fatty acid(%) B. licheniformis NB109 B. licheniformis DSM13 ^T Saturated acid C _{16 : 0} 8.14 3.91 Branch chain mountain C _{14 : 0} iso 3.91 1.13 C _{15 : 0} iso 19.30 28.87 C _{15 : 0} anteiso 54.74 37.75 C _{16 : 0} iso 4.34 4.42 C _{17 : 0} iso 3.68 6.99 C _{17 : 0} anteiso 5.90 11.30

Example  4: Establish Optimal Media Component Conditions for Strain Culture

The minimum medium for the optimal medium review of the isolated strain B. licheniformis NB109 was M9 minimal medium (glucose 2%, MgSO.7H ₂ O 0.2%, Na ₂ HPO ₄ .7H ₂ O 6.4%, KH ₂ PO ₄ 1.5%, NH ₄ Cl 0.5% and NaCl 0.02%) were used. The medium dissolves each medium separately in order to prevent caramelization of sugar during autoclaving at high temperature, amino-carbonyl reaction caused by sugar reacting with ammonium ion or amino acid, and mineral precipitation. The mixture was then mixed with other media in aseptic mass. The inorganic salt was dissolved at room temperature, filtered through a 0.45 μm filter, and immediately mixed. Optimal media components of the isolated strain B. licheniformis NB109 were based on the M9 minimum medium, based on the carbon source mannitol, fructose, glucose, D-sorbitol, cellobiose, xylose, galactose, maltose, liquid starch, arabinose, lactose and Each of raffinose was added at a concentration of 2% and the inoculated strain B. licheniformis NB109 was cultured at 2% each, followed by shaking culture at 42 ° C for 20-60 hours at 180 rpm, and the turbidity of the culture was measured at 660 nm. . The effect of carbon source concentration was examined under the same conditions by adding selected carbon sources at 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0 and 5.0%, respectively. Nitrogen sources were yeast extract, malt extract, beef extract, NaNO ₃ , NH ₄ NO ₃ , L-asparagine, peptone, (NH ₄ ) ₂ HPO ₄ and NH ₄ Cl were added to each of the 0.5% by the method of examining the carbon source Experiment was the same as Inorganic salts were added in the same manner as in the method of examining carbon sources by adding CaCl ₂ , MgCl ₂ , AgNO ₃ , HgCl ₂ , ZnSO ₄ · 7H ₂ O, and MgSO ₄ · 7H ₂ O each at 0.05% (Table 2).

Raw materials used in industrial media according to nutrition sources Nutrition Raw material carbon - Glucose Corn Sugar, Starch, Cellulose Sucrose Sugar cane, sugar beet molasses Glycerol - Starch - Maltodextrin - Lactose Whey Fat Vegetable oil hydrocarbon Petroleum fractions nitrogen - protein Soy flour, corn steep liquor, distilled water ammonia Pure Ammonia or Ammonium Salt Element nitrate nitrate Phosphorus source phosphate Vitamins and Growth Factors - - Yeast, Yeast Extract - Wheat malt flour, cottonseed - Beef extract - Corn Soak

Example  5: Establish Optimal Culture Conditions

Optimal culture conditions of the isolate strain B. licheniformis NB109 were examined by adding 200 ml of liquid medium to 500 ml Erlenmeyer flask and inoculating 2.0% (50: 1) of the whole culture of the isolated strain. Optimal incubation temperature was incubated at 180 rpm for 24 hours while varying in the range of 30-60 ℃ and the absorbance of the culture at 660 nm was measured. The initial pH of the medium was adjusted after the initial pH was adjusted to 4-8 by adding 0.5 N HCl and 0.5 N NaOH, and the effect of shaking speed was controlled by adjusting the initial pH of the optimum medium to 7.0 and then shaking speed of 100, The absorbance of the culture was measured at 660 nm by incubating at 42 ° C. for 24 hours while changing at 140, 180, and 220 rpm.

5-1. Carbon source  effect

As a result of examining the effect of carbon source on the growth of B. licheniformis NB109, liquid starch, fructose and arabinose, galactose and glucose were shown to be higher in order, whereas lactose and raffinose were less affected to growth. (FIG. 2). As a result, it was confirmed that the optimum carbon source of B. licheniformis NB109 was liquid starch, and the growth rate of bacteria was different depending on the type of carbon source. In particular, xylose showed a slow rate of use of the nutritional component of the bacteria, and other carbon sources appeared to have a high rate of use for growth (Fig. 2). Qingzhao Wang et al. Reported that when more than 10% of the carbon source was added, growth was inhibited and sugar conversion was low, and Jeong et al. Reported the highest cell mass in galactose.

5-2. Effect of Nitrogen Sources

The results of examining the effect of the nitrogen source on the growth of B. licheniformis NB109, an antimicrobial microorganism, are shown in FIG. 4. Incubation with 2% glucose and 0.5% nitrogen in the M9 minimal medium resulted in growth of NaNO ₃ and organic nitrogen sources except for malt extract in inorganic nitrogen sources, and L-asparagine growth in 24 hours of culture. This was found to be the highest. Yeast extract was found to have a lasting effect on growth for more than 48 hours incubation, it was confirmed that yeast extract is most suitable as a nitrogen source at the time of mass culture (Fig. 5). Jeong et al. Selected the yeast extract as an optimal nitrogen source and reported that the longer the incubation time, the greater the effect on growth.

5-3. Inorganic  effect

B. licheniformis, whose inorganic salt is an antimicrobial microorganism The result of having examined the effect on the growth of NB109 is shown in FIG. MgSO ₄ 7H ₂ O was found to have a significant effect on the growth of bacteria by adding 0.05% inorganic salt and minimizing the growth at 660 nm.

5-4. Influence of temperature

B. licheniformis , an isolated antimicrobial microorganism The influence of the culture temperature on the growth of NB109 was examined and shown in FIG. 7. As a result of incubating the culture temperature in the range of 30-65 ° C., it was confirmed that the growth of bacteria was high at the culture temperature of 42 ° C., but there was little growth of the bacteria above 60 ° C.

5-5. Early pH Influence of

Initial pH B. licheniformis The influence on the growth of NB109 was examined and shown in FIG. 8. As a result of confirming the growth of bacteria by pH, it was confirmed that the growth of bacteria was lower than 5.0 and above pH 10, while it was the most stable in the range of pH 5.0-9.0. These results indicate that the antimicrobial bacterium B. licheniformis NB109 has a wide range of growth and excellent environmental adaptability.

5-6. Of shaking speed  effect

Shaking speed B. licheniformis The influence on the growth of NB109 was examined and shown in FIG. 9. As a result of measuring the growth of the fungi by adjusting the shaking speed to 100-220 rpm at the initial pH of 7.0 and the culture temperature of 42 ° C., the growth of microorganisms increased as the shaking speed was increased. Ishwar B is B. licheniformis It has been reported that the production of poly (γ-glutamic acid) in NCIM2324 cultures is very sensitive to the agitation rate.

Example  6: Establishment of mass production conditions for antimicrobial active substances

In order to determine the optimal condition of the antimicrobial active material of the isolated strain B. licheniformis NB109, 200 ml of optimal medium was added to a 500 ml Erlenmeyer flask, and the whole culture of the isolated strain was inoculated by 2.0% (50: 1). After culturing for 48 hours at the optimum culture temperature of 42 ° C and shaking speed of 180 rpm, the clear zone was measured by paper disk diffusion method using the filtrate containing the antimicrobial active material recovered after centrifugation of the culture medium for each time period. Investigate.

In order to determine the optimal condition of the antimicrobial active material of the isolated strain B. licheniformis NB109, 200 ml of the optimum medium was added to a 500 ml Erlenmeyer flask, and the whole culture solution of the isolated strain was inoculated 2.0% (50: 1) to incubate the optimum culture temperature. The clear zone was measured by paper disk diffusion method using filtrate containing the antimicrobial active substance peptide recovered after centrifugation of the culture medium at each time period while incubating for 48 hours at 180 rpm with shaking speed. The highest antimicrobial active clear areas appeared in the time-cultured filtrate. As the incubation time increased, the cell population increased but the antimicrobial activity tended to decrease somewhat.

Example  7: evaluation of biological activity

7-1. Disk diffusion method ( Disc diffusion method )

B. licheniformis by Disk Diffusion The antimicrobial effect of NB109 strain was made by making NA agar medium and sterilizing it, pouring 4 mm on the sterilized flat medium, and hardening it. Then, agar medium and 0.7% and 1% of each test strain were added to 4-5 mm thickness. 50 μl of the fraction extracted from the paper disk (8 mm, Toyo, Japan) was absorbed on the surface, and the solvent was completely evaporated and then placed on the surface to be adhered and dropped, followed by incubation at 37 ° C. for 24 hours. The antimicrobial activity was measured by examining the clear area of the suppressed paper disk.

B. licheniformis by Disk Diffusion The antimicrobial effect of the strain NB109 was investigated for the minimum inhibitory concentration against three Gram-positive bacteria, three Gram-negative bacteria and two fungi (FIG. 10). MIC in gram positive bacteria ranged from 0.78-1.58 μM and MIC in gram negative bacteria ranged from 0.78-3.25 μM. And in two fungi, the same was 12.5 μM. Bacillus in Gram-positive Bacteria It showed strong antimicrobial activity against pathogens of strains, and Gram-negative bacteria showed strong antibacterial activity against Pseudomonas strains.

7-2. Microdilution method ( Micro Dilution method )

Antimicrobial Experiments Standard microbial strains were incubated for 18-24 hours at 30-37 ° C. in NB (Nutrient Broth, Difco, USA) liquid medium, and then the culture was diluted to 2 × 10 ⁴ cfu / mL with fresh NB medium. B. licheniformis The culture filtrate of the NB109 strain was step diluted with sterile NB medium that was sterile. 100 μl of the antimicrobial test standard microbial strain was then placed in each hole of a 96-well polypropylene microtiter plate (Costar 3790, Corning, USA) and the concentration of B. licheniformis prepared in each well. Culture dilutions of the NB109 strain were mixed. B. licheniformis Minimal Inhibition Concentration (MIC) was confirmed by reading the absorbance at 620 nm using an ELISA reader (Molecular Devices, USA) after incubation at 30 ° C. for 18 hours to see the antimicrobial activity of the NB109 strain.

B. licheniformis Dilution of the cultured filtrate of the strain NB109 by plant concentration pathogenic bacteria Erwinia carotovora ( softwood ), Erwinia chrisanthemi (blight), Ralstonia solacearum (foot blight), Pseudomonas fluorescence , Pseudomonas The minimum inhibitory concentration for antimicrobial activity was examined by microdilution method for six syringae ( diabetic ) and Erwinia tracheliphila ( dry blight) (Fig. 11). Although the antimicrobial activity was slightly lower than that of kanamycin and ampicillin, the broad-spectrum antibiotics used as controls, the antimicrobial activity was significantly higher than that of the control ampicillin against Pseudomonas strains. And E. carotovora and E. tracheliphila showed very high antimicrobial activity similar to control antibiotics.

7-3. Electron microscope Electron Microscopy )

B. licheniformis C. albicans and yeast-like pathogenic fungi treated with culture filtrate containing the antimicrobial active substance peptide of NB109 strain Ralstonia To observe the morphological changes of solacearum (foot blight), fix for 2 hours in 2.5% glutaraldehyde solution (0.1 M cacodyl hydrochloric acid buffer, pH 7.4, 4 ° C) and then 20 minutes with 0.1 M cacodyl hydrochloride buffer. After washing three times in the liver, it was fixed for 2 hours at room temperature with 1% osmotic acid (0.1M cacodylate hydrochloric acid buffer, pH 7.4), followed by a series of dehydration with ethyl alcohol solution, and the coating on the gold ion particles. It was observed with a DSM 940A scanning electron microscope (Hitachi S-4300, Japan).

B. licheniformis C. albicans group without NB109 culture filtrate had smooth surface but B. licheniformis C. albicans group treated with NB109 culture filtrate (20 ㎍ / 20 L) was confirmed that the hole in the surface (Fig. 12). In addition, the Ralstonia solacearum (foot blight) group, which causes foot blight ( blue eye disease ), exhibited cell destruction similar to that of the chemical pesticide tebuconazole (20 μg / 20 L) treatment group (FIG. 13). These phenomena are thought to affect cell depolarization, cell permeability, and cell lipid leakage.

7-4. Efficacy and Weak test

To investigate the efficacy and weakness of B. licheniformis NB109 strain, the experiment on the antimicrobial activity (control effect) and the analysis on the presence or absence of weakness by concentrations in peppers, which are the public crops, were carried out in the experimental crop of Jeonnam Biocontrol Center. (272, Changjeon-ri, Gokseong-gun, Jeollanam-do) The test batches were divided into three treatment groups: no treatment, sample dispensing, and sample recommendation. Twenty weeks per treatment were performed in three replicates of the egg mass method. Drug treatment was treated with groundwater for untreated plot, 500 times (drainage) for undiluted solution, and 1,000 times (standard amount) for undiluted solution.

7-4-1. Pepper seed sowing

Pepper seeds were sterilized with 70% ethanol and 2% NaOCl, and then thoroughly washed with sterile water. Sterilized pepper seeds were sown in sterilized plant cell culture MS (Murashige and Skoog, Duchefa, Netherland) medium, and then the seeded seeds were transferred to 12-row seedlings and subjected to primary sowing. Seed germination and primary growth were carried out in a sterile tissue culture room and the growth temperature was maintained at 25 ℃.

7-4-2. Chili Pepper and Sample Processing

In the first seedling transplant, young seedlings with leaves from 12 ball nurseries were transplanted into the first small pots. The transplanted soil was a mixture of soil, vermiculite and perlite 5: 1: 1. In the second soil formulation, seedlings with uniform growth were selected among the seedlings that had completed their swelling in small pots and transplanted into the mainland soil (12 m ^{2 per} treatment). Late blight ( Phytophthora) capsici ) was grown at 72.5 g / L oatmeal (Oatmeal, Difco, USA) medium for one week at 25 ° C., and then mycelium was scraped with sterile water and then treated with light in a growth chamber maintained at 25 ° C. for 24 hours. After using the spreader (Spreader) was scraped with sterile water in sterile water stored at 4 ℃ and then diluted again with sterile water was adjusted to 1 × 10 ⁴ sporangium / ml. After diluting the zygote sac at 4 ° C for 4 hours to release the spermatozoa, irrigation was performed on the roots of the plants and examined for disease. The samples were treated three times at weekly intervals by dividing the sample into 1,000-fold and 500-fold volumes, and the untreated plots were treated with groundwater in the same amount as the sample treatment to investigate the final disease occurrence (Table 3).

Sample processing and survey date and time Exam schedule Processing date Remarks Seed sowing June 21, 2011 Sterilization Room Port port June 28, 2011 Clean room (flower pot) Bonfo transplant July 12, 2011 Mainpo Bottle inoculation Aug 09, 2011 Irrigation and foliage treatment Sample Primary Processing August 10, 2011 Foliage treatment Sample secondary processing August 17, 2011 Foliage treatment Sample Tertiary Processing August 24, 2011 Foliage treatment Final control investigation August 31, 2011 Incidence count investigation

7-4-3. Control and weakness investigation

The number of disease-causing disease was calculated from the total number of bottles, and the ratio was statistically processed.Then, the sample was divided into 24, 48, and 72 hours after adequate application of the sample processing volume and the volume of pepper. It was visually determined whether the weakness occurred. Statistical analysis was performed using Duncan's Multiple Range Test (DMRT) with a significance level of 95%.

As a result of analyzing the disease control effect against pepper blight, the disease control effect was about 64.09% in 500 times of the cultivation group and 63.02% in 1,000 times the standard amount (Table 4). The control value survey was conducted at the significance level of 95%, and it was confirmed that each treatment group was significant compared to the untreated group. In order to analyze whether or not crops were harmed to the sample, pepper was treated at 500 times the sample volume and 1,000 times the standard ratio, which did not cause any damage at all (Table 5).

Control against pepper blight exam
drugs Treatment drugs
Concentration (times) Disease Incidence (%) valence
(DMRT) The control
(%) 1 repetition 2 repetitions 3 repetitions Average underground water No treatment - 79.61 68.52 70.47 72.87 a - sample recommendation 1,000 23.62 28.29 28.93 26.95 b 63.02 Quantity 500 23.41 28.63 26.45 26.16 b 64.09

Weakness for peppers exam
drugs Test crop Weak degree (0 5) Remarks Non-treatment 1,000 times 500 times sample pepper 0 0 0 No weakness

Example  8: Toxicology Assessment

The MTT (3- (4,5-dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide, Sigma, USA) analysis is according to Mosmann (1983) method. MTT assay is a method of measuring cell survival by measuring the activity of mitochondrial succinate dehydrogenase in cell organelles. As a measure of the absorbance of soluble yellow MTT tetrazolium to soluble succinate dehydrogenase by insoluble purple MTT formazan, it is more sensitive than the pigment exclusion test by trypan blue. The advantage is that the survival rate can be measured more accurately. MTT is colorless in aqueous solution, but is converted into a purple pigment that is insoluble in water by succinic acid dehydrogenase, an enzyme in the mitochondria of living cells, so the concentration of the pigment is measured by an ELISA (Enzyme-linked immunosorbent assay) colorimetric system. The proliferation and toxicity of living cells can be measured. For cytotoxicity assay, cells were placed in D-MEM (GIBCO Co, USA) in 10% FBS (Fetal bovine serum, Sigma, USA), 25 units / ml penicillin G (Sigma, USA), 25 μg / ml streptomycin (Sigma). , USA), Fungizone (3 μl / ml) and 10 μl / ml antibiotics were incubated to divide 4 × 10 ³ cells / ml into 96-well polypropylene microtiter plates at 200 μl each to allow cells to per well. Each antimicrobial peptide is treated by concentration when it is about 70% full. After 24 hours, the cells were exchanged with a culture medium containing MTT for 3 hours, followed by dissolution of promazan (Sigma, USA) with DMSO (Dimetylsulfoxide, Sigma, USA) and absorbance at 570 nm with ELISA reader (Molecular Devices, USA). Was measured and compared with the control.

B. licheniformis Toxicity evaluation of NB109 culture filtrates was determined by identifying the extent of growth inhibition in the three cells. B. licheniformis IC ₅₀ of the NB109 culture filtrate was obtained by confirming cell viability against the concentration of the culture filtrate (FIG. 14). B. licheniformis NB109 culture filtrate showed high cytotoxicity in all breast cancer related cell lines (MDA-MB-361), immune cancer related cell line (Jurkat) and kidney cancer related cell line (K-562), and normal cell line (NIH). 3T3) showed no cytotoxicity. B. licheniformis NB109 culture filtrate was investigated to cause cytotoxicity only in cancer cell lines. These results suggest that antimicrobial peptides in B. licheniformis NB109 culture filtrate may act as anticancer agents. B. licheniformis In the cytotoxicity evaluation of NB109 culture filtrates, B. licheniformis was determined by Facscan method to determine the extent of cell growth inhibition, especially in renal cancer-related cell lines. Cell growth was inhibited more closely to the control (negative control) hydrogen dioxide (H ₂ O ₂ ) than to the normal control without NB109 culture filtrate (FIG. 15).

Example  9: Establish antimicrobial active substance high productivity mass production condition

B. licheniformis NB109 strain was found in the optimum medium to 50 L fermenter (COBIOTECH, Korea), the final volume was adjusted to 30 L, sterilized at 121 ℃, 20 minutes, and then connected to the air supply pipe and the outlet, cooling water and stirred While cooling down to the optimum culture temperature while inoculating the seed was searched for the optimum culture conditions. The effect of oxygen supply and the stirring speed is a temperature control problem according to the mass culture conditions. The culture temperature is 42-45 ° C., the oxygen supply is 0.5 and 1.0 vvm, and the stirring speed is 150, 200, 250, 300 rpm, respectively. The growth rate with supply was examined. The pH and DO changes according to the culture were observed without changing the pH based on the pH 7.0 of the initial medium to observe the pH change over time and the relationship between the growth curve and pH change. The relationship between growth and activity was investigated by investigating the activity of antimicrobial active substances produced over time according to the culture conditions.

9-1. Carbon source  effect

Industrial carbon source medium B. licheniformis The effect on the growth of NB109 was investigated and shown in FIG. 16. As in the experimental medium, the growth of bacteria was higher in order of glucose, fructose, sucrose, maltose, and the like. In particular, it was confirmed that liquid starch had a high influence on bacterial growth, while corn starch and flour were low. In particular, as a result of confirming the effect on the growth by the concentration of glucose as a carbon source, the range of 1.5-2.0%, incubation temperature 42 ℃, incubation time 24 hours was the most suitable.

9-2. Effect of Nitrogen Sources

Industrial Nitrogen Source Medium B. licheniformis The effects on the growth of NB109 were investigated and shown in FIGS. 17 and 18. As in the experimental medium, yeast extract and CSL (corn steep liquor) showed the highest bacterial growth, while molasses was found to have low effect on bacterial growth. In particular, as a result of confirming the effect on the growth by cultivating the yeast extract, a nitrogen source by concentration, 1.0 ~ 1.5% range, the culture temperature 42 ℃, the incubation time 24-36 hours appeared that the most suitable, whereas CSL (corn steep liquor) It was shown to affect growth in a concentration-dependent manner, but the growth period was somewhat low (Fig. 19).

9-3. pH , DO  And O.D change

30 L medium was prepared in 50 L fermenter (COBIOTECH, South Korea) by using industrial optimum medium (glucose, yeast extracts, inorganic salt) and oxygen supply (1.0 vvm), stirring speed (200 rpm) Incubation under the condition of was confirmed as the optimum conditions for growth. The change in pH, DO, OD during the culture is shown in FIG. 20.

9-4. Production of antimicrobial active substances by incubation time

Optimal medium composition for industry, 1.0 vvm, 0.5 kg / cm ² , in 50 L incubator B. licheniformis incubated at 42 ° C and 200 rpm for 24 hours NB109 was recovered and centrifuged at 8000 rpm for 10 minutes to obtain a supernatant filtered with 0.45 탆 filter paper. Phytopathogenic plants were soaked in 100 μl supernatant of 7 mm diameter disc paper with corker borer on V10 juice agar plate medium, and placed 1 cm away from one end of plate medium, and then incubated for 24-72 hours at 28 ° C. Mycelial growth inhibition (inhibition zone) of the strain was observed. As a result, it was confirmed that the supernatant of the strain recovered in the logarithmic growth period in which the number of cells increased rapidly (Fig. 21).

Example  10: Exploration and Development of Mutant Strains for Mass Production of Antimicrobial Active Substances

External factors that increase the rate of mutation are called mutagens. Mutants include ultraviolet (Ultraviolet) and ionizing radiation as physical factors, and chemical substances include base-like substances, alkylated substances, pigments and other substances. In this experiment, we investigated the antimicrobial activity of wild type B. licheniformis by the disk diffusion method by incubating the colonies surviving in the plate medium after irradiation with UV, which is a physical factor for 0, 10, 20, 30 seconds. The comparison was made with the NB109 strain.

B. licheniformis NB109 mutants were obtained by culturing ultraviolet light, which is a mutagen, to investigate antimicrobial activity by mass dilution. B. licheniformis NB109 Mutants No mutants exhibiting superior antimicrobial activity than wild-type B. licheniformis NB109 were found in 96 independent communities. Bacillus in general In the case of strain microorganisms, gram-positive bacteria have been reported to be very difficult to induce mutants caused by chemical or physical external factors.

Example  11: microorganism Formulation

11-1. microbe Liquid Formulation

B. licheniformis NB109 strains were cultured in an independent colony cultured in NA medium for 24 hours at 42 ° C. under 5 mL NB medium under the same conditions, and then transferred to 500 mL NB medium and cultured under the same conditions. 300 ml of cultured 500 ml cultures were transferred to 30 L optimal medium containing 2% liquid starch and 0.5% yeast extract, and 1.0-1.2 × 10 ⁷ cultured at 50 L incubator under the optimum culture conditions and the highest antibacterial activity. cfu / mL B. licheniformis NB109 strain 100% molasses [(Hydex Storage Co., Ltd., Gunsan, South Korea) and yeast extract as a food additive Yeastex LSP [Yeast Extract, Leiber GmbH, Germany, Import Source Co., Ltd. VISION BIOCHEM] was formulated by mixing in various composition ratios.

B. licheniformis NB109 strain culture medium (1.0-1.2 × 10 ⁷ cfu / mL) cultured under optimal conditions for the production of antimicrobial active substances. Formulated by formulating a yeast extract Yeastex LSP, a 4% molasses 1% food additive, as a product potentiator. In this case, it was confirmed that the antimicrobial activity was maintained for a long time (Table 6).

Microbial liquid composition costs Raw material Composition ratio Remarks Microbial Strain Culture 95.0% of weight ^- molasses 4.0% (by weight) Yeast extract 1.0% (by weight) <Komaichi (liquid)> 100.0% Microbial cell count
Bacillus licheniformis
1 × 10 ⁷ cfu / mL

Molasses Ingredients Ingredients content Total nitrogen
Total P ₂ O ₅
Full K ₂ O
Full Sugar (as invert)
Brix
Moisture 0.94%
0.25%
3.18%
50.5%
83.0 Degrees
24.7%

Yeast extract ingredients Ingredients content unit Dry matter (4h, 105 ℃)
pH (5% solution)
Protein
Bitterness / 100g
NaCl 95
6.0
68
3
0.3 %

% dm
BU / 100g
% dm

11-2. microbe Granulation Formulation

B. licheniformis NB109 strains were cultured in an independent colony cultured in NA medium for 24 hours at 42 ° C. under 5 mL NB medium under the same conditions, and then transferred to 500 mL NB medium and cultured under the same conditions. 300 ml of cultured 500 ml cultures were transferred to 30 L optimal medium containing 2% liquid starch and 0.5% yeast extract, and 1.0-1.2 × 10 ⁷ cultured at 50 L incubator under the optimum culture conditions and the highest antibacterial activity. Cfu / ml B. licheniformis NB109 strain cultures and products were formulated by applying microorganisms with humic acid (Humic Acid, Yantai Humus Agrotech, China) in various composition ratios.

B. licheniformis NB109 strains were cultured in an independent colony cultured in NA medium for 24 hours at 42 ° C. under 5 mL NB medium under the same conditions, and then transferred to 500 mL NB medium and cultured under the same conditions. 300 ml of cultured 500 ml culture solution was transferred to 30 L optimal medium containing 2% liquid starch and 0.5% yeast extract, and 1.0-1.2 × 10 ⁷ cultured at 50 L incubator under the optimum culture conditions and the highest antibacterial activity. cfu / ml B. licheniformis NB109 strain cultures and products were formulated by applying microorganisms in various composition ratios with humic acid (Humic Acid, Yantai Humus Agrotech, China) as a preparation carrier (Table 9).

Microbial granulation composition ratio Raw material Composition ratio Remarks Containing microbial strains
Liquid culture 30% (by weight) ^- Humic acid 70% of volume CAS No. 68131-04-4
EPAlist 4A <Komaichi (entrance)> 100% Microbial cell count
Bacillus licheniformis
1.0 × 10 ⁶ cfu / g

Example  12: Microbial product safety assessment

12-1. Acute Oral Toxicity Assessment

① test animal

The test animal species (systems) are SPF mice (ICR system) supplied by Hallym Laboratory Animal Research Institute Co., Ltd., and the mouse is used for the toxicity test standard and method of agricultural chemicals notification No. 2010-29 of the Rural Development Administration. It is widely used in toxicological tests, and since the basic data are sufficiently accumulated, it is easy to interpret and evaluate the experimental results. The age and body weight of the test animals were 4 weeks and 17.7-19.1 g at the time of purchase, and the age and weight at the time of administration were 5 weeks and 19.2-21.5 g. The age and body weight of the test animals were 4 weeks and 16.0-18.7 g at the time of purchase, and the age and weight at the time of administration were 5 weeks and 18.1-20.2 g. Healthy individuals were published in the test after observing general health while purifying in the environment of the animal laboratory for 2 days after purchasing the test animals.

② Breeding environment

The breeding environment for this test is fed under laboratory conditions of temperature 23 ± 2 ° C, relative humidity 50 ± 10 ° C, ventilation system (air conditioning), lighting time 12 hours (7 am-7 pm) and roughness 200-300 Lux. And drinking water were fed and quarantined during the purification and testing period. During the period of acclimation and testing, 5 animals were put in a polycarbonate breeding box (26 × 20 × 12 cm) and straw was raised. The feed was fed freely by UV sterilization of the experimental animal solid feed [EP Superfeed Co., Ltd.], and the water was fed ground water (water quality test: November 25, 2010) passing through the water filter.

③ Dose level setting and drug preparation

In the basic experiments, the doses of all male and female test animals were set to 5,000 mg / kg. The number of published animals per dose level was 10 males and 5 males and 5 females, respectively, and individual identification was indicated by pigments on the hair. The solvent control was not set because the test substance was well suspended in distilled water when the drug was administered in liquid form. Since the test material was well suspended in distilled water in the liquid phase, the choice of solvent was selected as distilled water as a solvent, and a predetermined amount of the preparation was prepared by suspending it in a 50 ml volumetric flask and adding distilled water. The amount of administration was set to 10 mg / kg body weight for each dose level.

④ Administration of test substance

The feed was fasted for 3-4 hours before the start of the test substance administration and for 3 hours after the test substance administration, and the prescribed amount of the test substance preparation was calculated based on the weight measurement using an oral injection needle for the mouse, and then the oral route of administration. Only once in the stomach was forced to administer.

⑤ Observation and Inspection Items

On the day of dosing, the general poisoning symptoms and the number of killed animals were observed for 14 days, every hour from 1 hour to 4 hours after administration and once daily from the following day. All animals tested were body weighted on the day of dosing and on days 1, 7 and 14 post dose. At the end of the observation period, anesthesia was performed with ether to observe the presence of gross abnormalities of internal organs. The LD ₅₀ calculation was terminated in the baseline test and no statistical treatment was performed.

⑥ Evaluation result

As a result of the basic experiment, the dose was 5,000 mg / kg, and there were no lethal animals, and the LD ₅₀ value of acute oral toxicity was 5,000 mg / kg in both sexes and classified as Class IV (low toxicity) according to the pesticide control method. Table 10). No symptoms of general intoxication were observed after oral administration in this study (Table 11). Changes in body weight decreased until day 1 in both males and females, depending on the number of days after drug administration, but recovered from day 3 and increased (Table 12). The autopsy was performed by anesthesia with ether at the end of the study, followed by visual observation of each major organ, and no specific symptoms were observed by the administration of the drug (Table 13).

Investigate daily number of lethal animals after test substance treatment gender Dosage amount (mg / kg) Number of lethal animals after administration minute time Days Sum 10 30 One 2 3 4 One 2 3 4 5 6 7 ~ 14 Male cut 5,000 0 0 0 0 0 0 0 0 0 0 0 0 0 ~ 0 0/5 female 5,000 0 0 0 0 0 0 0 0 0 0 0 0 0 ~ 0 0/5

Clinical observation after treatment with test substance gender Dosage amount (mg / kg) Symptoms of poisoning after administration minute time Days 10 30 One 2 3 4 One 2 3 4 5 6 7 ~ 14 Male cut 5,000 - ^* - - - - - - - - - - - - ~ - female 5,000 - - - - - - - - - - - - - ~ -

- ^* : Normal

Weight change measurement result after test substance treatment gender Dosage amount (mg / kg) Days Since Dose 0 One 3 7 14 cock 5,000 20.3 ± 0.9 (5) ^* 19.7 ± 0.9 (5) 21.4 ± 0.7 (5) 24.4 ± 0.8 (5) 28.3 ± 0.8 (5) female 5,000 19.2 ± 0.9 (5) 18.6 ± 0.9 (5) 20.0 ± 0.7 (5) 22.3 ± 1.1 (5) 25.9 ± 1.3 (5)

() ^* : Horses, (unit: g)

Autopsy findings after test gender Dosage amount (mg / kg) Major organ name Gross findings Male cut 5,000 kidney N ^* Rain cabinet N Soy sauce N Heart N Digestive system N Lung intestine N Other organs N female 5,000 kidney N Rain cabinet N Soy sauce N Heart N Digestive system N Lung intestine N Other organs N

N ^* : normal

12-2. Acute Transdermal  Toxicity Assessment

① test animal

The test animal species (systems) are SPF rats (SD system) supplied by Hallym Laboratory Animal Research Institute, and the rats are to be used in the standard and method for testing toxicity of pesticides published by Rural Development Administration Notification No. 2010-29. It is widely used in toxicological tests, and since the basic data are sufficiently accumulated, it is easy to interpret and evaluate the experimental results. The age and body weight of the test animals were 240-260 g at 7 weeks of purchase, and 248-270 g at 8 weeks of body weight. The age and body weight of the test female were 7 weeks and 160-180 g at the time of purchase, and the age and weight at the time of administration were 8 weeks and 170-190 g. After purchasing test animals, apparently healthy animals were selected and randomized for 3 days in the environment of the animal laboratory and published in the test.

② Breeding environment

The breeding environment for this test is fed under laboratory conditions of temperature 23 ± 2 ° C, relative humidity 50 ± 10 ° C, ventilation system (air conditioning), lighting time 12 hours (7 am-7 pm) and roughness 200-300 Lux. And drinking water were fed and quarantined during the purification and testing period. During the purifying and testing period, 5 dogs were placed in a polycarbonate breeding box (26 × 20 × 12 cm) and placed on a pulp dryer. The feed was fed freely by UV sterilization of the experimental animal solid feed [EP Superfeed Co., Ltd.], and the water was fed ground water (water quality test: November 25, 2010) passing through the water filter.

③ Establishment of dosage level and preparation of drug

In the basic experiments, the doses of all male and female test animals were set to 4,000 mg / kg. The number of published animals per dose level was 10 males and 5 males and 5 females, respectively, and individual identification was indicated by pigments on the hair. The solvent control was not set because the test substance was well suspended in distilled water when the drug was administered in liquid form. Since the test material was well suspended in distilled water in the liquid phase, the choice of solvent was selected as distilled water as a solvent, and a predetermined amount of the preparation was prepared by suspending it in a 50 ml volumetric flask and adding distilled water. The amount of administration was set to 5.0 mg / kg body weight for each dose level.

④ treatment of test substance

Before the test material is treated, the epigastric abdomen is epilated to the chest abdomen using a hair removal device at a size of 5 × 6 cm or more, and a predetermined amount of the test substance prepared in a gauze having a size of 4 × 5 cm is calculated based on the weight measurement. After it was evenly applied, it was applied to the depilated area and then fixed with a medical band-aid.

⑤ Observation and Inspection Items

On the day of treatment, the general poisoning symptoms and the number of animals killed were observed for 14 days for 10 minutes, 30 minutes, 4 hours after 1-4 hours, and once every day after the next day. All animals tested were body weighted on the day of treatment and on days 1, 3, 7 and 14 post treatment. At the end of the observation period, anesthesia was performed with ether to observe the presence of gross abnormalities of internal organs. The LD ₅₀ calculation was terminated in the baseline test and no statistical treatment was performed.

⑥ Evaluation result

As a result of the basic experiment, the treatment dose was 4,000 mg / kg, and there were no lethal animals, and the LD ₅₀ value of acute dermal toxicity was more than 4,000 mg / kg in both sexes and classified as class IV (low toxicity) according to the pesticide control method. Table 14). In this study, no general poisoning symptoms were observed after transdermal treatment (Table 15). Changes in body weight decreased until day 1 in both males and females, depending on the number of days after drug administration (Table 16). Autopsy was performed with ether anesthesia at the end of the study, followed by macroscopic observation of each major organ, with no specific symptoms observed by drug administration (Table 17).

Investigate daily number of lethal animals after test substance treatment gender Dosage amount (mg / kg) Number of lethal animals after administration minute time Days Sum 10 30 One 2 3 4 One 2 3 4 5 6 7 ~ 14 Male cut 4,000 0 0 0 0 0 0 0 0 0 0 0 0 0 ~ 0 0/5 female 4,000 0 0 0 0 0 0 0 0 0 0 0 0 0 ~ 0 0/5

Clinical observation after treatment with test substance gender Dosage amount (mg / kg) Symptoms of poisoning after administration minute time Days 10 30 One 2 3 4 One 2 3 4 5 6 7 ~ 14 Male cut 4,000 - ^* - - - - - - - - - - - - ~ - female 4,000 - - - - - - - - - - - - - ~ -

- ^* : Normal

Weight change measurement result after test substance treatment gender Dosage amount (mg / kg) Days Since Dose 0 One 3 7 14 cock 5,000 258.7 ± 9.0
(5) ^* 251.8 ± 9.6
(5) 266.8 ± 9.1
(5) 287.0 ± 8.9
(5) 317.8 ± 8.7
(5) female 5,000 183.1 ± 6.2
(5) 176.5 ± 6.5
(5) 192.9 ± 5.4
(5) 210.6 ± 5.4
(5) 228.8 ± 5.0
(5)

() ^* : Horses, (unit: g)

Autopsy findings after test gender Dosage amount (mg / kg) Major organ name Gross findings Male cut 4,000 kidney N ^* Rain cabinet N Soy sauce N Heart N Digestive system N Lung intestine N Other organs N female 4,000 kidney N Rain cabinet N Soy sauce N Heart N Digestive system N Lung intestine N Other organs N

N ^* : normal

12-3. Acute Fish toxicity  evaluation

① test animal

The test animal species were lined with carp, Cyprinus carpio ) is to use carp in the standard and method of pesticide toxicity test No. 2010-29, and it is widely used in the toxicity test of pesticide, so it is easy to interpret and evaluate the test result. Because.

② Breeding environment

The breeding environment of this test was purified for 2 months under laboratory conditions of temperature 22-24 ° C and light 200-300 Lux. Cylindrical glassware 12.5 ℓ (25.5 × 25.5 cm) containers were used during the acclimation and testing periods. The feed was prepared from solid foods for experimental animals (Bio Meal, Cheil Feed Co., Ltd.). Salary was paid twice. The test water was ground water (water quality test: November 25, 2010) that passed through the water filter.

③ Establishment of dosage level and preparation of drug

10,000 mg / L stock solution was prepared by accurately weighing 1.05 g of the test substance into a 100 ml volumetric flask and adding test water to 100 ml, then suspending until completely suspended. The prepared 10,000 mg / L stock solution was placed in a 1,000 ml beaker, and 900 ml of test water was added thereto to suspend sufficiently to prepare a test substance solution (1,000 mg / L stock solution). 100 ml of the test substance solution (1,000 mg / l stock solution) was accurately taken and placed in a 12.5 L test bath with 9,900 ml of test water, followed by sufficient stirring until complete suspension before use as the test solution.

④ treatment of test substance

After the preliminary test, two tests were conducted at a single concentration of 10 mg / L. Feeding was stopped and fasted 24 hours before treatment of the test substance to the end of the test and 10 fish (concentration) were used. A negative control test was performed with the groundwater, which is test water, and PCP-Na salt (90%) was tested for acute fish toxicity to carp. The test was terminated in the basic test (10 mg / L) with LC ₅₀ The output was not statistically processed.

⑤ Observation and Inspection Items

General poisoning symptoms and lethality were recorded 1 hour after the start of the test and at 24 hour intervals. The determination of the lethal was fatal if there was no movement or gill breathing was stopped when the rat was touched with a glass foil. Water quality measurement was investigated by measuring the DO, pH, and water temperature by Orion 1 star model before the start of the test and at the end of the test at 24 hour intervals.

⑥ Evaluation result

Carp test drug (Cyprinus Acute fish toxicity test for carpio ) was performed for 96 hours to observe the number of live death and general poisoning symptoms, and to investigate the body weight and overall length. General poisoning symptoms and other specific symptoms were not observed (Table 18). The average body weight was 1.91 ± 0.09 g and the overall length was 4.20 ± 0.12 cm (Table 19). The water pH of the test tank averaged 7.52 (lowest 7.31-highest 7.78) and this averaged 6.90 mg / l (lowest 5.3-highest 9.1). The average water temperature for the test period was 22.7 ° C. (lowest 22.4-highest 23.1) (Table 20). The 48 and 96 hour half lethal concentrations (LC ₅₀ ) for carp of this test substance were all 10 mg / l or more and were classified as Class III by toxicity according to the pesticide control method.

Number of fatalities by elapsed time after test substance treatment Concentration (mg / l) Carp water (mari) Cumulative dimension (mari) Lethality (%) 24h 48h 72h 96h 48h 96h 10.0 10 0 0 0 0 0 0

LC ₅₀ and intoxication symptoms of the test substance division LC ₅₀ (Mg / l) Poisoning symptoms Weight (g) Full length (cm) 48h 96h Test substance > 10 > 10 N 1.91 ± 0.09 ^*** 4.20 ± 0.12 PCP-Na salt ^* 0.116
(0.103-0.129) ^** 0.108
(0.096-0.119) SUR 1.70 ± 0.13 4.08 ± 0.60 Negative control group - - N 1.90 ± 0.10 4.18 ± 0.09

※ Symptoms of poisoning: N (normal), SUR (sleep injury)

* Positive control trial period: 4 days

** 95% confidence limit

*** Mean ± Standard Deviation

Water quality test over time division Immediately after treatment of the test substance
/ Before inputting test organism Test substance treatment
After 48 hours 96 hours after treatment of test substance Test substance 8.1 7.1 5.3 DO PCP-Na salt ^* 8.2 6.5 4.6 Negative control group 8.0 6.6 5.1 Test substance 7.78 7.46 7.31 pH PCP-Na salt ^* 7.55 7.16 7.22 Negative control group 7.71 7.52 7.21 Water temperature 23.1 22.4 22.4

12-4. Pathogenic microbial testing

① Escherichia coli test

E. coli (Escherichia coli) the E. coli only agar medium to determine (Merck, USA) were inoculated to the test substance was determined as a positive single colonies stand the single colonies formed on the plate medium cyan or red. A single colony with cyan or red color was not detected and was determined to be pathogenic E. coli negative (FIG. 22).

② Pathogenic Salmonella Test

Pathogenic Salmonella sp . After inoculation of the test substance to SS agar (Difco, USA), which is a dedicated medium for determining), a single colony formed on a flat medium was determined to be positive. No dark brown single colonies were detected, resulting in pathogenic Salmonella sp . ) Negative result (FIG. 22).

③ Staphylococcus aureus inspection

Staphylococcus After inoculation of the test substance to the mannitol salt agar (Difco, USA), which is a medium for determining aureus , a single colony formed on a flat medium (red) was determined as positive in a single colony in which the surrounding medium color turned yellow. A single colony formed on a plate medium (red) was detected and a single colony in which the surrounding medium color turned yellow was detected. Confirmation experiment by 16S rRNA gene analysis was confirmed by Bacillus. 99.56% homology with licheniformis . The single colony formed on the plate medium is Bacillus Staphylococcus as the medium color changed to yellow due to a single colony of licheniformis It was judged as aureus negative (FIG. 22).

④ Bacillus cereus inspection

Bacillus After inoculation of the test substance to MYP agar (Difco, USA), a medium for determining cereus , a single colony formed on a flat medium was determined to be positive. Single pink colonies formed on plate medium were detected and confirmed by Bacillus 16S rRNA gene analysis. 99.56% homology with licheniformis . The pale pink single colony formed on the plate medium is Bacillus Since it is licheniformis, it was determined as Bacillus cereus negative (FIG. 22).

⑤ Listeria monocytogenes test

Listeria After inoculating the test substance into Listeria thickening agar (Difco, USA), a medium for determining monocytogenes , a single colony formed on a flat medium was determined to have a positive cyan colony. A single colony with a turquoise color was not detected and was judged to be Listeria monocytogenes negative (FIG. 22).

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that such a specific technology is only a preferred embodiment, and the scope of the present invention is not limited thereto. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Korea Gene Bank KCTC12256BP 20120802

<110> Nambo <120> Antimicrobial Composition Comprising Bacillus licheniformis NB109          As an Active Ingredient <130> PN120483 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 1384 <212> RNA <213> Bacillus licheniformis NB109 16S rRNA sequence <400> 1 cgacgggagc ttgctccctt aggttagcgg cggacgggtg agtaacacgt gggtaacctg 60 cctgtaagac tgggataact ccgggaaacc ggggctaata ccggatgctt gattgaaccg 120 catggttcaa ttataaaagg tggcttttag ctaccactta cagatggacc cgcggcgcat 180 tagctagttg gtgaggtaac ggctcaccaa ggcgacgatg cgtagccgac ctgagagggt 240 gatcggccac actgggactg agacacggcc cagactccta cgggaggcag cagtagggaa 300 tcttccgcaa tggacgaaag tctgacggag caacgccgcg tgagtgatga aggttttvgg 360 atcgtaaaac tctgttgtta gggaagaaca agtaccgttc gaacagggcg gtgccttgac 420 ggtacctaac cagaaagcca cggctaacta cgtgccagca gccgcggtaa tacgtaggtg 480 gcaagcgttg tccggaatta ttgggcgtaa agcgcgcgca ggcggtttct taagtctgat 540 gtgaaagccc ccggctcaac cggggagggt cattggaaac tggggaactt gagtgcagaa 600 gaggagagtg gaattccacg tgtagcggtg aaatgcgtag agatgtggag gaacaccagt 660 ggcgaaggcg actctctggt ctgtaactga cgctgaggcg cgaaagcgtg gggagcgaac 720 aggattagat accctggtag tccacggccg taaacgatga gtgctaagtg ttagagggtt 780 tccgcccttt agtgctgcag caaacgcatt aagcactccg cctggggagt acggtcgcaa 840 gactgaaact caaaggaatt gacgggggcc cgcacaagcg gtggagcatg tggtttaatt 900 cgaagcaacg cgaagaacct taccaggtct tgacatcctc tgacacccct agagataggg 960 cttccccttc gggggcagag tgacaggtgg tgcatggttg tcgtcagctc gtgtcgtgag 1020 atgttgggtt aagtcccgca acgagcgcaa cccttgatct tagttgccag cattcagttg 1080 ggcactctaa ggtgactgcc ggtgacaaac cggaggaagg tggggatgac gtcaaatcat 1140 catgcccctt atgacctggg ctacacacgt gctacaatgg gcagaacaaa gggcagcgaa 1200 gccgcgaggc taagccaatc ccacaaatct gctctcagtt cggatcgcag tctgcaactc 1260 gactgcgtga agctggaatc gctagtaatc gcggatcagc atgccgcggt gaatacgttc 1320 ccgggccttg tacacaccgc ccgtcacacc acgagagttt gtaacacccg aagtcggtga 1380 ggta 1384

Claims (4)

Bacillus licheniformis NB109 (KCTC 12256BP) strain having cytotoxicity against cancer cells.
The strain of claim 1, wherein the strain has a 16S rRNA sequence of SEQ ID NO: 1.
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