KR100868385B1 - Novel strain Bacillus vallismortis TB40-3 producing biosurfactant with oil degrading activity and antifungal substance - Google Patents

Abstract

The present invention relates to a novel Bacillus vallismortis TB40-3, which produces a biodegradable surfactant and an antifungal substance having excellent oil resolution, and has excellent crude oil degradability and biodegradable surfactant generation ability separated from an oil pollution area. In addition, there is an excellent effect of providing a novel Bacillus vallismortis TB40-3 strain exhibiting high antibacterial activity against phytopathogens. In addition, the present invention has a surface tension of as low as 29 mN / m, a CMC value of 0.0030% (w / v), the emulsification activity in soybean oil, the emulsification activity in most substrates, including crude oil, There is an excellent effect of providing a biodegradable surfactant separated and purified from Bacillus vallismortis TB40-3 strain having better emulsion stability than a synthetic surfactant. In addition, the present invention is strongly derived from Bacillus vallismortis TB40-3 strain that strongly inhibits the growth of B. cineria that causes leek fungal disease of leek and promotes the growth of leek It has an excellent effect on providing antifungal substances.

Bacillus valismoltis, biodegradable surfactants, oil resolution, antifungal substances

Description

Novel strain Bacillus vallismortis TB40-3 producing biosurfactant with oil degrading activity and antifungal substance}

1 is a scanning electron micrograph of the Bacillus vallismortis TB40-3 of the present invention.

Figure 2 shows the antimicrobial activity of the Bacillus vallismortis TB40-3 against the Botrytis Ceria (A) and Fusarium oxysporum (B) of the present invention will be.

Figure 3 shows the phylogenetic tree according to 16S rDNA analysis of the Bacillus vallismortis TB40-3 of the present invention.

Figure 4 is a graph showing the change in surface tension of the biodegradable surfactant produced over time in the Bacillus vallismortis TB40-3 of the present invention.

5 is a graph showing the emulsification activity of various substrates by the present invention biodegradable surfactant solution.

Figure 6 is a graph showing the surface tension of the surfactant separated and purified from Bacillus vallismortis TB40-3 of the present invention.

Figure 7 shows the stability of the emulsification activity by the present invention biodegradable surfactant solution.

8 is a graph comparing the emulsion stability of the present invention biodegradable surfactant and chemical synthetic surfactant.

Figure 9 is a photograph showing the antagonistic effect of phytopathogenic bacteria of the present invention Bacillus vallismortis TB40-3 in its natural state.

Figure 10 shows the activity of the enzymes involved in cell wall degradation of phytopathogenic fungi produced by Bacillus vallismortis TB40-3 of the present invention.

Figure 11 shows a partial sequence of 16S rDNA of Bacillus vallismortis TB40-3 of the present invention.

The present invention relates to a novel Bacillus vallismortis TB40-3 that produces an oil-degradable biodegradable surfactant and an antifungal substance. More specifically, the present invention relates to a novel Bacillus valismoltis TB40-3 strain that produces an antifungal substance that inhibits the growth of phytopathogens as well as a biodegradable surfactant having excellent oil resolution.

Biodegradable surfactants (Biosurfactant) is produced by microorganisms such as yeast, mold, bacteria, etc., depending on the strain is produced extracellularly or intracellularly. The outstanding advantages of biodegradable surfactants compared to chemically synthesized surfactants are that they are non-toxic, easy to biodegrade and therefore not secondary pollution when used and can be used for special purposes due to the complex chemical structure that is difficult to synthesize by conventional methods. In terms of the physical and chemical performance of surfactants such as surface tension lowering ability, stability to temperature and pH, the results are almost the same as those of conventional chemical synthetic surfactants (MacFaddin, JF 1984. Biochemical Tests for Identification for Medical Bacteria, 2nd ed. Williams and Wilkins Co., Baltimore, USA.). Globally, the surfactant market reached $ 2 billion in 1988 and about $ 9.4 billion in 1994, growing more than 400% in just six years (Shaw, A. 1994. Soap Cosmet.Chem). Specialities 70, 24-30). However, most of them are chemically synthesized surfactants that are not biodegradable by themselves and cause environmental pollution. Therefore, in order to solve these problems, non-toxic, biodegradable, eco-friendly biodegradable surfactants can replace environmentally friendly surfactants, thereby reducing environmental pollution. Furthermore, biodegradable surfactants are used in cosmetics, medicine, food, detergents It can be widely applied in various fields such as pulp and paper, secondary recovery of crude oil and environmental purification. Recently, sulfur and the like have been used for the industrial use of biodegradable surfactants by performing the measurement, isolation and structural analysis of the biodegradable surfactants produced from P. aeruginosa and Nocardia sp . Basic research is being carried out for Hwang, K. A et al. 1999. Kor. J. Appl. Microbiol. Biotechnol . 27, 159-165; Kim, SH et al. 2000. Biotech. Appl. Biochem . , 249-253; Suk, WS et al. 1999. J. Microbiol. Biotechnol. 9, 56-61.).

Currently, biodegradable surfactants are used in various industries, for example, cyclo-alkane-based hydrocarbon sources and crude oils, which are biological treatments for the control of soil and marine oil pollution. ( Cim , HJ et al. 1999. J. Biotechnol. Bioeng . Isolates and identifies strains that degrade substrates of various hydrocarbon sources for crude oil, petroleum products such as diesel oil and heavy oil.) 14, 192-197; Kim, HJ et al. 1997. Korean J. Biotechnol.Bioeng. 24, 443-448; Mibas, W. and DL Gutnick. 1993. Appl.Environ Microbiol. 59, 2807-2816; Suk, WS et al. 1999. J. Microbiol. Biotechnol. 9, 56-61). As a marine microorganism that these oils resolution erotic Monastir (Aeromonas), are Tropez bakteo (Arthrobacter), Corolle Los Fora (Corollospora), Den'll Pilar (Dendryphiella), and the like Pseudomonas (Pseudomonas), actually an emulsifier to produce these strains (Kim, HJ et al. 1997. Korean J. Biotechnol. Bioeng . 24, 443-448; Rheinheimer, G. 1981. Microbiologic der gewalsser. 3rd. Ed. P. 251. gustav Fischer Verlag Stuttgart; Suk, WS et al. 1999. J. Microbiol. Biotechnol. 9 , 56-61).

Next, many studies have been conducted to control various pests affecting the yield and quality of crops, and various kinds of pesticides have been developed and used. In particular, in order to control plant pathogenic fungi, chemical synthetic pesticides are mainly used, or biological control methods using antagonistic microorganisms having antagonistic action against specific pathogenic fungi are used. The use of chemical pesticides has resulted in an innovative increase in crop yields, but has caused a number of problems. In comparison, biological control using microorganisms is less harmful to livestock, does not cause damage to crops, and has a low load on the environment and high stability. In addition, it can be applied to heating pathogens, which are difficult to control with chemical pesticides, are less likely to develop resistance, are selective to harmful organisms, do not disturb the ecosystem greatly, and have little adverse effects on natural and useful organisms. These advantages have led to greater interest in antifungal substances produced by microorganisms, and many studies have been made and commercialized and sold (Attafuah, A., and JF Bradbury. 1989 J. Appl. Bacteriol. 67, 567-573; Tanaka , YT, and Omura S. 1993 Annu. Rev. Microbiol . 47 , 57-48; Fruh, T., P. Chemla, J. Ehrler, and Farooq. 1996. Pestic. Sci . 46, 307-316; Knight, SC et al. 1997. Annu. Rev. Phytophathol . 35, 349-372). However, biological control methods using microorganisms also have some problems. When the microbial agent is used, it is slow-acting and the effect is slow, and when the use time is narrow and the time is missed, the effect is difficult to appear, the mass production and preservation are difficult, and the use scale is limited. Moreover, they are susceptible to chemical pesticides. In order to solve these problems, continuous research must be continued, and many microorganisms or materials produced by the microorganisms that can satisfy these conditions will have to be found.

Bacteria creating a lot of cellulolytic enzymes include Bacillus (Bacillus), A claw host Lee Stadium (Clostridium) in Pseudomonas (Psedomonas), A Cellulofine Mona three (Cellulomonase) in such is known and (Fukumori, F., N. et al. 1986. J. Bacteiol . 168, 479-485; Conet, P. et al. 1983. FEMS Microbiol. Leett. 16, 137-141; Hall, J. and HJ Gilbert. 1988. Mol. Gen. Genet . 213, 112-117;.. Wong , WKR et al 1986. Gene 44, 315-324), and they continued the study of enzyme that produces, among these, Bacillus (Bacillus) in strains inhibit the growth of pathogenic fungi Not only does it have the effect of promoting plant growth. Inhibiting the growth of phytopathogenic fungi is synergistic due to the combination of antifungal substances and cell wall degrading enzymes secreted by Bacillus sp. Conventional research has been focused on the growth inhibition of phytopathogenic fungi through antifungal substances. Now, however, both enzymatic and non-enzymatic parts should be studied simultaneously for more effective phytopathogenic growth inhibition.

Accordingly, an object of the present invention is to develop bioemulsifiers for various uses, such as environmentally friendly control of oil-contaminated soils and biologics for environmentally friendly control of phytopathogens. It is intended to provide new marine strains that produce substances.

Another object of the present invention is to provide a biodegradable surfactant and an antifungal substance having excellent oil degradability from the strain.

The object of the present invention is to isolate and identify the strains excellent in the production of biodegradable surfactants and antifungal substances in the soil of the mountainous region and oil pollution area at the same time, to identify the location of taxonomics, the characteristics of oil degradation, cell growth and The characteristics of the resulting emulsifier, the growth inhibition against phytopathogens and the inhibition of the growth of pathogens in plants and the effect of promoting plant growth were investigated, and the activity of enzymes related to cell wall degradation of phytopathogenic fungi was achieved.

The present invention provides a biodegradable surfactant and an antifungal substance in the soil of the mountainous region and the oil pollution area at the same time, the separation and identification of the excellent strain; Investigating the characteristics of oil decomposition, cell growth, and the resultant emulsifier of the surfactant separated and purified from the strain; And inhibiting the growth of phytopathogens of the strain culture, inhibiting the growth of pathogens and promoting plant growth in plants, and investigating the activity of enzymes related to cell wall degradation of phytopathogenic fungi.

In order to isolate the biodegradable surfactant-producing strain of the present invention, C-medium (Carbon-minimal medium) lacking a carbon source in the medium was used, and the composition of the medium was (NH ₄ ) ₂ SO ₄ 5g / L, K ₂ HPO _{4 2g / L, MgSO 4 · 7H} 2 O 0.2g / L, KH 2 PO 4 1g / L, CaCl 2 10mg / L, FeSO 4 · 7H 2 O 10mg / L, NaCl 30g / L, yeast extract (yeast extract) 0.2 g / L and 2 mL of trace element solution (MoO ₃ 1 mg / L, ZnSO ₄ · 7H ₂ O 7 mg / L, CuSO ₄ · 5H ₂ O 0.5 mg / L, H ₃ BO ₃ 1mg / L, CoCl ₂ · 6H ₂ O 6 mg / L, NiSO ₄ .6H ₂ O 1 mg / L), and used to adjust to pH 7.0.

In order to identify the strains of the present invention, the morphological, physiological and biochemical characteristics were examined and then primarily identified on the basis of Bergey's manual of systematic bacteriology, and based on the analysis of 16S rDNA for more reliable identification. As a result, secondary identification was carried out through a flexible relationship with other species.

Hereinafter, the specific method of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited only to these Examples.

Example 1 Isolation and Identification of Strains Producing Biodegradable Surfactants and Antifungal Agents with Excellent Oil Degradability

In order to isolate the strain producing the biodegradable surfactant and antifungal substances with excellent oil resolution, the soil of the mountainous region and the contaminated area is collected and the crude oil and edible oil as a carbon source in 200 mL of C-medium lacking a carbon source. % Was added and shaken for 7 days at 37 ° C. and 200 rpm, followed by growth of bacteria having crude oil resolution and emulsifier production capacity. The strains grown three times in this way were plated on LB-solid medium and incubated at 37 ° C. The single strains selected were pre-cultured in LB liquid medium, and then cultured in a medium containing tributyrin, and a single colony was selected because it was an excellent strain. These single colonies were inoculated in LB liquid medium and pre-incubated at 37 ° C, and several strains with crude and tributyrin degradability were isolated. Among them, strains with broad antifungal spectrum against various phytopathogens (TBM 40 -3) (see FIG. 2: A: B. cineria ; B: F. oxysporum ).

Antifungal activity tests against phytopathogens were carried out using a solid medium diffusion method and a paper disc, in which phytopathogenic fungi Botrytis cineraria ( B. cineria ) and Fusarium oxysporum ( P. oxysporum ) were added to TB40-3, respectively. Inhibition zones were identified by replacement culture in a solid medium of potato dextrose agar, and PDA was prepared by sterilizing 39 g of potato dextrose agar in 1 L of sterile water and sterilizing at 121 for 15 minutes using an autoclave. Then cooled to moderately poured into plates (plates) and used to cool.

In order to identify the selected TBM 40-3 strains, Gram staining, morphological observations and biochemical experiments were performed and the results are shown in Table 1, and the exact form of the strains was observed in a scanning electron microscope as shown in FIG. . In Figure 1, the strain was confirmed by typical Bacillus (Bacillus) in the form of rods. Gram staining was identified as Gram-positive bacteria, and microscopic observation identified it as a strain with active motility. Biochemical experiments were identified according to Bergey's manual of systematic bacteriology using the API 20 NE Kit, a microorganism identification kit. As a result, it was found to be Bacillus sp. The sequence was confirmed. As a result, it showed 99% homology with Bacillus vallismortis . Therefore, the selected TBM 40-3 strain was named Bacillus vallismortis TB40-3, and deposited with the accession number KACC 91101 dated April 29, 2004 to the Center for Agricultural Microorganisms.

In addition, the relationship with other species is shown in FIG. In Korea and foreign countries, strains reported as oil degradation and emulsifier-producing bacteria include Pseudomonas sp., Aeromonas sp., Acinetobacter sp., Klebsiella sp., Bacillus sp. And Arthrobacter sp., But Bacillus vallismortis strains have not been reported.

Morphological and Physiological Characteristics of Crude Bacillus Ballismoltis TB40-3 Characteristic Bacillus vallismortis TB40-3 Morphological features Bacillus Gram Dyeing + motility + Optimum temperature 37 ℃ Aerobic + Physiological features Ortho-nitrophenyl ??-D-galactopyranoside + Arginine dehydrolase - Lysine Dicarboxylase - Ornithine dicarboxylase - Simmons citrate - H ₂ S generating ability - Urease - Tryptophan deaminase - Indole - Gelatin hydrolysis + Glucose + maltose + Lactose + Ribose + Glycerol + Erythritol - Mannitol + Inositol + Sorbitol + Xylitol - Cellobiose + Rhamnose - Saccharose + Saccharose + Melibiose + Amygdalin + L (T) arabinose + 2-ketogluconate - 5-ketogluconate -

+: Positive reaction,-: negative reaction

Experimental Example 1: Growth characteristics of the strain of the present invention

In order to investigate the growth temperature of the novel Bacillus vallismortis TB40-3 strain isolated from above, 200 mL of C-medium containing 1% of oil (crude oil) was added to a 500 mL Erlenmeyer flask, and LB 1% of the precultured strain in liquid medium was inoculated and shaken at 200 rpm at 25 ° C, 32 ° C, 37 ° C and 42 ° C, respectively. At this time, the growth of the cells according to the incubation time was determined by taking a certain amount of the culture medium shaken on 1, 3, 5 days and measured the absorbance (optimal density) at 600nm using a UV-VIS spectrophotometer (U-1100, Hitachi, Japan). .

As a result, at 27 ℃, 32 ℃ and 37 ℃ showed a similar state of vigorous growth. In addition, the growth was relatively good in the carbon source and salt concentration of 3% medium of various oils, showed a maximum growth in the culture of about 3 days (not shown).

Example 2: Extraction and Characterization of Biodegradable Surfactants from the Strains of the Invention

In order to extract biodegradable surfactant from the novel strain isolated in Example 1, a method such as sulfur was used (Hwang, K. A et al. 1999. Kor. J. Appl. Microbiol. Biotechnol . 27, 159- 165). First, a solution in which the Bacillus vallismortis TB40-3 strain was pre-cultured was reinoculated into a 2 L culture medium and shaken at 37 ° C. for 4 days. The culture solution was centrifuged (10,000 rpm, 4 ° C.) and the pH was 2.0 while slowly adding concentrated hydrochloric acid to the supernatant from which the cells were removed, and the biodegradable surfactant was allowed to stand at 4 ° C. overnight. The precipitate was recovered by centrifugation, dissolved in alkaline aqueous solution (pH 8.0 with NaOH) and lyophilized. The dried material was extracted with methanol and concentrated to serve as partially purified biodegradable surfactant.

Experimental Example 1: Emulsifying power of the present invention biodegradable surfactant

Surface tension was measured over time to determine the properties of the biodegradable surfactants isolated and purified from Bacillus vallismortis TB40-3 strain. The surface tension was measured by taking a certain amount of the solution incubated every 6 hours for 90 hours and measuring the absorbance at 600 nm using a UV-VIS spectrophotometer (U-1100, Hitachi, Japan). The average value of the culture supernatant from which cells were removed by centrifugation was repeated three times using the ring method of DeNouy Tensiometer (Itoh Seisakusho, Japan).

As shown in FIG. 4, the surface tension of the medium rapidly decreased from 12 hours after incubation, and after 36 hours, the surface tension of the medium decreased from 57 mN / m up to about 29 mN / m, and this phenomenon continued until 72 hours after the growth was stopped. It became. These results were somewhat different from those produced by the marine oil-degrading bacteria Pseudomonas sp., Which were most produced early in the stationary phase, two days after culture (Mulligan, CN, G. Mahmourides, and BF). Giggs. 1989. J. Biotech. 12, 199-210).

Therefore, emulsifying activity was measured using various hydrophobic hydrocarbons and oils as substrates of biodegradable surfactants produced in the solution cultured for 36 hours with the maximum surface tension reduction. The emulsifying activity was expressed as the measured absorbance at A _{540 nm} after 10 minutes of vigorous mixing of biodegradable surfactant and substrate.

As shown in Figure 5, the biodegradable surfactant produced by the strain of the present invention showed a significant emulsification in crude oil, soybean oil showed the highest activity as about 2.6 units, crude oil, tributyrin (tributyrin) and The hydrophobic hydrocarbons decane, tetratradecane and hexadecane also showed relatively high activity. On the other hand, kerosene showed relatively low activity. However, it was found that it has high activity against all substrates. These results showed opposite activities to those produced by Pseudomonas aeruginosa (Hwang, K. A et al. 1999. Kor. J. Appl. Microbiol. Biotechnol . 27, 159-165).

The CMC of the biodegradable surfactant partially purified through acid precipitation and solvent extraction from the culture was found to be 30 mg / L as shown in FIG. 6. The minimum surface tension at this time was 29 mN / m, almost similar to the value measured with the culture supernatant. This result shows a similar tendency of the surface tension lowering ability of Bacillus sp. And Norcardia sp. To be reported in the range of 25-40 mN / m. : Kim, SH et al. 1997. J. of Ferment. And Bioengin. 28, 71-75; Kim, SH et al. 2000. Biotech. Appl. Biochem . 31, 249-253).

Experimental Example 2: Emulsification Stability According to the Substrate of the Surfactant of the Invention

The emulsification activity and emulsion stability based on hydrophobic hydrocarbons and various oils of biodegradable surfactants isolated and purified from the strains of the present invention were investigated. To this end, emulsification activity and emulsion stability tests of biodegradable surfactant solution samples for the commonly used chemical surfactants and oil components of hydrophobic hydrocarbons were carried out according to the methods of Cirigliano and Carman (Cirigliano, MC and GM Carman. 1984. Appl.Environ.Microbiol . 48, 747-750; Cirigliano, MC and GM Carman. 1985. Appl.Environ.Microbiol . 50, 846-850).

The culture solution was sterilized by passing through a Millipore 0.2 μm filtration membrane as a biodegradable surfactant solution. 2 mL of the filtrate is placed in a stoppered test tube, 1 mL of substrate is added to a solution (concentration of CMC) mixed with 2 mL of 0.1 M sodium acetate buffer at pH 3.0, followed by vortex mixing at full speed for 2 minutes. After standing for 10 minutes, the suspension at 540 nm was measured. Emulsification stability is treated by the same method as in the measurement of emulsification activity, and then measured at 10 minutes and measured in suspension at 540 nm, converted into a log value, while standing at room temperature, and the slope at that time is the constant Kd. (The slope value of the emulsifying power to disintegrate per hour). Emulsification activity and emulsion stability based on the hydrophobic hydrocarbon and various oils of the production biodegradable surfactants of the isolates are shown in FIG. 7 and Table 2, and measured by using soybean oil as a substrate. The results of comparative investigation of the stability of the emulsion activity are shown in FIG. 8 and Table 3.

As shown in FIG. 7 and Table 2, as a result of using various emulsified substrates, the emulsification stability was the best in soybean oil with the best activity, and hexadecane maintained similar stability. The rest was relatively inferior in stability, but was confirmed to maintain high stability.                     

In addition, as shown in FIG. 8 and Table 3, the surfactant of the present invention maintained similar activity and stability as the surfactant of Tween and Triton X-100, and emulsified compared to Span and SDS. It was confirmed that the activity and stability were extremely high. This result was very similar to the biodegradable surfactant produced by Norcardia sp. L-417 (Kim, SH et al. 2000. Biotech. Appl. Biochem . 31, 249-253). .

Thus, the biodegradable surfactants produced by the Bacillus vallismortis TB40-3 strain showed very good activity and stability compared to currently available emulsifiers and stabilizers. In order to use this material industrially, investigation of physicochemical properties, biodegradability and environmental toxicity should be carried out.

Emulsification Activity and Stability of Biodegradable Surfactants for Various Substrates temperament Emulsifying Activity (OD ₅₄₀ ) Decomposition Constant (K _d , 10 ^-3 ) Soybean oil 2.65 -0.00 Kerosene 0.84 2.89 Tributyrin 1.59 1.70 crude oil 1.96 -3.48 Hexadecane (C ₁₆ ) 1.30 -1.86 Tetradecane (C ₁₄ ) 1.54 -4.66 Dodecane (C ₁₂ ) 1.17 -5.03 Deccan (C ₁₀ ) 1.62 -3.39

Comparison of Emulsification and Stability of Biodegradable and Commercial Surfactants Surfactants Emulsifying Activity (OD ₅₄₀ ) Decomposition Constant (K _d , 10 ^-3 ) Biodegradable surfactants 2.65 -0.00 Twin 20 2.71 -0.14 Twin 40 2.63 -0.12 Twin 80 2.60 -0.14 Span 40 1.80 -0.43 Span 85 0.88 -5.68 Triton X-100 2.36 -0.18 SDS 1.51 -16.05

Example 3: Bacillus Balymoltis of the present invention ( Bacillus vallismortis Antifungal Effect of TB40-3

Bacillus vallismortis (Bcillus vallismortis ) TB40-3 in order to confirm the antagonism and the growth of the leek in nature to infect B. cineria ( ca. B. cineria ) causing leek fungal disease, negative In the negative control (FIG. 9A), sterilized water (sterile water) in the positive control (positive control (FIG. 9B), the Smirrex hydration effect that is effective in gray mold disease, and the experimental culture TB40-3 culture (FIG. 9C). Sterile water was sprayed every 5 days, and sterile water was used as it was in each sample. Similex hydration was used at a concentration of 1 g / L, and a culture solution of TB40-3 had a turbidity of 1.5 at OD ₆₀₀ of 50 mL each. The foliar spray was carried out every 5 days. After processing each sample, what was observed on day 0 (a), day 4 (b), and day 10 (c) is shown in FIG.

As shown in Figure 9, the initial subject each leek was a lesion due to the infection of the gray mold, so the lesions did not disappear during the entire experiment. However, negative control almost died after 10 days, and positive control and TB40-3 prevented disease progression, and thus, growth of leek was promoted.

Experimental Example 1: Bacillus Balymoltis of the present invention ( Bacillus vallismortis Production Characteristics of Enzymes Associated with Cell Wall Degradation of Phytopathogenic Fungi of TB40-3

CMCase (Fig. 10A), pectinase (pectinase,) using the agar diffusion method, which can visually observe the production of cell wall lyase of Bacillus vallismortis TB40-3. Figure 10B), chitinase (chitinase) and protease (protease, Figure 10C) was confirmed the activity. For this purpose, 0.5% of each substrate was added to LB medium (polypeptone 10g, yeast extract 5g, NaCl 5g, agar 15g) to confirm the characteristics of secretion.

As shown in Figure 10, the secretion of chitinase was not confirmed, other enzymes showed a strong activity. From this, the strain of the present invention was determined to produce an antifungal substance exhibiting a stronger antimicrobial effect than cell wall degradation-related enzymes, thereby showing the effect of inhibiting the growth of pathogens.

As described through the above Examples and Experimental Examples, the present invention relates to a novel Bacillus vallismortis TB40-3 producing a biodegradable surfactant and an antifungal substance with excellent oil resolution, There is an excellent effect of providing a novel Bacillus vallismortis TB40-3 strain having excellent crude oil degrading ability and biodegradable surfactant generating ability and high antibacterial activity against phytopathogens. In addition, the present invention has a surface tension of as low as 29 mN / m, a CMC value of 0.0030% (w / v), the emulsification activity in soybean oil, the emulsification activity in most substrates, including crude oil, There is an excellent effect of providing a biodegradable surfactant separated and purified from Bacillus vallismortis TB40-3 strain having better emulsion stability than a synthetic surfactant. In addition, the present invention is strongly derived from Bacillus vallismortis TB40-3 strain that strongly inhibits the growth of B. cineria causing the fungal disease of leek and promotes the growth of leek It has an excellent effect on providing antifungal substances. Therefore, the present invention is a very useful invention for the environmental industry and agriculture.

Claims (3)

Bacillus vallismortis TB40-3 KACC, which produces biodegradable surfactants and has antifungal activity against phytopathogens B. cineria and F. oxysporum . 91101. Centrifugation of Bacillus vallismortis TB40-3 KACC 91101 strain according to claim 1 to obtain a supernatant from which the cells were removed, and added to the supernatant with hydrochloric acid, left to obtain a precipitate, and centrifuged to recover the precipitate. It was dissolved in an alkaline aqueous solution, lyophilized, the dried material was extracted with alcohol, concentrated and purified to obtain a material having an emulsification activity (OD ₅₄₀ ) of 1.17 to 2.65 and a minimum surface tension of 29 mN / m. A composition for biodegradable surfactants, which is contained as a component. Bacillus vallismortis (Bcillus vallismortis ) TB40-3 KACC 91101 strains described in claim 1 to the phytopathogens B. cineria and F. oxysporum obtained by the isolation and purification An antifungal composition comprising a substance having antifungal activity as an active ingredient.

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