KR101990820B1 - Method for providing information for predicting the antibacterial activity of a culture medium and antibacterial composition comprising the culture medium - Google Patents

Abstract

The present invention relates to a method for providing information for predicting the antimicrobial activity of a culture broth of a strain and an antimicrobial composition comprising the culture broth of a strain, which comprises the step of measuring the surface tension of a culture solution of a lipopeptide-producing strain, And it is possible to accumulate antimicrobial activity prediction data of the target strain, which can be used as a basic index for the optimal use of the biological control agent, and can provide an information providing method capable of selecting antibacterial bacteria having excellent antibacterial activity, And an antibacterial composition suitable for controlling various pathogens.

Description

TECHNICAL FIELD The present invention relates to a method for providing an information for predicting the antimicrobial activity of a culture medium of a strain and an antimicrobial composition comprising the culture medium for a microorganism,

The present invention relates to a method for providing information for predicting the antimicrobial activity of a culture broth and an antimicrobial composition comprising the culture broth.

Among the numerous microorganisms present in plant rhizosphere, the Bacillus genus, useful for plants, is a biological bacterium that inhibits the growth of plant pathogenic microorganisms by indirectly interacting directly with plants to promote plant growth or secrete secondary metabolites Play an important role both agriculturally and environmentally. The genus Bacillus produces chitinase, glucanase, and protease, enzymes that degrade cell walls, and secretes various lipid peptide antimicrobials and volatile organic compounds to inhibit the growth of plant pathogens.

Lipid peptide biosurfactants such as surfactin, iturin and fengycin produced by the strain of Bacillus genus exhibit various biological activities and exhibit a strong antimicrobial activity against plant pathogenic bacteria as a synergistic effect at the time of simultaneous production, and they have been refined and used as means for controlling plant diseases. In particular, B. amyloliquefaciens FZB42 is known to be a representative microbial control for controlling plant pathogenic microorganisms and nematodes by synthesizing lipid peptides such as surfactin, bacillomycin and fengycin, and polyketides such as difficidin and macrolactin . In addition, the biosurfactants produced by the Bacillus sp. Strains have direct antimicrobial activity against various pathogenic pathogens such as Magnaporthe oryzae, Botrytis cinerea, Colletotrichum gloeosporioides, Fusarium oxysporum and Rhizoctonia solani, induced systemic resistance, which is known to play an important role in inhibiting the development of crop disease.

In previous studies, B. amyloliquefaciens KB3 and LM11 strains isolated from insect larvae produced lipid peptides such as iturin A and surfactin and inhibited the growth of various phytopathogenic fungi. In particular, the surfactin produced by the two strains differs depending on the growth stage and the medium components, and the biological control ability is similar to that of the representative bacteriostatic B. subtilis QST-713. Generally, lipid peptide antimicrobials produced by Bacillus sp. Strains exhibit strong biosurfactant activity and inhibit the growth of various kinds of plant pathogens. It is also known that rhamnolipid, a biosurfactant produced by some Gram-negative bacteria, effectively inhibits host germination and mycelial growth of Botrytis cinerea, a tomato gray mold bacterium, and Phytophthora capsici, a contraceptive. In addition, some biological surfactants from Bacillus and Enterobacter spp. Inhibited spores of plant pathogens M. grisea, Aspergillus niger, Penicillium chrysogenum, P. roqueforti and Sclerotinia sclerotiorum.

Studies on the biosurfactants produced by various biocidal bacteria have mainly been carried out on the antimicrobial activity of plant pathogens by the amount and concentration produced by culture conditions or medium. However, there are few reports showing a clear relationship between the surface tension of the biosurfactant and the characteristics of the antimicrobial activity of the biosurfactant strain.

Therefore, in this study, we determined the growth stage of the lipid peptides of B. amyloliquefaciens strain LM11, which showed high antimicrobial activity against various plant pathogens, and found that the surface tension of the extracellular supernatant containing these antimicrobial substances and various plant pathogens And the spectrum of antimicrobial activity.

Korean Patent Publication No. 10-2014-0048745

It is an object of the present invention to provide a method for easily and accurately anticipating antimicrobial activity.

It is an object of the present invention to provide a method for accumulating antimicrobial activity prediction data of a target strain.

It is an object of the present invention to provide a method capable of selecting an antibacterial agent having excellent antibacterial activity.

It is an object of the present invention to provide a composition having excellent antimicrobial activity.

The present invention aims at providing a composition suitable for controlling various pathogens.

1. A method for providing information for predicting antimicrobial activity, comprising the step of measuring a surface tension of a lipopeptide-producing strain.

2. The method of information 1 above, wherein the lipopeptide is at least one selected from the group consisting of Surfactin, Iturin and Fengycin.

3. The method according to item 1 above, wherein the strain is a Bacillus species.

4. The information providing method according to the above 3, wherein the Bacillus strain is Bacillus amyloliquefaciens LM11 (KACC91643P).

5. The information providing method according to claim 1, further comprising the step of determining that the diluted solution diluted to a concentration of 30% by weight of the culture solution is a good product when the surface tension is 38.5 mN / m or less.

6. The information providing method according to 5 above, wherein said good product has a mycelial growth inhibition rate of 50% or more.

7. The method of claim 6, wherein the fungus is selected from the group consisting of Colletotrichum gloeosporioides (KACC40003), Fusarium oxysporum (KACC40032) and Rhizoctonia solani (KACC 40101) And at least one selected from the group consisting of:

8. Lipopeptide production A composition for the antimicrobial use comprising a culture of a Bacillus strain, wherein the 30% by weight dilution of the culture solution has a surface tension of 38.5 mN / m or less.

9. The antimicrobial composition according to the above 8, wherein the culture liquid is a culture medium of late stage index of the strain.

10. The antimicrobial composition according to the above 8, wherein the Bacillus strain is Bacillus amyloliquefaciens LM11 (KACC91643P).

According to one embodiment of the present invention, the method of the present invention can predict antimicrobial activity easily and accurately.

According to one embodiment of the present invention, the method of the present invention can accumulate antimicrobial activity prediction data of a subject strain, which can be used as a basic indicator for optimal use of a biological control agent.

According to one embodiment of the present invention, the antibacterial activity of the antibacterial activity can be selected through the method of the present invention.

According to one embodiment of the present invention, the composition of the present invention is excellent in antibacterial activity.

According to one embodiment of the present invention, the composition of the present invention is suitable for controlling various pathogens.

Figure 1 shows the results of the transcription and biochemical analysis of Bacillus amyloliquefaciens LM11 biosurfactant.
Figure 2 shows the effect of cell-free supernatant of Bacillus amyloliquefaciens LM11 on inhibitory mycelial growth or spore germination of plant pathogenic fungi.
Fig. 3 shows the correlation between the surface tension and the antifungal activity of Bacillus amyloliquefaciens LM11 bacterial culture grown as a stopper.

The present invention relates to a method for providing information for predicting the antimicrobial activity of a culture broth of a strain and an antimicrobial composition comprising the culture broth of a strain, which comprises the step of measuring the surface tension of a culture solution of a lipopeptide-producing strain, And it is possible to accumulate antimicrobial activity prediction data of the target strain, which can be used as a basic index for the optimal use of the biological control agent, and can provide an information providing method capable of selecting antibacterial bacteria having excellent antibacterial activity, And an antibacterial composition suitable for controlling various pathogens.

Hereinafter, the present invention will be described in detail.

The method for providing information for predicting the antimicrobial activity of the present invention includes a step of measuring the surface tension of the culture medium of the lipopeptide-producing strain.

The antimicrobial lipid peptides produced from the strains are closely related to the surface tension of the culture medium. Therefore, the antimicrobial activity against pathogenic bacteria can be predicted by measuring the surface tension of the culture solution, and if the antimicrobial activity is predicted to be excellent, the target strain can be selected as the biological antibacterial bacteria.

In measuring the surface tension of the culture liquid in the above step, the surface tension can be measured by setting a constant concentration condition (for example, a concentration of 50% by weight) without concentration. In this case, it is possible to measure the surface tension inherent to the culture medium itself, not to change the surface tension according to the increase or decrease of the concentration, so that the antimicrobial activity can be more accurately predicted and the reliability of the screening bacteria selection can be improved as a result.

Since lipopeptides are substances exhibiting antibacterial activity and lipopeptides have surface activity, the surface tension may vary depending on the content of lipopeptides. Therefore, antimicrobial activity can be predicted by measuring the surface tension.

The lipopeptide may be any known substance produced in a strain exhibiting antimicrobial activity. But are not limited to, for example, penicillin, bacillomycin, surfactin, iturin A, and the like.

According to one embodiment of the present invention, the lipopeptide may be at least one selected from the group consisting of Surfactin, Iturin and Fengycin. These lipopeptides are representative lipopeptides known to have antimicrobial activity, and by measuring the surface tension of the culture medium for producing them, it is not necessary to firstly select a strain having no antimicrobial activity, Lt; / RTI >

According to one embodiment of the present invention, the strain producing the lipopeptide may be a Bacillus species. Bacillus sp. Strains are known to have antimicrobial activity. Therefore, the antibacterial activity of the strain of Bacillus subtilis can be confirmed by measuring the surface tension of the culture medium of the strain of Bacillus subtilis, and the excellent antibacterial effect can be quickly and effectively selected for various uses based on the thus-confirmed results.

Examples of Bacillus strains include Bacillus amyloliquefaciens, Bacillus subtilis and Bacillus sapensis, Bacillus subtilis, Bacillus pumilus, Bacillus amyloliquefos, Bacillus ricinifomis, Bacillus megaterium and Bacillus ricinifomis, Simplex, and the like, but are not limited thereto.

According to a specific embodiment of the present invention, the strain of Bacillus subtilis may be Bacillus amyloliquefaciens LM11 (KACC91643P).

According to an embodiment of the present invention, it may further comprise determining that the diluted solution diluted to a concentration of 30% by weight of the culture solution is a good product when the surface tension is 38.5 mN / m or less.

In one embodiment of the present invention, when the surface tension of the diluent is 38.5 mN / m or less, the ability of the three types of fungi to inhibit mycelial growth is 50% or more. When the surface tension is 38.5 mN / m or less, It is expected that the ability to inhibit the growth of an excellent strain will appear.

The lipopeptide producing strain may be a strain to be measured for antimicrobial activity, for example, a strain of Bacillus sp., A strain of Pseudomonas sp., A strain of Pennibacillus, or the like, but is not limited thereto and may include any strain producing lipopeptide. Furthermore, even when a strain is transformed into a strain producing lipopeptide, the strain can be a lipopeptide producing strain according to the present invention.

In the present invention, the good product means a strain having an excellent antimicrobial activity and means a strain having an excellent antimicrobial activity activity measured according to various antimicrobial activity measuring methods.

According to one embodiment of the present invention, the good product may be a strain having a mycelial growth inhibition rate of 50% or more.

50% of the mycelial growth inhibition rate means that more than 50% of the mycelia grown from the fungi in a given time period reach to death, and the above criteria are the information providing method of the present invention for use in selecting strains having excellent biological control ability Is suitable as a basis of.

The fungi include, without limitation, strains belonging to fungi. For example, the fungi include Colletotrichum gloeosporioides (KACC40003), Fusarium oxysporum (KACC40032) and Rhizoctonia solani , KACC 40101).

The surface tension can be measured by measuring the degree of rise of the liquid surface in the canal, measuring the contact angle, and other surface tension measuring devices. In an embodiment of the present invention, the surface tension is measured by using a surface tension measuring device, but it is also possible to measure by a known surface tension measuring method.

According to one embodiment of the present invention, the method of the present invention may further include the step of measuring mycelial growth inhibition ability against a plant pathogenic fungus.

The method of the present invention can measure the antimicrobial activity only by measuring the surface tension of the culture liquid, but it is impossible to accurately measure due to the mechanical error of the measurement or the error of the experimenter, or an additional experiment The reliability of the antimicrobial activity measurement can be increased.

In the case of phytopathogenic fungi, the mycelial growth inhibiting ability can be used as an indicator for exhibiting the antimicrobial activity, so that the antimicrobial activity can be evaluated more accurately since the prediction by the surface tension can be supplemented by evaluating the mycelial growth inhibiting ability , The reliability of the evaluated antimicrobial activity can be increased.

Also, according to one embodiment of the present invention, the method of the present invention is characterized in that the expression amount of at least one lipid peptide selected from the group consisting of Surfactin, Iturin and Fengycin The method may further comprise the step of measuring.

As described above, the method of the present invention can increase the reliability of the antimicrobial activity measurement by further performing an experiment to support the measurement, as a method for supplementing the measurement of the surface tension of the culture liquid.

In this regard, sulphate, iturin and penicillin are lipid peptides known to exhibit antimicrobial activity and are included in the culture medium of the strain exhibiting antimicrobial activity.

Therefore, the antimicrobial activity can be evaluated by measuring the amounts of sulfatin, itulin and penicin contained in the culture broth, that is, the amount of expression thereof in the target strain. By measuring the expression level, the reliability of surface tension measurement and Accuracy can be increased.

As a method for measuring the expression level of sulphate, iturin and penicillin, a method of measuring the mRNA level of the gene expressing them, a method of measuring the protein level, and the like can be used. Can be used to measure their expression levels.

RT-PCR, Competitive RT-PCR, Real-time RT-PCR, and RNase protection (RPA) were used for the measurement of mRNA levels. assay, Northern blotting, DNA chip, and the like.

As the agent for measuring the mRNA level is a primer pair or a probe, and the nucleic acid information of the genes is known by GeneBank, a person skilled in the art can design a primer or a probe that specifically amplifies a specific region of these genes based on the above sequence . For example, agents that measure mRNA levels may include primer pairs, probes, or antisense nucleotides that specifically bind to the gene.

Specifically, the agent for measuring the mRNA level for measuring the expression level of sulphate, iturin and penicillin may be a primer pair that specifically binds to a gene expressing at least one of sulphate, iturin and penicillin, Antisense oligonucleotides.

A primer pair is a primer pair that contains all combinations of primer pairs consisting of forward and reverse primers that recognize the target gene sequence, but specifically provides analysis results with specificity and sensitivity.

The term " probe " means a substance capable of specifically binding to a target substance to be detected in the sample, and means a substance capable of specifically confirming the presence of a target substance in the sample. The probe molecule may be a peptide nucleic acid (PNA), a peptide nucleic acid (LNA), a peptide, a polypeptide, a protein, an RNA, or a DNA. Specifically, it is PNA. In addition, the probe may be, for example, an enzyme, a protein, an antibody, a microorganism, an animal or animal cell or an organ, a nerve cell, DNA, and RNA DNA includes cDNA, genomic DNA, oligonucleotides, RNA includes genomic RNA, mRNA, oligonucleotides, and examples of proteins include antibodies, antigens, enzymes, peptides, and the like.

Antisense oligonucleotides are DNA or RNA or derivatives thereof containing a nucleic acid sequence complementary to the sequence of a specific mRNA, and bind to a complementary sequence in the mRNA to inhibit the translation of the mRNA into a protein. Antisense oligonucleotide sequences refer to DNA or RNA sequences that are complementary to and capable of binding to the mRNAs of the genes. This can interfere with essential translational activities of translation of gene mRNA, translocation into the cytoplasm, maturation, or any other overall biological function. The length of the antisense oligonucleotide may be 6 to 100 bases, specifically 8 to 60 bases, more specifically 10 to 40 bases.

Protein level measurement is the process of determining the presence and degree of expression of at least one protein of sulphate, iturin and penigenin. An antibody that specifically binds to these proteins can be used to confirm the expression level of the protein, and the protein expression level itself can be measured without using the antibody.

Protein level assays or comparative assay methods include protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Desorption / Ionization Time of Flight Mass Spectrometry) analysis, SELDI-TOF (Sulface Enhanced Laser Desorption / Mass spectrometry analysis, radioimmunoassay, radial immunodiffusion, Oucheroton immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, complement fixation, two-dimensional electrophoresis, liquid chromatography-mass spectrometry But are not limited to, spectrometry, LC-MS, liquid chromatography-mass spectrometry / mass spectrometry (LC-MS / MS), Western blotting, and enzyme linked immunosorbent assay (ELISA).

According to one embodiment of the present invention, when the surface tension of the diluted solution of 30% by weight in the culture medium of Bacillus strain measured by the method of the present invention is measured, the antibacterial activity is also highest when the measured surface tension is 38.5 mN / m or less (Fig. 1).

Accordingly, in another aspect, the present invention provides an antimicrobial composition comprising a culture of a Bacillus strain, wherein the 30% by weight dilution of the culture solution has a surface tension of 38.5 mN / m or less.

When the surface tension of the 30 wt% diluted solution of the culture solution is 38.5 mN / m or less, the growth of various pathogens can be suppressed by 50% or more, and the ability to inhibit mycelial growth of the phytopathogenic fungus can be suppressed by 50% But is not limited thereto.

According to one embodiment of the present invention, the surface tension of the culture medium of the late-log phase of the Bacillus strain was higher than that of the stationary phase, and in particular, the expression of iturin among the antibacterial activity proteins The antimicrobial agent was more effective than the suspension culture medium of Bacillus strains as a late phase index culture medium.

Thus, according to one embodiment of the present invention, the antimicrobial composition may comprise a culture medium of late index of Bacillus strains. By including the late exponential phase culture fluid, the antimicrobial activity is superior to the case of using the culture medium of the stationary phase, and thus the pathogen can be effectively controlled.

The late exponents and stationary phases of Bacillus strains were determined based on the absorbance measured after 15 days of incubation on TSB medium. When the absorbance was measured at OD _600nm , it was judged to be the late exponential period when the value was 1.5 to 2.1, It was judged as a stopping machine.

Late exponents and stoppers can also be determined through colonies formed by nutritional cells and endospores, specifically stunners where the colony forming unit of the nutrient cells is log 10 or higher and the colony forming unit of endogenous spores exceeds the log 2 , And the late exponential period is the period when the colonization unit of the nutrient cells is less than 8 to 10 logs and the colony forming unit of the endospore is less than 1 to 2 logs (see Table 1).

The Bacillus strain may be the above-mentioned Bacillus strain, specifically, Bacillus amyloliquefaciens LM11 (KACC91643P).

The composition of the present invention may further comprise a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" as used herein means that the composition is free of toxicity to cells or animals or plants exposed to the composition.

Compositions comprising a pharmaceutically acceptable carrier can be of various oral or parenteral formulations. In the case of formulation, it can be prepared using diluents or excipients such as fillers, extenders, binders, humectants, disintegrants, surfactants and the like which are usually used. Examples of the carrier, excipient and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, But may be at least one selected from the group consisting of polyvinylpyrrolidone, physiological saline, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, dextrin, calcium carbonate, propylene glycol and liquid paraffin, But are not limited to, ordinary carriers, excipients or diluents.

Solid formulations for oral administration may include tablet pills, powders, granules, capsules, and the like, which may contain one or more excipients, such as starch, calcium carbonate, sucrose or lactose, gelatin, Can be prepared. In addition to simple excipients, lubricants such as magnesium stearate, talc, and the like may also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the non-aqueous solvent and the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. Examples of the suppository base include witepsol, macrogol, tween 61, cacao paper, laurin, glycerogelatin and the like.

The pharmaceutical composition of the present invention may also be in the form of tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, Or a pharmaceutically acceptable salt thereof.

The pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount. There is no particular restriction on the dosage, and it may vary depending on the body's absorption, body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, severity of disease and the like. The pharmaceutical composition of the present invention is prepared in consideration of an effective dose range, and the unit dosage formulations thus formulated are classified according to the judgment of the expert who monitors or observes the administration of the drug, if necessary, Or may be administered several times at a predetermined time interval.

Hereinafter, the present invention will be described in more detail with reference to the following examples.

Example

Materials and methods

Cultivation of biological control strains and phytopathogenic fungi

B. amyloliquefaciens LM11 (KACC91643P), isolated and isolated from intestinal cells of the larvae of beetle larvae, was used in this study. The LM11 strain was used in a Tryptic Soy Broth (TSB; Becton Dickinson GmbH, Heidelberg, Germany) medium while shaking for 5 days at 28 ° C and 120 rpm. The phytopathogenic fungi used in this study were Fusarium oxysporum f. sp. lycopersici KACC 40032, red pepper anthracnose fungus Colletotrichum gloeosporioides KACC 40003, rice sheath patterned diamond pathogen Rhizoctonia solani AG-1 strains with KACC 40101 are RDA National Academy of Agricultural Sciences Agricultural Genetic Resources Information Center (Korean Agricultural Culture Collection (KACC) , National Agrobiodiversity Center , Jeon-ju, Korea). Plant pathogens were cultured on a Potato Dextrose Agar (PDA, Becton Dickinson GmbH) medium.

Antibacterial activity gene expression and production analysis

Expression patterns of biosurfactants derived from LM11 were analyzed by quantitative real time reverse transcription polymerase chain reaction (qRT-PCR) and thin-layer chromatography (TLC). Total RNA was cultured in TSB medium at OD _600nm = 0.6 (mid-log phase), 1.8 (late-log phase), 2.2 (stationary phase) and then with NucleoZOL reagent (Macherey-Nagel, According to the company's protocol. The final RNA samples were purified by DNase I (Qiagen Inc., Hilden, Germany). qRT-PCR was performed using QuantiTect SYBR Green RT-PCR (Qiagen). RT-PCR was performed after synthesizing cDNA by reacting with moloney murine leukemia virus reverse transcriptase (MNLV-RT, Enzynomics, Daejeon, Korea) at 42 ° C for 30 minutes. The ituD using primers (forward, 5'-TTGAAYGTCAGYGCSCCTTT-3 '; revers, 5'-TTGAAYGTCAGYGCSCCTTT-3', 482 bp), ituC (forward, 5'-CCCCCTCGGTCAAGTGAATA-3 '; reverse, 5'-TTGGTTAAGCCCTGATGCTC-3' , 594 bp) and srfA (forward, 5'-TCGGGACAGGAAGACATCAT-3 '; reverse 5'-CCACTCAAACGGATAATCCTGA-3', 201 bp). 16S rDNA primer was constructed by using forward primer 5'-GAGGAACACCAGTGGCGAAG-3 'and reverse, 5'-TAAACCACATGCTCCACCGC-3' using the program primer 3 in the ITS (intergenic species region) sequence of B. amyloliquefaciens LM11 Respectively. The amplification reaction was performed by reacting 20 μl of QuantiTect RT Mix containing cDNA with 10 μM of each primer for 10 min at 95 ° C for 30 min, at 95 ° C for 30 sec, at 60 ° C for 55 sec, at 72 ° C for 30 sec cycle was amplified 40 times and repeated 3 times each. Stratagene Mx3,000P qPCR system (Agilent Technoloogies Inc., Santa Clara, CA, USA) and software. The 2 ^-ΔΔCT method was used to analyze the relative expression of each gene.

The lipopeptides produced by LM11 strain were thin layer chromatography (TLC). And cultured at 28 ° C in TSB medium, and extracted with n- butanol for each growing period. The mobile phase (chloroform - methanol - H ₂ O = 65: 25: 4) was eluted on a TLC (silica gel 60, Merck, Germany) plate after complete concentration with a vacuum concentrator. , v / v / v) water. The surfactin (CAS 24730-31-2) used as a reference material was purchased from Sigma-Aldrich (St Louis, MO, USA). In addition, iturin A and fengycin were searched by the previously reported Rf values, and they were performed 3 times independently and 3 repetitions were performed.

Analysis of surface tension and antibacterial activity of cell culture filtrate

B. amyloliquefaciens LM11 strain was cultured in TSB medium at 28 ° C and 120 rpm for 15 days [OD _600nm = 0.6 (mid-log phase), 1.8 (late-log phase), 2.2 (stationary phase)]. Growth of LM11 was measured at an absorbance of 600 nm using a spectrophotometer (UV-1601, Shimadzu, Japan). The number of endogenous spores and nutrient cells was counted by counting viable cell counts in the heat treated and untreated media at 100 ° C for 15 minutes. The culture broth was diluted with sterilized water 10 times serial dilution and plated on tryptic soybean agar (TSB agar; Becton Dickinson GmbH) medium. The number of live cells was measured three times independently, and three repetitions were performed.

The supernatant was used after removing cells using a 0.22 μm filter (Millipore Filter Corp., Bedford, Mass., USA) for each growing season. To confirm the contamination of the culture supernatant, TSB agar medium was used to confirm the presence of the strain. The changes in surface tension of the culture filtrate over time were measured three times using the ring method using a surface tensiometer (K6, KRUSS GmbH, Hamburg, Germany) and three times in total. Sterilized water and medium were used as control for surface tension.

The surface tension and antimicrobial activity of the LM11 strain were investigated by culturing the strains in TSB medium at 28 ° C and 120 rpm for 5 days. The surface tension of B. amyloliquefaciens LM11 culture supernatant was diluted to 40% of the sterilized TSB medium and 60% of the PDB broth containing 0 to 40% of the culture filtrate, and the value of each surface tension was measured. To measure the antimicrobial activity, 40% of the sterilized TSB medium containing 0 to 40% of the culture filtrate and 60% of the PDB and 1.5% of the agar were mixed with the sterilized PDA. The mycelial growth length of mycelial growth was measured on the PDA mixed medium prepared by supernatant containing 5 mN of mycelial fragments from the hyphae of F. oxysporum , C. gloeosporioides , and R. solani , which were cultured on a full length PDA for 5 days. Respectively. The percent inhibition of mycelial growth was expressed as the length of mycelial growth in the PDA contained in each supernatant by percentage of mycelial growth length in PDA without strain supernatant.

[Equation 1]

Inhibition rate of mycelial growth (%) = (control strain length - treatment strain length) / control strain length x 100.

Each treatment was performed 3 times independently, and 3 repetitions were performed.

Inhibition of sprouting of pepper anthracnose by culture filtrate

C. gloeosporioides KACC 40003 was inoculated on PDA medium and incubated at 25 ° C for 7 days. Then, 20 ml of sterilized water was added to collect conidia and the spore suspension was recovered using a 4-fold sterilized cheese close. Spores were used by adjusting the spore suspension concentration using a hemacytometer (Paul Marienfield GmbH & Co., Lauda-Konigshofen, Germany). Spore germination rate was determined by diluting 1 ml of sterilized TSB medium (40%) and PDB broth (60%) in a 12 well plate (SPL Life Sciences, Korea) containing 0 to 40% culture filtrate. The germination rate of 200 μl spore suspension (1 × 10 ⁴ spores / ml) was suspended in the prepared culture filtrate and incubated at 25 ° C for 20 hours at 120 rpm. The germination length of germination tube was longer than that of spore length under light microscope (Leica Microsystems, Wetzlar, Germany), and germination was observed on 100 spores of each repetition. For the control, sterilized TSB medium and 60% PDB medium were used.

Statistical analysis

Statistical analysis was performed by ANOVA using SPSS 23.0 software (IBM Corporation, Somers, New York, USA). Duncan's multiple range test and linear regression analysis were used to test statistical significance at significance level 0.05 only if F value was significant.

result

Expression and Generation of Antibacterial Lipopeptide Biosynthetic Gene by Growth Stage

Figure 1 shows the results of the transcription and biochemical analysis of the biosurfactant of Bacillus amyloliquefaciens LM11. Figure 1A shows the result of separating the lipid peptide fraction of B. amyloliquefaciens LM11 into thin-layer chromatography plates (silica gel 60) Distance Rf.

Figure 1B shows qRT-PCR analysis performed twice using primers specific for the itRC, ituD and sfrA genes, and their expression levels were generalized for 16s rRNA gene levels. The transfer amount of these was measured by the 2- ^ΔΔCT method (Table 1).

Growth stage Somatic cells (= nutrient cells)
(Log colony forming unit / ml) ^x Resistant spore
(Log colony forming unit / ml) ^y Surface tension of culture medium (mN / m) ^z Middle-term exponent 4.12 ± 0.77 ^a - 34.2 ± 0.52 ^b Late period index machine 8.33 ± 0.31 ^b 1.12 ± 1.33 ^a 30.0 ± 0.12 ^a Stopper 9.66 ± 1.09 ^c 2.95 + - 6.60 ^a 30.2 ± 0.06 ^a

Specifically, cells of Bacillus amyloliquefaciens LM11 were grown at 28 ° C under TSB conditions to a medium-term exponential period (OD _{600 nm} = 0.8), a late exponential period (OD _{600 nm} = 1.8) and a quiescent period (OD _{600 nm} > 2.2). A 10-fold serial dilution of the solution was prepared and the colony forming unit ^x of B. amyloliquefaciens LM11 was measured and 0.1 ml of each dilution was inoculated on an LB agar plate.

The culture medium boiled at 100 ° C for 15 minutes was diluted 10-fold and prepared, and the colony-forming unit ^y of the resistant spores was measured. Then, 0.1 ml of each colony forming unit was dispensed onto the LB agar plate. The surface tension ^z of the cell-free supernatant was measured by a surface tension meter (K6, KRUSS GmbH, Hamburg, Germany) using the principle of the Ring method.

The ± values in Table 1 represent the mean of the standard errors calculated by performing three independent experiments for each growth step and corresponding measurement value. Data were analyzed by one-way ANOVA (P <0.05), and when the P-value of the F-test was significant, Duncan's multiple range test further revealed the difference. Other characters showed significant difference at P <0.05. If the superscripts in Table 1 are other characters, P <0.05 was significant.

FIG. 1C shows the antimicrobial activity of the biosurfactant fraction extracted from B. growth phase of B. amyloliquefaciens LM11 against phytopathogenic fungal pathogens. The cell-free supernatant was acidified, extracted with n-butanol and dissolved in methanol. Fractions of the culture filtrate of LM11 strains of different growth stages were loaded onto paper discs. The photograph of Figure 1C represents three independent experiments with similar results. The bacterial cells were shake-cultured in TSB medium at 28 DEG C and 120 rpm. Total RNA and lipopeptide were extracted at each growth stage.

As a result of TLC analysis of the culture filtrate, furfurin group with Rf 0.08 - 0.2, iturin group with Rf 0.3 and surfactin group with Rf 0.7 - 0.75 were observed in culture filtrate of LM11 strain. The amount of these antimicrobial lipopeptides was detected in a large amount in the culture medium of late log and stationary phase, but a small amount was detected in the culture filtrate of the exponential phase (FIG. 1A).

The time of generation of the antibacterial lipopeptides and the expression timing of the biosynthetic genes of these substances were the same (Fig. 1B). The gene expression of the antimicrobial lipopeptides of the LM11 strain was statistically significantly higher in the late-log and stationary phase than in the mid-log LM11 strain (FIG. 1B). The expression of ituC, ituD and sfrA genes related to the biosynthesis of iturin and surfactin was also significantly higher in the late-log phase and the stopper (Fig. 1B). Interestingly, iturin was statistically significantly higher than late-log period.

The antimicrobial activities against the phytopathogenic fungi of the LM11 isolates were inhibited by mycelial growth of various phytopathogenic fungi in late-log and stagnant culture filtrates, but the exponential culture filtrate showed no inhibitory effect on mycelial growth 1C).

Correlation between surface tension and antimicrobial activity of cultured filtrate

B. amyloliquefaciens The effect of cell-free supernatant of LM11 on the mycelial growth and spore germination of plant pathogenic fungi and the correlation between the surface tension and the antifungal activity of the culture medium of Bacillus amyloliquefaciens LM11 strain grown on a stationary medium were investigated. The results are shown in Figs. 2 and 3.

In Figures 2 and 3, the symbols A to C are as follows: (A) Fo; Fusarium oxysporum f. sp. lycopersici KACC 40032, (B) C8; Colletotrichum gloeosporioides KACC 40003, (C) R5; Rhizoctonia solani AG-1 KACC 40101.

2 A to C show cell-free supernatant from B. amyloliquefaciens LM1 grown as a stopping group showing antifungal activity against phytopathogenic fungi. The supernatant was added to potato dextrose agar at different concentrations, and then the LM11 strain Fungal mycelial growth inhibition was measured.

Figure 2D shows spore germination measurements in 1/3 PDB medium with different concentrations of LM1 cell-free supernatant. Spore germination was measured by optical microscope after culturing at least 100 spores per experiment for 24 hours. Spores producing bacterial tubing longer than the diameter were considered germinated. The proposed error bars are the mean of ± standard errors for three independent experiments. Data were analyzed using ANOVA (P <0.05), and if the F test was significant, Duncan's multiple range test further revealed differences. In Figure 2D, the letters on the different bar graphs indicate significant differences at P &lt; 0.05. In the experiments described above, the independent variable X was the cell-free supernatant concentration of the LM11 stasis culture, and the dependent variable Y was the linear regression curve indicating the spore germination inhibition of C. gloeosporioides. The Spearman correlation coefficient (R value) and the corresponding P value were calculated.

FIG. 3 shows the result of continuous dilution (0 to 40%) of the cell-free culture medium of the stator with sterilized TSB broth and measurement of the surface tension. Also, the culture filtrate of the stan- dard LM11 strain at different concentrations (From a 5-day-old PDA plate) was placed at the center of a PDA plate containing the LM11 strain to measure mycelial growth inhibitory ability according to the concentration of the culture filtrate of the strain LM11.

Measurement of mycelial growth inhibition ability was carried out specifically as follows. First, all of the PDA plates described above were incubated at 28 DEG C for 5 days. Growth of the fungus was confirmed by increasing the colony diameter. Three replicated plates were used per analysis. The mycelial inhibition rate of the culture filtrate of LM11 strain was calculated by the following equation (2).

&Quot; (2) &quot;

Mycelial growth inhibition rate (%) = (A - B) / A x 100

Where A is the diameter (mm) of the mycelium grown for 5 days in the control PDA plate and B is the diameter (mm) of the mycelia grown for 5 days on the PDA plate containing the culture filtrate of strain LM11.

Each point represents the dilution of the cell-free supernatant of B. amyloliquefaciens LM11 grown in TSB to different concentrations to different concentrations.

Three independent clinical trial data were obtained and statistically analyzed using this to obtain a linear regression graph in which the independent variable X is the surface tension and the dependent variable Y is the mycelial growth inhibition. The Spearman correlation coefficient (R value) and the corresponding P value were calculated.

As shown in FIG. 2, the mycelial growth inhibition ability of various plant pathogens of STEM culture broth of LM11 strain was directly related to the concentration of cultured filtrate. In other words, the inhibition of mycelial growth of the pathogenic fungi as disclosed in the PDA medium containing 40% of the culture filtrate showed a strong antibacterial activity of 75.3 to 89.7% (FIG. 2). Above 20% by weight of culture filtrate, mycelial growth of plant pathogenic bacteria was inhibited by more than 50%, but when it was less than 20% by weight, mycelial growth inhibition ability was remarkably decreased.

The higher the stagnant culture filtrate content of LM11 strain, the more negative correlation was observed with the C. gondoporioides spore germination rate (R = 0.761, P <0.001, Fig. 2D). The lowest germination rate was 10% in the treatment containing 30% culture filtrate.

As shown in FIG. 3, the correlation between the surface tension of the LM11 strain suspension culture and the inhibition of mycelial growth of the plant pathogenic fungus strain was R = 0.973 ^** for C. gloeosporioides, F. oxysporum and R. solani, respectively , 0.951 ^** , and 0.977 ^** at 0.01% level (Fig. 3). C. gloeosporioides and R. solani showed a mycelial growth inhibition ability of about 50% or more at a concentration of 10 wt% of the stationary culture filtrate having surface tension of 42.0 and 51.8 mN / m, but F. oxysporum showed 37.1 (30 wt% Concentration) mN / m showed 50% or more mycelial growth inhibition ability. This means that when the surface tension of the culture filtrate exceeds 38.5 mN / m, it does not exhibit an ability of inhibiting mycelial growth of 50% or more, that is, excellent antimicrobial activity, against the strain of F. oxysporum.

conclusion

Bacillus amyloliquefaciens LM11, isolated from intestinal cells of long beetle larvae, produced a biosurfactant lipopeptide such as surfactin, iturin, and fengycin and strongly inhibited the growth of phytopathogenic fungi. There was a significant difference in biosurfactant production and surface tension according to the LM11 strain growth stage. The biosynthesis genes of surfactin, iturin, and fengycin, which are antimicrobial substances, were expressed intensively while reaching the stopper, and their production was also high. In addition, there was a high negative correlation with the spore germination of the anthracnose anthracnose of pepper according to the concentration of culture supernatant of LM11 strain (R = 0.761, P <0.001). The minimum surface tension level for inhibiting mycelial growth of phytopathogenic fungi was 38.5 mN / m (R = 0.951 to 0.977, P <0.001). The results of this study show that the biosurfactant of B. amyloliquefaciens LM11 acts as an important antifungal metabolite for biological control of plant diseases and that surface tension measurement of culture medium can be used as a basic index for optimal use of biological control agents.

Claims (10)

Measuring the surface tension of the diluted solution diluted to a concentration of 30% by weight in the culture filtrate of the lipopeptide-producing strain; And
And judging that the product is good when the measured surface tension is 38.5 mN / m or less,
The strain is Bacillus amyloliquefaciens LM11 (KACC91643P)
Wherein the diluent comprises the sterilized TSB medium containing the culture filtrate in a ratio of 40: 60 with the PDB medium.
The information providing method according to claim 1, wherein the lipopeptide is at least one selected from the group consisting of Surfactin, Iturin and Fengycin.
delete delete delete The information providing method according to claim 1, wherein the good product has a mycelial growth inhibition rate of 50% or more.
The method according to claim 6, wherein the fungus is selected from the group consisting of Colletotrichum gloeosporioides (KACC40003), Fusarium oxysporum (KACC40032) and Rhizoctonia solani (KACC 40101) Wherein the at least one information providing method is at least one of the following.
delete delete delete

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