WO2015072603A1 - Ebn-tk3 de bacillus amyloliquefaciens et agent antibiotique ou antifongique le comprenant - Google Patents

Ebn-tk3 de bacillus amyloliquefaciens et agent antibiotique ou antifongique le comprenant Download PDF

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WO2015072603A1
WO2015072603A1 PCT/KR2013/010377 KR2013010377W WO2015072603A1 WO 2015072603 A1 WO2015072603 A1 WO 2015072603A1 KR 2013010377 W KR2013010377 W KR 2013010377W WO 2015072603 A1 WO2015072603 A1 WO 2015072603A1
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ebn
bacillus amyloliquefaciens
kctc
fusarium oxysporum
amyloliquefaciens
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PCT/KR2013/010377
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English (en)
Korean (ko)
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조정섭
송홍규
임태빈
권혁도
정택경
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주식회사 두산에코비즈넷
강원대학교 산학협력단
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Priority to PCT/KR2013/010377 priority Critical patent/WO2015072603A1/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/30—Feeding-stuffs specially adapted for particular animals for swines
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
    • A23K50/75—Feeding-stuffs specially adapted for particular animals for birds for poultry

Definitions

  • the present invention relates to a newly isolated microorganism and an antibacterial or antifungal agent using the same, and more particularly, to Bacillus amyloliquefaciens EBN-TK3 and an antibacterial or antifungal agent comprising the same.
  • Atrophic rhinitis is known to be due to the interaction of Bordetella and Pasteurella. Broncho sepsis Bordetella bronchiceptica is a fundamental pathogen, and Pasteurella multocida also plays a secondary role in promoting lesions. Bordetella acts to stimulate the nasal mucosal epithelium, allowing it to settle pasteurella.
  • the atrophic rhinitis is a chronic wasting disease that occurs in piglets and causes rhinitis (sneezing), deformation of the nose and facial bones (nose crookedness, snout atrophy, etc.), lowering of body weight.
  • fowl typhoid is a sudden and chronic infectious disease occurring in birds such as chickens and turkeys, and is characterized by high mortality caused by sepsis.
  • the causative agent is known as Salmonella gallinarum , a kind of Salmonella bacteria.
  • Poultry typhoid occurred worldwide in the early 1900s and caused huge losses to the poultry industry. Domestic outbreaks were first reported in 1992, and since 1994, have been reported nationwide and have caused huge economic losses for poultry farmers.
  • the present invention provides Bacillus amyloliquefaciens EBN-TK3 (KCTC 12382BP).
  • the present invention provides an antimicrobial agent comprising Bacillus amyloliquefaciens EBN-TK3 (KCTC 12382BP) or a culture solution thereof as an active ingredient.
  • the antimicrobial agent may be preferably for inhibiting growth of Bordetella bronchiseptica or Salmonella gallinarum .
  • the antimicrobial agent may be an additive added to a detergent or a feed for cleaning purposes.
  • the present invention provides a feed additive or feed characterized in that it contains Bacillus amyloliquefaciens EBN-TK3 (KCTC 12382BP) or its culture.
  • the feed additive or feed may preferably be a feed additive or feed of pigs or chickens. Livestock are susceptible to pathogenic microorganisms through feed, but when the Bacillus amyloliquefaciens EBN-TK3 (KCTC 12382BP) or its culture solution is added to the feed, it inhibits the growth of pathogens and prevents the livestock from infecting the pathogen. I can protect it.
  • the present invention provides an antifungal agent characterized in that it contains Bacillus amyloliquefaciens EBN-TK3 (KCTC 12382BP) or its culture as an active ingredient.
  • the antifungal agent is preferably Rhizoctonia solani , Fusarium oxysporum F. Esp. Fusarium oxysporum f.sp.raphani , Fusarium oxysporum f . Esp. May be for any one or inhibition of the growth of the selected nibe Titanium (Fusarium oxysporum f.sp. niveum).
  • the antifungal agent may be preferably for controlling any one or more plant diseases selected from leaf blight, wilted stalk and vine cutting disease.
  • the antifungal agent may be an additive added to cleaning agents, pesticides or fertilizers for cleaning purposes.
  • the present invention provides a fertilizer additive or fertilizer characterized by containing Bacillus amyloliquefaciens EBN-TK3 (KCTC 12382BP) or its culture.
  • Bacillus amyloriquafaciens EBN-TK3 (KCTC 12382BP) is the cause of foliar blight in agriculture, Rhizoctonia solani , the cause of wilting disease Fusarium oxysporum F. Esp. Fusarium oxysporum f.sp.raphani and Fusarium oxysporum F, the causative agent of vine pecking disease . Esp. It was confirmed that the antifungal effect of inhibiting and inhibiting Nusarium Fusarium oxysporum f.sp.
  • Figure 1 shows the growth inhibitory effect of Bordetella bronchiseptica , a causative agent of swine atrophic rhinitis caused by candidate strains.
  • ⁇ control, ⁇ : 10 ⁇ l / ml treatment, ⁇ : 30 ⁇ l / ml treatment, ⁇ : 50 ⁇ l / ml treatment).
  • Figure 2 shows the inhibitory effect of the growth of Salmonella gallinarum , the causative agent of poultry typhoid by the candidate strain.
  • ⁇ control, ⁇ : 10 ⁇ l / ml treatment, ⁇ : 30 ⁇ l / ml treatment, ⁇ : 50 ⁇ l / ml treatment).
  • EBN-TK3 Bacillus amyloliquefaciens EBN-TK3 (KCTC 12382BP) strain isolated purely in the present invention.
  • Figure 4 is purely isolated Bacillus amyloliquefaciens in the present invention ( Bacillus amyloliquefaciens )
  • the culture results in NB medium of EBN-TK3 (KCTC 12382BP) strain are shown.
  • Bacillus amyloliquefaciens purely separated in the present invention ( Bacillus amyloliquefaciens )
  • the culture results in LB medium of EBN-TK3 (KCTC 12382BP) strain are shown.
  • FIG. 7 is an antifungal test result of Bacillus amyloliquefaciens EBN-TK3 (KCTC 12382BP) of the present invention against Rhizoctonia solani .
  • EBN-TK3 Bacillus amyloliquefaciens EBN-TK3 (KCTC 12382BP) of the present invention against Rhizoctonia solani .
  • the left side is the control group
  • the right side is the experimental group.
  • FIG. 8 is Fusarium oxysporum f. Esp. Bacillus amyloliquefaciens EBN-TK3 (KCTC 12382BP) antifungal activity test results of the present invention against Fusarium oxysporum f.sp.raphani .
  • the left side is a control group and the right side is an experimental group.
  • FIG. 9 is Fusarium oxysporum f. Esp. Niveum ( Fusarium oxysporum f.sp. niveum
  • the present invention Bacillus amyloliquefaciens () Bacillus amyloliquefaciens ) EBN-TK3 (KCTC 12382BP) Antifungal test results.
  • the left side is the control group and the right side is the experimental group.
  • FIG. 10 shows the formation of clear zones by the siderophore activity of Bacillus amyloliquefaciens EBN-TK3 (KCTC 12382BP) of the present invention. (Left: 1 day later, Right: 3 days later).
  • Figure 11 shows the change in the sidelophore production of Bacillus amyloliquefaciens EBN-TK3 (KCTC 12382BP) in KB medium over time.
  • FIG. 13 shows whether a clear zone is formed by rhamnolipid generation of Bacillus amyloliquefaciens EBN-TK3 (KCTC 12382BP) strain.
  • the left side is the result in "MS + 2% glucose” medium and the right side is the result in "MS + 2% glycerol” medium.
  • Figure 14 shows the formation of a clear zone according to the production of biosurfactant (Biosurfactin) of the Bacillus amyloliquefaciens EBN-TK3 (KCTC 12382BP) strain in the blood agar (Blood agar).
  • Biosurfactin biosurfactant
  • EBN-TK3 Bacillus amyloliquefaciens EBN-TK3 (KCTC 12382BP) strain in the blood agar (Blood agar).
  • Left Strained culture of isolated strain after smearing Salmonella gallinarum . Clear zone at 3 o'clock is due to TK3.
  • Bordetella bronchiseptica was smeared, and the isolated strains were decultivated, and the clear zone at 9 o'clock was caused by TK3.
  • candidate strains were to be isolated.
  • the samples were to be separated into the nasal cavity of the pigs being raised. After rubbing the nasal passages five times with a sterile swab, the swabs were washed with sterile saline solution (0.85% NaCl) and the saline solution was diluted to 10 -6 steps.
  • 100 ⁇ l of each diluted saline solution was plated in LB agar medium (10 g sodium chloride, 5 g yeast extract, 10 g tryptone, 15 g agar powder, 1 L distilled water) and incubated at 30 ° C. in dark conditions. When colonies were formed, they were visually classified according to shape, color, or properties, and cultured in fresh LB agar medium. Subcultures were continued in LB agar medium until single colonies resulted in isolation of pure strains.
  • candidate strains were screened through an antimicrobial activity test (clear zone test). That is, candidate strains were finally selected by performing antibacterial and antifungal tests on the purely isolated strains.
  • a first screening 100 ⁇ l of each pathogenic bacterium Bordetella bronchiseptica and poultry pathogenic bacterium Salmonella gallinarum ( S. gallinarum ) were plated on LB agar medium, and then Candidate groups were selected by culturing purely isolated colonies and observing the presence of clear zone formation.
  • the candidate strains selected above were incubated in LB medium under conditions of 24 h, 160 rpm and 30 ° C. OD 600 was corrected to 1 and then cultured for 36h. Then, it centrifuged on 10000xg, 4 degreeC, and 20min conditions. The pH of the supernatant was adjusted to 2 with 6N HCl and left at 4 ° C for 30 minutes. Thereafter, the mixture was dissolved in 15 ml of methanol, and then centrifuged at 10000 ⁇ g, 20 min, and 4 ° C. After centrifugation, the pellets were collected and discarded.
  • the prepared samples were treated with various concentrations of Bordetella bronchiseptica and Salmonella gallinarum to confirm the degree of growth inhibition.
  • Figure 1 shows the growth inhibitory effect of Bordetella bronchiseptica , a causative agent of swine atrophic rhinitis caused by candidate strains.
  • ⁇ control, ⁇ : 10 ⁇ l / ml treatment, ⁇ : 30 ⁇ l / ml treatment, ⁇ : 50 ⁇ l / ml treatment).
  • Figure 2 shows the inhibitory effect of the growth of Salmonella gallinarum , the causative agent of poultry typhoid by the candidate strain.
  • ⁇ control, ⁇ : 10 ⁇ l / ml treatment, ⁇ : 30 ⁇ l / ml treatment, ⁇ : 50 ⁇ l / ml treatment).
  • Figure 3 shows the form of Bacillus amyloliquefaciens EBN-TK3 (KCTC 12382BP) strain isolated and deposited in the present invention
  • Table 1 shows the simple characteristics of the present invention strain.
  • Example 5 Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) Culture Characterization of EBN-TK3 (KCTC 12382BP) Strain]
  • Bacillus amyloliquefaciens of the present invention Bacillus Amyloliquefaciens
  • EBN-TK3 KCTC 12382BP
  • Figure 6 shows the Bacillus amyloliquefaciens according to the pH in the LB medium ( Bacillus amyloliquefaciens) EBN-TK3 (KCTC 12382BP) showed the change in viable cell count.
  • ⁇ is a result of pH 4
  • ⁇ is pH 6
  • ⁇ is pH 7
  • ⁇ is pH 8.
  • Bacillus amyloliquefaciens The viable cell count of EBN-TK3 (KCTC 12382BP) ranged from 8.5 to 8.8 Log CFU / ml.
  • Example 6 Bacillus amyloliquifaciens B. amyloliquefaciens ) Confirmation of antifungal efficacy of EBN-TK3]
  • the Bacillus amyloliquifaciens of the present invention ( B. amyloliquefaciens) Lizatonia solanie, a causative agent of crop leaf blight caused by EBN-TK3 Rhizoctonia solani ), Fusarium oxysporum F. Esp. Raphani ( Fusarium oxysporum f.sp. raphani ), Fusarium oxysporum f. Esp. Niveum ( Fusarium oxysporum f.sp. niveum To determine the inhibitory capacity of the fungus was tested for fungal activity.
  • FIG. 7 shows Ligutonia Solani ( Rhizoctonia solani
  • Bacillus amyloliquefaciens Bacillus amyloliquefaciens
  • Antifungal activity test results of EBN-TK3 (KCTC 12382BP) (left control group, the right experimental group).
  • 8 is Fusarium oxysporum f. Esp. Raphani ( Fusarium oxysporum f.sp. raphani
  • the present invention Bacillus amyloliquefaciens () Bacillus amyloliquefaciens ) EBN-TK3 (KCTC 12382BP) antifungal activity test results (left control group, the right experimental group).
  • 9 is Fusarium oxysporum f. Esp.
  • Niveum Fusarium oxysporum f.sp. niveum
  • Bacillus amyloliquefaciens Bacillus amyloliquefaciens
  • EBN-TK3 Bacillus amyloliquefaciens
  • Results of antifungal activity test (left side control group, right side experimental group).
  • Example 7 Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) Confirmation of Siderophore Production of EBN-TK3 (KCTC 12382BP) Strain]
  • siderophore Side to the fore (siderophore) are a type of antibiotic in order to produce by micro-organisms, including pathogenic bacteria and other microbes compete for quick growth and proliferation in a given environment, an iron ion (Fe 3+) and then combined with the withdrawal transport system (high- It is transported into cells using an affinity iron-transport system, and the iron ions (Fe 3+ ) are used to grow microorganisms.
  • Microorganisms produce sideropores in order to competitively use iron ions (Fe 3+ ), and growth of other microorganisms may be suppressed by many microorganisms that produce sideropores.
  • the siderophore activity was measured and quantified using the "chromee azurol S (CAS) agar plate assy” method.
  • step 3 After mixing the production solution of step I and the solution of step II, the mixture was mixed slowly so as not to foam. Thereafter, a hole of 5 mm diameter was inserted, and 70 ⁇ l of the strain of the present invention in culture in KB (King's B) medium was added thereto. Thereafter, the CAS solid medium changed from blue to yellow (clear zone) (cm) to measure the activity of the sidelopores.
  • 10 shows the results of the experiment. As shown in FIG. 10, formation of a clear zone was confirmed. 10 shows the formation of clear zones by the siderophore activity of Bacillus amyloliquefaciens EBN-TK3 (KCTC 12382BP) of the present invention. (Left: 1 day later, Right: 3 days later)
  • Bacillus amyloliquefaciens Bacillus amyloliquefaciens (Bcillus amyloliquefaciens ) EBN-TK3 (KCTC 12382BP) strain of the present invention in KB (King's B) medium prepared as shown in Table 2 was pre-corrected at 1.0 nm at 600 nm. .
  • the highway solution was prepared by adding 1 ml of 0.1 M FeCl 3 and 1 ml of 0.1 M potassium ferric cyanide to 100 ml of distilled water, using 0.1 M HCl solution.
  • FIG. Figure 11 shows the change in the sidelophore production of Bacillus amyloliquefaciens EBN-TK3 (KCTC 12382BP) in KB medium over time.
  • Bacillus amyloliquefaciens EBN-TK3 (KCTC 12382BP) of the present invention showed superior siderophore generation ability as compared to other strains having excellent siderophore generation ability. Therefore, the strain of the present invention was expected to have an excellent effect on the prevention, such as the spread, settlement of pathogenic fungi.
  • Example 8 Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens ) Confirmation of Rhamnolipid Production of EBN-TK3 (KCTC 12382BP) Strain]
  • Rhamnolipid is composed of glycolipid glycolipids and is generally a beta-hydroxydecanoic acid with one or two lipid chains in one or two rhomboses. In addition to having an antibacterial function, it has a function as a natural surfactant (Biosurfactant).
  • EBN-TK3 strains were pre-cultured in MS medium (Tables 4 and 5), and then inoculated in RBSSM medium (Table 6) with a diameter of 0.5 cm and inoculated to confirm the clear zone.
  • MS medium Tables 4 and 5
  • RBSSM medium Table 6
  • the rhamno lipid was measured through an Orcinol assay.
  • FIG. 12 shows the amount of rhamnolipid produced by Bacillus amyloliquefaciens EBN-TK3 (KCTC 12382BP) over time.
  • the present invention was compared with the production of the rhamnolipid of Bacillus amyloliquefaciens (Bcillus amyloliquefaciens) EBN-TK3 (KCTC 12382BP) compared with the conventional strain having excellent production capacity, the results are summarized in Table 7 below.
  • Bacillus amyloliquefaciens Bacillus amyloliquefaciens
  • EBN-TK3 KCTC 12382BP
  • Figure 13 MS + 2% Glucose
  • MS + 2% Glycerol solid media resulted in a zona pellucida caused by the activity of the TK3 strain of the invention.
  • the difference in rhamno lipid generation ability was confirmed depending on the carbon source.
  • Example 9 Bacillus amyloliquefaciens Bacillus amyloliquefaciens ) Generation of Surfactin of EBN-TK3 (KCTC 12382BP)]
  • Surfactin (M.W .: 1036 Da) is a cyclic lipopeptide biosurfactant containing heptapeptide (LLDLLDL) and beta hydroxy fatty acid.
  • This biosurfactant is a versatile biological agent with anti-mycoplasma, anti-viral, anti-fibrinogenic, anti-hypercholesterolemic and anti-inflammatory functions. It is known as a natural antibacterial substance having activity.
  • the blood agar was tested for the formation of a clear zone according to the production of biosurfactin of Bacillus amyloliquefaciens EBN-TK3 (KCTC 12382BP) strain of the present invention. .
  • EBN-TK3 and two pathogens S. gallinarum and B. bronchiseptica were preincubated in LB liquid medium and each pathogen was smeared into blood agar. Thereafter, EBN-TK3 was decanted at the same interval and cultured at 30 ° C. to determine whether clear zones were formed.
  • FIG. Figure 14 shows the formation of a clear zone according to the production of biosurfactant (Biosurfactin) of the Bacillus amyloliquefaciens EBN-TK3 (KCTC 12382BP) strain in the blood agar (Blood agar).
  • Biosurfactin biosurfactant
  • the left side of FIG. 14 is a culture of isolated strains after smearing Salmonella gallinarum ( Salmonella gallinarum ), the clear zone at 3 o'clock is due to TK3.
  • the Bordetella bronchiseptica was plated, and the isolated strains were decultivated, and the clear zone at 9 o'clock is caused by TK3.

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Abstract

La présente invention concerne l'EBN-TK3 de Bacillus amyloliquefaciens, dont on a vérifié qu'il possédait un excellent effet antibactérien d'inhibition de bactéries, spécialement, la Bordetella Bronchiseptica, qui est un organisme qui provoque la rhinite atrophique du porc, et la Salmonella gallinarum, qui est un organisme qui provoque la typhose aviaire, dans les industries du porc et de la volaille. On a également vérifié que l'EBN-TK3 de Bacillus amyloliquefaciens possédait un effet antifongique de suppression et d'inhibition du Rhizoctonia solani, qui est un organisme qui provoque le flétrissement de la gaine causant des maladies dans les cultures, Fusarium oxysporum f.sp. raphani, qui est un organisme qui provoque une flétrissure, et Fusarium oxysporum f.sp. niveum, qui est un organisme qui provoque l'excoriose.
PCT/KR2013/010377 2013-11-15 2013-11-15 Ebn-tk3 de bacillus amyloliquefaciens et agent antibiotique ou antifongique le comprenant WO2015072603A1 (fr)

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CN104336091A (zh) * 2014-09-28 2015-02-11 句容市农业技术推广中心 一种水稻纹枯病防治药剂
CN105733982A (zh) * 2016-02-24 2016-07-06 青岛农业大学 用于防治蓝莓毛色二胞枝枯病的解淀粉芽孢杆菌、菌剂及其制备方法
KR101773342B1 (ko) 2015-10-21 2017-09-01 대한민국 항진균 활성을 갖는 바실러스 아밀로리쿼페이션스 dh1 및 이의 용도
CN108676758A (zh) * 2018-07-06 2018-10-19 四川农业大学 一种巨大芽孢杆菌bm22及其液体制剂的制备和应用
CN110257309A (zh) * 2019-08-08 2019-09-20 广东省农业科学院蚕业与农产品加工研究所 一株芽孢杆菌sem-2、包括sem-2的复合菌剂、复合微生物肥及其应用

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CN104336091A (zh) * 2014-09-28 2015-02-11 句容市农业技术推广中心 一种水稻纹枯病防治药剂
KR101773342B1 (ko) 2015-10-21 2017-09-01 대한민국 항진균 활성을 갖는 바실러스 아밀로리쿼페이션스 dh1 및 이의 용도
CN105733982A (zh) * 2016-02-24 2016-07-06 青岛农业大学 用于防治蓝莓毛色二胞枝枯病的解淀粉芽孢杆菌、菌剂及其制备方法
CN105733982B (zh) * 2016-02-24 2019-12-03 青岛农业大学 用于防治蓝莓毛色二胞枝枯病的解淀粉芽孢杆菌、菌剂及其制备方法
CN108676758A (zh) * 2018-07-06 2018-10-19 四川农业大学 一种巨大芽孢杆菌bm22及其液体制剂的制备和应用
CN108676758B (zh) * 2018-07-06 2020-08-21 四川农业大学 一种巨大芽孢杆菌bm22及其液体制剂的制备和应用
CN110257309A (zh) * 2019-08-08 2019-09-20 广东省农业科学院蚕业与农产品加工研究所 一株芽孢杆菌sem-2、包括sem-2的复合菌剂、复合微生物肥及其应用

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