AU7709094A - New antifungal antibiotic, and new (bacillus sp.) microorganism which can produce the above antibiotic and its manufacturing method - Google Patents
New antifungal antibiotic, and new (bacillus sp.) microorganism which can produce the above antibiotic and its manufacturing methodInfo
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- AU7709094A AU7709094A AU77090/94A AU7709094A AU7709094A AU 7709094 A AU7709094 A AU 7709094A AU 77090/94 A AU77090/94 A AU 77090/94A AU 7709094 A AU7709094 A AU 7709094A AU 7709094 A AU7709094 A AU 7709094A
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- antibiotic
- manufacturing
- bacillus
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/16—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing two or more hetero rings
- C12P17/162—Heterorings having oxygen atoms as the only ring heteroatoms, e.g. Lasalocid
-
- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01S—DEVICES USING THE PROCESS OF LIGHT AMPLIFICATION BY STIMULATED EMISSION OF RADIATION [LASER] TO AMPLIFY OR GENERATE LIGHT; DEVICES USING STIMULATED EMISSION OF ELECTROMAGNETIC RADIATION IN WAVE RANGES OTHER THAN OPTICAL
- H01S5/00—Semiconductor lasers
- H01S5/30—Structure or shape of the active region; Materials used for the active region
- H01S5/34—Structure or shape of the active region; Materials used for the active region comprising quantum well or superlattice structures, e.g. single quantum well [SQW] lasers, multiple quantum well [MQW] lasers or graded index separate confinement heterostructure [GRINSCH] lasers
Description
New antifungal antibiotic, and new Bacillus sp. microorganism which can produce the above antibiotic and its manufacturing method
Technical Field
The present invention relates to new antifungal antibiotic which is described the below general formula(l), new Bacillus sp. microorganism which can produce the above antibiotic, and its manufacturing method.
wherein each R denotes -H, -OH, -NH2, alkyl group having 1 to 6 carbon atoms, allyl group having 5 to 20 carbon atoms, or alkylene group having 2 to 6 carbon atoms and, subscript 1, m, or n is an integer less than 3.
Background Art
After discovering of Penicillium notatum inhibiting growth of Staphylococcus aureus by Fleming on 1929, study concerning antibiotic had been progressed actively, and effective antibiotics against pathogenic microorganisms had been developed.
Conventionally, the study of antibiotic against procaryotic cells such as pathogenic microorganisms had been progressed but effective antibiotics against eucaryotic cells such as fungi had not been progressed up to now.
Study of antifungal antibiotic for cure and prevention of human and plant diseases was begun by Elizabeth and Brown. They isolated fungicidin showing antifungal activity against Candida albicans, Cyptococcus neoformans. And then griseofulvin which showed an activity against plant pathogens was discovered, and amphotericin B which had an antibiotic effect against yeast was developed by Gold et al. Pyrrolnitrin, which was produced by Pseudoαonas pyrrocinia with exhibiting activity against yeast and gram positive bacteria and showing little virulency, was discovered by Arima et al. Polyoxin discovered by Isono et al. had an activity against fungi causing rice diseases and had little harmful effect against men, beasts, fishes, and crops.
As an antibiotic against fungi causing rice blast disease, kasugamycin, which had a prevention effect on Pyricularia oryzae and penetrated easily to rice plant, was discovered by Umezawa et al. on 1965. Validamycin having a prevention effect on Pellicularia filamentosa was developed by Iwasa et al.
Thereafter, aculeacin which inhibited fungi or yeast and had a low toxicity, lipopeptin and neopeptin which had an activity against Pyricularia oryzae, and mycoversilin which had a high activity against filamentous fungi were developed.
As new nucleoside antibiotic, dapiramicin which was produced by Micromonospora and had a superb effect on Pellicularia filamentosa was developed as well as albopeptin A, fengycin, and octacosamicin. Among the above antibiotics, kasugamycin, validamycin, and polyoxin have been generally used as agricultural antibiotics, but production cost of these antibiotics is too high resulting in an economic problem.
The synthetic organic compounds have caused the side effects such as
agricultural poisoning and inflow of the residual chemical agents into soil. In the current work, most of the residual chemical agents of synthetic organic compounds in soil have caused an agricultural pesticide poisoning. Especially, destruction of the ecological system by means of pesticide toxicity has arisen many serious social problems including the environmental pollution. Therefore, various kinds of synthetic organic chemical compounds have been prohibited to use as pesticide because of carcinogenic possibility and the residual toxicity.
Disclosure of Invention
It is an object of the present invention to provide new antifungal antibiotic and its derivatives which have an excellent antifungal activity and do not cause the environmental pollution because of its non-toxicity.
Another object of the present invention is to provide new Bacillus sp. microorganism producing the above antibiotic and manufacturing method of antibiotic.
And, another object of the present invention is to provide composite which contains the above antifungal antibiotic as an effective ingredient.
In order to obtain other object, new Bacillus sp. microorganism which has a high antibiotic effect on plant fungi, especially Pyricularia oryzae and Pellicularia filamentosa, is isolated from the soil and identified. This microorganism is cultured in YS medium and centrifuged. The supernatant is extracted with ethyl acetate. Organic
layer is collected and evaporated. The active fraction is dissolved in methanol, followed by running silica gel column chromatography using methanol. Methanol is removed and then the residue is dissolved in dichloromethane:methanol (1:5) mixture and put on Sephadex LH-20 column chromatography. Solvent is removed and then the active fraction is collected and dissolved in water and lyophilized. Finally powdery antibiotic which has an excellent antifungal activity is obtained. Thus, the present invention has succeeded in providing a very simple and economical manufacturing method of new antibiotic.
Brief Description of Drawings
Fig.1 is an electron microscope photograph of Bacillus sp. SY-414, Fig.2 is an electron microscope photograph of spore formed by the invented Bacillus sp. SY-414, Fig.3 is the graph representing the cell growth and the antibiotic production according to culture time, Fig.4 is the graph representing the antibiotic production according to culture temperature, Fig.5 is the graph representing the antibiotic production according to initial pH, Fig.6 is the graph representing the cell growth and the antibiotic activity against Pyricularia oryzae in NB and YS media, Fig.7 is the graph representing the cell growth and the antibiotic activity against Pellicularia filamentosa in NB and YS media, Fig.8 is the photograph of inhibition zone against Pyricularia oryzae, Fig.9 is the photograph of inhibition zone against Pellicularia filamentosa, Fig.10 is the photomicrograph of the crystallized antibiotic obtained
in example 4, Fig.11 is the UV spectrum of the antibiotic obtained in example 4, Fig.12 is the IR spectrum of the antibiotic obtained in example 4, Fig.13 is the *H NMR spectrum of the antibiotic obtained in example 4, Fig.14 is the 13C NMR spectrum of the antibiotic obtained in example 4, Fig.15 is the mass spectrum of the antibiotic obtained in example 4, Fig.16 is the HPLC sugar analysis of the antibiotic obtained in example 4.
Best Mode for Carrying Out the Invention
The embodiments of the present invention will be described in detail as follows.
The antifungal antibiotic of the present invention (hereinafter referred to "Trihexocin") represents the below general formula(l).
wherein each R denotes -H, -OH, -NH2, alkyl group of Ci - Cε, allyl group of C5- C20, or alkylene group of C2- Cβ and, subscript 1, m, or n is an integer less than 3.
The antibiotic of the present invention showed a needle shape and a low Rf value in general solvents, and its melting points was 127-1281C. As the results of its positive color reaction with alkaline permanganate, 10% sulfuric acid and iodine including *H NMR analysis, it was elucidated that new antibiotic contained the sugar moiety. 13C NMR analysis showed that it did not contain any aromatic compounds. From all the above results, we decided the antibiotic of the present invention as the new material, and named it as "Trihexocin".
Trihexocin of the present invention had an activity at the concentration of 50-100 tg/ml against Pyricularia oryzae and Pellicularia filamentosa, but did not against gram positive and negative bacteria, and yeast. The antibiotic activity against Pyricularia oryzae was stable at pH 6, 7 and 8, but represented 68* at pH 5 and 70* at pH 10. The antibiotic activity against Pellicularia filamentosa was stable at pH 6,7 and 8, but represented 0* at pH 5, 82* at pH 9 and 0* at pH 10.
And also, the antibiotic activity against Pyricularia oryzae was stable at 301C and 40 X but represented 80* at 501C and 60* at 601. Against Pellicularia filamentosa, the antibiotic activity was stable at 30C and 401C but represented 70* at 501C, and disappeared above 60 TC.
With the above results, trihexocin of the present invention showing an antifungal antibiotic is the material having sugar moiety, and its derivatives and salts having the biological activity are easily obtained.
Trihexocin is produced by Bacillus sp. SY-414 (KCCM-10043) and its manufacturing method will be described in detail as follows.
Soil collected from the Chunchon area, Kangwon-do was suspended in distilled water and cultured in plate and then the microorganisms were isolated.
The isolated strain was inoculated in nutrient broth (NB medium: meat extract 0.3*, peptone 0.5*) and cultured at 30 : for 3 days.
The culture broth was centrifuged at 12,000xg for 20 minutes and the antibiotic activity of supernatant was detected by paper disc method using Pyricularia oryzae IF0 30517 and Pellicularia filamentosa IF0
8985 as a test microorganism.
The isolated strain which showed the highest activity against the above test microorganisms was selected and identified.
The above microrganism was similar to Bacillus licheniformis but confirmed as a new microorganism, thus it was named Bacillus sp. SY-414 and deposited as deposit number KCCM-10043 at Korea Federation of Culture Collections (KFCC) on September 14, 1993.
Bacillus sp. SY-414 of the present invention was identified by general identification methods that were commonly used and, the results were in Table 1-5.
Table 1. Morphological properties of strain SY-414
Shape Rod
Cell size 1.5im x 3.6/im
Motility Positive
Gram stain Positive
Fragella Positive
Spore shape Ellipsoidal
Spore position Central
Sporangium swollen Negative
As shown in Table 1, Bacillus sp. SY-414 of the present invention was rod and motile, and the cell size was 1.5/M x 3.6 μ as shown in Figure 1. The spore of Bacillus sp. SY-414 was ellipsoidal and located in central(Figure 2) and the sporangium was unswellen.
Table 2 Cultural characteristics of strain SY-414
Form Circular
Surface Smooth
Edge Undulate
Elevation Umbonate
Opacity Opaque
Color Creamy
Brilliancy Glistening
Nutrient broth (30T , 1-2 days) :
Growth abundant, turbid with pellicle and sediment
Table 3. Physiological properties of strain SY-414
Temperature range for growth 15-50°C pH range for growth 3-10
NaCl tolerance for growth < 6%
Catalase +
Oxidase -
Urease +
Lipase(Tween 80) - β-Galactosidase +
Arginine dihydrolase -
Phenylalanine deaminase -
Hydrolysis of: Starch +
Casein +
Cellulose -
Esculin +
Indole production -
H∑S production on TSI agar -
Levan formation from sucrose +
NH3 production from peptone +
NH3 production from arginine +
Gelatin liquefaction +
Utilization of citrate -
Utilization of propionate +
Methyl red test +
Voges-Proskauer reaction +
Nitrate reduction +
Denitrification +
Action on milk : Coagulation -
Peptonization +
Hemolysis, human blood +
0-F test Fermentative
Degradation of tyrosine ~
Positive Negative
Table 4. Sugar uti l ization by strain SY-414
Sugar Utilization
+
Arabinose
+
Cellobiose
Cellulose -
Dextrin +
Fructose +
Galactose +
Glycerol +
Inositol
+
Inulin
+ Lactose
+
Mannitol
+
Mannose
Raffinose +
Soluble starch +
Sorbitol +
Sucrose +
Utilized Not Utilized
Table 5. Sugar fermentation by strain SY-414
Sugar Acid Gas
Arabinose _ .__.
Celloblose + -
Cellulose - -
Dextrin - -
Fructose + -
Galactose - -
Glycerol + -
Inositol - -
Inulin - -
Lactose - -
Mannitol + -
Mannose + -
Raffinose + -
Soluble starch - -
Sorbitol - -
Sucrose + —
Positive Negative
As shown in the above tables, the growth temperature of Bacillus sp. SY-414 was within the range of 15-50TC, the pH range was 3-10, and it was able to grow up to 6* salt concentration. Bacillus sp. SY-414 represented catalase positive and oxidase negative, utilized propionate and hydrolyzed starch. And also, as the results of sugar
utilization, Bacillus sp. SY-414 utilized most of sugars except cellulose for the growth of microorganism. As the results of sugar fermentation, an acid was produced from some sugars but a gas was not produced from all the sugars.
Bacillus sp. SY-414 was inoculated in 20ml nutrient broth and cultured at 30"C for 48-96 hours. The seed culture was prepared under the above conditions.
Figure 3 represented the growth of Bacillus sp. SY-414 and the antibiotic production according to the culture time in nutrient broth. The growth of Bacillus sp. SY-414 reached the stationary phase after 4-days cultivation and the production of the antibiotic was the maximum at the same culture time and decreased after 4-days of cultivation.
To measure the antifungal activity of Bacillus sp. SY-414, Pyricularia oryzae IFO 30517 and Pellicularia filamentosa IFO 8985 were selected as a test microorganism, and the activity of the antibiotic was measured by the cup method.
Potato dextrose agar (PDA) plate was prepared with the test microorganism which was layered in PDA soft agar plate. The cup (innner diameter 6mm, outer diameter 8mm, height 10mm) was put on the above plate and the culture supernatant was added into the cup. The plate was incubated at 30O for 48 -72 hours, and then inhibition zone was measured.
To examine the antibiotic production by Bacillus sp. SY-414 according to culture temperature, the seed culture (1.0*,v/v) was inoculated in 20ml nutrient broth and cultured at various temperatures for 96 hours. The activity of supernatant was measured.
Figure 4 represented the production of the antibiotic was the maximum at 30"C.
And, to examine the antibiotic production by Bacillus sp. SY-414 with the various initial pH, medium pH was adjusted by HC1 and NaOH. The seed culture (1.0*, v/v) was inoculated and cultured at 30O for 96 hours.
As shown in Figure 5, the activity was the maximum at the initial pK 8.
To investigate the effect of carbon sources on the antibiotic production by Bacillus sp. SY-414, the basic medium containing carbon source (1.0*. w/v) was cultured at 30"C for 96 hours. Table 6 described the results of the antibiotic production.
Table 6. Effect of carbon sources on the antibiotic production
Inhibition zone size (mm)
Carbon source
Pyricularia oryzae Pellicularia filamentosa
Control 14.0 12.8
Dextrin 11.2 8.0
Glucose 14.3 12.5
Mannito1 15.5 13.5
Sucrose 13.9 11.5
Fructose 14.2 12.3
Lactose 13.7 11.3
Raffinose 13.8 11.8
Cellulose 14.0 9.7
Galactose 13.5 10.0
Maltose 13.2 12.0
Soluble starch 16.5 14.0
Xylose 14.1 12.2
As shown in Table 6, the production of the antibiotic was the best in the case of soluble starch. The optimum concentration of starch was 3.0* (w/v).
To examine the effect of nitrogen sources on the antibiotic production by Bacillus sp. SY-414, organic and inorganic nitrogen (1.0*. w/v) were added to basic medium. The seed culture was inoculated and cultured at 30 ! for 96 hours and the antibiotic production of supernatant was measured and the results were described in Table 7.
Table 7. Effect of nitrogen sources on the antibiotic production
As shown in Table 7, organic nitrogen sources including beef extract was fairly good for the production of the antibiotic. The antibiotic production was increased remarkably when yeast extract was added and 0.8* (w/v) yeast extract was the optimal concentration. However, all inorganic nitrogen sources had no effect on the production of the antibiotic.
The effect of mineral sources on the antibiotic production by Bacillus sp. SY-414 was investigated. The mineral source (0.1*. w/v) was added and the seed culture (1.0*. v/v) was inoculated and cultured at 301C for 96 hours. The antibiotic production was measured and the results were described in Table 8.
Table 8. Effect of mineral sources on the antibiotic production
As described in Table 8, KCl was the best mineral source for the production of the antibiotic. The antibiotic production was not considerably affected with MgS04 «7H2θ, CaCl2, NaCl, or K2HPO.1. The others had no effect on the production of the antibiotic. The optimum concentration of KCl was 0.2* (w/v).
The optimal broth for the production of the antibiotic by Bacillus sp. SY-414 as the above was designated as 'YS medium'.
Through culturing Bacillus sp. SY-414 at 30C in YS and NB media, pH change, the growth, and the production of the antibiotic were examined (Figure 6 and 7).
When Bacillus sp. SY-414 was cultured in YS and NB media, the growth reached the stationary phase at 3 and 4 days, respectively. In each medium, the production of the antibiotic was the maximum at the same
culture time.
The antibiotic production in YS medium was twice in NB medium and the range of pH was within 1.0.
Figure 8 and 9 represented the size of inhibition zone against
Pyricularia oryzae and Pellicularia filamentosa in YS medium at the optimal culture conditions.
The activity of the antibiotic of the present invention showed 24mm inhibition zone against Pyricularia oryzae and 21mm inhibition zone against
Pellicularia filamentosa.
As described in the above, Bacillus sp. SY-414 (KCCM-10043) was cultured at 301C for 96 hours in YS medium and then centrifuged at
12,OOOxg for 20 minutes and the supernatant was extracted with the equal volume of ethyl acetate.
The organic layer was collected and removed by rotary evaporator under the reduced pressure.
The condensed active fraction was collected and dissolved in water and lyophilized.
The above lyophilized powder was dissolved in methanol and loaded in silica gel column, and eluted with methanol.
The active fraction was concentrated in vaccuo and dissolved in water and lyophilized.
The above lyophilized powder was dissolved in dichloromethane methanol(l:5) mixutre and Sephadex LH-20 column chromatography was carried out. Elution solvent was a dichloromethane:methanol (1:5) mixture and the solvent was removed.
The active fraction was dissolved in water and lyophilized.
The above powder was dissolved in methanol and stored at 41C, and then the crystallized antibiotic was obtained.
As described in the above, for the antibiotic production by Bacillus sp. SY-414 of the present invention, soluble starch, mannitol, xylose and glucose could be used as carbon sources and beef extract, yeast extract, peptone and soybean meal could be used as nitrogen sources. And NaCl, KCl, MgS04 and KH2PO4 could be used as mineral sources. Preferably, medium containing 3* soluble starch, 0.8* yeast extract and 0.2* KCl was the optimum for the antibiotic production. The optimal condition was 25-361C for 48-96 hours with shaking.
The following examples describe the present invention in detail, but the present invention is not limited within the following examples.
Example 1 : Screening
Soil collected from Chunchon area was suspended in distilled water, streaked onto nutrient plate (beef extract 0.3*. peptone 0.δ*. agar 1.2*), and incubated at 301C for 1-2 days.
The above isolated microorganism was inoculated in 10ml nutrient broth and cultured at 30*C for 76 hours, and then centrifuged at 12,000xg for 20 minutes. The activity of supernatant was measured. With Pyricularia oryzae IFO 30617 and Pellicularia filamentosa IFO 8986 as a test microorganism, Bacillus sp. SY-414 represented the highest activity against the two test microorganisms.
Example 2 : Seed culture
The isolated microorganism was cultured in 20ml nutrient broth at 30 1C for 72 hours. The cell at the stationary phase was used as the seed of the present invention.
Example 3 : Fermentation
3.0* soluble starch, 0.8* yeast extract, and 0.2* KCl. Fermentation was carried out at 30 . for 72 hours with shaking. The culture broth was centrifuged at 12.000xg for 20 minutes. The supernatant was used for the purification.
Example 4 : Purification
The supernatant obtaind in example 3 was extracted with the equal volume of ethyl acetate. The organic layer was collected and evaporated under the reduced pressure. The residue was dissolved in water and lyophilized.
The lyophilized powder was dissolved in methanol and silica gel column chromatography was carried out. The active fraction was eluted with methanol, and then concentrated in vacuo. The concentate was dissolved in water and lyophilized.
The lyophilized powder was dissolved in dichloromethane : methanol (1:6) mixture and loaded in Sephadex LH-20 column. The solvent was removed and dissolved in water and lyophilized. The lyophilized powder was dissolved in methanol and stored at AX_ . lOOmg of the purified trihexocin was obtained from 201 culture. Figure 10 represented the photograph of the purified crystalline trihexocin.
Example δ : Characteristics of the antibiotic
The above trihexocin was dissolved in water and the thin layer chromatography (TLC) was carried out using n-Bu0H:Ac0H:Hz0 (4:l:δ) as a developing solvent. Single spot appeared through alkaline potassium permanganate reaction. Rf value of trihexocin with TLC was measured by spraying alkaline
- 20 -
potassium permanganate.
Table 9 represented the Rf value of the antibiotic of the present invention.
Table 9. Rf value of the antibiotic
Solvent system Rf value
0.00
CβH6 = EtOAc (1:1)
0.00
CβHe = EtOAc = MeOH (2=2:1)
0.80
MeOH : Acetone : HzO (4:2=1)
0.15 n-BuOH = AcOH : HzO
(4:1:5, upper phase) n-BuOH = AcOH = H20 (3=1 = 1) 0.34
CHC13 MeOH = Hz0 (4=1=1.25) 0.00
MeOH = H20 = AcOH (16=3=1) 0.78
Benzene = BuOH = HzO (9=1:10)
0.86
As shown in Table 9, 0.78 Rf value was shown in MeOH : H20 : AcOH (16:3:1) mixture solvent and 0.86 Rf value in benzene = BuOH = H2O (9=1=10) mixture solvent. Trihexocin was dissolved well in polar solvents such as water, methanol, and ethanol but, was not dissolved in nonpolar solvents such as chloroform, benzene, and hexane.
The color reactions of the antibiotic spotted in TLC were observed by spraying reagents. Table 10 showed the results of the color reactions.
Table 10. Color reactions of the antibiotic
Color reaction Result
Positive
Iodine
Negative
Ninhydrin
Negative
Biuret
Ehrlich Negative
Sakaguchi Negative
10* H2S04 Positive
Alkaline KMn04 Positive
Ferric chloride Positive
Dragendorff
Negative
As described in Table 10, the antibiotic of the present invention showed a positive reaction with alkaline potassium permanganate, 10* sulfuric acid and iodine. Thus it was concluded that the antibiotic contained the sugar moiety in structure.
Melting point was within the range of 127-128TC. The antibiotic obtained in example 4 was dissolved in water and measured to get UV absorption spectrum using UV/Vis spectrophotometer. Any absorption band did not appear as shown in Figure 11. By means of IR spectrophotometer, absorption bands appeared at 3660-3300"1, 2970-1700 cm"1, 1694cm"1, and 1422cm"1 as shown in Figure 12. The signal of aromatic compound near 7.5 ppm did not appear in JH NMR spectrum and the signals of sugar at 70-78 ppm were shown in 13C NMR spectrum, as shown in Figure 13 and 14.
And also, mass spectrum of trihexocin after acetylation was shown in Figure lδ. The molecular weight of the antibiotic obtained example 4 was 604.
Example 6 = Hydrolysis of the antibiotic
The antibiotic (lOmg) obtained in example 4 was hydrolyzed at 110*0 for 10-12 hours with 0. IN HC1. The hydrolysate was analyzed on HPLC to identify the sugar components. As shown in Figure 16, the antibiotic consisted of fructose only. Thus, this antibiotic was named as trihexocin.
As shown in results of the above, antibiotic obtained in example 4 was described as follow,
wherein a is CH20H ; b is (0H)2 ; and b ' is (0H)ι.
Example 7 = Activity of the antibiotic
The inhibition zones against Pyricularia oryaze varying antibiotic concentration were 9.8mm at δOμg/ml, 14.6mm at lOO/zg/ml, 19.5 mm at 200ig/ml, and 23.3mm at 400/ig/ml and, against Pellicularia filamentosa, 12.3mm at lOOjug/ml, 16.2mm at 200ig/ml and 20.2mm at 400i g/ml.
Antimicrobial spectrum was tested through the following paper disc agar diffusion method against the test microorganisms to observe the antibiotic activity of the present invention.
In the case of bacteria and Candida, 0.1ml of test microorganism was
inoculated in soft agar (agar 0.8*) and overlayed. In the case of fungi, the spores were suspended homogeneously and 0.1ml suspension was inoculated in PDA (Potato Dextrose Agar) and overlayed.
The paper disk (lOOjug/ml) was put on plate and incubated at 30 ! for
24-48 hours. The formation of inhibition zone was observed.
Table 11 represented the antimicrobial spectrum of the antibiotic.
The antibiotic of the present invention did not have an effect on gram positive and negative bacteria, and yeast, but showed an activity against fungi.
Example 8 : Stability of the antibiotic
To observe the pH stability of the antibiotic of the present invention, the antibiotic (lOOμg/ml) was treated with 0.06M sodium citrate-HCl buffer, potassium phosphate buffer, and ammonium hydroxide-ammonium chloride buffer for two hours.
The antibiotic solution was added into cup and incubated at 30*0 for
48 hours. The size of inhibition zone was compared.
The antibiotic was stable at pH 6-8 against Pyricularia ozyzae and
Pellicularia filamentosa.
As the above, the antibiotic activity was stable around the neutral
PH.
To examine the temperature stability of the antibiotic of the present invention, the antibiotic solution (100/g/ml) was treated for 1 hour at 30 -2 , 401C, δOlC, 60TC and 70 O. The antibiotic solution was added into cup and incubated at 30"C for 48 hours. The size of inhibition zone was measured.
The activity of the antibiotic against Pyricularia oryzae was stable at 301C-40 ; but represented 80* at 50"C and 60* at 60"C. The activity
of the antibiotic against Pellicularia filamentosa was stable at 30*0 -401C but represented 70* at 50"C.
Example 9 : In vivo test of the antibiotic
The antimicrobial activity of the present invention was observed through the following method.
Rice plant which had become 3-4 leaves was infected by Pyricularia oryzae or Pellicularia filamentosa, and then treated with the antibiotic. The effect of the antibiotic was observed for 7 and 14 days with comparison to the control. The infected rice plant was grown well at the concentration of 50-100 g/ml.
Example 10 = Toxicity of the antibiotic
To examine the toxicity of the antibiotic of the present invention, the antibiotic was prescribed to 5 ICR mice (age: 6-8weeks) for intraperitoneal injection or oral administration. After two weeks, all the tested mice were survived, indicating the survival of 100*. It was proved that this antibiotic had no toxicity.
The antibiotic of the present invention can be used alone or in a mixture with fillers, conventionally in the field of the present invention. The composite as an effective ingredient can be manufactured.
Claims (11)
1. New antifungal antibiotic represented below the general formula(I)
wherein each R denotes -H, -OH, -NH2, alkyl group of Ci - Cβ , allyl group of C5 - C20, or alkylene group of C2 - Cβ, and subscript 1, m, or n is an integer less than 3.
2. New antifungal antibiotic as defined in claim 1, wherein formula(I), all the R groups are -OH, subscripts 1, m, and n are 2, 1, and 2, respectively.
3. New Bacillus sp. SY -414 (KCCM-10043) having an antifungal activity.
4. A method for manufacturing the antifungal antibiotic as described in the below, culturing Bacillus sp. SY-414 with shaking in liquid medium and centrifuging; extracting the supernatant with ethyl acetate and collecting organic layer and removing solvent : collecting the active fraction and removing solvent and dissolving in solvent and carrying out the first column chromatography ; collecting the active fraction and removing solvent and dissolving in solvent and carrying out the second column chromatography ; collecting the active fraction and removing solvent and dissolving in water and lyophilizing ; and dissolving in methanol and storing at 41C and obtaining the crystalline antibiotic.
5. The method for manufacturing the antifungal antibiotic as defined in claim 4, wherein the liquid medium contains one or more materials selected from soluble starch, glucose, xylose, or mannitol as carbon sources.
6. The method for manufacturing the antifungal antibiotic as defined in claim 4, wherein the liquid medium contains one or more materials selected from beef extract, yeast extract, peptone or soybean meal as nitrogen sources.
7. The method for manufacturing the antifungal antibiotic as defined in claim 4, wherein the liquid medium contains one or more materials selected from sodium chloride, potassium chloride, magnessiu sulfate, calcium chloride or potassium phosphate as mineral sources.
8. The method for manufacturing the antifungal antibiotic as defined in claim 4, wherein the fermentation is carried out at 25-35TC, pH 6-9 for 48-96 hours.
9. The method for manufacturing the antifungal antibiotic as defined in claim 4, wherein the first chromatography is silica gel column chromatography and the second chromatography is Sephadex LH-20 column chromatography.
10. The metohd for manufacturing the antifungal antibiotic as defined in claim 9, wherein the solvent is methanol for silica gel column chromatography, and dichloromethane methanol (1:5) mixture for Sephadex LH-20 column chromatography.
11. The composite which contains the antibiotic of claim 1 as an effective ingredient.
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR9318746 | 1993-09-17 | ||
| KR930018746 | 1993-09-17 | ||
| KR1019940019211A KR970010603B1 (en) | 1993-09-17 | 1994-08-03 | New antifungal antibiotic and new bacillus sp. microorganism which can produce the above antibiotic and its preparing method |
| KR9419211 | 1994-08-03 | ||
| PCT/KR1994/000123 WO1995007997A1 (en) | 1993-09-17 | 1994-09-15 | New antifungal antibiotic, and new bacillus sp. microorganism which can produce the above antibiotic and its manufacturing method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| AU7709094A true AU7709094A (en) | 1995-04-03 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU77090/94A Abandoned AU7709094A (en) | 1993-09-17 | 1994-09-15 | New antifungal antibiotic, and new (bacillus sp.) microorganism which can produce the above antibiotic and its manufacturing method |
Country Status (7)
| Country | Link |
|---|---|
| JP (1) | JPH09510862A (en) |
| KR (1) | KR970010603B1 (en) |
| CN (1) | CN1135238A (en) |
| AU (1) | AU7709094A (en) |
| CA (1) | CA2171947A1 (en) |
| GB (1) | GB2296716A (en) |
| WO (1) | WO1995007997A1 (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1055310C (en) * | 1995-06-22 | 2000-08-09 | 华中农业大学 | Supper toxic strain YBT-1520 of thuricin brood cell and its zymosis process and products |
| KR100385152B1 (en) * | 2001-03-14 | 2003-05-23 | 씨제이 주식회사 | Process for the purification of capreomycin |
| KR100514430B1 (en) * | 2002-12-12 | 2005-09-14 | 대한민국 | Isolation method of secondary product having weeding efficacy against white clover released from culture floid of sclerotinia trifoliorum bwc98-105 |
| WO2007016680A2 (en) * | 2005-08-02 | 2007-02-08 | University Of Missouri Board Of Curators | Phage-display plant defense peptides directed against phakopsora pachyrhizi and uromyces appendiculatus |
| EP2098536A1 (en) | 2008-03-05 | 2009-09-09 | 4-Antibody AG | Isolation and identification of antigen- or ligand-specific binding proteins |
| CN110954625A (en) * | 2019-12-21 | 2020-04-03 | 潍坊科技学院 | Method for detecting antibiotic residues in soil |
| JP2022094481A (en) * | 2020-12-15 | 2022-06-27 | 大阪瓦斯株式会社 | Method for producing hydroxyalkanoic acid crystal and crystalline polymorph of hydroxyalkanoic acid |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3885655T2 (en) * | 1987-05-30 | 1994-06-16 | Kaken Pharma Co Ltd | Antibiotic 6270B, process for its preparation and its use as an anticoccidial agent and feed additive. |
| HU203789B (en) * | 1988-02-08 | 1991-09-30 | Pfizer | Process for producing polycyclic acidic ether-type antibiotica of anticoccidial and growth-stimulating activity |
| DD270542A1 (en) * | 1988-04-12 | 1989-08-02 | Adw Ddr | PROCESS FOR THE PREPARATION OF 26-DESOXYLAID LOMYCIN |
| US5278053A (en) * | 1991-09-16 | 1994-01-11 | Eli Lilly And Company | Method of producing a polyether antibiotic from actinomadura fibrosa sp. nov. NRRL 18348 and actinomadura sp. NRRL 18880 |
-
1994
- 1994-08-03 KR KR1019940019211A patent/KR970010603B1/en not_active IP Right Cessation
- 1994-09-15 WO PCT/KR1994/000123 patent/WO1995007997A1/en active Application Filing
- 1994-09-15 CA CA002171947A patent/CA2171947A1/en not_active Abandoned
- 1994-09-15 CN CN94193799A patent/CN1135238A/en active Pending
- 1994-09-15 GB GB9605096A patent/GB2296716A/en not_active Withdrawn
- 1994-09-15 AU AU77090/94A patent/AU7709094A/en not_active Abandoned
- 1994-09-15 JP JP7509095A patent/JPH09510862A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| KR970010603B1 (en) | 1997-06-28 |
| GB9605096D0 (en) | 1996-05-08 |
| JPH09510862A (en) | 1997-11-04 |
| KR950008689A (en) | 1995-04-19 |
| CA2171947A1 (en) | 1995-03-23 |
| GB2296716A (en) | 1996-07-10 |
| WO1995007997A1 (en) | 1995-03-23 |
| CN1135238A (en) | 1996-11-06 |
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