JPS6160840B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention is a novel antibiotic planothiocin A.
(planothiocin A) and its production method. The antibiotic planothion A of the present invention is as follows:
This is a novel sulfur-containing peptide antibiotic having the physicochemical and biological properties shown in the table.

【table】

【table】

【table】

[Table] Due to the above properties, the antibiotic planothiocin A of the present invention is recognized as a sulfur-containing peptide antibiotic, and known similar sulfur-containing peptide antibiotics include thiostrepton, thiopeptin thiopeptins, sulfomycins, siomycins,
A-59, pepthiomycins, thermothiocin, sporangiomycins, althiomycin, A-7413, nosiheptide, L-13365, actinothiocin ), A-10947 are known, and among these, nocyheptide is a substance having the same amino acid composition as the antibiotic planothiocin A. Regarding the antibiotics pranothiocin A and nosiheptide, nosiheptide is
416 mμ (E ¹ % ₁ cm = 124) and 440 mμ (E ¹ % ₁ cm
=
104.8), but the antibiotic planothiocin A has no absorption, and nosiheptide is a pale yellow crystal, and the antibiotic planothiocin A is a colorless or white crystal. The Rf values are clearly different [Rf of nosiheptide CHCl ₃ :methanol (3:1) = 0.87, ethyl acetate:methanol:water (10:2:1) Rf = 0.71], and as mentioned above, the antibiotic pranothiocin A does not match any known sulfur-containing peptide antibiotics, and therefore, the antibiotic planothiocin A of the present invention is recognized as a novel substance. The novel antibiotic planothiocin A-producing actinomycete of the present invention is an actinomycete isolated from the soil of a chili pepper field in Mihama-cho, Mikata-gun, Fukui Prefecture, and was identified as belonging to the genus Actinoplanes.・It was decided to call it P.A12526 (Microorganism accession number notification microorganism accession number "Feikoken Bacteria No. 5063 FERM-P No.
5063”). The mycological properties of this bacterium are described below. [] Morphological properties Starch/inorganic salt agar medium, 30℃, 10-14
The findings observed after culturing for days are as follows. The basal hyphae are wavy or curved and branched.
It has a diameter of 0.8 to 1.0μ and does not form aerial mycelia.
The sporangium grows on a short sporangial stalk produced from the basal hyphae. Forms numerous sporangiospores. Sporangium spores are approximately spherical or subspherical, with a diameter of 1
It is ~1.5μ, has many flagella, and is a zoospore that moves in water. [] Composition of Diaminopimelic Acid Diaminopimelic acid was detected as a main component in the meso form by whole cell analysis, and a small amount of the hydroxy form was detected. []Culture properties The findings observed after culturing on various media at 30°C for 14 days are shown in Table 2. On many media, small amounts of undeveloped aerial mycelium were observed to form. Colors are shown in the Color harmony manual 4th edition (1958) (Container Corporation of
America) color classification.

【table】 [] Physiological properties The physiological properties are as follows. (1) Assimilation of carbon sources

【table】

【table】

[Table] −: Negative
(2) Growth temperature range: 7 to 40℃ (3) Skimmed milk: Peptonization; positive, coagulation: positive (4) Production of melanin-like pigment Tyrosine agar medium: negative Peptone yeast iron agar medium; positive, (5 ) Starch hydrolysis: Positive (6) Gelatin liquefaction: Positive (7) Cellulose decomposition: Negative (8) Casein hydrolysis: Positive (9) Tyrosine decomposition: Positive (10) Xanthine decomposition: Negative (11 ) Decomposition of hypoxanthine: Negative (12) Production of hydrogen sulfide: Positive (13) Reduction of nitrate: Positive As mentioned above, the A12526 strain of this bacterium has a spherical sporangium produced from basal hyphae with branches. It forms sporangia that are spherical or subglobular, have flagella and are motile, and mesodiaminopimelic acid can be detected in the bacterial cells. Judging from these properties, it is recognized as belonging to the genus Actinoplanes. Next, in carrying out the present invention and obtaining the antibiotic planothiocin A, the above-mentioned actinoplanes
Collection from a culture using S.P. A12526 strain is preferred. In addition, the above-mentioned bacteria are one example of the bacteria that can be used, and in addition to this bacteria, all bacteria that produce the antibiotic planothiocin A (hereinafter simply referred to as plaquethiocin A) can be used in the present invention, such as Actinoplanes spp. Planothiosin A-producing bacteria or mutant strains thereof are used. Next, in carrying out the present invention, for example, a planothiocin A-producing bacterium belonging to the genus Actinoplanes is cultured by a conventional method for producing antibiotics. The form of culture may be liquid culture or solid culture, and from an industrial perspective, it is advantageous to perform deep aeration agitation culture. As the nutrient source for the medium, a wide variety of nutrients commonly used for culturing microorganisms can be used. The carbon source may be any assimilable carbon compound, such as glucose, sucrose, lactose, maltose, starch, molasses, and glycerin. The nitrogen source may be any available nitrogen compound, such as corn steep liquor, soybean flour, cottonseed flour, wheat gluten, peptone, meat extract, yeast extract, dried yeast, casein hydrolyzate, ammonium salt, Nitrates and the like are used, and salts such as phosphates, carbonates, magnesium, sulfates, calcium, potassium, sodium, copper, iron, zinc, manganese, cobalt, etc. are used as necessary. The culture temperature can be changed as appropriate within a range that allows the bacteria to grow and produce planothiocin A, but is particularly preferably 25 to 30°C. The culture time varies depending on the conditions, but it is sufficient to determine the time when the maximum yield of planothiosin A is reached and terminate the culture at an appropriate time.
It usually takes about 2 to 4 days. Planothiosin A is collected from the culture of planothiosin A-producing bacteria obtained in this way, but most of the planothiosin A is accumulated within the bacterial cells, and some is also present in the supernatant liquid. . Furthermore, to give an example of a method for separating and purifying planothiosin A, first, planothiocin A-producing bacteria are cultured as described above, and bacterial cells containing planothiosin A are separated from the culture obtained by overseparation using a drum filter or by centrifugation using a basket centrifuge. A wet bacterial cell and a culture solution are obtained by separating the cells using a method. This culture solution contains a small amount of planothiocin A, and it may be extracted and separated from this, but preferably it is extracted and separated from the bacterial cells. Next, when extracting and separating and collecting the wet bacterial cells, for example, acetone is added to the wet bacterial cells for extraction, and the acetone extract is concentrated under reduced pressure. The syrup was collected and concentrated under reduced pressure to obtain a brown oily syrup, and hexane was added to this to obtain a brown precipitate containing the target substance, which was collected and further dissolved in a chloroform-methanol mixture and concentrated under reduced pressure. The chloroform is removed,
When it becomes a methanol solution, it produces a grayish white precipitate,
This is collected to obtain a planothiocin A-containing mixture. This crude product is then treated with e.g. chloroform:
Dissolve and charge with the same solvent on a silica gel column prepared with methanol:acetic acid (13:1:0.1), elute with this solvent, and collect the active fraction. During elution, an active fraction containing a new antibiotic having antibacterial activity and different physicochemical properties (named planothiocin B) was obtained, and then an active fraction containing planothiocin A of the present invention was obtained. It will be done. The fractions are then collected and concentrated to produce white crystals, which are then collected, dissolved in a chloroform-methanol mixture, concentrated under reduced pressure to remove chloroform, and purified white needle-like crystals of planothiocin A are obtained. is obtained. Furthermore, these may have their crystal systems exchanged.
For example, planothiocin A obtained as described above
The white needle-like crystals of were dissolved in a mixture of acetic acid: chloroform: methanol (3:1:1), and this was left overnight at room temperature to precipitate colorless columnar crystals, which were collected and dried under reduced pressure. What is necessary is to obtain colorless columnar crystals of planothiocin A. Furthermore, the thus obtained planothiocin A can be used as a crude or purified product containing at least planothiocin A as an antibacterial therapeutic or prophylactic composition or a feed composition. Further, this composition may be a composition for livestock, poultry, or fish and shellfish, and is suitable as a feed composition for the purpose of improving pig feed efficiency and growth promoting effects such as weight gain. For chickens, the feed may contain about 2 to 20 ppm of planothiocin A, and as a feed composition aimed at promoting the growth of chickens, it may contain 1 to 20 ppm of planothiocin A.
It is sufficient to contain about 10 ppm, and for the purpose of promoting the growth of fish and shellfish, it can be added to their feed.
It may contain about 0.1 to 100 ppm, and commercially available feeds for livestock, poultry, fish and shellfish can be used as feeds, and the necessary amount of planothiocin A can be added as appropriate using these feeds. Furthermore, it may be added to feed for antibacterial treatment or prevention, and usually 1 to
It is sufficient to use about 500ppm. Further, these compositions may be appropriately processed into a form suitable for feeding livestock, poultry, fish and shellfish; for example, in the case of compositions for livestock, they may be simply mixed with the feed; It may also be a paste-like preparation containing A. Furthermore, for this paste preparation, thickeners such as carboxymethyl, cellulose, methyl cellulose, gum arabic, sodium alginate, and gelatin are used in an amount of about 0.5 to 5% based on water, and talc, calcium hydrogen phosphate, phosphorus, etc. magnesium oxyhydrogen,
Magnesium silicate, aluminum silicate, magnesium metasilicate, aluminum metasilicate,
Excipients such as titanium oxide, silicic anhydride, calcium stearate, and magnesium stearate that reduce the fluidity of the paste, give it appropriate hardness, and disperse the paste stably and uniformly are added to 10% of the total amount. ~50% is used, and additionally glucose, fructose, sucrose, D-
It may be obtained as an aqueous paste preparation by using 20 to 60% of a sweetener preferred by livestock, such as sorbitol, galactose, malt, or sorbitol, and further using a flavoring agent. Furthermore, the preparation is not limited to this paste-like preparation, and may be made into various oral dosage forms such as tablets, granules, pellets, and syrup by known formulation means. By administering the composition obtained in this manner to livestock, poultry, or fish and shellfish, it not only exhibits antibacterial therapeutic or preventive effects, but also exhibits a growth-promoting effect on livestock, poultry, and fish and shellfish. These effects have improved the spawning effect of fish and shellfish in particular. Furthermore, Planothiosin A does not remain in the organs or other tissue cells of livestock, poultry, or fish.
Moreover, no signs of acute toxicity were observed, and no teratogenic effects were observed. Next, the present invention will be specifically described with reference to Examples and Reference Examples, but the present invention is not limited thereto. Example 1 Glucose 2%, soluble starch 2%, yeast extract 1%, casein hydrolyzate (product name: NZ-
An aqueous medium having a composition of Amine Type A) 1% and CaCO ₃ 0.2% was prepared and sterilized. 100 ml of the medium was dispensed into a 500 ml Erlenmeyer flask, and cells from the slanted medium of Actinoplanes sp. strain A12526 were transplanted into this, and this was cultured with shaking on a rotary shaker at 250 rpm and 30°C for 4 days. I got a seed mother. Next, 3% sucrose, 2% soybean flour,
_CaCO3 0.3%, _CoCl2・_6H2O0.001 %, _MgSO4・
Place the above seed mother into a 30 volume jar fermenter containing 20 sterile medium with a composition of 0.001% _7H2O .
Transfer 200 ml, stir at 30°C, 200 rpm, 20 min.
The cells were cultured for 4 days while aerating sterile air.
The obtained culture is separated into wet bacterial cells and supernatant liquid by centrifugation, and acetone is added to the wet bacterial cells.
After stirring for 3 hours, the mixture was filtered to obtain an acetone extract. The obtained acetone extract was concentrated under reduced pressure to obtain 2. After adjusting the pH to 6.5, ethyl acetate 2 was added and vigorously stirred, the ethyl acetate extract was collected, and this extract was further concentrated under reduced pressure. A brown oily syrup was obtained. Next, about 200 ml of hexane was added to this oily syrup to produce a brown precipitate, which was collected by centrifugation.The precipitate was further dissolved in 200 ml of chloroform:methanol (5:1) and concentrated under reduced pressure. death,
As a result, a grayish white precipitate was produced by distilling off the chloroform component. This precipitate is collected by centrifugation and dried to form a mixture containing planothiocin A and the antibiotic planothiocin B.
240 mg (approx. 60% purity) was obtained. Furthermore, planothiocin A and the antibiotic planothiocin B are separated and purified from this mixture as follows. Example 2 170 mg of a mixture containing planothiocin A and antibiotic planothiocin B obtained in Example 1
was dissolved in the same solvent and charged onto a silica gel column (120 ml) prepared in advance with chloroform:methanol:acetic acid (13:1:0.1).
Further elution was performed with the same solvent. 1 fraction 18g
Active fractions containing the antibiotic planothiocin B were obtained in fractions No. 9 to 15, and active fractions containing planothiocin A were obtained in fractions No. 16 to 46.
Next, each active fraction was collected and concentrated to produce white crystals, which were collected by centrifugation, dissolved in 20 ml of a chloroform:methanol (2:1) mixture, and left at room temperature to separate the chloroform. was gradually removed to precipitate white needle-like crystals of planothiocin A and antibiotic planothiocin B, and these crystals were collected and dried.
Planothiocin A 59mg, antibiotic planothiocin
Obtained 23mg of B. Furthermore, these may have their crystal systems exchanged as follows. 50 mg of the white needle-like crystals of planothiocin A obtained above were mixed with acetic acid:chloroform:methanol (3:
1:1) Dissolved in 10 ml of the mixed solution and allowed to stand overnight at room temperature to precipitate colorless columnar crystals, which were collected and dried to yield colorless columnar crystals of planothiocin A.
Obtained 29 mg. Reference example 1 35ml of 2% carboxymethylcellulose aqueous solution
40 g of maltite was dissolved in the solution, and 2 g of calcium carbonate, 22 g of talc, and 12 g of fine powder of white needle-like crystals of planothiocin A obtained as in the above example were thoroughly mixed and kneaded, and then further stearic acid was added. 11 g of magnesium was added, thoroughly mixed and kneaded, and the mixture was filled into a tube to obtain a paste preparation containing planothiocin A. This product is used as a paste preparation for pigs, and as an antibacterial therapeutic or preventive composition for pigs.
15g (planothiosin A content: approximately 0.5-1.5g) is orally administered to pigs. Reference example 2 Commercially available pig feed (product name: Nihon Nosan Co., Ltd.,
1 kg of Maruei complete feed) was thoroughly mixed with 1.5 g of fine powder of white needle-like crystals of planothiocin A, and this was further mixed with 99 kg of the same feed for pigs to obtain a feed composition for pigs containing planothiocin A. Ta. When this product was fed to piglets (5th week) for 10 weeks, it was found to have a growth promoting effect of about 8-14% compared to the control (same pig feed that does not contain planothiocin A). In addition, in the same manner as above, 1.5 g of the antibiotic planothiocin B was used instead of planothiocin A to obtain a pig feed composition having a growth promoting effect for pigs. Reference Example 3 600 g of lignin powder and 0.15 g of fine powder of white acicular crystals of planothiocin A were thoroughly and uniformly mixed with 20 kg of commercially available chicken feed (manufactured by Kumiai Chemical Co., Ltd., fine powder of No. 17 pine nuts for adult chickens).
Furthermore, 8% water was added to this and mixed uniformly, and then made into pellets with a diameter of 8 x 4 mm using a tablet machine.
This was stirred at 105℃ for 8 hours, and the planothiocin A
A feed composition for chickens containing the following was obtained. When this product was fed to chickens, no residual planothiocin A was observed in the organs or tissues of the chickens, and planothiocin A remained in the chickens' feces.
A considerable amount of the intake amount was excreted. Reference Example 4 1 g of the mixture containing planothiocin A and the antibiotic planothiocin B (purity approximately 60%) obtained as in the above example was uniformly mixed with 100 kg of feed for young fish (manufactured by Nippon Compound Feed Co., Ltd.). , a feed composition for yellowtail was obtained. Reference Example 5 A mixture containing planothiocin A and planothiocin B obtained as in the above example (ratio; planothiocin A: planothiocin B = 93:
7), feed (piglet artificial milk; composition: crude protein
22.44%, crude fat 5.14%, crude fiber 0.74%, crude ash
6.16%, calcium 1.25, phosphorus 0.95; DCP21.38
%, TDN87.41%), the first area is a control area with no additives, 0.5ppm added area, 5ppm added area, and the second area is an unadded control area, 10ppm added area, and 20ppm added area. 25-day-old piglets (10 pigs per group) were fed these feeds for 4 weeks. As a result, as shown in Table 3, the average weight per piglet, average weight gain, and weight gain index during the breeding period showed a good growth promoting effect.

[Table] Reference Example 6 A mixture containing planothiocin A and antibiotic planothiocin B (ratio: planothiocin A: antibiotic planothiocin B = 93:7) obtained as in the above example was added to feed (feed for young chicks). Composition: Crude protein 20.0%, crude fat 3.4%, crude fiber 3.4
%, crude ash 5.5%, calcium 1.03%, phosphorus 0.83
%, inorganic phosphorus 0.43%; TDN 70.3%,
GE349Cal/1000g), a control group without additives as a female group, a control group with addition of 0.5ppm, a group with addition of 5ppm, a group with addition of 10ppm, a group with addition of 20ppm, a control group with no additives as a male group, a control group with addition of 0.5ppm, a group with addition of 5ppm, The feed was added at a ratio of 10 ppm added and 20 ppm added, and young chicks (25 birds per sex, separated by sex) were raised with these feeds for 4 weeks. As shown in Table 4, the results showed a good growth promoting effect.

[Table] Although a significant difference was observed in the female group, some variation peculiar to hens was observed.

[Brief explanation of the drawing]

Figure 1 shows the ultraviolet absorption spectrum of planothiocin A in methanol, Figure 2 shows the ultraviolet absorption spectrum of planothiocin A in methanol-acidic conditions, and Figure 3 shows the ultraviolet absorption spectrum of planothiocin A in methanol-alkaline conditions. Figure 4 shows the infrared absorption spectrum of planothiocin A.

Claims (1)

[Claims] 1. A novel antibiotic, Planothiocin A, having the following physicochemical properties. Melting point: 266-269°C (decomposition) Elemental analysis: C = 49.47%, H = 3.53%, N = 13.25%, S = 13.73% Molecular weight: 1438 (minimum molecular weight based on amino acid analysis that contains one molecule of threonine in one molecule) Optical rotation: [α] ²³ _D = +19.6 (C = 0.7, pyridine) Ultraviolet absorption spectrum: 1st As shown in Figure (in methanol), Figure 2 (in acidic methanol), and Figure 3 (in methanol-alkaline), Infrared absorption spectrum: As shown in Figure 4 Color reaction: Potassium permanganate decolorization reaction , iodine color reaction is positive, ferric chloride reaction, ninhydrin reaction, Moritsch reaction is negative, Solubility in solvents: Soluble in CHCl ₃ - methanol mixture, pyridine, DMSO, glacial acetic acid, benzene, acetone, petroleum ether, hexane Insoluble in. Distinction between acidic, neutral, and basic: Weakly acidic substance Color property: White crystal 2 After inoculating the antibiotic planothiocin A-producing bacteria belonging to the genus Actinoplanes into a medium containing assimilated carbon and nitrogen sources and inorganic substances. 1. A method for producing the antibiotic platyocin A, which comprises culturing and then collecting the antibiotic planothiocin A from the culture. 3 The antibiotic planothiocin A-producing bacteria belonging to the genus Actinoplanes is Actinoplanes S.
The production method according to claim 2, which uses P. A12526 bacteria.