JPS6237019B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention is a novel antibiotic planothiocin A.
(palnothiocin A), planothiocin B
The present invention relates to a composition containing (planothiocin B). The antibiotics planothiocin A and planothiocin B of the present invention are novel sulfur-containing peptide antibiotics having the physicochemical properties and biological properties shown in Table 1 below.

【table】

【table】

[Table] From the above properties, the antibiotics planothiocin A and planothiocin B of the present invention are recognized as sulfur-containing peptide antibiotics. ), thiopeptins, sulfomycins, siomycins,
A-59, pepthiomycins, thermothiocin, sporangiomycins, althiomycin, A-7413, nosiheptide, L-13365, actinothiocin ), A-10947 are known, and among these, nosiheptide is a substance that has the same amino acid composition as the antibiotic planothiocin A, and there is no substance that has the same amino acid composition as the antibiotic planothiocin B. Regarding the antibiotic planothiocin A and nosiheptide, nosiheptide is 416 mμ (E ¹ % _{1 cn} = 124) and 44
0
It has maximum absorption at mμ (E ¹ % _{1 cn} = 104.8),
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.
Furthermore, silica gel thin layer chromatography
The Rf values are clearly different [CHCl ₃ of nosiheptide:
Rf of methanol (3:1) = 0.87, ethyl acetate:
methanol:water (10:2:1) Rf = 0.71], as mentioned above, the antibiotics planothiocin A and planothiocin B do not match any known sulfur-containing peptide antibiotics, and therefore, the present invention Both the antibiotics pranothiocin A and B are recognized as new substances. The novel antibiotic planothiocin A or planothiocin B-producing actinomycetes of the present invention are actinomycetes isolated from the soil of a chili pepper field in Mihama-cho, Mikata-gun, Fukui Prefecture, and were identified as belonging to the genus Actinoplanes. Planes S.
It was decided to call it P.A12526.
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. Among the spores, the spores are approximately spherical or sub-spherical, 1 to 1.5 microns in diameter, have numerous flagella, and are zoospores that move in water. [] Composition of Diaminopimelic Acid Diaminopimelic acid was detected as a main component in the meso type, and a small amount of the hydroxy type was detected in diaminopimelic acid by whole cell analysis. []Culture properties The findings observed after culturing on various media at 30°C for 14 days are shown in Table 2. A small amount of undeveloped aerial mycelium was observed to form on many of the media. Colors are shown in the Color harmony manual 4th edition (1958) (Container Corporation Of
America) color classification.

【table】

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

【table】

[Table] (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 is a sporangium produced from basal hyphae with branches. The sporangium is spherical or subglobose, has flagella and is 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 order to obtain the antibiotic substances planothiocin A and B of the present invention, it is preferable to collect them from a culture using the above-mentioned Actinoplanes sp. strain A12526. In addition, the bacteria mentioned above are examples of the bacteria used, and in addition to this bacteria, the antibiotic planothiocin A (hereinafter simply referred to as planothiocin A) or the antibiotic planothiocin B
(hereinafter simply referred to as planothiosin B) can be used in the present invention; for example, planothiosin A or planothiosin B-producing bacteria belonging to the genus Actinoplanes or mutant strains thereof are used. Next, to obtain planothiosin A or planothiosin B, for example, a planothiosin A or planothiosin B 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 planothiosin A or planothiosin B, but is particularly preferably 25 to 30°C. The culture time depends on the conditions, but for planothiosin A
Alternatively, the culture can be terminated at an appropriate time by determining the time when planothiocin B reaches its maximum yield.
It usually takes about 2 to 4 days. Planothiosin A or planothiosin B is collected from the culture of planothiosin A or planothiosin B produced in this way, but most of the planothiosin A or planothiosin B is accumulated within the bacterial body, and some of it is It is also present in the supernatant. Furthermore, to give an example of means for separating and purifying planothiosin A or planothiosin B, first, from the culture obtained by culturing planothiosin A or planothiosin B producing bacteria as described above, bacterial cells containing planothiosin A or planothiosin B are filtered using a drum filter. The cells are separated using an over-separation method such as a centrifugal separation method or a centrifugation method using a basket centrifuge to obtain wet bacterial cells and a culture solution. This culture solution contains a small amount of planothiocin A and planothiocin B, and 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. When the chloroform is removed and the mixture becomes a methanol solution, an off-white precipitate is produced, which is collected to obtain a mixture of planothiosin A and planothiosin B. This crude product is then mixed with, for example, chloroform:methanol:acetic acid (13:1:
Dissolve and charge with the same solvent on the silica gel column prepared in 0.1), elute with this solvent, and collect the active fraction. During elution, an active fraction containing planothiocin B is first obtained, and then an active fraction containing planothiocin A is obtained. 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 crystals of pranothiocin A and pranothiocin B are obtained. Needle-shaped crystals are obtained. Furthermore, their crystal systems may be exchanged; for example, the purified white needle-like crystals of planothiocin A obtained as described above may be dissolved in a mixture of acetic acid: chloroform: methanol (3:1:1). This may be allowed to stand overnight at room temperature to precipitate colorless columnar crystals, which are collected and dried under reduced pressure to obtain colorless columnar crystals of planothiocin A. Furthermore, the thus obtained planothiosin A or planothiosin B is used as a crude or purified product containing at least planothiosin A or planothiosin B 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. The feed may contain about 2 to 20 ppm of planothiocin A or planothiocin B.
In addition, feed compositions intended to promote the growth of chickens may contain approximately 1 to 10 ppm, and furthermore, feed compositions intended to promote the growth of fish and shellfish may contain approximately 0.1 to 100 ppm. Commercially available feeds for livestock, poultry, fish and shellfish may be used, and the necessary amount of planothiocin A or planothiocin B may be added to these feeds. It may also be added to feed for antibacterial treatment or prophylaxis, usually at 1 to 500 ppm.
Just use it to a certain degree. 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 into their feed; Alternatively, it may be a paste preparation containing pranothiocin B.
Furthermore, for this paste preparation, a thickener such as carboxymethylcellulose, methylcellulose, gum arabic, sodium alginate, gelatin, etc. is used in an amount of about 0.5 to 5% based on water,
It also reduces the fluidity of pastes such as talc, calcium hydrogen phosphate, magnesium hydrogen phosphate, magnesium silicate, aluminum silicate, magnesium metasilicate, aluminum metasilicate, titanium oxide, silicic anhydride, calcium stearate, magnesium stearate, etc. At least 10 to 50% of the total amount of excipients is used to give appropriate hardness to the paste and to disperse the paste stably and uniformly, and in addition, excipients such as glucose, fructose, sucrose, D-sorbitol, galactose, and malt are used. and sweeteners that livestock like, such as sorbit.
An aqueous paste preparation may be obtained by using 20 to 60% and further adding a fragrance. 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 and planothiosin B did not remain in the organs or other tissue cells of livestock, poultry, fish and shellfish, and did not show any signs of acute toxicity, nor did they exhibit teratogenic effects. . 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 35 ml of 2% carboxymethylcellulose aqueous solution
40 g of Maltit 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 reference example described below 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. Example 2 Commercially available pig feed (product name: Nihon Nosan Co., Ltd.,
1.5 g of fine powder of white acicular crystals of planothiocin A was thoroughly mixed with 1 kg of Maruei complete feed, and this was further added to 99 kg of the same feed for pigs and mixed 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 approximately 8-14% compared to the control (same pig feed not containing planothiocin A). Further, in the same manner as above, 1.5 g of planothiocin B was used instead of planothiocin A to obtain a feed composition for pigs having a growth promoting effect for pigs. 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. Example 4 1 g of a mixture containing planothiocin A and planothiocin B (purity about 60%) obtained as in the reference example below was uniformly mixed with 100 kg of feed for young fish (manufactured by Japan Mixed Feed Co., Ltd.), A feed composition was obtained. Example 5 A mixture containing planothiosin A and planothiosin B obtained as in the reference example below (ratio; branothiocin A: planothiosin 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 was a control area with no additives, 0.5ppm added area, 5ppm added area, and the second area was 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] Example 6 A mixture containing planothiosin A and planothiosin B obtained as in the reference example below (ratio; planothiosin A: planothiosin B = 93:
7), 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
%; TDN70.3%, GE349Cal/1000g), female plot with no additive, 0.5ppm added plot, 5ppm added plot, 10ppm added plot, 20ppm added plot, male plot with no additive control plot, 0.5ppm The feed was added at the following ratios: additive, 5ppm, 10ppm, and 20ppm, and young chicks (25 per sex, 25 per sex) were raised with these feeds for 4 weeks. As shown in Table 4, the results showed a good growth promoting effect.

[Table] Reference example 1 Glucose 2%, soluble starch 2%, yeast extract 1%, casein hydrolyzate (product name: NZ-
An aqueous medium having a composition of 1% Amine.TypeA) and 0.2% CaCO ₃ was prepared and sterilized. the medium
Dispense 100 ml into a 500 ml Erlenmeyer flask, transplant cells from the slanted medium of Actinoplanes sp. I got my 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 and 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 was collected by centrifugation and dried to obtain 240 mg of a mixture containing planothiocin A and planothiocin B (purity approximately 60%). Furthermore, planothiocin A and planothiocin B are separated and purified from this mixture as follows. Reference Example 2 170 mg of the mixture containing Planothiocin A and Planothiocin B obtained in Reference Example 1 was mixed in advance with chloroform:methanol:vinegar residue (13:
1:0.1) silica gel column (120ml)
On top of that, it was charged and dissolved with the same solvent, and further eluted with the same solvent. Fractionation was carried out using 18 g per fraction, and active fractions containing 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, white needle-like crystals of planothiosin A and planothiosin B were precipitated, and these crystals were collected and dried to obtain 59 mg of planothiosin A and 23 mg of planothiosin B. Furthermore, these may be obtained by converting the crystal system as described below. 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.

[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, Figure 5 shows the ultraviolet absorption spectrum of planothiocin B in methanol, Figure 6 shows the ultraviolet absorption spectrum of planothiocin B in methanol-acidic conditions, and Figure 7 shows the ultraviolet absorption spectrum of planothiocin B in methanol. The ultraviolet absorption spectrum in methanol-alkalinity, and FIG. 8 shows the infrared absorption spectrum of planothiocin B.

Claims (1)

[Scope of Claims] 1. An antibacterial therapeutic or preventive composition or feed composition containing the antibiotic planothiocin A or planothiocin B. 2. The composition according to claim 2, wherein the composition is a composition for livestock, poultry, or fish and shellfish.