KR800000966B1 - Process for preparation of 31559 rp antibiotic - Google Patents

Abstract

Title compd. and its salts, effective against coccidia and bacteria, were prepd. by fermentation. Thus, streptomyces hygroscopicus DS24367 was grown in a medium contg. sol. distillates, bean oil, glycerin, glucose monohydrate, NaCl, CoCl36H2O and tap water. The fermn. brew was filtered, extd. with CH2Cl2 and concd. under reduced press. For purifn. hexan was slowly poured into the CH2Cl2 conc. at roon temp. and filtrate. The filtrsate was concd under reduced press., stirred for 2 hr at 0≰C and held at 4≰C for 24 hr for crystallization. The final products were obtained by purifying with active carbon.

Description

Method for preparing antibiotic 31559 RP

Figure is a graph of the infrared spectrum of the antibiotic of the present invention.

The present invention relates to methods for preparing antibiotics and compositions containing them.

The present invention provides a novel antibiotic 31559RP and its metal salts and nitrogen-containing basic salts which are useful for anti-cordicia activity and anti-bacterial activity. The compounds of the invention are also useful as inhibitors of growth factors in ruminants and bacterial etiosis in pigs.

The 31559RP of the present invention can be obtained by culturing a novel microorganism called Streptomyces hygroscopic picose DS 24367 (NRRL5787) belonging to the genus Streptomyces.

31559RP of the present invention contains carbon, hydrogen and oxygen, and the elemental composition of the soda salt thereof is C = 63.2%, H = 8.8%, O = 24.3% Na = 2.9%.

The neutralization equivalent of the antibiotic of the present invention is 836 by titration of sodium salt with perchloric acid in glacial acetic acid. From the elemental analysis and the NMR spectra of both carbon 13c, the empirical formula of the sodium salt of 31559R.P of the present invention was found to be C ₄₄ H _73-75 O ₁₄ Na. Furthermore, the NMR spectra of both protons and carbon 13c indicate the presence of four methoxy groups in their molecules.

The sodium salt of 31,559RP of the present invention has the following properties.

Form: Crystalline White Powder

Solubility: Water insoluble, especially soluble in hydrocarbon solvents such as hexane, soluble in alcohols such as methanol, ketones such as acetone, dimethylformamide and ethyl acetate, and easily soluble in chlorinated solvents such as methylene chloride and chloroform. being.

Melting Point: 220 ℃ (Decomposition Temperature)

Beneficiation (in C = 1.050 methanol):

임.

being.

Ultraviolet spectrum: Absorption characteristic up to 210 mm (measured at a solution concentration of 0.950 mg / dl in ethanol)

Infrared spectra: Measured with tablets made by mixing with potassium potassium bromide, the results are as shown in the figure (However, the wavelength is indicated in microns and the upper scale of the graph is indicated, and the wave number is in cm- ¹ . The lower scale of the graph is indicated by the abscissa and the optical density is indicated by the ordinate.

The main band of the infrared absorption of the product of the present invention is shown in Table 1 by the frequency (cm ^-1 ).

TABLE 1

only,

Sodium salt of 31,559 RP may be characterized by an upward curve of thin layer chromatography of silica gel using two solvent systems.

1) Ethyl acetate, cyclohexane, water and butanol (50-50-25-5 volume ratio): The Rf of the sodium salt of 31,559RP in this system is 0.5.

2) Methylene chloride and methanol (94-6 volume ratio): The Rf of the sodium salt of 31,559RP in this system is 0.7. The structural formula of the sodium salt of antibiotic 31,559RP is as follows.

[Ebacterial Activity in Vitro]

31,559RP has a particular bactericidal activity against some Gram-positive bacteria.

The bactericidal activity of the sodium salt of 31,559 RP against some microorganisms is measured by one of the dilution methods commonly used for these purposes. The minimum concentrations of agonists that interfere with the visible growth of the microorganisms under various conditions were measured, and their measurement results are shown in Table II below.

In Table II, the minimum bactericidal concentrations are shown as microorganisms of agonists per test medium.

TABLE II

[toxicity]

The 50% lethal dose (LD ₅₀ ) of 31,559 RP of sodium salt in a single dose of chick is 150 mg / kg.

[Anticodicia activity]

Anticodicia activity of 31,559 RP sodium salt was measured in chicks infected with either Emerian Tenella or Emeria aseburina. Substantial anticordician activity of 31,559RP is obtained when 31,559RP of sodium salt is formulated into the feed at a nontoxic concentration of 0.0005-0.04 wt% relative to the feed.

The microorganism producing 31,559 RP is a Streptomyces strain isolated from a sample taken from soil in India, which was given number DS 24367. A sample of this was registered as NRRL 5787 in the Northern Research Research Laboratories of USDA in Peoria, Illinois, USA.

The strain is suspended in a small amount of soil in sterile distilled water, which is diluted by various concentrations and isolated by a general method of spraying a small amount of each diluent on the surface of the petri dish containing nutrient agar medium.

After culturing for several days at 25 ° C., the microorganisms were grown, these were isolated to examine bacterial colonies, and transplanted onto a tetratrophic agar so that they were sufficiently cultured.

Intrinsic properties of the Streptomyces strain DS 24367 belonging to the Streptomyces hygroscopicus strain are characterized by H, D Trasner and E, J Backcus Low Applied Microbiology 4,243-250, 1956 and S, A Wakesman Low Actinomycetes II 1961 , P. 230-231, and was designated Streptomyces hygroscopicus strain DS 24367.

The Streptomyces hygroscophysics DS 24367, defined as the strain Streptomyces hygroscophytes by H. D. Trasner, E. J. Backcus and S.A Wakeman, has three characteristics:

A) The tips of their spores are solid spirals containing several helices, whose spiral spores are concentrated along the filaments to form more or fewer colonies.

B) When its spore-forming aerial mycelium reaches the developmental stage of development, it appears to be a unique dark gray color of the strain Streptomyces hygroscopyus.

C) Some media appear on the spore-forming surface over time in a black area with a wet appearance that shows good development of airborne mycelium.

In the case of Streptomyces hygroscopyus DS 24367, the formation of airborne mycelium occurs frequently but not on any medium. When the gray spore forming aerial mycelium appears, a black region is formed over time, and when it occurs, it has a variety of forms, and it occurs in a local narrow area rather than the whole of the aerial mycelium. This is particularly noticeable on Freedham East Extract Agar, Freetham Oat and Tomato Colloidal Agar, and on Glucose-asparagine Agar.

The strains of Streptomyces hygroscophytes described in `` Radiation Fungi '', which bound the morphological appearance on some mediums authored by S.A Wakeman, differed slightly compared to strain DS 24,367.

In other words, strains of Streptomyces hygroscophycus form aerial mycelium on sucrose-containing nitrate agar and nutrient agar, but strain 24,367 is biphasic and does not form any aerial mycelium. Moreover, on medium containing sucrose as a carbon source, Streptomyces hygroscopycus does not reduce nitrate, but under the same conditions strain DS 24,367 turns to nitrite with a large decrease in nitrate. However, these minor differences are not sufficient to distinguish between strains DS 24,367 and Streptomyces hygroscopicus species. Streptomyces hygroscopic picospheres DS 24,367 form spore chains with rigid helix with a limited number of turns at the tip, i.e. 3 or 4 turns, which in some cases may contain more or more turns. A few spore chains that are not formed but simply curled up, or more or fewer loose spirals, are observed. The spore generator contains colony tissue and spiral spores, which have a small number of branches inserted along the basic filament, and the length of these branches is very constant. Spores are ovoid to columnar, 0.8-1.0μ / 0.6-0.8μ. The organization of spore generators on Bennett agar and Freedam oat-tomato agar showed the same results when viewed under a microscope. By the sporulation method, Streptomyces hygroscopic picose DS 24,367 belongs to the spiraxanthone of the Freedam classification.

Streptomyces hygroscopic picose DS 24,367 develops very well at 25 ° C., moderately at 37 ° C. and not at 50 ° C. When cultured at 25 ℃ it has the following biochemical properties.

Production of melanin: negative

Generation of hydrogen sulfide: negative

Tyrosinase: Voice

Liquefaction of gelatin: positive

Use of cellulose: positive

Production of nitrite from nitrates: marked positive

Hydrolysis of Starch: Positive

Culture on milk: peptonized without coagulation.

PH changes slightly during 1 month

The culture characteristics of Streptococcus hygroscopicus DS 24,367 are shown in the table below.

These show the cultures when they have reached a good stage of development, ie 2-3 weeks at 25 ° C unless otherwise noted. Their properties were observed on nutrient agar and meat juice used to determine the morphological characteristics of Streptomyces and agar cultures were performed on sloped agar. Some of the cultures used were prepared according to the prescriptions listed in `` Radiation Fungi '' (S. A. Wakesman, pages 193-197, Chronica Botanica Campani, Wallam, Mass, USA, 1950). This is indicated by the letter W along the numbers given in this document. Reference symbols of other cultures or their components are as follows.

A-'' Bennet's agar ''-S.A. Wakeman-Actinomycetes-Volume 2-Page 331-30-Williams & Wilkins Company, Baltimore 1961

B-'' Hike and Tresner Agar-TG.Freedam and Co-Investigators Antibiotic Yearbook, pp. 1956-1957, 950

To C-prescription W-23, 2% agar was added.

D-'' Yeast Extract Agar ''-T.G Freedam and Co-Investigators Antibiotic Yearbook 1956-1957, p. 950

F-'' Melanin Formation Medium ''-S.A Wakeman-Radiant Fungi, Vol. 2, p. 333, -42-Williams & Wilkins Company, Baltimore, 1961

G-W. E Grundi and Collaborators, Antibiotics and Chemistry, 2,401, 1952,

H-'' Inorganic Salt Starch Agar ''-T. G Freedam and Co-Investigators, Antibiotic Yearbook, 1956-1957, pages 951

30g sucrose was replaced by 15g glucose, corresponding to I-prescription W-1

Equivalent to J-prescription W-1, 30 g sucrose was replaced with 15 g glycerin

30 g sucrose was replaced with 15 g of glucose, corresponding to K-prescription W-18

Sucrose was removed as corresponding to L-prescription W-18 and three pieces of filter paper partially impregnated with the solution were added.

M-'' Manual of Pure Culture Research in Bacteria '' American Bacterial Association-Geneva, NY, II ₅₀ -18

N- '' plain gelatin '- hayeoteum prepared according to the study of pure cultures of bacteria Handbook - American Association of bacteria, Geneva, NY, II ₅₀ -18

P-derived milk powder prepared commercially according to manufacturer's instructions.

Q-H, D, Tressner and F. Units converted test medium to hydrogenated medium-Bacterial Society magazine. 76, 239-244, 1958.

In the table, the degree of development was classified in the order of very good, good, slightly good, moderate, very moderate, poor, very poor, nothing. The ability of Streptomyces hygroscopic picosphere DS24,367 to utilize various carbon and nitrogen sources for its development was measured by the Freedam and Gotliv method (Antibiotics Magazine, 56, 107-114, 1948). One of the various test carbon sources was replaced with glucose or one of the various test nitrogen sources with ammonium sulfate and incubated at 25 ° C. for a suitable period, and the degree of development was observed on the basic medium indicated by the authors and the results are shown in the following table.

According to the present invention, 31,559 RP is an aerobic incubation of Streptomyces hygroscopic picose DS 24,367 or a mutant (31,559) RP) in the medium, and the 31,559 RP formed during the culture itself It can be prepared by isolating in the form of a salt thereof.

Incubation of Streptomyces hygroscopic picose DS24,367 can be performed by any method of aerobic surface culture or impregnation culture, but is preferable because of the convenience of impregnation culture. For this purpose, various types of incubators commonly used in the fermentation industry can be used.

In particular, it is good to advance the culture in the following order.

Fermentation media must contain anabolic carbon sources, anabolic nitrogen sources, inorganic elements, in particular chlorides and suitable growth factors, which can be introduced in the form of well-known products or in various biological co-formulations.

Suitable assimilar carbon sources are glucose, dextrins, carbohydrates such as starch, other carbohydrates such as sugar alcohols (including glycerin) and some organic acids such as lactic acid or citric acid.

Some vegetable or animal oils, such as pig oil and soybean oil, may be substituted for or added to various carbohydrate sources.

Suitable sources of assimilation nitrogen are very wide. These are very simple chemicals-inorganic or organoammonium salts, urine and other amino acids. Nitrogen is primarily a complex of nitrogen-containing compounds in the form of proteins, namely casein, lact Almin, gluten, and their hydrolysates, soy flour, peanut flour, fish meal, meat extracts, yeast extract soluble varieties and corn. Can be introduced in the form of a tip.

Among the inorganic elements to be added, they may be buffered or neutralized, such as alkali metal or alkaline earth metal phosphate or calcium or magnesium carbonate. Other additives, such as chlorides and sulfides of alkali and alkali metals, provide the ionic equilibrium required for the development of Streptomyces hygroscopic picos DS24,367 and the production of 31,559RP. Finally, some act as activators of the metabolic reactions of Streptococcus hygroscopicus DS24,367, such as zinc, cobalt, iron, copper and magnesium salts. Suitable growth factors are vitamin-type natural products such as ribohulabin, folic acid and pantothenic acid.

The pH of the fermentation broth at the beginning of the culture must be between 5.8-7.8 and strictly between 6.2-7.4. The optimum temperature for fermentation is 25-30 ° C, but satisfactory results are obtained even at temperatures between 23-33 ° C. Aeration volume of fermentation is very wide. However, aeration rate of 0.3-3 liters of air per minute per liter of meat juice is very suitable. The highest yield of 31,559 RP is obtained after 2-8 days of culture, depending on the medium used.

From these facts, it can be inferred that the general conditions of culturing Streptomyces hygroscopic picose DS24,367 to produce 31,559 RP are very extensive.

31,559 RP can be isolated from the culture product by the following method.

The medium is filtered at an acidic pH, generally between 3-6 and preferably 5. The active substance remaining in the filter cake can be extracted from them with a lower alcohol, i.e., a metasol or a chlorinated solvent, a suitable solvent such as methylene chloride. After a suitable alkali treatment, 31,559 RP is diluted under reduced pressure to a suitable concentration in solvent or salt, and then isolated from the above-mentioned solution in the form of sodium salt by crystallization and stored in a cold place.

31,559RP or its sodium salt can be purified by known methods such as crystallization, chromatography on various adsorbents or by countercurrent distribution.

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

Example 1

A) fermentation

The following ingredients were injected into a 170 L fermentor.

1,200 g peptone

Yeast extract 600g

Glucose Carbohydrate 1,200g

240g agar

Tap water, enough 110ℓ

The pH was adjusted to 7.5 by adding 10N caustic soda solution (70CC). The embryo was sterilized with superheated steam at 122 ° C. for 40 minutes. On cooling, the volume of meat juice is reduced to 120 l. This is due to the condensation of steam in the sterilization process. At this time, the pH is 6.85. Meat juice was then inoculated with a culture solution (200CC) of Streptomyces hygroscopic picus DS24,367 produced in a stirred Erlenmeyer flask. If the culture solution is grown at 27 ° C. for 25 hours and aerated with sterile air, a culture medium suitable for inoculation is produced.

The resulting culture was incubated in an 800 L fermentor and the following materials were injected.

Soluble 2 kg

Soybean oil 10㎏

Glycerin 6 kg

2 kg of salt

Cobalt Chloride Heptahydrate

0.4 l solution (concentration of 20 g / l)

Tap water q.s.p 365ℓ

The pH of the medium is adjusted to 7.50 by adding 10N caustic soda solution 200CC, sterilized with superheated steam at 122 ° C for 40 minutes, and cooled.The volume of meat juice is reduced to 390ℓ due to condensation of steam during sterilization. 10 liters of sterile aqueous solution is added to make 400 liters.

The pH of the medium is 6.65. Inoculate 40 liters of inoculum culture from the above-mentioned 170 liter fermenter. The culture solution is stirred at a turbine of 205 r.p.m while being grown at 27 ° C. for 113 hours while ventilating sterile air at a rate of 20 m 3 / hr. At the end of the operation, the pH of the culture is 7.65 and the volume is 380 liters.

B) extraction

The medium (400 L) obtained by the above-mentioned method was added with 2.8 L of 6N hydrochloric acid to adjust the pH to 5 and stirred for 30 minutes. After adding the filter aid (20 kg), the medium is filtered with a filter press, and the filter cake is washed with water (100 l) on a filter and then the pH is adjusted to 5 with 6N hydrochloric acid. Remove filtrate and wash.

The filter cake is triturated in methanol (300 L), and the resulting mixture is adjusted to pH 7 with 6 N hydrochloric acid and stirred for 30 minutes. The mixture is filtered through a filter press and the filter cake is washed on the filter with methanol (80 L). The filtrate and the liquor are mixed and concentrated under reduced pressure (5-10 mmHg) to produce an aqueous concentrate (30 L). The concentrate was adjusted to pH 3 by adding 6N hydrochloric acid (290 kPa) and stirred for 10 minutes in the presence of methylene chloride (30 L) to separate the two layers. The aqueous layer is extracted two more times using methylene chloride (30 L) in this way. The methylene chloride extract extracted three times was combined, and 20 N of 6N hydrochloric acid was added while stirring with water (9 L) to adjust the pH of the aqueous phase to 3. The organic layer is separated, washed with water, and caustic soda is added until the pH of the aqueous phase (20N of 6N sodium hydroxide) reaches 9.5. The methylene chloride layer is concentrated under reduced pressure (5-10 mmHg) until 1 l.

c) tablets

The methylene chloride concentrate thus obtained is slowly added to hexane (10 L) at room temperature. The solution is filtered to remove inert insoluble matter (63 g). The stock solution is concentrated under reduced pressure (5-10 mmHg) until it is about 0.5 L. The solution is stirred at 0 ° C. for 2 hours and then left at 4 ° C. for 24 hours. The crystals thus obtained are separated from the solution by centrifugation, washed with hexane (250 Pa) at 4 ° C, and dried under reduced pressure. This isolates 31,559 RP of sodium salt (103 g).

D) recrystallization

31,559 RP of sodium (57 g) obtained by the above method is dissolved in 1.2 L of acetone and stirred for 15 minutes. Activated carbon washed with hydrochloric acid (2.5 g) was added thereto, and the mixture was stirred for another 30 minutes, followed by filtration of decolorized activated carbon. Distilled water (750 cc) was added to the filtrate, and the mixture was slowly stirred. Sodium salt of 31,559 RP slowly crystallizes at 0 ° C. The crystals were filtered off, washed with 150 1: 1 of a 1: 1 solution of acetone and water, and dried under reduced pressure at 35 ° C. to obtain 31,559 RP of pure sodium salt (35 g).

The mother liquor is concentrated to half of the stock and then cooled to 4 ° C. to further yield 31,559 RP of sodium salt (13.2 g).

The present invention includes animal-raising compositions in which 31,559 RP or its salts are contained within 0.0001-99.9% of the metal or nitrogen-containing base and materials that can be consumed by the animal. (Of course, all salts used should contain cations which are non-toxic during concentration and oral administration).

Such compositions may constitute a combined feed of animals with 31,559 RP or its salts and metal or nitrogen containing-bases and other suitable anticosidia.

Of course, the dosage required to achieve a moderate effect depends on the feed, but is very wide. Typical animal feed content is 0.005-0.04% by weight of 31,559 RP or its salts.

31,559 RP or its salts may be distributed in the form of a homogeneous dispersion in the feed at the above concentrations. It can also be dispersed in supplements at a dosage of 0.025-1% with additives such as vitamins and mineral salts. These supplements are mixed 5-20% of the base feed.

`` Premixes '' used to make supplemental feeds typically contain 0.05-20% of 31,559 R.P or salts thereof in diluted feed. These consist of suitable intermediate feeds that can distribute the active product evenly throughout the feed. The premix itself contains 99.9-20% of 31,559 RP or salts thereof, with the addition of food, flavor, dispersing or flocculant and feed diluent.

Generally, the concentrate and the premix are in powder.

Supplementary feeds and complete feeds may be prepared in powder or granular form by conventional methods.

The following examples illustrate the compositions of the present invention and percentages refer to weight percentages unless otherwise indicated.

Example 2

Prepare basic feed having the following composition.

Medium grain 13.41%

Barley Flour 13.41%

Corn flour 13.41%

31.32% flour

Fish meal 8.92%

Soy flour 8.92%

Dehydrated Feed Powder 4.56%

Iced Extract 2.33%

Dry milk powder 2.68%

Salt 0.09%

Calcium Chloride 0.89%

Inorganic element 0.06%

Vitamin-Compound

Vitamin A 4.000 I.U / kg

Vitamin D ₃ 1.000 IU / kg

Choline chloride 11.5 mg / kg

Ribohulabin 2.24mg / kg

31,559 RP of sodium salt is added to these feeds 0.02% and distributed evenly.

The present invention also provides a composition of 31,559 RP or salts thereof, which can be used as a growth factor for ruminants (bovine, sheep, goat) and as a bactericidal agent in pigs.

These compositions containing 31,559 RP or salts thereof constitute a mixed feed of ruminant or swine and concentrated feed for animal breeding.

Of course, the dosage required to produce a moderate effect is not limited and depends on the type of animal and the nutritional value of the feed.

It is recommended to administer 50,250 mg of 31,559 RP per animal per day.

31,559 RP should be evenly distributed throughout the supplement containing 0.01–0.1% of antibiotics and other food additives such as vitamins and inorganic salts. Since these supplements are usually mixed 1-10% of the basic feed per day, the amount of 31,559 RP administered per day will eventually be 0.0001-0.01%.

The `` premix '' used to prepare complete or supplemental feeds contains 0.1-5% 31,559 RP of the diluted feed.

These consist of suitable intermediate feeds that can evenly distribute sterile products throughout the feed. The premix itself contains 99.9-5% of 31,559 RP with the addition of edible feed, flavors, dispersants or anticoagulants and feed diluents.

In general, the concentrate and the premix are in powder form. Supplementary feeds and complete feeds may be prepared in powder or granular form by conventional methods. 31,559 RP or salts thereof in the composition may be dragee microparticles.

The following example illustrates the use of 31,559 RP as a growth factor.

Example 3

Twelve Holstein calves were placed in separate cages with a surface area of 48 m2 and weighed. The feed consumption of the survey subjects was zero. After 28 and 56 days the calf was weighed and the amount of feed consumed during this period was measured. The average body weights and the average conversions of the feeds were increased at time intervals of 0-28 and 28-56 days.

Feed was fed twice a day. The daily ration was determined to be the maximum amount of food that can be consumed per day with no calf left. During the experiment, calves were divided into three groups.

Each group of calf was fed a basic diet consisting of corn, hay and protein.

Animals of group 1 were fed basic diets, and animals of group 2 were fed basic diets of protein containing 100 mg of antibiotics 31,559 RP, and animals of group 3 were fed protein containing 200 mg of antibiotics 31,559 RP. Basic feed was taken.

The feed consumed per day per animal has the following composition:

Protein supplements use `` Pro Brand 50 '' from Methus Landmark Company, whose composition is as follows:

Total protein equivalent〉 50%

Fat〉 1%

Vegetable fiber ≤ 9%

Vitamin A〉 65.000 units / ㎏

The results obtained here are described in the following table.

Therefore, animals that consumed 31,559 RP per day for 28 days for the first 28 days had a weight gain of 3% or more, a conversion of less than 1.3%, and a percentage of 31,559 RP per day for 200 mg per day compared to the control animals. Intake of animals corresponds to 15.3% and 13.4%, respectively.

During the second period (28-56 days), animals who consumed 100 mg of 31,559 RP a day overwhelmed those who ate more. Higher weight gain (13.4%) and lower degree of conversion (9.8%) than control animals at the 100 mg / day dose, these percentages were 9.6% and 7.6% for the animals adjusted to the 200 mg / day dose, respectively. Corresponds to

During the entire test period (56 days), animals fed 31,559 RP 100 mg and 200 mg per day had average weight gain rates of 7.8% and 12.2% and feed conversions of 6.4% and 10.2%, respectively, compared to control animals.

Claims (1)

A method for preparing antibiotic 31559 RP and its salts, characterized by incubating Streptomyces hygroscopicus DS 24367 (NRRL 5787) in aerobic media to produce and isolate antibiotic 31559 RP.

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