DE2028986C3 - Selenomycm - Google Patents

Description

2 G 2 S 9 6 6

V.

S -.:;■. η c u- ;;
>. .;> ^ nn-Asparagi
N'idium η. 5.
>': -. ir 11 η g and

c; τ I i e b ■.

N '; id: u-ii n. <X
H . r 1 i π g and ο ο: t! ieb ■ '!

• Medium η. ~.

S hir !; η c and G ο 111 i eb *

Gi uc-osi-asparagine agir

N her-A car

Kar; open! -Agar

Xanthine agar

Ciiciummalai agar

Maeemilch

Hickey and Tresner's Agar

Bennett Agar

L? • vii ^ es U measure ;;:, dait. searched; =; LM Sruren. white LP none V. " Well. slightly wrinkled, cream-colored LNi none LP none W. Well. smooth, cream-colored to pale yellow LNi 'icichirosa LP none W. moderate, smooth and flat, translucent LNl White" LP none W. moderate, smooth and after, translucent

Table II

none

2Ut. smooth, creamy to light yellow

Tracks, white

none

Well. wrinkled, cream to amber in color

Physiological Eigensc stick of none

cm. -unzehg. cream-colored to light yellow

do not know any

rasiliense n. sp. ATCC Z \ . '*.'

Result

Solution of Calci malate i negative

N:; rat reduction positive

-N-rosinase formation negative

Melanin formation negative

H-.drolysis of starch 'positive

Test method

H, S formation

Liquid gelatin Liquorice milk

Casein HydroKse

positive

positix

not coagulated.

Peptonization

positive

Table St.
Utilization of carbon sources

Carbon qucllc behavior

Inositol ■] -

Fructose r

Rhamnose r +

Mannitol -

Xylose

Raffinose

Arabinose

Cellulose

Sucrose

Glucose (positive control)

No carbon
(negative control) -

- + Strongly positive recovery; Growth similar or stronger than with a positive control. Positive recovery; Growth much stronger than in the case of a negative control (no carbon), although somewhat less than in the case of glucose.

Negative recovery; Growth similar to negative control (no carbon).

From the comparative information in Table IV it can be seen that the strain Streptosporangium brasiliense n. sp. ATCC 21 393 differs from all other Streptosporangium species described so far.

During the breeding of the strain Streptosporangium brasiliense n. Sp. ATCC 21 393 used Inorganic salts are alkali metal and alkaline earth metal chlorides, nitrates, carbonates, sulfates or

ίο salts of magnesium, iron, manganese, zinc with the same anions. As sources of nitrogen and carbon are used: natural or synthetic Amino acids, peptides and proteins and their hydrolysates, such as peptone or tryptone; Meat extract; the water-soluble components of cereal grains such as corn or wheat; soluble residues from alcohol distillation, yeast extract, corn steep liquor, glucose, sucrose, lactose, starch, Dextrins.

ao Cultivation is generally carried out for about 24 to about 96 hours under aerobic conditions at about 25 to about 35 ° C carried out in a submerged culture, with air or oxygen supply stirs.

Table IV Comparison with other species of the genus Strepiosporangium

Size of
Sporangic, Length of
Sporangio
phorcn, Width of
Aerial myccls, Shape and size
the spurs, Color of
Aerial myccls Physiological cn sheep
GcIa-
tine-
ver en
Ni
kicked H ₂ S-
BiI-
manure Art Prop
Strength
kc-
hy- liquid- rcduk- μ μ μ μ dro- supply tion 6 to 8 NB 0.7 to 1.0 1.0 to 1.3 White lysis + NB S. album χ 1.5 to 1.9 6 to 8 NB 0.7 to 1.0 1.0 to 1.3 Pale pink + + NB S. amethy- x 1.5 to 1.9 + stogenes *) 5 to 9 NB 0.7 to 1.0 spherical Pale pink + + NB S. roseum 1.0 to 1.3 + χ 1.5 to 1.9 7.7 to 11.0 1.6 to 27.0 0.8 spherical Dark NB S. rubrum 1.0 to 1.1 Gray 6 to 8 NB 0.7 to 1.0 1.0 to 1.3 Greyish + NB S. viridi- χ 1.5 to 1.9 knows or + album Yellowish Gray 6 to 8 NB 0.7 to 1.0 1.0 to 1.3 Pale pink + ΝΪ S. vulgar χ 1.5 to 1.9 + 20 to 48 25 to 64 0.4 to 0.6 spherical Light green + + NI S. viridog- (d. 29) (d. 38) 0.2 to 0.4 lich, gray + riseum 3.5 to 14.0 'until 24 0.9 to 1.0 cylindrical White + + + S. brasi (d. 12.0) (d .5,0) 2.3 to 2.8
X 1.1 + liense

*) Purple crystals on oatmeal agar.

d. = Average. NB = Not determined.

To obtain the selenomycin from the fermentation liquid, this is mixed with butanol or ethyl acetate or chloroform extracted. The extraction is done after the fermentation liquid wedge up an acidic pH above about 5 is carried out.

According to a preferred method, c

Fermentation medium at alkaline pH

trated to prevent something microbiological

active substance reverberated in the filter cake

will. The filtrate is acidified to pH 5.5 a

r

extracted twice with chloroform. The combined extracts are concentrated under reduced pressure narrowed small volume. Selenomycin is obtained by adding a large volume of light petroleum to the concentrated extracts. The product filled out in this way can be treated with a Mixture of 30% chloroform and 70% benzene can be purified at 35 "C. The insoluble fraction is filtered off, and the concentrated to a small volume Solution is added to a large volume of hexane and the product precipitates. The The product obtained in this way is dissolved in hot benzene, decolorized with activated charcoal, filtered and cooled to 4C. The antibiotic is obtained as a white amorphous powder with a melting point of 135 to 138 ° C.

The antibiotic selenomycin is soluble in alcohols, chloroform, ethyl acetate, acetone and dioxane and hot benzene; insoluble in water at neutral and acidic pH; soluble in alkaline water and insoluble in diethyl ether.

The UV absorption spectrum shows the following maxima: in 0.1 n-HCl at 245 to 257 ηιμ (E!?. = - 41); in buffer solution of pH 7.3 at 295 ΐυμ (E! r " - 88); in 0.1 N NaOH at 295 πιμ (El *. = 88).

The presence of a functional group of pKa 5.5 was confirmed by spectrophotometric Ti'ration proven.

The IR absorption spectrum of selenomycin in N'uj oil shows absorptions at the following frequencies: 3400, 1720, 1650, 1550, 1520, 1350 (Sch), 1200, 1170, 1100, 1040, 980, 950, 912, 870,

25th

1240, 1200, 1170,,

835, 815, 785, 770, 740 and 720 cm ¹ . The IR absorption spectrum of selenomycin is shown in FIG. 1 shown.

Selenomycin gives a positive Tollens and Fehling reaction, a negative Schiff, FeCl ₃ and Molish reaction; It quickly removes the color of potassium permanganate in acidic and neutral solutions, forms a phenylhadrazone with 2,4-dinitro-phenylhadrazine and gives a brown color with concentrated sulfuric acid and concentrated hydrochloric acid.

The paper chromatographic behavior of selenomycin with different solvent systems is given in Table V below

Table V

Paper chromatographic behavior of selenomycin

Solvent mixture Rf value

1. Butanol saturated with water 0.84

2. Butanol saturated with water

+ 2% p-toluenesulfonic acid 0.95

3. Butanol saturated with water

+ 2 ⁰ I ₀ NH ₄ OH 0.69

4. Water saturated with butanol 0.78

5. NH ₄ Cl, 20 ° / _o aqueous solution ... 0,00

6 75 _^0/0 phenol - 25 ° / ₀ water 0.96

7. Butanol to methanol to water

(4: 1: 2) + 0.75 g methyl orange ... 0.82

8. Butanol to methanol to water

(4: 1: 2) 0.92

9. Acetone to water (1: 1) 0.34

10. Ethyl acetate saturated with water 0.79

The selenomycin becomes visible on paper strips through microbiological development on agar plates inoculated with B. subiiiis.

Selenomycin is stable in neutral and alkaline solution, but inconsistent in acidic solution. Watery Antibiotic solutions with a pH of 3.0 lose 80% of their effectiveness within 24 hours.

Selenomycin is effective against various gram positive bacteria, but much less effective against gram negative bacteria. Yeasts and fungi. The smallest inhibitory concentrations against various Microorganisms are compiled in Table VI.

Table V!
Antimicrobial Effectiveness of Selenomycin

Smallest

inhibiting

concentration

in micrograms

Organism per ml

Staphylococcus aureus

ATCC 6538 0.2

Staphylococcus aureus Tour 2.0

Staphylococcus aureus

ATCC 9144 0.5

Staphylococcus albus

ATCC 12228 2.0

Streptococcus faecalis

ATCC 10541 2.0

Streptococcus faecalis

ATCC 7080 5.0

Streptococcus hemolyticus C 203 0.1

Streptococcus agalactiae

ATCC 7077 0.2

Streptococcus bovis ATCC 9809 1.0

Diplococcus pneumoniae UC 41 0.5

Sarcina lutea ATCC 9341 0.5

Micrococcus flavus ATCC 10240 0.5

Bacillus subtilis ATCC 6633 0.5

Bacillus cereus ATCC 10876 0.5

Bacillus anthracis M 401 0.5

Clostridium perfringens

ATCC 3626 0.5

Corynebacterium diphtheriae

mitis ATCC 11051 0.2

Proteus vulgaris> 100

Escheridia coli ATCC 10536> 100

Pseudomonas aeruginosa

ATCC 10 145> 100

Candida albicans SKF 10231> 100

Trichophyton mentagrophytes

SKF 8757> 100

Mycobacterium tuberculosis

H 37 Rv ATCC 9360> 100

45 Selenomycin is of interest in the treatment of infections caused in animals by Staphylococa, Streptococcus, Diplococcus and, more generally, by Gram-positive bacteria. The following ED ₆₀ values were obtained in mice that were treated subcutaneously with the antibiotics for three days after they had been infected with the following strains:

S. aureus ED ₆₀ ~ 30 mg / kg

S. hemolyticus ED ₈₀ 2: 20 mg / kg

D. pneumoniae ED ₆₀ ^ 15 mg / kg

The LD ₄₀ in mice when administered intraperitoneally is 400 mg / kg. Usable therapeutic blood levels! are used in subcutaneous, rectal and int!

309647 /:

pcritoneal administration of M) nip kg selcnomycin echo to Miiuse. The blood levels are in the table VII compiled.

after subcutaneous rectal intra- Table VII Mice 27 4.92 perilonea Ululspicgcl of selenomycin in y / ml Appointment type 25th 6.10 37.20 Administration of 50 nig / kg 18th 3.10 23.70 hours
after 21.25 Ver submission 1 3 5

Dilution using Bacillus subtilis and Slaphyloccoeus aureus as indicator cultures examined. The following results were obtained :

Microorganism

Bacillus subtilis
Staphylococcus aureus

Antibiotic c

Effectiveness in

Dilution";-

cinhcitcn

(72slündigc

Fermentation)

1/2560 1/320

Example 2

Example I. A. Flask fermentation

I. Manufacture of the vaccine

With Streptosporangium brasiliense n. Sp. ATCC 21393 inoculated oatmeal agar Schrägnährböden 7 are held up to 10 days at 32 ⁰ C and then used to inoculate 100 of a peptone-glucose-yeast extract ml medium in a 500 ml Erlenmeyer Kolbcn used, which had a single lateral baffle plate.

This medium had di? the following composition:

Peptone 5.0 g

Meat extract 5.0 g

NaCl 5.0 g

Yeast extract 1.0 g

Glucose 10.0 g

Tap water to top up .. 11

The medium is adjusted to pH 7.2 and ^{sterilized at 121 °} C. for 20 minutes. The bottles were ^{kept on a rotary shaker at 28 °} C. for 72 hours, with a deflection of 6 cm and 240 rpm. M. is operated. The pH of the medium is then adjusted to 8.5. The percentage mycelium volume (pMv) is 12. The percentage mycelium volume is determined by centrifuging a 10 ml sample at 3000 r.p.m. for 10 minutes. M. determined.

II. Fermentation Conditions

10 ° transfers were made from the culture obtained to 500 ml Erlenmeyer flasks, which are equipped with a side baffle and contain 100 ml of the following medium:

Meat extract 4.0 g

Peptone 4.0 g

NaCl 2.5 g

Yeast extract 1.0 g

Soybean meal 10.0 g

Glucose 50.0 g

CaCO, 5.0g

Tap water to fill up aui .. Il

The pH of the medium is adjusted to 7.3 and the medium is ^{sterilized at 121 °} C. for 20 minutes. The flasks are inoculated and kept under the same conditions as indicated above. After 72 hours the Mycei is centrifuged off and aa clear supernatant liquid is removed by streaking. As in Example 1, a vaccine is prepared using a fermentation medium similar to that in Example 1, but instead of the glucose

also uses various other carbohydrates. To For 72 and 96 hours, samples of the fermentation medium are collected using B. subtilis examined as an indicator strain for their antibiotic effectiveness. The following results were obtained:

Culture medium of Rnkniels 1 with

Glucose (control)

Xylose

Galactose

Inhibition zones (in mm) against B. subtilis using

of standard filter paper

fail with one

Diameter of 9 mm

72 hours

96 hours

18 22 12

16 20

12th

Example ..

I. Manufacture of the vaccine a) First vegetative stage

Vaccine source: Streptosporangium brasiliense n. Sp. ATCC 21 393 on oatmeal agar. The medium and the growth conditions are identical to those of Example I.

b) Second vegetative stage

Vaccine source; 2.5 ° ' _o (25 mil of the first vegetative stage. The medium used for the second vegetative stage is the same as in the first stage. The medium, 4 liters in a 10-1 glass fermentation vessel, is brought to pH Set 7.2 to 7.3, sterilized at 12 ° C for 30 minutes , inoculated and kept at 28 ° C for 72 hours.

ss During the incubation, the culture medium is aerated at a rate of 11 air per 1 culture liquid per minute and at 800 rpm. M stirred. After the end of the growing season, the pH value is 8.5 and the percentage mycei volume is 12 ° / *

H. Fermentation Conditions Vaccine source: 3GOmI (7.5 · /,) of the second vege

6 $ tative stage. The medium used is the gkkb

as in example 1. Ar * pH 7.2 Ws 7.3 thin and sterilized for 30 minutes at 121 ° C. The inoculated medium is treated under the same conditions as sowing

hatches as they are for the production of the second vegetative Stage were applied. Maximum antibiotic titers are usually obtained after a fermentation period received from 72 hours. Typical changes that occur during fermentation are graphically in FIG. 2 shown.

Example 4

Extraction of the selenomycin from the fermentation medium

40 1. Culture medium are filtered at pH 8.5. The mycelial cake is discarded. The pH of the culture filtrate is reduced to 5.5 by adding 1 η aqueous hydrochloric acid. Sodium chloride is added to a concentration of 20 ° / ₀ (Gew./V) was added. The filtrate is extracted twice with chloroform, one volume part of chloroform being added for each extraction.

form je '/ ■, volume part Filtnii used. The chloroform extracts are combined. The combined Chloroforme.'Urakte are dried over sodium sulfate and concentrated in vacuo at 30C to a volume of 50 ml. The sclcnomycin is precipitated from the concentrated extract by adding 21% light petroleum. 4 g of crude selenoinycin are obtained. The raw sclenomycin is in 200 m! a mixture of 30 percent by volume of chloroform suspended in benzene. The suspension is shaken at 35 to 40 ^° C., the insoluble fraction is filtered off, the filtrate is concentrated to a volume of 80 ml, and 500 ml of hexane are added. The precipitate is dissolved in 30 ml of hot benzene, the hot solution with active carbon is be filtered and cooled 10 to 12 hours at 4O ⁰ C. 800 mg of white amorphous product are obtained, which has been found to be effective against Staphylococcus aureus in a concentration of 0.2 μg / ml.

1 sheet of drawings

Claims (1)

Claim: Selenormcin, with the melting point 135 to 138 C: (E _r 41!) The UV-absorption maxima in 0.01 N HCl at 245 to 257 ma: m.mu. in buffer solution of pH 7.3 at 295 (E] ■ "■ = 88) and in 0.1 n NaOH at 295 ηΐμ (egg.:;.- 88); 1R absorption maxima in Nujo'l at 3400, 1740, 1650, 1570, 1550, 1350, 1245, 1200, 1170, 1100, 1040, 1000, 980, 950, 912, 870, 835, 812, 783, 770, 738, 720 and 680 cm '; an acid group with a pKa value of 5.5: a positive Tollens and Fehling reaction and a negative Schiff. Ferric chloride and Molisch reaction, which is soluble in lower alkanols, chloroform, ethyl acetate, acetone, dioxane and hot benzene and insoluble in diethyl ether; Potassium permanganate decolorized in neutral and acidic solution; forms phenylhydrazone with 2,4- dinitronhenylhydrazine; with concentrated sulfuric acid and concentrated hydrochloric acid results in a brown color reaction and by cultivating Streptosporangium brasiliense n. sp. ATCC 21 393 in an aqueous nutrient medium under aerobic conditions at a temperature between about 25 and 35'C, freeing the medium from the mycelium, acidifying the mycelium-free medium to a pH of about 5 and extracting the medium with butanol, ethyl acetate or chloroform is available. Most antibiotics, so far described, are from the genera Streptomyces and Micromonospora and nocardia formed. While studying the extraction of new antibiotics from the genus Streptosporangium a new microorganism was formed from a soil sample obtained from Brazil isolated, the one antibiotic complex effective in vitro and in vivo against several gram-positive bacteria forms. Numerous fractions were isolated from this complex. It was found that one of these fractions, which was designated selenomycin, practically all of the antibiotic activity having. The new microorganism was given the name Streptosporangium brasiliense n. Sp. given. It is deposited with the American Type Culture Collection under number 21 393. The subject of the invention is selenomycin with a melting point of 135 to 138 "C; the UV absorption maxima in 0.01 N HCl at 245 to 257 ιτιμ (El * ^ 41); in buffer solution of pH 7.3 at 295 ιπμ (EU ; = 88) and in 0.1 n-NaOH at 295 πιμ (Eil = 88); IR absorption maxima in Nujol at 3400, 1740, 1650, 1570, 1550, 1350, 1245, 1200, 1170, 1100, 1040, 1000 , 980, 950, 912, 870, 835, 812, 783, 770, 738, / 20 and 680 cm "¹; an acid group of pKa 5.5; a positive Tollens and Fehling reaction and a negative Schiff, ferric chloride and Molisch reaction; which is soluble in lower alkanols, chloroform, ethyl acetate, acetone, dioxane and hot benzene and insoluble in diethyl ether; Potassium permanganate decolorized in neutral and acidic solution; forms a phenylhydrazone with 2,4-dinitrophenylhydrazine; with concentrated sulfuric acid and concentrated hydrochloric acid results in a brown color reaction and by cultivating Streptosporangium brasiliense n. sp. ATCC 21 393 in an aqueous nutrient medium under aerobic conditions at a temperature between about 25 and 35 C. Removal of the mycelium from the medium. Acidification of the mycelium-free medium to a pH value above about 5 and extraction of the medium with butanol, ethyl acetate or chloroform can be obtained. To determine the growth characteristics de-, Streptcsporangium brasiliense n. Sp. ATCC 21,393 were grown on a variety of media cultures. The breeding took place for up to 3 weeks at 28 to 29 C. Most of the taxonomic ones Methods used were the investigation Recommended by S h i r 1 i η g and G ο t t 1 i e b. General characteristics: The culture grows on plentiful on oatmeal agar. On this medium the individual colonies reach a diameter of 5 to 6 mm and are abundant with white aerial mycelium which, when the formation of sporangia has ended, takes on a light pink color. Iodinine-like crystals common to some of the Streptosporangium species described are derived from not educated in this culture. On some culture media, mainly soil agar, will be incomplete Sporangia are formed in which more or less tightly rolled spirals do not form a spore container aufwe.sen. The optimum growth temperature was found to be 28 to 32 ^C C. Morphology of the Luftmycels: Microscopic examination of the Luftmycels revealed short, heavily branched ones Hyphae, at the tip of which a spherical sporangium develops. The average diameter the air mycelium is 0.9 to 1.0 microns. The sporangia * vary quite a bit in size, im generally from 3.5 to 11 microns in diameter. For fully ripe crops, the average is However, it is 12 microns in diameter. The spore carrier is quite short, 6 to 24 microns, with an average 15 microns in length and with a diameter equal to that of the air mycelium. The spore bearers are regularly rolled up inside the sporangia. Most of the spore bearers are cylindrical, always immobile, measuring 2.3 to 2.8 by 1.1 microns, but occasionally spherical spore carriers with an average diameter of 1.1 microns are also found. On the rolled up hyphae that are not surrounded by a visible wall of sporangia however arthrospore-like spores are formed, which is quite common on certain poor media, such as soil agar. These fragment spores are typically 3.2 to 3.5 in size at 1.0 micron. Cultural and Physiological Characteristics: The cultural and physiological characteristics of the Are summarized in Tables I, II and III, On most media, the vegetative mycelium was a light cream color with white aerial mycelium, the became very slightly pink when the sporangia was strong. The Anyalse on the cleaned cell wall of the new tribe identified this as "Type 11" according to Lechevalier. From all the given characteristics it must be concluded that the new strain belongs to the genus Streptosporangium, since the genera of the family Actinoplanaceae the sporangiophores are immobile.