DE1061964B - Manufacture of the antibiotic phyllomycin - Google Patents

Description

GERMAN

The invention relates to the production and extraction of a new streptomycete antibiotic with bacteriostatic and fungistatic properties. It is in the culture solution as well as in a small amount in the mycelium contain new Streptomycetes and was phyllomycin because of its effectiveness against phyllomycin fungi called.

The Streptomycete isolated from the river mud, which received the name Streptomyces umbrosus and under this Designation in the Centralbureau voor Schimmelkultures Baarn, Holland, has been deposited with none of the known Streptomycetes identical.

After "Actinomycetes and their Antibiotics" by S. A. Waksman and H. A. Lechevalier, 1953, p. 66, it is a chromogenic type with a pigment, Streptomyces viridoflavus, which is soluble in all organic media similar, but differs from it in essential features. According to the diagnostic methods of W. Lindenbein (Archiv f. Mikrobiologie, 17, pp. 361 to 381 [1952]) results for the new species in the following Table 1 compiled characteristics.

Streptomyces umbrosus can be based on nutrient solutions with z. B. glycerol or malt extract as a carbon source, peptone, corn steep liquor, brewer's yeast or casein hydrolyzate as a nitrogen source, with the addition of inorganic salts such. B. sodium chloride, DikaUumhydrogenphosphat, iron (II) sulfate and magnesium sulfate are grown. The culture medium should also contain the essential trace elements that are usually found in tap water and in natural nutrient additives, e.g. B. brewer's yeast are included. The _pH value of the broth prior to inoculation should be between 6.5 and 7.5. It was observed that the medium slowly becomes acidic at the beginning of almost all culture experiments. The formation of the antibiotic starts very early compared to other active ingredients and already reaches its maximum after 3 days of cultivation. Small amounts of phyllomycin can be produced in the shake flask or in the top culture. For the production of larger quantities of the antibiotic, the stirred, submerged, aerobic culture is preferred. To achieve the fastest possible growth and a shortened fermentation time as a result, there is always plenty of inoculation with a freshly prepared spore suspension or a corresponding submerged preculture. Streptomyces umbrosus can be cultivated at temperatures between 26 and 32 ° C, preferably at 29 ° C.

However, the production of the active ingredients is not limited to the use of Streptomyces umbrosus, but also includes mutations of this strain, which by the action of mutation agents, - z. B. from Ultraviolet rays, X-rays and chemical substances.

Manufacture of the antibiotic
Phyllomycin

Applicant:

Farbenfabriken Bayer Aktiengesellschaft, L everkus en-B ay er werk

Dr. Günter Schmidt-Kastner, Dr. Johannes Schmid,
Wuppertal-Elberfeld,
Dr. Ferdinand Grewe ₁ Cologne-Stammheim,
and Dr. Victor Flück, Leverkusen,

have been named as inventors

The concentration of the antibiotic in culture filtrates and culture extracts is determined by Rhodotorula flava CBS 331 was determined as the test organism. A typical culture filtrate inhibits in the plate hole test on a for Yeast suitable nutrient medium for growth in a dilution of 1:50.

The antibiotic can be obtained from the culture filtrates by extraction or adsorption processes. In particular, ethyl acetate and butyl acetate are suitable for extraction, but chlorinated hydrocarbons, ethers, ketones and alcohols can also be used. The extract of the culture liquid is evaporated in vacuo, accompanying substances are precipitated with hydrocarbons and, after the hydrocarbons have been removed in vacuo, the oily residue is mixed with a lower alcohol, primarily methanol. After separating off the methanol-insoluble fractions, a brown oil (unsaponifiable fraction of the oil = 20%; iodine number 290) is obtained after concentration, which is still effective against Rhodotorula flava in a dilution of 1: 1,000,000 in the plate hole test. The biological tests below were carried out with this oily phyllomycin concentrate. For further purification of the fungistatic, the phyllomycin concentrate with an effectiveness of 1: 1000,000 is distributed in countercurrent with the system: 13 parts by volume of ligroin, 6 parts by volume of butyl acetate, 2 parts by volume of di-n-butyl ether, 14 parts by volume of N-dimethylformamide and 7 parts by volume of water, distributed over ten stages , the fraction five distilled to dryness in vacuo and from the remaining solid residue after crystallization from methyl propyl ketone the phyllomycin in rosette-shaped needles from mp 177 to 178.5 ° C (uncorrected), [afj + 75 ° (c = 0.834 % in chloroform), 57.70% C, 5.77% H, 5.88% N and

909 578/404

3 4

Table 1

Cultural characteristics of Streptomyces umbrosus

medium growth Color of
Culture surface
JJZ W. UCS jUUI LlllyCClb Color of
bottom
ίΛΎ * Tv Π 1 4 ~ Π f
L-Cl JiUlLUl Pigment formation
and color Remarks ^! tm τη f * ti cr * Ti fvp
Oy Il LlXc LloL-Ilt- 1
Agar ■ Faiti Imicficr
XCJLll HlLlSLlg cr * n τη 11 τ 7 i er—
O Ul III 1 U. LZ / lg
brownish yellow,
no aerial mycelium ViTimncTf ⁱ IH
Ul «. Lille: CIL / rvran ti Cf ¹ IVi
Ul CLU-llgClL? Synthetic
solution weak,
only at the bottom White without Cr Il 1 i "a G P» —Wί π P ca ti 7 Cff ¹ Ti τι c
enough,
only at the bottom IIlLill L XCo lO LCli-IJCLl. without Irin pnco- A OTQt ¹
VTlULUbC ΛΚαΐ Ertlt
gUL J_ / Lll Lilly L-Cl
grey black er * Ti τη π ■ f * ?! & -
oLilllllU. Lilg
brownish Glucose-
Asparagine agar well, thin Aerial mycelium
Gray brownish brownish yellow I ^ o TVffolo + Δ tror
^ d-lVldlcLX-X \ gd .r slim,
slimy SCllIirLÄlg-
brownish, no
Aerial mycelium SLl IlIlU ΙΛΙκ-
brownish without gelatin good, velvety
pillow-shaped Aerial mycelium,
White yellow not detectable deep brown no
liquefaction Starch agar not good,
slim without aerial mycelium,
brownish not detectable black-brown Nutrient agar not good,
slim without aerial mycelium,
gray-brown gray-brown Brown "Co , -4. , -.- Cf _n I
Cardboard box not good without aerial mycelium,
Gray J & artoitel tiei
dark colored carrot no growth Cellulose medium weak,
at the bottom White without no growth
on cellulose
strip Liquorice milk
Agar Well no aerial mycelium,
dirty-
brownish without Lakmus discolored

30.74% 0; X _max 322 ιημ with α = 9.70 and X _max 225 πιμ with a = 60 (a = specific extinction coefficient = extinction for c = 1 g / 1 and i - 1 ecm).

Phyllomycin has characteristic absorption bands in the UR at the following wavelengths: (KBr-Press-Iing; the values in brackets indicate the percentage permeabilities)

2.97 (50.7%), 3.37 (54.8%), 5.72 (7.1%), 5.97 (27.7%), 6.10 (34.6%), 6.21 (37.0%), 6.29 (47.3%), 6.55 (17.1%), 6.87 (47.8%), 6.98 (48.4%), 7.36 (30.0%), 7.52 (43.2%), 7.64 (48.8%), 7.84 (36.3%), 7.98 (24.2%), 8.49 (17.0%), 8.66 (17.2%), 9.28 (36.9%), 9.30 (37.1%), 9.49 (49.0%) and 9.85 μ (44.6%) . The exact course of the absorption curves in the ultra-violet and ultra-red range of the Spectrum is shown in Figs. 1 and 2.

Phyllomycin is temperature resistant within certain limits; at 20 ° C, the tested against Rhodotorula flava effectiveness over 7 hours is maintained at 50 ° C, the efficacy is under comparable test conditions after 3 hours of 100%, after 7 hours 95%, at IOO ⁰ C to _V2 hour, 100% after 3 hours 80%, after 7 hours 18%, at 150 ° C after ^{1 and} ₂ hours 3%, after 3 hours 0%. Phyllomycin is alkali-sensitive.

In the ring distribution chromatogram, the following R _f values are found for phyllomycin with the distribution systems given below: 0.13 with 1 part by volume of water, 1 part by volume of ethylene glycol, 4 parts by volume of N-dimethylformamide, 3 parts by volume of di-n-butyl ether, 3 parts by volume of ligroin; 0.31 with 1 part by volume of ethylene glycol, 2 parts by volume of N-dimethylformamide, 1 part by volume of di-n-butyl ether, 2 parts by volume of ligroin; 0.68 with 1 part by volume of water, 2 parts by volume of methanol, 2 parts by volume of N-dimethylformamide, 2 parts by volume of butyl acetate, 3 parts by volume of ligroin, and 0.88 with 1 part by volume of water, 3 parts by volume of ethylene glycol, 2 parts by volume of N-dimethylformamide, 3 parts by volume of butyl acetate, 2 Parts by volume ligroin. To carry out the chromatogram, filter papers cut in the round (Schleicher & Schüll 2043b) are sprayed with the hydrophilic phase of the respective distribution system, previously equilibrated by continuous shaking, and developed with the hydrophobic phase. After the chromatogram has dried, a 0.5 mm wide strip is cut out radially and placed on an agar plate inoculated with Rhodotorula flava. At the point where the active ingredient is located, a clear zone of inhibition arises during incubation, from which the R _f value is calculated as the quotient of the distance traveled by the antibiotic to the distance traveled by the hydrophobic phase (= solvent front).

The crystallized phyllomycin still inhibits Rhodotorula flava CBS 331 in a dilution in the plate hole test 1: 50 OOO OOO to 1: 100,000,000.

On the other hand, the phyllomycin can be obtained from the culture medium by centrifuging the or brings together filtered culture solution with an adsorbent, in particular with activated carbon, the adsorbate separates and the active ingredient with organic solvents, especially with chlorinated hydrocarbons and alcohols, desorbed.

In addition to phyllomycin, the culture nitrates also contain another antibiotic, which is also used against fungi, z. B. against Rhodotorula flava and Cladosporium cucumerinum, is effective. When extracting with organic Solvents, such as butyl acetate or ethyl acetate, it remains in the aqueous phase.

Phyllomycin is extremely effective against plant pathogenic organisms in vivo and in vitro. A phyllomycin concentrate (activity against Rhodotorula flava 1: 1,000,000) was used for the test, its effectiveness in vitro in the tube dilution test and in the plate hole test against fungi and bacteria in Table 2 and its effectiveness in vivo on the plant in Table 3. However, the effectiveness of the antibiotic is not limited to the organisms listed in Tables 2 and 4. Phyllomycin can be used as a systemic crop protection agent. The infestation quotient of Omphalia flavida on Coleus plants after drenching the soil with a l ° / ₀ Phyllomycinlösung strength is 52, after impregnation with a 0, l% solution 68 (control 100); (see Table 3).

The phyllomycin concentrate 1: 1,000,000 has only a slight phytotoxicity (see Table 4). Without exception, l ° / ₀ solution was treated with a 0, observed no damage. Also, the results obtained with a l ° / ₀ solution are insignificant.

As fungistatically effective Streptomycetenantibiotika a group of compounds, such as. B. Fungicidin (= Nystatin) (J. D. Putcher, G. Boyack, S. Fox, Antibiotics Annual, 1953 to 1954, p. 191), Eurocidin

(Y. Okami, R. Wahara, S. Nakomura, H. Umezawa, journal of Antibiotics, Japan, 7A, p. 98, 1954) Flavacid (J. Takahashi, Journal of Antibiotics, Japan, 6A, p. 117, 1953), Candicidin (H. A. Lechevalier, R. F. Acker, C. T. Corke, C. M. Haenseler, S. A. Waksman, Mycologia, 45, p. 155, 1953) that chemically characterized by a chain of conjugated double bonds and a typical for this constitution Absorption spectrum with three closely spaced

ίο have maxima, e.g. B. Fungicidin (= nystatin) at 292, 304 and 318 πιμ, Eurocidin at 318, 333 and 351 πιμ, Flavacid bei 341.358 and 379πιμ and Candicidin at 359.5, 379.5 and 401.5 πιμ. As can be seen from the spectrum, the new antibiotic phyllomycin does not have such a typical absorption spectrum and should be clearly different from the compounds mentioned. Actidion (AD Whiffen, RL Emmerson, N. Bohonos, USA.-Patent 2,574,519; Upjohn Company) and phyllomycin also exist

ao clear difference, since Actidion in the tube dilution test against Cladosporium cucumerinum only in a dilution of 1: 50,000 inhibits growth, while phyllomycin is still effective at 1: 10,000,000. Also between phyllomycin and the well-known streptomycete antibiotics streptomycin, tetracycline, viomycin, Erythromycin, chloramphemcol and novobiocin are marked differences in biological effects too record. The phyllomycin concentrate in the plate hole test against Candida utilis CBS 840 still yields in a dilution of 1: 200,000 produces an inhibition zone of 19 mm, while streptomycin with the dilution 1: 400, tetracycline with 1: 400, viomycin with 1: 400, erythromycin with 1: 4000, chloramphemcol with 1: 4000 and novobiocin at 1: 6000 are ineffective. There are also differences between phyllomycin and the Antibiotic Antimycin A discovered by C Leben and G. W. Keitt (Phytopathology, 37, p. 14 [1947], and 38, p. 899 [1948]).

Table 2

Efficacy in vitro of the phyllomycin concentrate (activity against Rhodotorula flava 1: 1,000,000)

Type of test

Effective limiting dilution; Inhibition zone diameter during the plate hole test (-> -)

Test organism

1: 10,000,000
1: 500,000

500,000
750,000
750,000
2000 -
4000 -
500 -
1000 -

1: 500

1000 -
500 -
1000 -
10,000 500,000 ■
1: 200,000
ineffective

20 mm
14 mm
14 mm
8.5 mm
11.5 mm
8.0 mm
22.5 mm
15.0 mm

> 38 mm
-> 17 mm
-> 19 mm

Cladosporium cucumerinum
Cladosporium sp. (Hop)
Bortrytis cinerea

Xanthomonas malvacearum, strain RI
Xanthomonas malvacearum, strain R II

Cerospora musae

Xanthomonas malvacearum, strain RI

Xanthomonas malvacearum, strain R II

Xanthomonas malvacearum, strain 1002
Sarcina lutea

Rhodotorula flava CBS 331
Candida utilis CBS 840
Staphylococcus aureus

R = tube dilution test. P = plate hole test.

As can be seen from Table 5, Streptomyces NRRL 2288 shows clear culture differences compared to the phyUomycin-former Streptomyces umbrinus. A direct comparison between an authentic antimycin A preparation and phyllomycin revealed differences in the melting point (uncorrected): Antimycin A 136 to 137 ° C, PhyUomycin 177 to 178.5 ° C, mixed melting point 129 to 130 ° C, and, as shown in Fig 3 can be seen, differences in the R _p values in the ring distribution chromatogram when using a distribution

systems of 6 parts by volume of ligroin, 1 part by volume of din-butyl ether, 3 parts by volume of butyl acetate, 7 parts by volume of N-dimethylformamide, 3 parts by volume of water and using the technique described above (see Fig. 3). The chromatogram was developed with diazotized sulfanilic acid. Since there are also qualitative and quantitative differences in the biological effects of the two fungistatic agents (see Table 6), phyllomycin should undoubtedly be addressed as a new antibiotic ίο .

Table 3

Effectiveness in vivo of the PhyUomycin concentrate (activity against Rhodotorula flava 1: 1 000 000)

concentration pest host Infestation quotient 0.2% Phytophtora infestans Tomato plant 52 0.1% Phytophtora infestans Tomato plant 59 Phytophtora infestans Tomato plant 100 o, i% Alternaria solani Tomato plant 19th 0.01% Alternaria solani Tomato plant 51 Alternaria solani Tomato plant 100 0.05 «/ ο Downy mildew Vines 20th 0.01% Downy mildew Vines 37 0.005% Downy mildew Vines 40 Untreated Downy mildew Vines 100 0.025% Pericular orycae Rice plant 66 Pericular orycae Rice plant 100 0.05% Omphalia flavida Coffee plant 0 0.005% OmphaUa flavida Coffee plant 1.2 0.0005% OmphaUa flavida Coffee plant 78 Untreated Omphalia flavida Coffee plant 100 1%*) Omphalia flavida Coleus 52 0.1% *) Omphalia flavida Coleus 68 Omphalia flavida Coleus 100

*) The soil was soaked with the solution to be tested (= testing of the systemic effectiveness).

Table 4

Phytotoxicity of the PhyUomycin concentrate
(Activity against Rhodotorula flava 1: 1,000,000)

Test plant injected
Conc
tration Finding Bean 1% Leaf margin necrosis; New growth normal Bean .... 04% no damages; New addition normal Bean 0.05% no damages; New addition normal Bean .... 0.01% no damages; New addition normal BaumwoUe 1% Leaf margin necrosis cotton o, i% no damages Coffee .... 0.1% no damages Banana ... 1% Leaf spots at the base of the Heart leaf Banana ... 0.1% no damages rice 0.1% no damages

Table 5

Differentiation of the "antimycin-producing" Streptomyces against Streptomyces umbrosus

“Antimycin
producing «
Streptomyces
NRRL 2288 Streptomyces
umbriosus Color of the air mycelium
on glucose asparagus
gin agar, glucose
Agar and nutrient agar know up
cream colored Gray Microscopic book
of the Luftmycels no spirals Spirals with
two to five
Turns Color of the substrate
on peptone
Culture media
Agar and gelatin
Agar) colourless
to weak
yellowish dark brown

Claims (1)

9 10 Table 6 Effectiveness of antimycin A and phyllomycin in the plate hole test
against three different yeasts Antimycin, A.
VerdiiTiniiTig (diameter now, Hemmhof) Phyilomycin
Thinning (diameter mm Hemmhof) Candida lipolytica (Harrison) 1 10,000 - > 21.8 1 640000 - - > 27.0 Diddens et Lodder 1 40,000 - - > 20.5 1 2,500,000 - - > 20.0 1 160,000 - 5 »· 18.0 1: 10,000,000 - > 12.5 Sporobolomyces holsaticus 1 10,000 > 22.2 1: 640000 - > 25.5 Windisch 1 40000 > 21.0 1: 2,500,000 - > 19.5 1: 160,000 > 18.0 1: 10,000,000 - > 15.0 Rhodotorula glutiuis (Fres.) 1: 10,000 - > 21.8 1: 160,000 - - > 29.0 Harrison 1 : 40,000 > 20.6 1: 640,000 - > 21.0 1: 160,000 > 18.0 1 2,500,000 - - > 14.0 Example 1 _ao phase refilled. Both separating funnels are getting longer 150 ecm of a nutrient time are shaken in 1-1 Erlenmeyer flasks. The process is in the sense of a Solution of the composition: 2% glycerine, 0.1 ° / ₀ countercurrent distribution repeated over eight stages. All Peptone, 0.25% glycocolla, 0.1% Na Cl, 0.1% K ₂ HPO ₄ , eight aqueous phases are drained, to the organic 0.02% FeSO ₄ -7 H ₂ O, 0.02% MgSO ₄ -7 H ₂ O, ruffled phases 21 ethyl acetate and once with fills, sterilized for 20 minutes at 120 ° C, shaken out after the waste 25 water (water discarded). The eight cool with a spore suspension of the strepto-aqueous phases are converted to the corresponding organic myces umbrosus nov. spec., the phases given on a nutrient medium, all eight fractions the same composition was grown with 2% agar, shake and the aqueous phases are separated. These inoculated and on a shaker at 240 rpm still contain small amounts of phyllomycin. The resulting Fermented at 29 ° C. After 72 hours of culture 30 animal organic phases are three times with each inhibits the fermentation broth Rhodotorula flava shaken out in 21 water (water discarded) and im a dilution of 1:50. With 21 of these in the growth vacuum distilled to dryness at a bath temperature of 60 ° C, The pre-culture is in a 1000-1 fermenter, which The fractions four, five and six contain the main with 7001 of the nutrient solution: 2% glycerine, 1% dry content of the active ingredient. Fraction five becomes after brewer's yeast, 0.25% glycine, 0.1% Na Cl, 0.1% K ₂ HPO _4, 35 three times recrystallization from methyl-propyl 0.02% FeSO ₄ -7 H ₂ O, 0.02% MgSO ₄ -7 H ₂ O, 0.1% 2 g PhyUomycin in rosette-shaped needles, mp. Silicone oil (commercially available goods), is charged and at 177 to 178.5 ° C (uncorrected), [af ¹ _D + 75 ° (c = 0.834% two consecutive days each 20 minutes in chloroform), 57.70% C, 5.77% H, 5.79% N and 120 ° C was sterilized, inoculated and with an air 30.74% 0, A _max 332 πιμ. with a = 9.70 and X _max 225 ταμ passage of 350 1 / minute, stirred vigorously for 72 hours. 40 with a = 60 obtained, which is still in a dilution of The effectiveness against Rhodotorula flava is then 1: 50,000,000 to 1: 100,000,000 the growth of 1:50. Then the fermentation liquid will inhibit Rhodotorula flava, freed from the mycelium on the centrifuge, the clear centri-. . fugate extracted once with 300 liters of butyl acetate, the yellow example 2 green-colored extract in vacuo at 50 ° C. on 4 1 45 101 culture solution of Streptomyces umbrosus nov. concentrated, mixed with 161 ligroin, a greenish spec. with an activity against Rhodotorula flava brown precipitate is separated off and the solvents of 1:50 are centrifuged, the clear centrifugate is distilled off in vacuo at 50 ° C. The remaining residue is stirred with 50 g of activated charcoal and the charcoal is thrown off, which is still used as an antifoam agent. In the culture containing silicone oil that has been freed from the adsorbent, approximately 10% of the effectiveness remains twice with 31 50 solution each time. The methanol is drawn off, the oily phase adsorbate which separates out is dried and separated off with 1 1 chloroform and the methanol is distilled off in vacuo. Stirred for 30 minutes at 50.degree. C., spun again, and about 30 g of brown oil result, which in the mixture of the supernatant chloroform solution to dryness, distilled 1: 1000,000, the growth of Rhodotorula. As a residue left 1.7 g of an oil which flava and inhibits the growth of Rhodotorula flava is still used in a try as starting material for plant 55th At a dilution of 1: 200,000 and the given For further cleaning of the antibiotic, if further cleaning can be carried out as above,
eight 10-1 separating funnels each 41 of the organic phase of the distribution system: 13 volume parts ligroin, 6 volume claim:
parts butyl acetate, 2 parts by volume di-n-butyl ether, 14 parts by volume 60 parts N-dimethylformamide and 7 parts by volume water, The use of Streptomyces umbrosus nov. filled, 400 g of the above phyllomycin concentrate spec, for the production of the fungistatic and bacterial with an effectiveness of 1: 1,000,000 in 41 of the aqueous static antibiotic phyllomycin Phase of said distribution system solved and with the usual biological way and extraction of the 41 of the organic phase of the first separating funnel 65 antibiotic by extraction or adsorption shaken for a long time. The aqueous phase is made up of the mycelium or the filtrate of the fermentation liquid separated, to the organic phase of the second separating and optionally purification by chromatographic funnel and in the first 41 fresh aqueous graphic processes. 1 sheet of drawings © 909 578/404 7.59