DE1792019B - Process for the preparation of rifamycin-SV - Google Patents

Description

Vegetative ATCC 13 685 Soluble Vegetative ATCC 21 271 Soluble fertile soil Mycelium air pigment Mycelium Air- pigment crystal clear mycelium ', green-yellow crystal clear to mycelium bright Starch agar to light brown pink ; until bright orange brown is missing green yellow White Brown until dark to brown Brown green yellow crystal clear light yellow crystal clear to bright green Ca malate to light brown pink orange yellow is missing yellow to Glucose agar White Brown green yellow crystal clear little crystal clear to very bright Ca malate to light orange White weak- orange-yellow to Brown Glycerin agar bright ; orange orange brown green yellow crystal clear to orange '■ very bright crystal clear to is missing brownish yellow Bennet agar orange yellow pink yellow-brown orange yellow to brown White until dark Red red-brown ^: missing

The nutrient media mentioned in the table above had the following composition:

Starch agar: 10 g soluble starch, Ig K ₂ HPO ₄ , Ig MgSO ₄ -7 H ₄ O, Ig NaCL. 2 g (NH _{4) 2} SO _4, 2 g of CaCO _3, depending 0.001 g FeSO ₄ .7H _ä O, MnCl ₄ .4H ₄ O, ZnSO ₄ 7H ₂ O, 20 g of Agar Difco, with distilled water to 1000 ml filled up. After 20minutiger sterilization at 12O ⁰ C the pH was 6.7 to 7.8.

Ca malate glucose agar: 20 g glucose, 10 g calcium

Malate, 0.5 g NH ₄ Cl, 0.5 g K ₂ HPO ₄ , 15 g Agar Difco,

made up to 1000 ml with distilled water. After sterilization at 115 ° ^{C. for} 1 minute, the pH was 6.5.

Cu malate glycerine agar: 10 g glycerine, 10 g Ca malite, 0.5 g NH ₄ Cl, 0.5 g K ₂ HPO ₄ , 15 g agar Difco, made up to Ϊ000 ml with distilled water. After 15minutiger sterilization at 115 ^C C the pH was at 6.5 to 6,6.

Bennet agar: 10g glucose, Ig yeast extract, Ig Meat extract, 2 g N-Z-Amin A, 15 g agar, made up to 1000 ml with distilled water. To After sterilization, the pH was 6.7 to 6.8.

The fermentation and extraction process is essentially the same as the process described for the production of rifamycin B and begins with the cultivation of Streptomyces mediterranei ATCC 21 271 in a nutrient medium containing an assimilable carbon and nitrogen source and important mineral salts until this nutrient medium has a considerable antibiotic Has effectiveness, and in the extraction of Rifamycin SV from the nutrient medium. This strain is grown under stirring and under aerated submerged conditions at a temperature between 25 and 37 ° C and preferably at 28 ^C C. The following carbohydrates and carbon derivatives can be used as the carbon source: glucose, galactose, lactose, sucrose, maltose, glycerine, mannitol. Usable nitrogen sources are e.g. B. Am: nosr iren and their mixtures, peptides, proteins and their breakdown products such as peptone, yeast extract, soybean meal, corn water, soluble fish residues, meat extracts, aqueous parts of the grain seeds. The fermentation can be carried out for 96 to 180 hours. The initial pH, which was generally around pH 6.2 to pH 6.4, fell to pH 5.5 to 6.0 during the fermentation. In general, the best results were observed after fermentation for 180 hours. After this time an excellent yield of the antibiotic was obtained.

After the fermentation, the rifamycin SV could be isolated by the following procedure: The fermentation medium is filtered at the final pH of pH 6.4 to pH 6.6. To be the best To guarantee the stability of the antibiotic substance, the filtrate is brought to a pH, which is preferred is below pH 5, acidified. The active substance is mixed with water immiscible solvents, such as chloroform, butanol, ethyl, propyl, butyl or amyl acetate, extracted. The volume ratio between the medium and the solvent varies depending on the solvent chosen; in general a ratio of 2: 1 up to 10: 1 is used.

The mycelium continues to retain a microbiological effectiveness that is remarkably higher than that observed in the fermentation of rifamycin B. With a water immiscible The active substance is extracted from the mycelium and then mixed with the organic solvent Combined phase that already contains most of the rifamycin. The active substance can come from the mycelium can also be extracted with a water-miscible solvent such as acetone. In this case the liquid is filtered, the acetone evaporated in vacuo, the rifamycin with one with water extracted immiscible solvent and the process as described above, carried out.

When the majority of the antibiotic substance has been converted into the solvent, distillation is preferably carried out at a temperature of below 30 ° C. in vacuo to dryness.
The rifamycin SV is purified by chromatography on a column prepared with a suitable adsorption material such as silica gel. The rifamycins are dissolved in a suitable organic solvent such as acetone on the

ίο column brought and mixed with a suitable solvent eluted as acetone and benzene. In general, the separation of rifamycin SV from other rifamycins quite sharp, as it has a much higher mobility and on The characteristic orange-yellow colored ring inside the column can easily be followed. The eluate containing the rifamycin SV is then concentrated to dryness in vacuo and a crystalline, orange-yellow residue obtained.

The following examples illustrate the invention.

example 1

A suspension of the mycelium of Streptomyces mediterranei ATCC 13 685 was made with N-methyl-N'-nitro-N-nitrosoguanidine treated and inoculated on Bennet agar. The colonies that received the mutagenic treatment survived, were isolated and assessed for their ability to stimulate the growth of Pseudomonas reptilivora NRRL-B6 grown on Penassay seed agar at pH 7.2 inhibit, checked. Rifamycin B is microbiologically inactive under these conditions, and rifamycin SV forming Streptomyces med-i'.erranei ATCC 21 271 was selected.

This strain was propagated on Bennet agar for 6 to 8 days and incubated ^{at 28 ° C.} With the culture obtained from the agar culture placed at an angle in the test tube, two 500 ml Erlenmeyer flasks were placed under sterile conditions. Conditions oculated. The flasks contained 100 ml of the vegetative medium of the following composition:

Meat extract 5 g

Yeast extract 5 g

Peptone 5 g

Casein hydrolyzate 3 g

Glucose 20 g

NaCl 1.5 g

made up to 1 liter _{with H 2 O.}

The pH was adjusted to 7.3 with NaOH.
The flasks oculated in this way were

SS placed on any shaking apparatus ^{at 28 ° C. for 72 hours.} The contents of the two EHenmeyei * flasks were used as a vaccine by pouring them into a 10 liter pre-fermenter containing 4 liters of the aforementioned vegetative medium.

Incubation was carried out at 28 ° C. with stirring at 750 rpm and with aeration at 1 V / V / min. After growth 30stündigem a 7- to 10 "/ ₀ sodium volume of packed cells obtained.

G5 In the next stage, a 10 liter, from Ola's existing fermenter used the 4 liters of the fermentation medium mentioned below contained:

Peanut flour 25 g

Soybean flour 5 g

(NH ₄ WaO ₄ 9.5 g

VIgSO ₁ -7H ₅ O 0.85 g

Glucose 95 g

Glycerin 40 g

KH ₂ PO ₁ Ig

Propylene glycol 5 g

CaCO, 8.5 g

Na diethyl barbiturate .... 1.7 g

CuSO ₁ -5 H, 0 2.8 mg

FeSOj-7H.rO 8.5 mg

ZnSO ₄ -7 H., 0.22.5 mg

VInSO ₄ -4HoO 3.4 mg

CaCl., - 6H., Ö 1.7 mg

(NH ₄ HMo ₇ O ₂₄ · 4 H ₂ O ... 0.85 mg

made up to 1 liter _{with H 2 O}

The pH was adjusted to pH 7.8 with NaOH. The sterilization was carried out at 120 ° C. for 60 minutes. After sterilization, the pH was 6.4. (A part of the contents of the prefermenter equal to 5 ° / ₀ of the contents of the fermenter were used as a vaccine).

The fermentation was carried out at 28 ^° C. with stirring at 750 rpm and with aeration at 1 V / V / min. Silicone A was used as an antifoam agent. During the fermentation, the culture broth acquired a characteristic red-brown color. After growth for 120 hours, a volume of 20 to 25% packed cells was obtained. The pH of the broth was pH 6.0. The highest antibiotic effectiveness (200Oy / ml rifamycin SV) was achieved after 180 hours. The broth was isolated at this point.

Example 2

A culture of Streptomyces obtained according to Example 1 meditcrranei ATCC 21 271 was prepared according to Example 1 in a flask with stirring. To the For pre-cultivation, it was poured into a 10-liter, glass fermenter that contained 4 liters of the following medium contained:

Glucose 5 g

Peanut flour 7.5 g

CaCO ₃ 1.65 g

MgSO ₁ -7 H., 0 0.33 g

KH ₂ PO ₄ 0.33 g

FeSO ₄ · 7 H ₂ O 3.3 mg

ZnSO ₁ -7H, 0.16.5 mg

MnSO ₄ -4H ₂ O 1.3 mg

made up to 1 liter _{with H 2 O}

The pH was adjusted to pH 7.5. The sterilization was carried out ^{at I2O Q C for 50 minutes.} After sterilization, the pH was 6.4. After 38stündigem growth, the packed cell volume of the total volume was 6 to 8 ° / _0th A vaccine equal to 10% was used for a 20 liter glass fermenter containing 10 liters of the following fermentation medium:

Corn water 20 g

Soybean flour 15 g

(NH ₄ ) S ₁ SO ₄ 6 g

MgSO ₄ -7 H ₂ O 0.85 g

Glucose 100 g

KHoPO ₄ Ig

CaCO ₃ 6 g

FeSO ₄ -7HoO 8.5 mg

ZnSO ₄ -7H "O 42.5 mg

MnSO ₄ -4HoO 3.4 mg

CuSO ₁ -5 HoO 2.8 mg

COCl ₂ -OH ₂ O 1.7 mg

made up to 1 liter _{with H 2 O}

The pH was adjusted to pH 7.8 with sodium. The sterilization was carried out ^{at 120 3 C for 50 minutes.} After sterilization, the pH was 6.4. The fermentation was carried out at 28 ° C for 150 hours. On isolation, the pH of the fermentation broth was pH 6.0. The final antibiotic activity was 1600 μg / ml rifamycin SV.

The product was extracted, purified by chromatography and then crystallized, as described above. Chromatography was performed with silica gel (Merck 0.05-0.20 mm) accomplished. The crude rifamycin was dissolved in acetone and then with a benzene-acetone mixture eluted in a ratio of 1: 1. The product purified in this way showed the chemico-physical Properties of Rifamycin SV.

Melting point: decomposition starting at 140 C. ² [\] o = -3.9 ° C ⁰ (c = 1 ° / ₀ in methanol).

The IR spectrum showed the following absorption bands: 3440, 2920 (Nujol), 2850 (Nujol), 1700, 1658. 1605, 1545, 1464 (Nujol), 1415, 1375 (Nujol), 1325, 1290, 1260, 1218, 1197, 1164, 1125, 1115, 1102, 1083. 1050, 1020, 976, 962, 950, 930, 915, 898, 871, 845.

830, 801, 785, 772, 760, 750, 717, 703 cm " ¹ .

The visible and UV spectrum were carried out with phosphate buffer pH - 7.3 and showed absorption maxima at 223.314 and 445 m / x.

Claims (1)

Claim: Process for the preparation of Rifamycin SV, characterized in that one Streptomyces mediterrane: ATCC 21 271 in an aqueous nutrient medium, the assimilable carbon and contains nitrogen sources and mineral salts, under aerobic submerged conditions breeds and the fermentation solution worked up in the usual way. 15th In an earlier publication (Antibiotics Annual, 1959/1960, p. 262) Se η si et al. the production of the antibiotic rifamycin, which is a mixture of many substances with similar Structure is made by fermentation of a strain of Streptotnyces mediterranei. The most interesting of these substances turned out to be rifamycin B: therefore one tried to improve the yield. In “Appl. Microbiol ", p. 325 (1961), is by Margalith tt al. one of the research results described; there it is said that there is yield and purity of rifamycin B by adding sodium diethylbarbiiurate to the fermentation broth leaves. Another publication (S e η s i et al., 11 Farmaco, Ed. Sci., 16, p. 165) relates to the production from rifamycin SV, which is one of the components of the rifamycin genus, a higher one Has antibacterial activity and an important link in the synthesis of other rifamycin derivatives represents. In general terms, the procedure is that one rifamycin B of a series chemical processes during which it first oxidizes to rifamycin O, then hydrolyzes to rifamycin S. and finally converted to rifamycin SV with a mild reducing agent. This Synthesis is obviously very tedious and involves quite difficult to solve in the various stages practical problems associated with probably frequent losses of the same Antibiotic. A simpler process was sought for economic reasons for the production of Rifamycin SV. The process according to the invention for the preparation of rifamycin SV is characterized in that one Streptomyces mideterranei ATCC 21271 in an aqueous nutrient medium that is assimilable Contains carbon and nitrogen sources and mineral salts, grows under aerobic submerged conditions and the fermentation solution is worked up in the usual way. Streptomyces mediterranei ATCC 21 271 will hold if you look at Streptomyces mediterranei the effects of physical (e.g. UV rays), chemical (Mustard gas, N-methyl-N'-nitrosoguanidine) or biological (e.g. actinophage) mutant agents. Streptomyces mediterranei ATCC 21 271 retains the original taxonomy of Streptomyces mediterranei at, and the characteristics concerning its culture are practically the same. Only with the Formation of aerial mycelium and some differences in the color of soluble pigments were observed, after Streptomyces mediterranei ATCC 21 271 had grown on appropriate nutrient medium. The following table lists the properties of Streptomyces mediterranei ATCC 13 685 with compared to those of Streptomyces mediterranei ATCC 21 271. Comparison of the characteristics of the cultures of Streptomyces mediterranei ATCC 13 685 and from Streptomyces mediterranei ATCC 21 271 on several nutrient media