DD248143B1 - PROCESS FOR PREPARING PRODUCTION MIXTURES WITH 1,4-ANDROSTADIENE-3,17-DION AND 4-ANDROST-3,17-DION - Google Patents

Description

Field of application of the invention

The invention relates to a process for the preparation of 1,4-androstadiene-3,17-dione and 4-androstene-3,17-dione by microbial transformation of sterols. Both compounds, known as sterol degradation intermediates, are key products for the production of steroidal drugs. By known syntheses, the products can be converted into the pharmaceutically valuable derivatives of the androstane and pregnane as well as the estran series with a wide range of applications. The field of application of the invention is thus preferably in the pharmaceutical industry.

Characteristic of the known state of the art

It is known that sterols, such as. As cholesterol, sitosterol, stigmasterol, campesterol and ergosterol, by microorganisms from numerous genera, such. B. Arthrobacter, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Nocardia, Pseudomonas, Protaminobacter, Serratia, Streptomyces and especially Mycobacterium among other substances via the intermediates 4-androstene-3,17-dione (hereinafter called AD for short) and 1,4 -Ndrostadien-3,17-dione (hereinafter called ADD for short) can be degraded. These products are enriched in the fermentation medium only if no 9-a-hydroxylation takes place or if both 1 (2) -dehydration and 9-a-hydroxylation can not take place. this will

inter alia achieved by using microorganism mutant strains with defects in said enzymatic performances for fermentation (see, for example, the patents JP 79 73191 or JP 80148083). The first processes using enzyme deficient mutants to produce AD (see US Pat. No. 3,759,791) with Mycobacterium spec. NRRL B-3805/1 / or ADD (see US Pat. No. 3,684,857) with Mycobacterium spec. NRRL B-3683/1 / from sterols reveal yields of 43% of theory AD and 40% of theory ADD, respectively, based on the proportion of fermentation when using 1 g of sitosterol / I and a fermentation time of 186 hours to 216 hours converted sitosterols. In the patent EP 8214, 30 g of sitosterol / I are transformed during a fermentation time of 336 hours using the strain Mycobacterium fortuitum CBS 313.79 to an undisclosed amount of AD with a low proportion of ADD. With Mycobacterium fortuitum NRRL B-8153 or Mycobacterium phlei NRRL B-8154 (see US Pat. No. 4,243,654), an unspecified amount of AD / ADD mixture is also obtained from 30 g of sitosterol / I. Thus, the hitherto known method has the deficiency of the fact that the effectiveness, due to low sterol use and / or long fermentation times with simultaneously low yields, is too low.

Also, various additives such as glycerides (see patent JP 54138192) or egg yolk (see patent JP 8608794) to the fermentation medium have not yet led to a significant improvement in the effectiveness of the method. Thus, for example, with the addition of 10 g / l of dry egg yolk to the fermentation medium with Mycobacterium vaccae NRRL B-3683, 16 g of fermentation time are followed by 15 g of cholesterol with a yield of 27% by weight of ADD. Another common disadvantage of the said and other methods known from the literature (see, for example, JP 80111796) is to be seen in the necessity of separating the fermentation products from unreacted sitosterol, since upon extraction of the fermentation broth both substrate and products in the extract are included. A first solution to overcome this difficulty was found by M. Aoki et al. (see JP 7757161, Kokai), which added non-polar adsorbers of the XAD type after completion of the fermentation and thus could selectively separate the products formed. By this subsequent step, the effectiveness of the actual fermentation could not be positively influenced.

The I. Sauerbaum et al. (1st European Congress for Biotechnology, Interlaken, 1978) described fermentation of sitosterol with Mycobacterium spec. NRRL B-3683 in the presence of styrene-divinylbenzene copolymers (XAD 2) as a nonpolar adsorber in which 10g of sitosterol / I is converted to a mixture of ADD and AD in 96 hours to 80% of theory is the most effective solution so far to watch. This gives AD in a space-time yield of about 16 g / m ³ h, ADD of about 41 g / m ³ . The calculation of the space-time yields was carried out as (g) formed ADD or AD per (m ³ ) fermentation broth and fermentation hours (h) without consideration of the fermentor changeover times. This method has at least the following disadvantages:

AD is only a by-product. The yield of AD can not be increased in the presence of XAD 2 as an adsorber not more than 25% by weight, based on the sitosterol used.

On the other hand, under the condition which gives the best yields (addition of 7g / ITween 20 as nonionic

Surfactant: polyethylene glycol sorbitan fatty acid ester), the proportion of ADD / AD of the product mixture ADD / AD is about 30%, and can not be lowered below about 20% even by decreasing the surfactant concentration even by assuming a decrease in overall yields.

- The resulting mixture in the fermentation ADD / AD in the specified concentration ratios can only by

consuming cleaning operations and thus be separated using additional process steps. In addition, the use of polar adsorbers of the XAD structure type in the microbial transformation of sterols with Nocardia spec. M29 (see Europ. J. Appl. Mikrobiol.2, 243 1976). According to their statement, however, such a process modification leads directly to a reduction in the yield of ADD or AD.

Object of the invention

The aim of the present invention is the preparation of product mixtures with 1,4-androstadiene-3,17-dione (ADD) and 4-androstene-3,17-dione (AD) by microbial transformation of sterols.

Explanation of the essence of the invention

The invention has for its object to describe a microbial process for the preparation of product mixtures with ADD and AD as the main components of sterols, in which after largely uniform technology both ADD / AD mixtures with an ADD content of at least 88Gew .-% and pure AD can be obtained in each case at high sterol throughput, short fermentation times and good overall yields

 According to the invention, this object is achieved as follows:

Sterols such as sitosterol, cholesterol, campesterol, stigmasterol or ergosterol or mixtures of these sterols are used in the form of a sterol surfactant preparation, so that the concentration of sterol is 1 g / l to 50 g / l, and fermented with bacteria of the genus Mycobacterium , The sterol surfactant preparations are obtained either by processes known per se (lyophilization or grinding, for example in corundum mills) or by the spray drying method described in an earlier application (for the surfactants used according to the invention, see below).

The microorganisms used are preferably bacterial strains of the species Mycobacterium vaccae. It is fermented in media containing carbon sources such as glycerol, glucose, sucrose, starch and / or molasses, etc., and nitrogen sources such as yeast extract, urea, ammonium salts, corn steep liquor, plant flours and / or other complex media ingredients (such as e.g. Meat extract, hydrolysates, etc.) as well as mineral salts of various compositions. The fermentation extending over 24 hours to 192 hours and at 20 ⁰ C to 45 ° C at pH values in the range of pH6bispH8 under aerobic conditions in Rührfermentoren or in fermentors, which after

1 According to JP 105289 (Kokai), these Mycobacterium spec. Strains were later classified as Mycobacterium vaccae.

Air-lift principle work, ζ. B. in loop reactors carried out. The fermentation mixture is from the beginning or after a time interval up to 48 hours in a conventional manner (see patent DD 232167) an adsorbent of the type of organic polymer containing in addition to aromatic groups polar groups in an amount of 2g / l to 200g / l too. The adsorber is separated after completion of fermentation by per se known separation methods such as filtration, centrifugation or separation of the ausfermentierten culture solution including biomass and optionally unreacted sterol (see a., Patent DD 127455). Subsequently, the product mixture present at the adsorber is eluted by treatment with an organic solvent, preferably with methanol, and the crude product is purified by per se known process steps, preferably by recrystallization of by-products.

The adsorbents to be used according to the method preferably contain acrylic acid ester monomer units (see below in more detail in this description of the invention); they can be prepared by polymerizing the corresponding monomers together with other bifunctional monomers (such as styrene or ethylstyrene) with polyfunctional monomers (such as divinylbenzene) under conditions which result in a porous structure of the polymer.

The adsorbers used thus contain at the same time both aromatic groups and acrylic acid derivative groups. Surprisingly, in the stated technical problem, polar adsorbers of this type are in terms of stimulating fermentation both in the direction of high overall yields and in influencing the desired ADD / AD product ratio of the known adsorber types (XAD1 to 12), either exclusively vinyl aromatic or exclusively Acrylic acid ester-based, superior.

It has further been found that by employing sterol preparations with surfactants both from the group of polyethylene oxide-polypropylene oxide (PEO-PPOJ block copolymers in a proportion of 30% to 70% polypropylene glycol and having molecular weights of 1000 to 3500, hereinafter called group A surfactants, and from the group of the polyethylene oxide-polypropylene oxide adducts of the N, N "-tetra (oxypropylpropyol) -N '- (2-oxypropyl) -diethylenetriamine with molecular weights of the polypropylene glycol around 1400 to 2000, hereinafter referred to as surfactants of group B, in In addition to a further increase in the fermentation yields, the proportion of the respective by-product of the ADD / AD product pair can also be suppressed by fermentations with the adsorbers mentioned above, since the particular advantage of the process is that the total yield has hitherto been increased by surfactants, but not a selective one Effect could be generated

 The process is advantageously carried out as follows:

a) seed culture

For fermentation to ADD (with a slight addition of AD), preferably Mycobacterium vaccae NRRL B-3683 or a NRRL-B-3683 selectin is used (one of the selectants has been deposited in the culture collection of ZIMET under registration number METETK113).

For the fermentation to AD, Mycobacterium vaccae NRRL B-3805 or alternatively a selectin from NRRL B-3805 is preferably used (one of the selectants has been deposited in the culture collection of ZIMET under the registration number IMET SK110).

The microorganisms are cultured in media containing carbon sources as glycerol or glucose, as nitrogen sources, for example, yeast extract, urea or ammonium salts, and mineral salts of various compositions.

The pH is around the neutral point. The incubation temperature is in the range of 20 ⁰ C to 45 ⁰ C; the incubation period is 12 hours to 77 hours.

With 2.5% to 20% of the submerged culture thus obtained, the fermentation medium, which may have the same or a different composition as the seed culture, is inoculated.

b) fermentation

Moist adsorber is expediently added to the fermentation medium even before sterilization; However, adding it up to 48 hours after the sterol addition does not cause any significant loss of effectiveness. The adsorbers used are advantageously copolymers of acrylic acid esters, ethylvinylbenzene and divinylbenzene having a particle size above 0.4 mm, referred to below as adsorbents of type I or II. In this case, type I adsorbent is a porous copolymer in the form of spherical particles, which is obtained by free-radically initiated polymerization of a mixture of about 45% by weight of ethylvinylbenzene and divinylbenzene and about 10% by weight of methyl acrylate or ethyl ester in suspension and by a specific surface area is characterized by at least 25OmVg and a total pore volume of at least 0.4cm ³ / g. Adsorber type II is a copolymer of similar composition, but has a specific surface area of at least 400m ² / g and a total pore volume of at least 1.0cm ³ / g.

The required adsorber amount is between 2g to 200g / l. The said amount is dependent on both the capacity and the adsorption rate of the selected adsorber as well as the sterol concentration used and the expected amount of product. For example, for the fermentation of 10 g / l sitosterol, the addition of 80 g / l type II adsorber has proven to be advantageous (case of sterol transformation to ADD).

The sterol preparation is added as a finely divided suspension together with a surfactant from group A or from group B in total (1 g / l to 50 g / l) or portionwise immediately or up to 48 hours after or else continuously after inoculation. Sterilization of the total amount with the medium is also possible.

The fermentation may take place in suitable vessels as a shake culture, in fermentors which are mechanically stirred and aerated, or in fermentors operating on the air-lift principle. Both ADD and AD are enriched at the adsorber.

Adsorber of the type II in the presence of a surfactant from group B is advantageously used for the production of ADD. Thus, space-time yields of up to 74g ADD / m ³ . h in addition to 7.3 g AD / m ³ h and 7.5 g of 1,4-C ₂₂ alcohol / m ^{3 achieved} · h.

For AD production, it is advantageous to ferment with type I adsorber in the presence of a group A surfactant. In this adsorber-surfactant combination, space-time yields of up to 68 g pure AD / m ³ · h were achieved.

The process control is carried out by known analytical methods, for example by HPLC, GC, TLC determination of the extracted by butyl acetate from the fermentation medium sterols as well as by GC, HPLC or TLC determination of the products eluted by the adsorber. For this purpose, a sample is taken from the fermentation mixture, the adsorber optionally separated by sieving, washed with water from adhering bacteria and medium components and weighed nutschefeucht. A defined amount is eluted with 80 to 100% aqueous methanol. By HPLC, GC or DC separation, coupled with UV measurement, the product mixture consisting mainly of ADD and / or AD and the corresponding C ₂₂ alcohols (21-hydroxy-20-methyl-4-pregnene-3 -on and / or 21-hydroxy ^ O-methyl-M-pregnadiene-S-on) as by-products can be quantitatively analyzed.

c) Chemical work-up

In fermentations to produce AD, the adsorber separated and washed from the fermentation medium is preferably transferred to a column and eluted with an organic solvent, preferably with 80% methanol.

After concentration of the eluate in vacuo at 30 ⁰ C to 40 ⁰ C to about 10%, the crude product precipitates and can be filtered off after cooling to about + 5 ° C and dried. After a single recrystallization from methanol, AD is obtained with a purity of about 95%. The by-products ADD and C ₂ 2 alcohols formed only in small quantities remain in the mother liquor.

In fermentations with predominant formation of ADD, the crude product is obtained in an analogous manner. From the crude product, the ADD / AD mixture is recovered by recrystallization with an ADD content of about 90%.

Both type I and type II adsorbers can be reused without loss of yield and quality of the products and without affecting the process.

The solution according to the invention has the following advantages:

According to the stated method, the fermentation with Mycobacterium vaccae NRRL B-3805 produces the desired product AD in high yield, which is bound quantitatively to the type I adsorber used. By simple elution of the adsorber and crystallization of the AD, this is then obtained in pure form. While according to the best known solution AD arises in a space-time yield of about 16g / m ³ · h in admixture with ADD, so still by consuming purification operations that are associated with yield losses must be separated, according to the invention space-time Yields up to 68 g of pure "AD / m ³ .h can be achieved (see Embodiment 3).

Here, the method is distinguished from the prior art mainly by a substantial increase in the effectiveness and an improvement of the technology.

In accordance with the method according to the invention fermentation with Mycobacterium vaccae NRRL B-3683 using a type II adsorber, the desired product ADD with a share of about 90% of the mixture of end products ADD / AD also results in high yields.

While according to the prior art (best solution) ADD in a space-time yield of about 41 g / m ³ · h is formed in a mixture with about 16g AD / m ³ · h, space-time yields of up to 74g ADD / m ³ - h can be achieved in addition to 7.3 g AD / m ³ h and 7.5 g of 1,4-C ₂₂ -AlkOhOlZm ³ · h (see Ausführungsbei'spiel 8).

Here, the method is distinguished from known solutions, in particular by an increased effectiveness while at the same time substantially improving the purity of the ADD achieved.

embodiments

example 1

a) seed culture

Mycobacterium vaccae NRRL B-3805 is cultivated on slant of the following composition for 48 hours to 96 hours at 28 ° C: 20g glycerol, 5g Bacto-peptone, 3g meat extract, 15g agar-agar, distilled water. ad 1 000ml; pH 7.0.

500 ml stalk flasks are inoculated with 50 ml nutrient solution of the following composition: 10 g glycerol, 10 g yeast extract, 0.5 g KH ₂ PO ₄ , 0.5 g K ₂ HPO ₄ , 1.5 g (NH ₄ I ₂ HPO _4, 0.2 g MgSO ₄ -7 H ₂ O, 0.01 g FeSO ₄ .7H ₂ O, ZnSO ₄ .7H ₂ 0,002g O, distilled water ad 1 000ml;. pH 7.0.

After shaking at 28 ° C for 48 hours, this submerged culture is used at a 1:10 vaccination ratio to inoculate further growth stages or fermentation stage.

b) fermentation

Sixty 500 ml stirrer flasks are charged with 50 ml of fermentation medium of the following composition and sterilized: 5 g glucose, 10 g yeast extract, 1 g K ₂ HPO ₄ , 0.3 g CaCO ₃ , 1.5 g (NH ₄ ) ₂ HPO ₄ , 0.2 g MgSO 4 ₄ 7H ₂ O, 0.01 g FeSO ₄ .7H ₂ O, ZnSO ₄ .7H ₂ 0,002g 0.60 g nutschenfeuchter adsorber type I Aqua dest. ad 1 000ml; pH 7.0.

After inoculation with 10% of the previously described seed culture, 6.5 g sitosterol / I is added with 0.5 g / l Group A surfactant and 1 g / l Antaphron NM40 silicone defoamer.

After shaking at 28 ° C for 96 hours, the fermentation is stopped by separating and washing the adsorber with water.

The adsorbent is eluted with 80% methanol, the eluate in vacuo at 30 ⁰ C to 40 ⁰ C concentrated.

10 g of crystalline crude product were obtained, the composition of which is shown in the table below.

By recrystallization from methanol, 7.5 g were obtained by gas chromatography and thin layer chromatography pure AD.

In the present example, the following yields were achieved:

commitment AD AD Products C ₂₂ alcohol Remarks 19.5 g G % ADD G sitosterol 9.71 100 G 1.23 EluatCII) 8.71 89.7 0.72 0.7 readings crude 0.51 readings (10g) 0.87 0.42 I.Mutterlauge 0.12 readings (2.6g) 7.5 77.2 0 crystals 0 gravimetr. (7.5 g) 1.15 0.57 ⁺¹ value 2. mother liquor 0.52 0.12 ⁺⁺⁾ readings (2.5 g)

+) гі-Нусігоху-го-теіпуІ-Д-ргедпеп-З-оп (4-C ₂₂ -AlkOhOl) ++) 21-hydroxy-20-methyl-1,4-pregnadien-3-one (1,4- C ₂₂ -Acool)

From the above table shows that at an egg rate of 6.5 gSitosterol / l 72% of theory of AD crude product and 54% of theory was obtained by gas chromatography pure AD. This corresponds to a calculated space-time yield of gas-chromatographically pure AD of 25 g AD / m ³ · h.

Example 2

20 round bottom flasks with 50 ml filling were charged for the fermentation according to Example 1. Adsorber type II is used in the same concentration. After 96 hours of fermentation at 28 ° C on the rotary table, the fermentation batch is stopped and worked up according to Example 1. When using 6.5 g of sitosterol / I, 3.6 g of crude AD product and 0.4 g of by-products (ADD + C ₂₂ alcohols) were obtained. This corresponds to a calculated space-time yield of 37.5 g of crude AD product / m ³ · h.

Example 3

20 round bottom flasks are charged according to Example 1 for the fermentation; the following changes compared to Example 1 are made: Type I adsorber is used at a concentration of 100 g / l. In addition, 7.25 g sitosterol / I are added immediately after inoculation of the fermentation culture, another 7.25 g sitosterol / I 24 hours later. After 96 hours of fermentation and work-up according to Example 1, 6.5 g of AD / 1, 5, 5 g of ADD / I and 1.5 g of C ₂₂ -alcohol / 1 were obtained. This corresponds to a calculated space-time yield of 67.7 g of AD (pure gas chromatography) / m ³ · h.

Example 4

A small fermentor of the type Chemap 30 with turbine stirrer is charged with 20 l fermentation solution according to Example 1 without adsorber. For inoculation (1:10), another submerged culture is applied in 2-liter vials with 400 ml of filling. After inoculation, 150 g of sitosterol preparation with Group A surfactant are added. The sitosterol surfactant preparation was prepared according to the method described in our application WP CO7J / 2408957. The aeration is carried out with 11 air / l fermentation solution and minute. The speed of the stirrer is 600 rpm during the first 6 hours, then after the addition of 1.2 kg type I adsorbers, up to 120 hours 150 rpm. After working up according to Example 1, 49.6 g of crude product and 13.2 g of by-products (ADD + C ₂₂ -AlkOhOl) were obtained. This corresponds to a space-time yield of 20.8 g AD (crude product) / m ³ · h.

Example 5

Starting from 300 ml of a 2-day-old seed culture according to Example 1, an air-lift fermenter is inoculated with 3I fermentation medium according to Example 1 (including Adsorbereinsatz). The air intake rate is 10 l / min in the first 52 hours and then adjusted to 12.5 l / min until the end of the fermentation. After 90 hours at 28 ° C, the employed 18.3 g of sitosterol were converted to the following products: 7.08 g of AD, 1.29 g of ADD, 1.35 g of 4-C ₂₂ -alcohol, 1.02 g of 1,4-C _M - Alcohol. This corresponds to a space-time yield of 26.2 g AD (pure gas chromatography) / m ³ · h.

Example 6

An air-lift fermentor with 31 medium (including adsorber) according to Example 1, wherein the glucose concentration of 5 g / l was reduced to 2.5 g / l and instead of Hefeextra kt1 g / l urea was used, according to Example 4 with 300 ml inoculated into a seed culture according to Example 1. After 96 hours, the following products were formed from 18.3 g of sitosterol / I: 6.39 g of AD, 1.35 gADD, 1.59 g of 4-C ₂₂ -alcohol, 0.33 g of 1,4-C ₂₂ -alkoHol. This corresponds to a space-time yield of 22.2 gAD (pure gas chromatography) / m ³ · h.

Example 7

Mycobacterium vaccae NRRL B-3683 is cultivated on slant of the following composition for 48 hours to 96 hours at 28 ° C: 20g glycerol, 5g Bacto-peptone, 3g meat extract, 15g agar-agar ad 1 000ml aqua dest; pH 7.0.

500 ml of nutrient solution with the following composition are inoculated with the appropriate cells of each tube: 10 g glycerol, 10 g yeast extract, 0.5 g KH ₂ PO ₄ , 0.5 g K ₂ HPO ₄ , 1.5 g (NH ₄ J ₂ HPO _4, 0.2 g MgSO ₄ · 7H ₂ O, 0.01 g FeSO ₄ .7H ₂ O, ZnSO ₄ 0,002g 7 H ₂ O, 000ml distilled water ad 1; pH 7.0.

After shaking at 28 ° C for 36 hours, this first submerged culture is used at a 1:10 inoculum to inoculate a second submerged culture, which is performed as the first submerged culture. After combining 5 stirrer flasks of the second submersed culture, a laboratory fermenter is inoculated with 2.5 l useful volume in the ratio 1:10. The fermentation solution has

following composition: 5 g glucose, 0.125 g urea, 2g KH ₂ PO _4, 8g _of K ₂ HPO _4, 3 g (NH ₄ J ₂ SO _4, 0.2 g MgSO ₄ · 7H ₂ O, 0.01 g FeSO ₄ · 7H ₂ O, 0.002 g ZnSO ₄ .7H ₂ O, distilled water ad 1 000 ml, pH 7.0.

The fermentation batch contains an additional 80 g / l wet-adsorber type I. After inoculation, a sterile sitosterol surfactant preparation (group B surfactant) containing 25 g of sitosterol is added. With aeration of 0.331 l / l fermentation solution and minute and a stirrer speed of 450 rpm, 12.1 g of ADD, 1.6 g of AD and 1.9 g of 1,4-C ₂₂ -alcohol are obtained after 96 hours of fermentation. This corresponds to a yield of 80% of the theory ADD / AD with an ADD content of 88.3% and 9.7% of theory 1.4-C ₂₂ -AlkOhOl. This results in a space-time yield of 50.4 g ADD / m ³ h.

Example 8

The cultivation of Mycobacterium vaccae NRRL B-3683 and the production of a seed culture is carried out analogously to Example 7. Two 500 ml stirrer flasks are each charged and sterilized with 50 ml of fermentation medium of the following composition: 5 g sucrose, 7.5 g yeast extract, 3 g (NH ₄ I ₂ SO _4, 1 g KH ₂ PO _4, 4 g K ₂ HPO _4, 0.2 g MgSO ₄ · 7H ₂ O, 0.01 g FeSO ₄ 7H ₂ O, ZnSO ₄ .7H ₂ 0,002g 0.120 g nutschenfeuchter adsorber of the type I, distilled water ad 1000ml, pH 7.0.

After inoculation with 10% of the previously described seed culture, a sterile sitosterol surfactant preparation (Group B surfactant) is added to the concentration of 14 g / L sitosterol. After shaking for 96 hours at 28 ° C., the fermentation is stopped by separating and washing the adsorbent with water. The adsorber is eluted with methanol. Gas chromatography showed the formation of 0.71 g ADD, 0.14 g AD and 0.1 g C ₂₂ alcohols; this corresponds to a yield of 74.5% of theory ADD in addition to 14.5% of theory AD and 8.8% of the theory C ₂₂ -AlkOhOIe. This results in a space-time yield of 74g ADD / m ³ · h.

Example 9

When 50 g of sitosterol and 350 g of adsorbents of type I are used, under the conditions described in example 7, inter alia when using a group B surfactant, after 94 fermentation hours 13.8 g ADD, 1.85 g AD and 0.7 g 1, 4-C ₂₂ -alcohol obtained; this corresponds to 46% of theory ADD / AD mixture with an ADD proportion of 88%.

When the fermentation time is extended to 100 hours, 17.7 g of ADD, 2.2 g of AD and 1.4 g of 1,4-C ₂₂ -alkoHOl are formed; that is 58% of theory ADD / AD mixture with an ADD content of 89%.

Example 10

Under the conditions mentioned in Example 9, 75% of the sitosterol immediately after the inoculation and 25% of the sitosterol are added to the fermentation batch after 76 fermentation hours.

After 94 hours of fermentation, 17.5 g of ADD, 1.7 g of AD and 1.8 g of 1,4-C ₂₂ -alcohol, ie 57% of theory ADD / AD with an AD content of 10.3%, are found. This corresponds to a space-time yield of 74 g ADD / m ³ · h.

An extension of the fermentation leads after 192 hours to the formation of 23.2g ADD, 2.2g AD and 1.7g 1,4-C ₂₂ alcohol; these are 74% of theory ADD / AD mixture with an AD content of 8.7%. This corresponds to a space-time yield of 48.4 g ADD / m ³ · h.

Claims (5)

1. A process for the preparation of product mixtures with 1,4-androsta-diene-3,17-dione and 4-androstene-3,17-dione as main components by aerobic Submersfermentation of bacteria of the genus Mycobacterium in culture media, in addition to sources of carbon and nitrogen and mineral salts containing sterols or sterol mixtures in concentrations of 1 g / l to 50 g / l, in the presence of surfactants at temperatures in the range of 20 ° C to 45 ° C for a period of 24 hours to 192 hours at pH values between pH 6.0 and pH 8.0, where a) from the beginning or until 48 hours after the addition, an adsorbent of the organic polymer type containing polar groups in addition to aromatic groups, in an amount of from 2 g / l to 200 g / l, b) this adsorber is separated from the culture solution after completion of the fermentation and from it c) the product mixture above labeling separated by elution with organic solvents and purified by means of known sub-steps is characterized in that for fermentation - As adsorber, a copolymer of acrylic acid esters, ethylvinylbenzene and divinylbenzene and either a PEO-PPO block copolymer or a PEO-PPO adduct of N, N "-tetra (oxypropylpropyol) -N '- (2-oxypropyl) -diethylenetriamine as surfactant is used. 2. The method according to claim 1, characterized in that as the adsorbent in each case a copolymer of ethyl acrylate or methyl ester, ethylvinylbenzene and divinylbenzene, prepared by free-radically initiated polymerization of a mixture of about 45Gew .-% ethylvinylbenzene and divinylbenzene and about 10Gew .-% methyl acrylate or ethyl acetate, is used, with either the specific surface area of at least 250 m ² / g and the total pore volume is at least 0.4 cm ³ / g (type I) or the specific surface area of at least 400m ² / g and the total pore volume is at least 1 , 0cm ³ / g (Type II). 3. The method according to claim 1 and 2, characterized in that as surfactant in each case a compound either from the group of PEO-PPO block copolymers having a polypropylene glycol content of 30% to 70% and having molecular weights of from 1 000 to 3500 (surfactants of group A) - or from the group of PEO-PPO adducts of the above labeling with molecular weights from 1 400 to 2000 (surfactants of group B) is used. 4. The method according to claim 3, characterized in that the surfactant used in each case in the form of a sterile preparation with the sterol provided autoclaved either together with the culture medium or at the time of main culture inoculation or portionwise in a period up to 48 hours or continuously Inoculation of the main culture is added. 5. The method according to claim 1 to 4, characterized in that the fermentation in Rührfermentoren or in fermentors, which operate on the air-lift principle is performed.