DE2308059B2 - Process for the biosynthetic production of griseofulvin - Google Patents

Description

There are already numerous processes for the biosynthesis of griseofulvin using various types of Genus Penicillium, such as B. by means of the P.patulum, P.griseofulvum and P.urticae strains known (USA. Patents 3 095 360, 3 069 329 and 3 069 328).

In these known methods, culture media are used, their composition in terms of the nitrogen, carbon and other component sources depending on the metabolic peculiarities the species or the strain used is varied. The physico-chemical growing conditions (Temperature, ventilation, stirring process, duration, etc.) are both the trunks in the known methods as well as the media used.

The known methods (U.S. Patents 3,095,360, 3,069,329 and 3,069,328), in the case of soft Strains of P.griseofulvum and P.urticae used have the disadvantage that low activities (Antibiotic concentration) can be achieved.

In the known processes according to the above-mentioned USA patents, inoculation (Inoculation) of the medium for obtaining the preculture (the vegetative inoculum) in agar-agar-containing Spore suspensions generated by culture media are used. These methods have in particular the Take advantage of the fact that the biosynthesis potential becomes unstable over time and that constant laboratory processing of cultures is necessary.

In some of the known processes (U.S. Patents 3,069,329, 3,069,328), with soft Penicillium patulum strains are used as stimulating methyl group donors, and the Carbon source is continuously added during the biosynthesis process, which has disadvantages in terms of additional expenses for the materials and plant costs required for continuous addition are connected.

According to the present invention, a method for the biosynthetic production of griseofulvin by growing Penicillium griseofulvum or Penicillium urticae in a culture medium is proposed that is characterized in that the culture medium consists of 5 to 15 g / l corn germs, 5 to 8 g / l corn extract , 100 to 140 g / l lactose, 7 to 15 g / l glucose, 10 to 14 g / l calcium carbonate, 1 to 2 g / l potassium chloride, 6 to 10 g / l monopotassium phosphate, 0.05 to O ₁ I g / t magnesium sulphate, 1 to 2 g / l urea and 2 to 10 g / l hydrogenated sunflower oil (Sonit).

The invention is explained using the following example, in which the strain Penicillium urticae was used: a) Preparation of the spore-bearing inoculum

Peeled off, soaked in water and sterilized Millet or rice grains were inoculated with a spore suspension obtained from one by selection obtained and pre-tested pure culture with high potential has been prepared.

The incubation was carried out until the spore formation was complete, the culture was then dried under vacuum and stored at +4 ° C for 1 to 2 years, being the viability of the spores and there. Biosynthetic potential good values during this time showed.

b) Preparation of the I. vegetative * inoculum I a medium of the following composition were prepared:

G/

g, g,

Corn extract (50 ",,)

Sodium acetate

Monopotassium phosphate

Potassium chloride υ ,.

Magnesium sulfate Ο, ί

Iron D-sulfate 0, (

Glucose (100% calculated) 28

Hydrogenated sunflower oil (Sonit) 1

pH 5.95 to 6.0
After sterilization, spore-bearing M-. material inoculated and stirred at 27 to 28 C; ··. with an aeration rate of II air / l / minute, b> an overpressure of 0.3 to 0.5 atm for 25 hours, with a biomass of 20 to 30 'characterized by the IL to III under optimal unfilling conditions. Degree metactmatic corpuscles, was obtained.

c) Preparation of the II. vegetative inoculum I of a medium of the following composition were prepared:

Soybean meal

Calcium carbonate ο

Monopotassium phosphate 4

Glucose (100% calculated) 20

Lactose 20

Sodium chloride 3

Ammonium sulfate 2

g / l g / l g / l g / l g / l g / I g / l

Ammonium sulfate

Magnesium sulfate 0.01 g / l

Hydrogenated sunflower oil (Sonit) 1 g / l pH value 5.95 to 6.0

After sterilization, 5 to 10% vol / vol des I. Inoculated vegetative inoculum and 18 to 25 hours grown under the conditions used for the I. vegetative inoculum.

d) The actual biosynthesis tons of the medium of the following composition were prepared:

Corn germ 5 to 15 g / l

Corn extract 5 to 8 g / l

Lactose 100 to 140 g / l

Glucose 7 to 15 g / l

Calcium carbonate 10 to 14 g / l

Potassium chloride 1 to 2 g / l

J 2

Monopotassium phate 6 to 10 g / l Magnesium sulfate 0.05 to 0.1 g / l Urinary torrent I up to 2 g / l Hydrogenated sunflower oil

(Sonit) 2 to 10 g / l

pH 5.8 to 7.5

6 tons of water were introduced into a 15 to 20 ton vessel, the corn flour and the corn extract were added, and the mixture was boiled for 15 to 60 minutes at 100 ° C. In another identical vessel, the lactose and glucose were dissolved in 6 tons of water at 55 to 65 C, then poured over the thermally treated corn flour and extract. The monopotassium phosphate, potassium chloride, magnesium sulphate and urea were dissolved in a 500-1 vessel intended for the preparation of the salts, and the resulting solution was then added to the other components. The calcium carbonate suspension was prepared in a 5-ton vessel, which was then also poured over the other components. The volume calculated for the mixing formula was made up with water, the pH was adjusted to 5.7 to 6.3 with 10 to 15% strength sodium hydroxide solution and the hydrogenated sunflower oil was added as an antifoam agent.

The medium prepared in this way was fed to sterilization by a continuous process. The sterilization was carried out at 120 to 125 ° C. In order to avoid the breakdown of the sugars, these can be sterilized separately at a lower temperature (1 15 to I20 ^c C) , but only after 059

the entire sterilization system is regulated with water became. It must be ensured that the sterilization time for a batch (30 t) does not exceed 4 to 5 hours. Both the extension of the sterilization time and the increase in the sterilization temperature lead to a partial degradation of the substances present, in particular the sugar, which caramelizes at temperatures above 120 ° C., which is negative affects.

. After seeding II vegetative Snokulum mild in the ratio of 5 to I0 _"o VoI / VoI the biosynthetic process with the following parameters has been used:

Temperature: 26 to 30 C.

Ventilation: during the first 12 hours between 0.8 and 1.2 l air / l / minute, after! 2 hours and until the end

the fermentation between 1.2 and 2 l air / l / minute.

PH value:

the natural pH value, which is between 5.8 and 7.0 '

The fermentation process lasted 300 to 350 hours and an activity of 12,000 gamma / ml was achieved.

The process according to the invention has the advantage that yields of 12,000 gamma / ml are achieved and that the cleaning process proceeds under comfortable conditions, since there are no intermediate products of the Biosynthesis are formed.

Claims (1)

Claim: Process for the biosynthetic production of griseofulvin by growing Penicillium griseofulvum or Penicillium urticae in a culture medium, characterized in that that the culture medium consists of S to 15 g / l corn germs. S to 8 g / l corn extract, 100 to 140 g / l lactose, 7 to 15 g / l glucose, 10 to 14 g / l calcium carbonate. to 1 to 2 g / l potassium chloride, 6 to 10 g / l monopotassium phosphate, 0.05 to 0.1 g / l magnesium sulfate, 1 to 2 g / l urea and 2 to 10 g / l hydrogenated sunflower oil (Sonit). »5