CS237553B1 - Process for making penicillin V and G - Google Patents

Abstract

The invention relates to a method for producing penicillin V and G by submerged cultivation of the microorganism Penicillium chrysogenum in a liquid nutrient medium containing sources of assimilable carbon, inorganic and organic nitrogen sources, precursors and nutrient salts.

Description

The invention relates to a process for the production of penicillin V and G by submerged cultivation of a penicillium chrysogenum microorganism in a liquid nutrient medium comprising assimilable carbon sources, inorganic and organic nitrogen sources, precursors and nutrient salts.

As is known in the fermentative production of penicillin V or G, both the qualitative and quantitative ratio of sources providing assimilable nitrogen is a decisive factor for the growth of the biomass of the production strain and for the biosynthesis of penicillin.

Biomass growth and production height are both dependent on the nature and quantity of assimilable nitrogen sources and on the rate of oxygen dissolution in the soil, which depends on the capabilities of the fermenter available.

It is also known that the limiting effect of oxygen dissolved in the soil on production already occurs at a lower concentration than at which the growth of biomass is reduced. As a result, the optimum concentration of assimilable nitrogen sources in the nutrient medium applies to fermenters with the given ability to achieve a certain rate of oxygen dissolution.

If this concentration is exceeded, there may be an increase in biomass at which the rate of oxygen dissolution may be insufficient for some metabolic pathway, in this case for the production of penicillin V and G.

It is further known that the production of penicillin V and G on soils with an organic nitrogen source (e.g., various flour, corn steep liquor, etc.) can be stimulated by an inorganic form of nitrogen sources (e.g. ammonium salts, nitrates.

In checking this, it was found that the use of an inorganic form of nitrogen sources resulted in non-reproducibility of the results in various apparatuses.

On the other hand, it has been found that these difficulties can be eliminated when the inorganic form of the nitrogen source is not added in arbitrarily estimated amounts, but in some proportion to the organic form of nitrogen.

On the basis of this finding, a process for the preparation of penicillin V and G according to the invention has been developed, characterized in that the fermentation is carried out in a culture medium in which the amount of nitrogen from the inorganic source is 20-30%, preferably 25% of the total. the amount of total nitrogen from the organic source, the optimum amount of nitrogen from the organic source being dependent on the rate of oxygen dissolution in the broth in the apparatus used.

The finding of these facts and dependencies is new and has been shown to have an unexpectedly beneficial effect on the substantial increase in penicillin V and G production by up to 25-30% compared to the yields of previous batches.

The basic calculation of the total amount of organic nitrogen results from experimental values optimal for the given apparatus. The mixing intensity, the degree of air dispersion and the viscosity of the medium are so variable that the general rules cannot be used in this case.

For given parameters the calculated amount of organic nitrogen is then in constant dependence on the concentration of inorganic nitrogen. Their sum according to the principle of the invention gives the optimum conditions of penicillin production.

By exceeding the limits of the ratio of nitrogen sources, and upwards or downwards, a noticeable reduction in production has been demonstrated.

Example 1

10 liters of fermentation broth containing 2.3% peanut flour, 1.55% corn steep liquor (60%), 0.66% micronized calcium carbonate, 0.24% sodium thiosulfate, 0.5% soybean oil are prepared into a fermentation tank. 0.3% phenoxyacetic acid (for penicillin V production) or 0.3% ammonium phenylacetate (for penicillin G production) added in six portions.

After sterilization, the soil is inoculated with 10% Penicillium chrysogenum inoculum and fermented at 25 ° C, 1 air volume and stirring at 450 rpm, dosing the carbon source as a 50% sucrose solution in a total amount of 5.8% / based to 100% sucrose], semi-continuously from 15 to 110 hours and with soybean oil defoaming.

Fermentation terminated at 115 hours, production of 5 620 IU / ml penicillin V (determined by iodometric method //).

Example 2

Procedure and soil composition as in Example 1 but without sodium thiosulfate. In the soil, the ratio of inorganic nitrogen to organic nitrogen was adjusted to 0.25: 1 by addition of 0.3% ammonium sulfate. *

In the same fermentation conditions as in Example 1, the fermentation was terminated at 115 hours to produce 9600 IU / ml penicillin V (determined by the iodometric method).

Example 3

The procedure and soil composition as in Example 2 except that the amount of phenoxyacetic acid was increased to 0.5%. Fermentation conditions as in Example 2, except for the dosing of sucrose, which was extended to 160 hours of fermentation to a total of 7.3%.

Fermentation was terminated at 162 hours with the production of 15,000 IU / ml penicillin V (iodometric) and 15,020 IU / ml · (biologically).

Example 4

31,500 liters of soil containing 2.3% peanut flour, 1.66% corn steep liquor (60%), 0.66% micronized calcium carbonate, 0.24% sodium thiosulfate, were prepared into a 50,000 liter fermentation tank. 5% soybean oil, 0.2% phenoxyacetic acid for the fermentation of penicillin V, or 0.3% ammonium phenyloctate for the fermentation of penicillin G.

After sterilization, the fermentation broth is inoculated with 3500 liters of Penicillium chrysogenum inoculum and fermented at 25 ° C, aeration with 1 volume of air / min. in the production phase, with an automatic feed of the carbon source in the form of a 50% sucrose solution in a total amount of 5.5% (based on 100% sucrose), depending on optimum pH values and antifoaming with soybean oil.

In the production of penicillin V, the precursor is added to the soil at the beginning of the fermentation, in the production of penicillin G it is dosed semi-continuously.

At 96 hours, the fermentation was terminated, producing 6,300 IU / ml penicillin V (determined iodometrically).

The average production of penicillin V on the apparatus ranged from 6,000 to 6,800 IU / ml.

Example 5

Procedure and soil composition as in Example 4, the inorganic nitrogen ratio was adjusted to 0.25: 1 in the soil by the addition of 0.185% ammonium nitrate. The inorganic nitrogen source was dosed semi-continuously during fermentation.

Fermentation conditions as in Example 4. Fermentation was terminated at 86 hours to produce 8,200 IU / ml penicillin V (determined iodometrically).

In further field trials, penicillin V production ranged from 7,500 to 8,500 IU / ml (determined iodometrically), and similar favorable results were obtained with penicillin G.

Claims (1)

SUBJECT OF THE INVENTION A process for the production of penicillin V and G by submerzary cultivation of a penicillium chrysogenum microorganism in a liquid culture medium comprising assimilable carbon sources, inorganic and organic nitrogen sources, precursors and nutrient salts, characterized in that the fermentation is carried out in a culture medium in which The amount of nitrogen from the inorganic source is 20 to 30%, preferably 25% of the total by weight of the total nitrogen from the inorganic source.