SU1735366A1 - Method for preparation of inoculum material - a producer of aminoglycoside antibiotic complex of streptomyces cremeus subsp "nebramycini" - Google Patents

Abstract

Usage: in the microbiological and medical industry in obtaining an aminoglycoside antibiotic complex consisting of carbamoyltrabracin, carbamoylcanamine, and apromycin. SUMMARY OF THE INVENTION: A vegetative inoculum is grown in a liquid nutrient medium for 24 hours with stirring and aeration followed by the selection of a conforming material based on the morphological characteristics of the culture, pH of the medium and dehydrogenase activity of the mycelium. An increase in the content of antibiotics in the culture fluid by 30% is achieved through the use of a vegetative inoculum at the fermentation stage, decolorizing a solution of methylene blue for 1-2 minutes at a pH of 7.2-7.5 and having a morphology in the form of separate hyphal plexuses. 1 tab.

Description

The invention relates to the microbiological and medical industry, in particular, to a method for producing an aminoglycoside antibiotic complex consisting of carbamoyl tamycin (tobramycin, a medicine, is of interest in chemotherapy of infections caused by Gram-negative bacteria, including Ps. Aeruginosa) , carbamoylcanamycin and apramycin (used in veterinary medicine in the treatment of gastrointestinal diseases of cattle and pigs).

There is a known method of obtaining seed material when the vegetative seed material of the culture of Streptomyces cremeus subsp. nebramycini is grown on a seed medium of the following composition, May% corn extract 2.0; glycerin 2.0;

ammonium chloride; sodium chloride 0.3; chalk 0.5; animal fat 0.75.

By the time of transfer to the fermentation stage, the seed mycelium must satisfy the following requirements: pH 5.5-7.0; the appearance of the culture; small mycelium, fills the entire volume of the sample, does not settle on standing mycelium. Microscopic characteristics of the mycelium; in a preparation stained with methylene blue, short weakly branching basophilic hyphae are observed.

The disadvantage of this method is the low antibiotic activity of the culture fluid at the end of the fermentation.

The purpose of the invention is to increase the antibiotic activity of the culture fluid, which will lead to a decrease in the cost of the antibiotics tobramycin and apramycin.

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The goal is achieved by growing the vegetative inoculum to determine the dehydrogenase activity of the mycelium by decolorizing the methylene blue solution and decolorizing for 1-2 minutes (pH 7.2-7.5) and the presence of separate hyphalum plexuses select the appropriate material.

In the known method, the physiological state of the culture of Streptomyces rimosus, the producer of oxytetracycline, is determined by the redox (dehydrogenase) activity of the mycelium. The dehydrogenase activity of the producer is measured by the rate of bleaching of the methylene blue dye. For a culture of Streptomyces cremeus subsp. The nebramycini dehydrogenase activity, as measured by a known method, can be characterized by the bleaching time of a 0.01% methylene blue solution. It was established that the conditioned seed has a dehydrogenase activity of 1-2 minutes. However, this parameter cannot be the only characteristic of the conditionality of the vegetative inoculum, since the same values of dehydrogenase activity are possible if there is a large amount of lysed biomass in the seed material. In order to accurately select a conditional seed, it is necessary to introduce an additional 2 parameters: the culture morphology during microscopy — individual hyphal flow and pH of the medium — 7.2–7.5. Only 3 parameters in total allow to obtain a conforming seed, the use of which later on at the fermentation stage gives the maximum antibiotic activity of the culture liquid. The value of dehydrogenase activity of more than 2 min indicates a lack of maturity of the producer’s mycelium, it will be unproductive at the fermentation stage. The value of dehydrogenase activity less than 1 unit indicates a strong autolysis of culture.

Example 1 (by a known method). The culture of Streptomyces cremeus subsp. nebramycini (strains 2242, 9871). The vegetative inoculum is grown at 37 ° C for 24 hours in flasks on a rocking chair with a speed of 240 rpm and in a seed apparatus with a capacity of 7 liters (filling factor 0.7) with a stirrer speed of 150 rpm on the medium of the following composition, wt.%: glycerin 2,0; corn extract 2.0; animal fat 0.75; ammonium chloride

0.2; sodium chloride 0.3; chalk 0.5; tap water the rest; pH of 7.4 ± 0.1.

Seed material (pH 6.0), not containing extraneous microflora, having

appearance: small mycelium, fills the entire sample volume, does not settle on standing. Microscopic characterization: in a preparation stained with methylene blue, there are short weak branches,

basophilic hyphae in the amount of 2% of the volume of the culture fluid are used when carrying out fermentation at 37 ± 1 ° C in apparatus with a capacity of 30 l (filling factor 0.5) and in flasks (100 ml of medium; Vioo 25 ml of medium) on a rocking chair with a number of turns 240 rpm on the medium of the following composition, wt.%: Soybean flour 3.5; animal fat 1.1; glucose 1,5; ammonium chloride 0.6; magnesium sulphate 1.1; chalk 0.7; tap water the rest; pH of the medium 6.2 ± 0.1.

The duration of fermentation is 72 hours. The antibiotic activity of the culture fluid is determined by the method

diffusion into agar using the test culture of Bacillus subtilis strain 6633 and expressed in units of 1 ml. The total antibiotic activity of the culture fluid is 2800 µg / ml for strain 9871 and

1400 µg / ml for strain 2242.

Examples 2-7. Growing vegetative inoculum is carried out on the seed medium of the prototype for 24 hours at 37 ± 1 ° C in flasks on a rocking chair with a speed of

240 rpm and in a seed drill with a capacity of 7 liters (filling factor 0.7) with a speed of 150 rpm.

The conditionality of the vegetative inoculum is determined by 3 parameters in

aggregates: microscopic morphologists — individual hyphal tangles; pH of the culture fluid 7.2; bleaching time of a 0.01% solution of methylene blue (1 ml of solution + 5 ml

culture fluid) 1 min.

Conforming seed material is used when carrying out fermentation in devices with a capacity of 30 l (filling factor 0.5), in flasks (100 ml

Wednesday) on a rocking chair with a speed of 240 rpm, adding it in the amount of 5% of the volume of the culture fluid. Fermentation was carried out at 37 ± 1 ° C on a fermentation medium type.

The duration of fermentation is 72 hours. The antibiotic activity of the culture liquid is determined by agar diffusion into the agar using the test culture of Bacillus subtilis strain 6633 and is expressed in

units per 1 ml. The total antibiotic activity of the culture fluid is 3640 µg / ml for strain 9871 and 1820 µg / ml for strain 2242.

The use of a vegetative inoculum, the conditionality of which is determined by one parameter (by morphology during microcopying, as in the case of the prototype), leads to a loss of activity at the fermentation stage due to the use of a still young or overgrown seed.

The obtained data on the effect of individual characteristics of the vegetative inoculum on the content of aminoglycoside comyx in the culture fluid are presented in the table.

The given examples show that only the determination of the conditionality of a seed by 3 parameters in total leads to an increase in

actibiotic activity of the culture fluid by 30%.

Claims (1)

The invention The method of obtaining seed - producer of the aminoglycoside antibiotic complex Streptomyces cremeus sub-sp. nebramycini, which includes growing the culture in a liquid nutrient medium, followed by selection of a conforming material based on the morphological characteristics of the culture and the pH of the medium, characterized in that, in order to increase the antibiotic activity of the culture fluid, the dehydrogenase activity of the mycelium is determined by bleaching by bleaching the methylene blue solution and bleaching within 1-2 minutes, a pH of 7.2-7.5 and the presence of separate hyphal plexuses select a suitable material.