CS245182B1 - Streptomyces fradiae CCM 3765 strain of microorganism - Google Patents

Abstract

Strain of the microorganism Streptomyces fradiae CCM 3765 producing tylosin. The strain according to the invention was obtained by the action of mutagens and repeated passive selections. The advantage of the strain according to the invention is increased production of tylosin /80 to 120 u/ and resistance to mechanical stress to which the culture is subjected during stirring in the fermenter.

Description

The invention relates to a strain of Streptomyces fradiae CCM 3765 (internally designated NMU 2/32) producing tylosin.

An increase in industrial production of tylosin can be achieved by increasing production capacity or by increasing production during its biosynthesis. The second method, i. increasing production in its biosynthesis, unlike the first, does not require large investment costs. It is based on the better production ability of the strain used for biosynthesis or on the better fermentation conditions that are ensured! improved fermentation control.

This latter method is currently the only possible way to increase yield and one of the main benefits is to improve the production properties of the strain. The production strains are obtained by a number of established procedures for long-term breeding programs.

The resulting strains show! then a number of distinct properties have been identified, the most important of which are those manifested in increased tylosin production in the fermenters in production.

The Streptomyces fradiae strain CCM 3765 was obtained by mutagenizing and repeated passive selection on the starting strain of Streptomyces fradiae EMS 98. The breeding pattern of the strain is shown in the following scheme where the genealogical line leading to the strain to which the patent application is applied is shown.

Streptomyces fradiae EMS 98 PS

115-10

UV-1 UV 1/31

PS

131 - 7

NMU-1

NMU - 1/19

NMU - 2

NMU - 2/32

Abbreviations PS - passive selection - selection without UV mutagen exposure - UV light exposure

MNU - nitrosomethyl urea multiplication

The last mutagenic multiplication was on nitrile methyl urea at a concentration of 0.15 M for 100 min. phosphate buffer pH 6.8 to a MNU 1/19 spore suspension, obtained by washing the cultured culture with water. During the multiplication, the mutation mixture was stirred on an electromagnetic stirrer at 25 ° V.

At intervals of 40, 60, 80 and 100 min. For the nitrosomethylurea treatment, samples were taken and diluted with a 10-fold dilution series while diluting the nitrosomethylurea to an ineffective concentration. Diluted samples were inoculated on Petri dishes with sporulation agar so as to avoid colony contact, and after spores were separated, single colonies were re-grafted to slant agar.

The spores for passive selection were suspended in distilled water, diluted with a Factor 10 dilution series, and seeded onto sporulated bottom petri dishes. After incubation for 14 days at 28 ° C, the isolates were sporulated and inoculated onto slant agar.

Incubation of this basic sporulation material took place at 28 ° C for 10 to 12 days. By applying the mutagens and passive selection method described above, the NMU 2/32 isolate was obtained. Isolates were evaluated according to the slope agar - veg inoculum - self fermentation scheme.

The strain of Streptomyces fradiae CCM 3765 exceeds the production of the strains used so far by 80 to 120%. The strain forms colonies 4-6 mm in diameter on a sporulated yeast extract, bovine extract, hydrolyzed casein and dextrin diameter, above the sporulated center substrate, with white spores in the center after cultivation at 28 ° C for 10-12 days.

The advantage of this strain is its resistance to the mechanical stresses to which the culture is subjected during agitation in a fermenter. The main and most important characteristic of Streptomyces fradiae CCM 3765 is increased tylosin production in submerged fermentation.

EXAMPLE

Fermentation in flasks on rotary shakers

The vegetatively inoculum was prepared submerged in 500 ml boiling flasks with 60 ml inoculum (corn extract, soy flour, soybean oil and CaCC> ₃ ). The fermentation broth after inoculation was inoculated with a sloping agar spore and incubated on a rotary shaker for 40 to 48 hours.

The vegetative inoculum thus prepared was seeded with 500 ml production flasks with a fermentation chamber volume of 60 ml (carbon source of beet molasses, soybean oil, fish flour nitrogen source, corn gluten, inorganic salts and CaCO 3). Fermentation takes 8 to 9 days to cultivate. Tylosin production under the above conditions reached 6,000 IU / ml.

Example 2

Fermentation in pilot plant fermenter

Fermentation in a 5,000 L pilot plant fermenter was two-stage. The inoculation stage contained corn extract, soy flour, feed yeast, soybean oil and CaCO 3. The fermentation pod contained corn extract, fish flour, corn gluten, beet molasses, soybean oil and CaCO 3.

The fermenter was filled with 2800 liters of inoculum and inoculated with 10% of the volume from the seed fermenter. Cultivation was carried out at 28 ° C, stirring 180 rpm, aeration 2500 rpm. for 200 hours cultivation. The production strain Streptomyces fradiae CCM 3765 produced 6,300 IU / ml.

Claims (1)

Incubation of this basic sporulating material took place at 28 ° C for 10 to 12 days. An NMU 2/32 isolate was obtained as described above by application of mutagens and passive selection. Isolates are evaluated according to the angled agar - inoculum - self fermentation scheme. Streptomyces fradiae strain CCM 3765 exceeds 80 to 120% of the production of previously used strains. The strain, when cultivated on a sporulation pod with yeast extract, bovine extract, hydrolyzed casein and dextrin, colonies 4 to 6 iron in diameter raised above the spore center substrate, with white spores on Wednesday after a 10-12 day culture at 28 ° C. The advantage of this strain is its resistance to mechanical stress, which is cultured during mixing in the fermenter. The main and most important feature of Streponthies fradiae strain CCM 3765 is the increased production of tylosin in submerged fermentation. Example 1 Fermentation in rotary shaker flasks Vegetatively inoculum was prepared submerged in 500 ml boiling flasks with 60 ml of inoculant / corn extract, soybean, soybean oil and CaC03. the nutrient stage, after sterilization, was inoculated with a sloping agar spore and incubated on a rotary shaker for 40 to 48 hours. The vegetative inoculum thus prepared was seeded with 500 ml production boiling flasks with a fermentation volume of 60 ml / carbon source beet molasses, soybean oil, nitrogen flour source, corn gluten, inorganic salts and CaCO 3. Fermentation takes place from 8 to 9 days of cultivation. Tylosin production under the above conditions reached 6,000 IU / ml. Example 2 Fermentation in a semi-operational fermenter Fermentation in a 5,000 l semi-operational fermenter was two-stage. The vaccine base contained corn extract, soybean poppy, feed yeast, soybean oil, and CaCO 3. The fermentation stage contained corn extract, poppy fish, corn gluten, beet molasses, soybean oil, CaCO 3. The fermenter was filled with 2800 liters of beer and inoculated with 10% of the volume of the inoculum fermenter. Cultivation was carried out at 28 ° C, stirring 180 rpm, aeration 2,500 rpm. for 200 hours of cultivation. The production strain Streptomyces fradiae CCM 3765 produced a production of 6 300 IU / ml. OBJECT OF THE INVENTION The tylosin-producing strain of Streptomyces fradiae CCM 3765.