CS267066B1 - Streptomyces aureofaciens CCM 3948 strain of microorganism - Google Patents

Abstract

Breeding of production strains of Streptomyces aureofaciens producing chlortetracycline is aimed at permanently increasing their production capacity and resistance to phage infection. According to the described procedure, the Streptomyces aureofaciens CCM 3948 strain was obtained from the production strain CCM 3820, which produces chlortetracycline in an increased amount without signs of sensitivity to actinophage B1. The production capacity of the strain is manifested with the unchanged cultivation regime.

Description

2 CS 267 066 B1

The invention relates to a strain of Streptomyces aureofaciens CCM 3948 (internally designated strain NMM 84/16) with increased production of chlorotetracycline resistant to phage indications. The preferred strain of the invention is the resistance to phage infection, the stability of the passage properties and the long-term storage in lyophilized form.

An increase in the production of chlorotetracycline antibiotic can be achieved by extending the production fermentation plants or by increasing the yield of the fermentation process. A second alternative, which does not require significant investment costs, is made possible either by improving the fermentation process or by increasing the production ability of the strains used for biosynthesis.

The strains used must also be characterized by resistance to the serious danger of fermentation, such as phage infection.

The NMM strain 84/16 was obtained from the strain Streptomyces aureofaciens CCM 3820 by a series of repeated mutagenic interventions (methylnitrosourea, N-nitrosomethylurea, DV-light + N-nitrosomethylurea, phage B1 treatment) and subsequent regular passive selection.

Isolates were evaluated according to the following scheme: isolated colonies on Petri dish 1 inclined agar and vegetatively inoculum as well as self fermentation

The production isolates were evaluated in two experiments before lyophilization and in three experiments after lyophilization. Lyophilized cans serve for long-term storage of spore material and allow for the maintenance of suitable growth and production properties. The production capability of the isolates was made using a phosphate method and measured on a Shimadzu instrument. This evaluation system has obtained the strain to which the claim is claimed.

The Streptomyces aureofaciens strain CCM 3948 forms a 2 to 5 mm size sporulation on the sucrose-peptone colony sporulation spore at 28 ° C for 12 to 14 days. The colonies are radially wrinkled at the edge, the center of the colonies is crinkled. Cultured sporula on the sporulation pad and on the natural substrate (millet).

In microscopic observation, mycelium is formed from thin, richly branched fibers. Compared to strain CCM 3820 in strain CCM 3948, a higher growth rate is observed. The color of the cultured culture is gray. The comparison of CCM 3948 strain characteristics with CCM 3820 production strain is shown in the following table:

Strain Production of chlorotetracycline in flask Phage display in culture (% of number of cultures) (/ g.ml-1, (%, CCM 3820 5 400 100 2 CCM 3948 (NMM 84/16, 6 230 115,4 0 CS 267 066 Bl The advantage of using strain streptomyces aureofaciens CCM 3948 is to create a more complete growth of chlorotetracycline production without compromising phage infection.

The production of chlortetracycline by the strain of the invention increased on average by 8 to 10 S.

The following examples illustrate, but do not limit, the strain of the invention. Example 1

Vegetatively inoculum was prepared submerged in 500 ml boiling flasks with a 60 ml refining vessel containing essential nutrients, i.e. a carbon source (sucrose), nitrogen (corn liquor in combination with an inorganic nitrogen source), and calcium carbonate. The vegetative inoculum was inoculated with a slant agar spore and cultured on a rotary shaker for 21 hours. The vegetative inoculum thus prepared was seeded with a 500 ml fermentation flask with 60 ml of fermentation. The source of carbon was sucrose, sources of nitrogen-inducing liquor, soy flour and ammonium salt. The pod further contained calcium carbonate, benzyl thiocyanate and mineral salts. The preparation of the vegetative inoculum and the fermentation were carried out with a rotary shaker at 240 rpm. 1, an eccentricity of 2.5 cm and a culture temperature of 28 ° C. The fermentation lasted 96 hours when the highest antibiotic production was achieved and samples were taken for determination.

The production evaluation of the isolates was repeated twice, the best isolates were lyophilized and, after vaccination, were evaluated at least three times.

The above fermentation method yielded chlorothetracycline production of 5,900 to 6,200 µg / ml of the poison. Example 2

According to the method of Example 1, except that the preparation of the vegetative inoculum and the fermentation took place in a fourth operating volume. The vegetatively inoculum was prepared in a 20 L inoculation laboratory tank, filled with 10 L of inoculum, inoculated with 100 ml of spore mill suspension. The nutrient stage in the fermented laboratory tanks was inoculated with a vegetative inoculum of 10% by volume. Fermentation was carried out at 28 to 29 ° C, stirring at 450 rpm. 90 to 92 hrs.

Samples were taken from the 44th hour of culture for sucrose inorganic nitrogen and production activity, which in the 92nd production index was 108-110%. Example 3

According to the method of Example 1, except that the preparation of the vegetative inoculum and the fermentation is carried out in a large volume apparatus. The inoculum was prepared in a 3 m inoculum tank with a volume of 5 m filled with a 3 m inoculum. The vegetative inoculum 3 3 was a seeded production broth in a 50 m fermenter filled with 29 m of production nutrient feed, the benzyl thiocyanate being dosed at 1.5 to 3 µg / ml of the feed, with the feed solution starting at consumption a carbon source of 26 to 25 mg'.ml of cathode. By the above procedure, the production of chlorotetracycline from a batch corresponding to an index of 108-110% is achieved.

Claims (1)

OBJECT OF THE INVENTION tetracycline and resistant to phage infection, in particular actinphage B1.