CS267066B1 - Strain of microorganism streptomyces aureofacines ccm 3948 - Google Patents

Abstract

Breeding Streptomyces production strains chlorothetracycline-producing aureofaciens it is aimed at continuously increasing them production ability and phage resistance infection. According to the procedure described above obtained strain of Streptomyces aureofaciens CCM 3948 from the production strain CCM 3820 it produces to an increased extent of hypertracycyclines without symptoms of actinophage B1 sensitivity. production the ability of the strain manifests itself unchanged cultivation mode.

Description

KVETKOVÁ MÁRIA RNDr., VIDO VLADIMÍR ing., ŽIGOVÁ EVELÍNA ing., BANSKÁ BYSTRICA, ŠEBEŠOVÁ VIERA, BRUSNO, BERTAN MILAN ing., BANSKÁ BYSTRICA

Streptomyces aureofaciens strain CCM 3948 (57) Breeding of chlortetracycline-producing strains of Streptomyces aureofaciens is aimed at permanently increasing their production capacity and resistance to phage infection. Following the procedure described, the Streptomyces aureofaciens strain CCM 3948 was obtained from the production strain CCM 3820, which increasingly produces chlortetracycline without signs of susceptibility to actinphage B1. The production abilities of the strain are manifested in the unchanged current cultivation regime.

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The invention relates to a phage-induced strain of Streptomyces aureofaciens CCM 3948 (internal designation of the strain is NMM 84/16) with increased production of chlortetracycline.

The advantage of the strain according to the invention is the resistance to phage infection, the stability of the properties during passage and during long-term storage in lyophilized form.

Increasing the production of the antibiotic chlortetracycline can be realized by expanding the production fermentation plants, or by increasing the yield of the fermentation process. The second alternative, which does not require significant investment costs, is made possible either by improving the fermentation process or by increasing the production capacity of the strains used for biosynthesis.

The strains used must also be resistant to serious hazards during fermentation, such as phage infection.

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

Isolates were evaluated according to the following scheme:

isolated colonies on petri dishes slant agar i

vegetatively inoculum own fermentation

Production isolates were evaluated in two experiments before lyophilization and in three experiments after lyophilization. Lyophilized cans are used for long-term storage of spore material and allow the preservation of suitable growth and production properties. The production capacity of the isolates was determined by the phosphate method and measured on a Shimadzu instrument. The patented strain was obtained by this evaluation system.

The Streptomyces aureofaciens strain CCM 3948 forms colonies of 2 to 5 mm in size in a monosporus seed on a sucrose-peptone sporulation medium during 12 to 14 days of cultivation at 28 ° C. The colonies are radially wrinkled at the edge, the center of the colonies is intersected. Culture spreads well on the sporulation medium and on the natural substrate (millet).

Upon microscopic observation, the mycelium is formed from thin, richly branched fibers.

Compared to strain CCM 3820, a higher growth rate is observed for strain CCM 3948. The color of the sporulated culture is gray. A comparison of the properties of the CCM 3948 strain with the properties of the CCM 3820 production strain is given in the following table:

Tribe Chlortetracycline production in a flask Proof of phage in culture (% of the number of cultures) / 7 I Qig.ml ) (%) CCM 3820 5 400 100 2 CCM 3948 (NMM 84/16) 6 230 115.4 0

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The advantage of using the strain streptomyces aureofaciens CCM 3948 is in creating the conditions for further growth of chlortetracycline production without endangering it by phage infection.

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

The following examples illustrate but do not limit the embodiment of the strain according to the invention.

Example 1

The vegetatively inoculum was prepared submerged in 500 ml boiling flasks with 60 ml of inoculum, which contained essential nutrients, i. j. carbon source (sucrose), nitrogen (corn steep liquor in combination with an inorganic nitrogen source) and calcium carbonate. The vegetative inoculum was inoculated with a spore agar slant and cultured on a rotary shaker for 21 hours. The vegetative inoculum thus prepared was inoculated into 500 ml fermentation flasks with 60 ml of fermentation broth. The carbon source was sucrose, the nitrogen source was corn steep liquor, soy flour and ammonium salt. The soil further contained calcium carbonate, benzyl thiocyanate and mineral salts. Vegetative inoculum preparation and fermentation were performed on a rotary shaker at 240 rpm. eccentricity 2.5 cm and a culture temperature of 28 ° C. The fermentation lasted 96 hours, when the highest antibiotic production was reached and samples were taken for determination.

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

The above fermentation process to achieve the production of chlortetracycline 5900-6200, ug.ml ¹ soil.

Example 2

According to the method described in Example 1, except that the preparation of the vegetative inoculum and the fermentation took place in quarterly operating volumes. The vegetatively inoculum was prepared in a 20 L inoculation laboratory tank filled with 10 L of inoculum, which was inoculated with 100 mL of a canned spore suspension.

The nutrient medium in the fermentation laboratory tanks was inoculated with vegetative inoculum in an amount of 10% by volume. The fermentation took place at 28 to 29 ° C, stirring at 450 rpm. for 90 to 92 hours

Samples were taken from 44 o'clock. cultivation for the determination of sucrose inorganic nitrogen and production activity which reached '92. production index 108 to 110%.

Example 3

According to the method described in Example 1, with the difference that the preparation of the vegetative inoculum and the fermentation are carried out in a large-scale apparatus. The inoculum was prepared in a 5 m seed fermentation tank with a volume of 5 m, filled with 3 m of inoculum. The vegetative inoculum 3 3 was inoculated with the production medium in a 50 m fermenter, which was filled with 29 m of production medium. 'carbon source to a level of 26 to 25 mg'.ml of soil. By the above procedure, the production of chloroxetracycline from the batch corresponding to an index of 108 to 110% is achieved.

CS 267 066 Bl

Streptomyces aureofaciens CCM 3948 strain with increased chlorine production

Claims (1)

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