Strain actinomycete streptomyces avermitilis producing avermectins

 

(57) Abstract:

Usage: the invention relates to the microbiological industry, the production of avermectins and relates to a new strain actinomycete for the production of avermectins. The inventive strain actinomycete Streptomyces avermitilis obtained by two-stage selection from a strain of Streptomyces avermitilis-198, processed nitrosomethylurea and ultraviolet radiation. The strain is deposited in the collection ARRIAM at number 56. When the cultivation of the strain in the underlying conditions, the yield of avermectins for 7 days. 300 to 600 µg/ml

The invention relates to the field of industrial microbiocides, namely the production of avermectins with a broad spectrum antiparasitic action. Preparations on the basis of the avermectins are used in crop production and animal health.

A known strain producer avermectines complex Str. avermitilis ADS 31272 [1] This strain is used to obtain anti-parasitic substances and is characterized by the activity of up to 500 µg/ml.

Also known producer of avermectins Str. avermitilis VKM AC 1301, which is stored in the national collection of microorganisms and has activity to 130 µg/ml [2]

To want to make up to 300 µg/ml A disadvantage of the known strains producing avermectins is a relatively low activity.

The aim of the invention is to expand the range of domestic strains producing avermectins and getting more active cultures on the biosynthesis avermectines complex.

To achieve the goal is a new strain of Streptomyces avermitilis (ARRIAM 56) producer of avermectins. The strain obtained by two-step selection of the strain Str. avermitilis 198, processed nitrosomethylurea and ultraviolet radiation. The proposed strain deposited the practical approach at number 56.

Cultural and morphological characteristics. Mycelium deceptively, forms a substrate and aerial mycelium gray, environment allocates the pigment from brown to dark brown. The average thickness of the hyphae of 0.6-0.8 μm, shporonosni spiral branches off on the sides of the hyphae of the aerial mycelium. Spores spherical, oval size of 0.95 μm and above.

On agar media culture forms a velvety colonies from white to dark gray in color. Colony radially folded with smooth edges, in the center there is a crater in the environment is secreted soluble pigment dark brown is em at a temperature of 20 40oC, the optimum temperature 28 32oC, at 50oC is not growing.

The interval pH-growth of 5.0 to 9.0.

Growth on synthetic media. On Chapek's medium with glucose or sucrose forms small colonies from white to gray in color, the surface is folded. Pigment light gray or light brown not abundant. Starch-ammonia agar: colonies are small, malaslahaty, slabopigmentirovannyh, white.

The growth in organic media: potato agar growth abundant mycelium gray, the reverse side of the agar dark brown or brown-purple. Soluble pigment brown.

On potato slices growth abundant aerial mycelium gray pigmentation of gray-brown color. On wort-agar growth medium, the colonies are small, gray, brown pigment. Mastopathy agar: colonies are white and grayish ring, folded, slabopigmentirovannyh.

Physiological and biochemical characteristics: aerobe, gelatino thins, peptonizing milk, starch hydrolyses. Nitrates restores, cellulose does not decompose, tyrosinase positive. Environment Tresner-Danga blackens.

Relation to carbohydrates: assimilates glucose, arabinose, fructose, lactose, maltose, mannose, begins to nitrogen sources: uses as mineral (salt nitric acid, ammonium salts) and organic nitrogen (urea, peptone, egg albumin, amino acids).

At cultivation depth conditions biosynthesis of avermectins starts with 24 h of growth and lasts up to 10 days. depending on the cultivation conditions.

The invention is illustrated by the following examples.

Example 1.

Lyophilized culture Str. avermitilis (ARRIAM-56) from the ampoule is transferred into catalogno flask 750 ml with 50 ml of sterile medium of the composition, glucose 4, PVC 0,6, corn extract 0,3, chalk 0,3, soy flour 1,2, water the rest. The initial pH value of 7.0.

The cultivation is carried out on a rocking chair with a speed of 180 rpm at a temperature of 28oC. After 24 h of cultivation, the growing mycelium used as inoculum for seeding katalozhnyh flasks with the above environment. To do this in 750 ml flasks with 50 ml medium add 2 ml of inoculum and grown at 180 rpm, 28oC. Each day of shoot growth by 3 bulbs for determination of avermectins in the culture fluid. The determination is performed by high performance liquid chromatography. To do this, take 5 ml of the culture fluid and placed in a centrifuge tube. The biomass is separated, zentrifugenbau is, peremeshivayte and again centrifuged. The supernatant liquid is drained to the precipitate and add 5 ml of ethanol for extraction of avermectins. The extraction was carried out with stirring for 20 minutes Biomass from the extract is separated by centrifugation. The supernatant alcoholic solution of avermectins used for chromatography. However 0.003 ml alcohol solution is applied to the column and analyzed in the following mode: temperature 20oC, flow rate of eluent of 0.1 ml/min, detector at 246 nm.

To construct the calibration graph use branded drug IVOMEC with a concentration of 0.01 g/ml of Calibration solution have a concentration of 0.001, 0,0008, 0,0006, of 0.0004, 0.0002 and 0.0001 g/ml calculation of the total content of avermectins in the culture fluid is conducted according to the formula

< / BR>
where the concentration of avermectins in the culture fluid, g/ml,

To the calibration factor,

Si Si the sum of the peak areas of avermectins, cm2,

Y volume of sample applied to the column, cm3.

The concentration of avermectins in the culture fluid on the first day of growth is 70 µg/ml Further accumulation dynamics of avermectins on the day is 90, 120, 200, 260, 290, 300 µg/ml.

Example 2.

Example 3.

All operations is carried out, as in example 1, but the producer is cultivated with aeration 280 rpm, and the output of avermectins is 500 µg/ml.

Example 4.

All operations is carried out, as in example 2, but as the fermentation environment using the following composition, starch 5,0, PVC 0,6, corn extract 0,3, chalk 0,3, soy flour 1,2, water to 100% Output of avermectins for 7 days. growth is 580 µg/ml.

Example 5.

All operations were carried out as in example 2, but the producer is cultivated at a temperature of 35oC. productivity of the strain is 300 µg/ml.

Example 6.

All operations is carried out, as in example 2, but the cultivation of the producer is carried out at a temperature of 25oC. the Productivity of the strain is 540 µg/ml.

Example 7.

To determine the biological activity of avermectins produced by the proposed strain, use rice nematode Ahelenchoides bessei. The determination is conducted in parallel with a proprietary IVOMEC. Take a 1% alcoholic solution of avermectins and microplankton. Then in cell put on 15 to 20 species of nematodes and microplanet incubated in a thermostat at 30oC. After 2 h examine the state of the nematodes. It is established that at a dilution of 1000 times both drugs cause paralysis (lack of movement) of the test object.

Thus, the presented examples demonstrate that a strain of Streptomyces avermitilis (ARRIAM-56) has a high activity in terms of the biosynthesis of avermectins and produced avermectines complex has nematocidal action. Therefore, the strain can be used to produce anti-parasitic drugs.

The strain is deposited in the collection of the all-Russia research Institute for agricultural Microbiology at number 56.

Sources of information

1. Bung R. W. Miller B. M. et. all. Avermectins, new family at potenf antyhelmintic agents, producing organism and fermentation. Antimicrob. Agents Chemother. 1979, 15, p. 361 367.

2. Chermen A. N. Adanin C. M. and other Avermectins: Biotechnological characteristics of the producer strain Str. avermitilis Wcmas 1301. Applied biochemistry and Microbiology, N 6, 1991.

Strain actinomycete Streptomyces avermitilis ARRIAM-56 producer of avermectins.

 

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FIELD: biotechnology, microbiology.

SUBSTANCE: invention proposes a method for preparing exopolysaccharide by culturing microorganisms in nutrient medium containing one or more carbon source assimilated by microorganisms and caruba seeds fraction as nitrogen organic source. Applying the proposed method provides preparing exopolysaccharide eliciting improved organoleptic, sensor and visual properties. Invention can be used in building, paper, textile, cosmetic, food, oil output industry and agriculture.

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15 cl, 4 ex

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