SU550423A1 - Strain actinomycete 243-11 - Google Patents

Description

The invention relates to the microbiological industry, in particular to the production of antibiotics, and can be used in the stabilization of semi-sweet wines. The known phsycidic antibiotic 243, related to the subsoil of organolentic heptaen-levorip, kandicidin, trichomycin, ascosine, used in medicine, wine maker and other industries 1. When using this antibiotic in winemaking, sulfurous anhydride and cold are not used. However, this strain is not sufficiently productive. The purpose of the invention is to obtain a fungicidal antibiotic with high productivity, namely, the antibiotic activity of the culture fluid in the tray 243-11 is 8230 units. in 1 ml, and the strain 243-6130 units. in 1 ml. The proposed strain obtained by mutation from the original strain as follows. The original actinomycete strain No. 243 of the species Actinomyces levoris is cultured in a liquid nutrient medium of the following composition Peat extract MgSO4-71120 Pe8O4-7P2O0.001 Distilled water1.0 L pH6.8 Before seeding the actinomycete, the chemical mutagen solution is introduced into the liquid nutrient medium NaNO2 , 0% of the volume of the medium is poured into 5 ml tubes and the spore suspension of actinomycete No. 243 in the amount of 0.5 million spores per 1 ml of medium is introduced into them. Incubation is carried out at 27 ° C for 14 days (I generation). The obtained culture of actinomycete is scattered on the surface of the agar nutrient medium of the same composition in Petri dishes so as to obtain individual colonies of actinomycete. Crops of culture are incubated at 27 ° C for 14 days (II generation). The obtained individual colonies of actinomycete are grouped according to morphological features, 100 colonies of plaits in test tubes are sown from each group. Sowing culture incubated at 27 ° C for 10 days. In this way, a total of 396 actinomycete strains (III generation) were obtained. The resulting actinomycete strains were studied for fungicidal activity against the Torula utilis culture test. To do this, the agar nutrient medium of the same composition is poured into standard Petri dishes

10 ml, placed on a horizontal surface. Each test strain is sown on three cups. Spore strokes taken from the spit are looped onto the surface of the agar medium and spatula evenly distributed over the entire surface. Incubation is carried out for 5 days at 27 ° C. The 6-mm-diameter blocks are cut out of the resulting actinomipet lawns and applied in three replications onto the surface of agar medium No. 44, previously inoculated with a deep method, with a test culture of Torula utilis at the rate of 100 million cells per 1 ml of medium. To ensure the same thickness of the medium layer, inoculated medium No. 44 is poured into standard Petri dishes placed on a horizontal surface, 10 ml each.

The composition of the medium N ° 44 the following,%:

Peptone1,0

Yeast Extract 3.0

iNaCl0,5

NasPOi0,3

Glucose1.0

, 0

Agar1,5 Piped water

up to 1 l, pH 6.2

Incubation of the Torula utilis culture test was carried out at 37 ° C for one day. According to the obtained zones of growth inhibition of the test culture, the fungicidal activity of the strain is judged, and by the size of the diameters and plume, adi zones, the productivity of this strain of actinomycete.

Of the 396 strains of actinomycete studied, only 40 strains, i.e. 10%, found fungicidal activity. The remaining 90% were inert.

From the identified 40 active strains of actinomycete LL 243, 12 strains with activity greater than those of the original strain were selected for further study. Their four generations were studied by this method for fungicidal activity. As a result of this, 4 strains were selected, having a superiority in activity of more than 20% compared with the original strain. These were the strains No. 243/8, 243/11, 243/34 and 243/47.

The indicated promising 4 strains were studied for the ability to produce antibiotic No. 243B fermentation conditions, for which they take a 850 ml shake flask, prevent 150 ml of the liquid nutrient medium and ferment it at 27 ° C for 5 days, while the fermentation process performed in a circular rocker making 229 rpm. The composition of the fermentation medium is as follows,%: Cornmeal 5.0 Soybean flour 1.0 reduced,

for

NaCl0.5

СОСОз1,0

The activity of the culture liquid is determined by the spectrophotometric method. Take 2 ml of the culture liquid, add 8.0 ml of Dimethylformamide, shake for 30 minutes, filter, dilute with 80% ethanol 1: 20 and remove the optical density at a wavelength of 380 nm. The obtained optical density data is inserted into a special formula developed by the Leningrad Institute of Antibiotics to determine the activity of the polyene antibiotic Levorin, and calculate the activity of the culture liquid

, 44.D-103.a,

where a is the activity of the test solution; D is the optical density of the solution; a is the multiplicity of dilution.

The study of these 4 strains of actinomycete No. 243 by the described method was carried out in triplicate. So they were studied by three generations, as a result of which the most active actinomycete strain No. 243/11 was selected, the productivity of which, compared with the original strain N ° 243, is 34% more.

Next, a toxonomic study of the mutant strain N ° 243/11 was carried out on the basis of the common method. To establish the identity of antibiotics produced by the original actinomycete strain of Act. levoris no. 243 and mutant strain no. 243/11 studied their chemical nature and spectrum of fungicidal action. It is established that the antibiotic substance produced by the original strain of Act. levoris No. 243 and mutant strain No. 243/11 are similar in their chemical irirode and antibiotic effects.

The resulting actinomycete strain Actinomyces levoris 243/11, producing antibiotic No. 243, has the following morphological and physiological characteristics.

The size of the mycelium of strain No. 243-II after the incubation period: length 5-9 microns, thickness 0.09-0.11 microns.

On the environment of čapek with starch - good growth, mealy, colonies nloskie, smooth, slightly crater. Aerial mycelium mauve. Substrate mycelium pale terracotta. Sporostsy straight, convoluted (wavy), collected in bundles. Spores are oval, round, smooth.

Glycerin-aspartic medium number 79 - good growth, nlosky colonies. Aerial mycelium is white. Substrate mycelium cream. Sporonosa direct, spores oval, smooth.

The ammonia starch medium is a good growth, the colonies are flat, smooth. Aerial whitish mycelium.

Szbstratny mycelium cream. Sporosts are straight, convoluted, spores are oval, round.

Potato agar - GROWTH good, mealy, flat colonies. Aerial mycelium whitish. Substrate mycelium bistrovy. Sporonosa direct, spores oval, smooth.

Wednesday Trespera (H2S) -growth good, smooth colopia. Aerial mycelium cream. Substrate mycelium snout evaty. Sporosts are straight, convoluted. Spores are oval, round.

Relation to carbon. Absorbs glucose, arabinose, xylose, mannitol, maltose, galactose, sodium acetate. Does not absorb sucrose and sorbitol. Poorly absorbs lactose.

Gelatin thins, milk energetically peptonizes, hydrolyzes starch, weakly restores nitrates. Can not grow on fiber.

By laboratory testing of the activity of the obtained mutant strain 243-11 in comparison with the original strain 243, it turned out that the antibiotic activity of the culture fluid in strain 243-11 is 8230 units in 1 ml, and in strain 243-6130 units in 1 ml.

The yield of antibiotic No. 243-11 from the culture fluid is 34-40% higher than that of the original strain 243. The resulting strain No. 243-11 is stored in the industrial crop museum of the antibiotics department in the form of sowing on test tubes of the agar medium.

Claims (1)

1. The journal "Antibiotics number 1, Nvar, volume XVII, publishing house". Medicine, M., 1972, p. 15-18 (prototype).