SU1342011A1 - Strain of claviceps purpurea fungus as producer of peptide ergoalcaloids - Google Patents

Description

t

The invention retracts to the micro-biohazard industry and will be an ergot gatamm producing pharmacologically priced, peptidic ergoalkaloids under saprophytic conditions during deep fermentation.

The aim of the invention is C lav leaps purpurea, which has a high productivity,

The strains of the fungus Clavlceps pu.rpurea K-41 were obtained by irradiation, with spores of spores C (5 rye, followed by selection on agaric nutrient media), using UV-radiation.

The strain of the fungus Clavlceps purpursa

 deposited in the l collection. The tour is M1 Isroo Gash-.31chov of the All-Union Scientific Research Institute of Antibiotics and i-neck. reh istraci- oHHbift number.

The strain Clavlceps purpurea VNIIA is characterized by the following characters.

Morphology culture signs, When grown. on T2 agar medium (medium composition, in g / lx. sucrose 100, yeast extract .0.55 asparagine 10 KN „Р04 G ,, 25, KCI QJ2, HgS0. 0.25 / SaSH 1, peSO -7Н: 0502, ZnSO THjO 0,015, agar 20 distilled water - YUSO cm, pH 5d2) 3x growth mi atft-ia lro vr per day, Obrazsposhles kolosh-sh at first light ;, transparently, later acquire 6ypo-Kopw4 -. The new color, 2-3 weeks long, has folds on a large mass of a clear edge, clearly oht ajchen, a ralef of a cocus, sometimes truncated, i growing a cockle-size column; from O, -. 8 cm TO 1, 5 CMg color brown-brown :; the reverse side of the colonies behsh. God color. The strain grows in the form of midi malignant cells rounded with a diameter of 3050 μm5. Strongly granular: ulified. Conidia under the specified conditions for non-forming. The colonies of this strain are easily separated from the agar medium at. when transferred into a liquid medium, small aggregates of them break down into separate cells; As they grow in liquid media, the strain of the strain into the medium ment of the medium stains in brown and light brown shrub; sometimes almost black about

Physiological signs. Strain: kulichshiruyut in aerobic conditions in

immersed culture in the liquid

2011. dak, containing as a source of carbon predominantly sucrose, glucose, makhnit, glycerin, organic acids - citric or succinic acid, ammonia asparasin, yeast extract, or ammonium salt may be used as a source of nitrogen , as the mineral lees, the nitrates, phosphates, salts of magnesium, iron and zinc can be used.

Itamm is stable and well reproducible by 1st productivity.

Cultivation can be maintained on media or by deep freezing in liquid nitrogen.

Culture vyrashvagat on Ketrn cups or in flasks on kacha.tsah with

20 rotation speed of 180-240 rpm at 20-30 sec., Cultivation period from 4 to 20 days. Strain grown on. environments at pH 3, 0-5 s5. B. Period. Intensive synthesis of pK alkaloids

2B of the medium ranges from 3.12 to 6.2, depending on the composition of the used media. Cult5-shearing can be carried out in one, two or three stages with the most favorable effect using the ratio of inoculum and medium from 1: 5 to 1 :20

Example 1, Strain UAHa C lair Iceps purpurea VNIIA K 312-A was expressed for 9 days on a T2s agarose bath nutrient medium. Then, colonies 1 were homogenized and transported B. B. Erle1-gmeier flasks with a volume of 500 cm containing 50 cm of the following media composition j g / l 5 Glucose

40 SHO, LG-helonic acid 10, KHgP04

OjS ,, Id504- N, 0 0.3, yeast extract 0.1. ResoN H About 0.007,

ZnSO,

O J 006 5 distilled

rocking temperature of 24 C in those

1000 cm. J pH to 5z2 ai-gmiakom ,.

The culture is grown on in the dark at

days, a 5-t-m pollen culture is inoculated in flasks of the same volume with 50 cm of medium T25 of a medium Dtero composition, g / l, sucrose, $ 300 .. shkka acid 15, O-3. 0.25, yeast extract Оj КС 1 0.12, FeS047Н 0 () s007j ZnSO 7Н ,, 0 0.006, districted water 1000 sk, pI - up to 5.2. Amino acid ом Cult d days

Sod: Neft of a snowy pept adn ergo; scales in micellas and culture liquid is determined by the Rumpel method.

To determine the alkaloids, the culture fluid is separated from the mycelial mass by filtration. The culture liquid is separated, the volume is measured, and the mycelium is washed twice with 0 m distilled water. 0.3 g of the crude filtered mycelium is ground in a porcelain mortar with a small amount of sand and 2 ml of a 4% tartaric acid solution in 50% is added. methanol. The resulting mass is transferred to a conical centrifuge tube. Extraction It is carried out in a water bath at a temperature of 50-60 ° C for 3 minutes with stirring. To the hot tartaric extract, add 2 ml of a 10% solution of zinc acetate, stir and settle for 10 minutes. Then centrifuged at 2000 rpm for 10 minutes. To 1 ml of the supernatant obtained, add 2 ml of Van-Urq reagent (paramete solution Laminobenzaldehyde in 60% sulfuric acid) and leave it in the light. After 30 min, blue-violet colored solutions are colorimetric on FEC-M with a green light filter (wavelength 540 nm) in a cuvette with a layer thickness of 10 mm. The content of alkaloids is calculated using a calibration curve constructed on the basis of mineramine-tartrate. The content of alkaloids in percent (X) is calculated by the formula:

., a.4.100.100

 about ЗПТоб-ь)

where a is the number of ergoalkaloids

i ml solution, g, found on the calibration graph; . b - moisture,%

To determine the content of 1L1 ergoalkaloids in the culture fluid (QL), 4 ml of Van-Urc reagent is added to 2 ml of QOL, stirred, left in the light and after 20 minutes. . colorimetric on FEC. The number of ergoalkaloids (X) in QOL is calculated by the formula

X a.1000-100 mg%

 ,

342011

where a is the number of ergoalkaloids in I ml of QOL, g, found from the calibration schedule. The total alkaloid productivity is calculated as the sum of the alkaloids found in QOL and mycelium. To identify alkaloids, you carried out: - division of the amount of alkaloids from QOL and 10 mycelium. QOL and micelles are alkalinized to pH 9-10, extracted with methylene chloride, then with a 2% aqueous solution of acid. The binnacid extraction of the sub is acidified to pH 9 and again. 15 is extracted with methylene chloride. The chloro-methyl selective extract is concentrated and hexane is added to precipitate the alkaloids. The obtained powdered amount of alkaloids is dried in a vacuum 20. ), by the method of liquid chromatography and the determination of the amino acid composition of the peptide part of the 30- molecule after acid hydrolysis.

Thus, the test showed that Ciaviceps purpurea VNIIA No. 3I2-A produces j-peptide ergoalkaloids, while its productivity in the fermentation process is 1657 / ml on the 8th day of cultivation.

40 11example2. The culture is grown on T2 agar medium and in Td medium as in Example I. 5 ml of the inoculum mass is inoculated with a 50 cm flask of medium 668 of the next flask.

45 tava, g / l: sucrose 60, glycerin 60, glucose 80, yeast extract 0.1, citric acid 15, KCl 0.125, KNgRO 0.5, 0.5, x7H, b 0.007, ZnSO. -0.006, bottom 5Q tilled water - 1000 cm, pH up to 5.2 with ammonia. After 10 days of cultivation, the strain produces 1701 y / ml of the amount of ergoalkaloids.

55 EXAMPLE 3. Colonies from the T2 agar medium, grown for 21 days, are homogenized, seeded with Erlenmeyer flasks with 50 cm of medium Td and grown under the same conditions as in

513420П

Claims (1)

Example 2, 5 ml of inoculum mass After II days of cultivation, the strain of the strain of the fungus Clavlceps purpurea produces 2315 y / np ergoap- amount. VNIIA W - producing peptide calonds., Ergoalkaloids.