The method of obtaining deuteromycota

 

(57) Abstract:

The invention relates to the microbiological industry, and in order to deuteromycota. The method involves culturing bacteria strain Methylobacillus flagellatum VKPM B-7823 on a nutrient medium containing watermethanol as a source of carbon, heavy water and mineral salts. Bacteria of the strain-producer, selected as a form adapted to heavy water environment, synthesize such environments datarepository consisting of monomers of deuteromycota mixed with deteroriate (2%). After selecting datereported from the culture fluid and their hydrolysis produce the target product - deuteromycota dry. The method allows to obtain up to 100 mg/l deuteromycota with the level of incorporation of deuterium is not less than 89%.

The invention relates to the field of Microbiology and relates to a method of obtaining deuteromycota (fructose containing in the molecule instead of hydrogen atoms1H atoms of stable isotope deuterium -2H or D).

Drugs deuteromycota used in the study of carbohydrate and lipid metabolism? to simplify the diagnosis of diseases associated with the violation of biologicheskogo synthesis datereported with their subsequent hydrolysis to deuteromycota. Information about getting deuteromycota by microbial synthesis in the scientific and patent information us not detected.

The invention is new and has no analogues.

In the present invention for microbiological synthesis of datereported use bacteria species Methylobacillus flagellatum. Based on the bacteria strain Methylobacillus flagellatum VKPM B-4295 by multistage breeding the resulting strain, characterized by the ability to grow on nutrient medium prepared on the basis of heavy water and containing watermethanol as a carbon source. Selected strain deposited in Russian national collection of industrial microorganisms as Methylobacillus flagellatum VKPM B-7823.

Bacteria strain Methylobacillus flagellatum VKPM B-7823 have the following morphological and physiological-biochemical features: movable wand 1,8h0,6 μm, spores do not form, after 2-3 days of growth on solid nutrient medium to form a smooth matte grayish-yellow colonies with smooth edges. Methylobacillus flagellatum VKPM B-7823 obligate methylotroph, aerobe, prototroph, neutrophil, growing at 35-40oC, absorbs nitrogen in form of ammonium salts. When growing on a nutrient medium containing watermethanol, heavy water and mineralientage strain can be stored on a solid nutrient medium of the same composition in backpatch 6-10oC for long term storage use lyophilized culture.

The method of obtaining deuteromycota as follows. Bacteria Methylobacillus flagellatum VKPM B-7823 cultivated in a nutrient medium containing watermethanol, as a source of carbon, heavy water and mineral salts. With the growth in such an environment, these bacteria produce extracellular datarepository that accumulate in the culture fluid. At the end of the process of cultivation of the culture fluid is separated from the biomass by centrifugation. Datarepository precipitated by an excess of acetone and hydrolyzing in acidic conditions to form deuteromycota, which distinguish by evaporation under vacuum to obtain a dry product. The output deuteromycota is 80-100 mg/l of culture fluid. The level of impurities determinisano determined by liquid chromatography. The level of deuteranomaly target product - deuteromycota determined by the method of proton nuclear magnetic resonance (NMR) spectroscopy.

The invention is illustrated by the following example.

Example. For culturing bacteria Methylobacillus flagellatum VKPM B-7823 use nutrient medium NaCl - 1,0, NH4Cl - 1,0, CaCl2- 0,02, MgSO4- 0,04, Na2EDTA - 0,015, Na2MoO4is 0.0001, MnCl2is 0.0001, ZnSO4- 0,0003, CuCl2- 0,0003, CoCl3- 0,0006, Fe2SO4- 0,0006, H3BO3- 0,0009, NiCl2- of 0.00006, heavy water (2H2O 99,85 at.%2H), pH to 7.0, and 7.1.

Seed culture grown in test tubes filled to 1/8 of the volume of nutrient medium on a shaker at 200 rpm overnight. Flask of 250 ml containing 50 ml of sterile nutrient medium, seeded, transferring the contents of the test tubes, and cultivated 50 h at 37oC. To prevent dilution of the deuterium label environment moisture atmosphere test tubes and flasks stoppered, equipped with a chlorine-calcium tube. The culture fluid contained in it deuterophlebiidae separated from the biomass by centrifugation at 10,000 rpm for 20 min Datarepository precipitated by adding with vigorous stirring chilled culture fluid to the double volume of chilled acetone. Fallen for 18 h white flakes of datereported separated by centrifugation (16000 rpm for 40 min). The output of datereported with 1 liter of nutrient medium is 110 mg. Thus, pin 15 ml of 2n. H2SO4for 2H at 100oC. the Hydrolysate is neutralized by Ca(OH)2deposited sediment CaSO4separated by centrifugation (5000 rpm, 10 min). Adosados is evaporated to dryness under vacuum. Weight obtained deuteromycota is 100 mg. Thus, the conversion of watermethanol in deuteromycota - 1,78%.

Determination of the monosaccharide composition of datereported carried out using high-performance liquid chromatography chromatograph "Waters", equipped with a differential Refractometer R401, column Separon SGX NH2(150 mm x 3.3 mm) in isocratic mode using as elution solvent system of acetonitrile:water in the ratio 3:1. Comparison of chromatograms of hydrolyzed monomers, deutero-polyfructanes with the chromatograms of the standards of the most common sugars (arabinose, ribose, fructose, glucose, sucrose, maltose) showed that the synthesized datarepository primarily consists of deuteromycota mixed with deteroriate - 2%.

The determination of the level of deuteranomaly deuteromycota by the method of proton NMR spectroscopy is carried out by research acetylated derivatives (pentaacetate) hydrolysis products of datereported. the ka of acetic anhydride was added an equal volume of hydrolyzate deuterophlebiidae, the mixture is vigorously stirred until complete dissolution of sugar (4-5 h), allowed to stand for 1 h at 20-25oC and 2 h at 50oC. the Solution was cooled in an ice bath, stirred with 10 ml of water with ice for 2 h and neutralized with excess sodium bicarbonate. The solution is shaken out three times with chloroform (10 ml), the combined extract is washed with water, dried with anhydrous sodium sulfate and evaporated under vacuum. Thus obtained syrupy residue is dissolved in 5 ml of absolute ether and left overnight at 10oC. Pentaacetate deuteromycota is isolated by recrystallization from ethanol with the addition of chloroform (2 vol%). The results of the analysis of the NMR range of pentaacetate received deuteromycota indicate that the level of deuteranomaly target product - deuteromycota is 89%.

Thus, the inventive method allows to obtain up to 100 mg/l deuteromycota with the level of incorporation of deuterium is not less than 89%. The mixture of deteroriate - no more than 2%.

The method of obtaining deuteromycota providing for the cultivation of bacteria strain Methylobacillus flagellatum VKPM B-7823 on a nutrient medium containing watermethanol as a source of carbon, heavy water and mineral salts, otherones to education deuteromycota and its allocation by evaporation to obtain a dry product.

 

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