Method for melanin production

FIELD: biotechnology, in particular production of melanin which represents pigment of high antioxidant and anti-tumor activity.

SUBSTANCE: claimed method includes providing by selection of yeast strain Nadsoniella nigra which under deep cultivation accumulates up to 25 g/l of yeast biomass after 96 hours from cultivation starting. Melanin is isolated both from yeast biomass (intracellular melanin) and from cultural liquid (extracellular melanin). Intracellular melanin is isolated by extraction with alkali solution followed by melanin deposition with hydrochloric acid. To isolate extracellular melanin cultural liquid is treated with ethanol followed by melanin deposition with hydrochloric acid.

EFFECT: method if increased effectiveness.

4 ex

 

The invention relates to the field of biotechnology and concerns a method for obtaining melanin - pigment with high antioxidant and anticancer activity.

Melanin is a large group of brown and black natural pigments that are widely present in the tissues of plants and animals, with a wide spectrum of biological activity. Cells of living organisms produce melanin to protect the body from exposure to adverse environmental factors, especially UV rays. Melanin possess antimutagenic properties (2-4 times reduce chromosomal damage in bone marrow cells, which occurs under the influence of mutagens), significantly inhibit the growth of tumor cells and metastas have radioprotective properties, inhibit the free-radical oxidation processes (1, 2).

A method of obtaining melanin from vegetable raw buckwheat husks (3). The buckwheat husks are processed cellulolyticus enzyme (CELLULARONE G3x) within 48 hours. Next, the extraction is carried out melanin solution of alkali, then melanin is precipitated with hydrochloric acid and the precipitate was separated by filtration. The output of melanin from buckwheat husk is 8-10%. However, the application of the technology of expensive enzyme, and the use of plant materials, when weather conditions and the effect of phytopathogens could the t significantly affect its quality, doing this method is economically inefficient.

Well-known examples of making melanin of the rare raw materials. For example, the company "Oligomers as raw materials for the production of melanin uses Pomor bees and premium teas. The use of such materials does not allow the melanin in sufficient quantity.

Solving these problems is possible by creating a biotechnological method of producing melanin. Melanin is synthesized various groups of microorganisms (fungi, actinomyces, yeast) (4).

Closest to the claimed invention are methods of obtaining melanin described in the patents of the Russian Federation№№2116036, 2069696 (5, 6). According to these patents to obtain melanin using yeast strains Nadsoniella nigra. The yeast is grown in a nutrient medium of the following composition:

To2HPO40.1 g/l

CaCl2- 0.03 g/l

MgSO40.05 g/l

NaCl - 0.05 g/l

Yeast autolysate - 1%

Glucose is 1.5-2%

pH of 3.0-3.5

The cultivation is carried out at a temperature of 26-28°C for 7-10 days depending on the strain of yeast.

To highlight melanin grown biomass was separated by centrifugation and autoclave at 0.5 ATM with 0.5 N. NaOH within 1-3 hours. Then cell mass is removed by centrifugation, centrifugal acidified with concentrated hydrochloric acid to pH 1-2. The precipitation chalk is Nina separated and dried.

The disadvantage described in the patents of the Russian Federation No. 2116036, 2069696 ways to obtain melanin is the long duration of the fermentation process (7-10 days), which significantly affects the cost of production. In addition, the RF patents №№2116036, 2069696 no quantitative characteristics of the process (the melanin content in the biomass, the biomass yield), which complicates the assessment of the efficacy of the yeast strains and the process.

The objective of this invention to provide a new highly efficient strain of yeast producer of melanin, the development of more effective method of its production, the expansion of the number of producers, cheaper process.

The essence of the invention lies in the fact that as a result of breeding and selection of the resulting strain of yeast Nadsoniella nigra var. heuselica B-3, which in submerged cultivation on a nutrient medium defined within 96 hours from the beginning of cultivation accrue to 20 g/l yeast biomass production.

Melanin is identified as the biomass of yeast (intracellular melanin), and from the culture fluid (extracellular melanin). To obtain intracellular melanin grown yeast biomass was separated by centrifugation. Next, the resulting biomass homogenized with a solution of KOH for 10-20 minutes at a temperature of 20-25°followed by extraction of the melanin in three the tupeni under the following conditions: homogenized heated to the boiling temperature of the solution in terms of excess pressure of 1 ATM and incubated for 3 hours. After the process is finished, the reaction mixture is cooled to a temperature of 35-40°and filtered in a nitrogen atmosphere to prevent oxidation of the target product. The precipitate is washed with a solution of caustic potash. Obtained at each step the extract melanin combine and add 10%solution of hydrochloric acid. The resulting solution was incubated under stirring for 3 hours and the temperature of the reaction mixture 10-15°C. the Obtained melanin precipitate is separated by filtration, washed successively 10%solution of hydrochloric acid and distilled water. The washed crystals intracellular melanin is dried at a temperature of 60-80°and the residual pressure of 0.2 ATM for 3-4 hours.

To obtain extracellular melanin in the culture fluid obtained after separation of the biomass of yeast is added to ethyl alcohol and kept under stirring for 20-30 minutes at a temperature of 35-40°C. Then the precipitate salts of melanin is separated from the aqueous solution of melanin. The precipitate is washed with ethanol. Next to the precipitate salts of melanin add distilled water. The reaction mixture was kept under stirring for 20-30 minutes at room temperature. Then to aqueous solution of salts of melanin poured 10%solution of hydrochloric acid and maintained is with constant stirring for 20-30 minutes at a temperature of 30-35° C. the precipitate extracellular melanin is separated by filtration, washed on the filter first with 10%hydrochloric acid, then with distilled water. The obtained crystals extracellular melanin is dried at a temperature of 60-80°and the residual pressure of 0.2 ATM for 3-4 hours.

The strain of yeast Nadsoniella nigra var. heuselica B-3 obtained from the collection of strain Nadsoniella nigra var. heuselica BKM-F-2137 in the consistent application of mutagenic factors: UV rays, diphenylamine and N-methyl-N-nitro-N-nitrosoguanidine with subsequent sieving on Petri dishes with nutrient medium wort agar (7°). The selection of strains was conducted at a long-term culture under aseptic conditions when Otieno-topping-up the way of cultivation on the basis of the highest rates of growth in liquid nutrient medium.

The morphological characteristics of the strain Nadsoniella nigra var. heuselica B-3 does not differ from the parent: cells have an oval shape, with a diameter of 5-8 μm. On the environment wort agar strain Nadsoniella nigravar. heuselica B-3 forms a convex, smooth, with smooth edge of colonies with a diameter of 4 mm (5 days of cultivation on agar medium at a temperature of 26-27°).

On biotechnological indices strain of yeast Nadsoniella nigra var. heuselica B-3 significantly superior to the parent strain as on growth rate, and the level of accumulation of biomass.

When growing the Tamm yeast Nadsoniella nigra var. heuselica B-3 liquid mineral medium for 4 days, the concentration of biomass reaches 25-26 g/l completely dry yeast. The melanin content in biomass is 10%. The parent strain Nadsoniella nigra var. heuselica F-2137 in optimal conditions in liquid mineral medium for 10 days accumulated only 2.0 g/l completely dry yeast.

The strain of yeast Nadsoniella nigra var. heuselica B-3 is stored in the collection of the company "Biosynthesis of MT on the beveled wort-agar and in dried form in ampoules.

Example 1.

The strain of yeast Nadsoniella nigra var. heuselica B-3 was grown on the sloping wort-agar for 10 days at 26-27°C. Then planted on mineral medium of the following composition:

(NH4)2SO4- 5.8 g/l

To2HPO4- 5,2 g/l

KN2PO2to 5.0 g/l

MgSO4·7H2O - 0.34 g/l

CaCl2- 0.03 g/l

ZnSO40.01 g/l

CuSO4- 0.03 g/l

gidrol (waste starch production) - 30 g/l

peptone - 5%

pH 5,5

The cultivation was conducted at 26-27°C. After 96 hours from the beginning of the fermentation the biomass content in the environment amounted to 25.2 g/l completely dry yeast.

Example 2.

Same as in example No. 1, but the composition of the nutrient medium instead of peptone was introduced yeast autolysate in the amount of 5%. After 96 hours of cultivation the biomass content in the environment amounted to 23.7 g/l completely dry yeast.

Example 3.

Obtained from fermenting biomass in the amount of 25 g of homogenized in 250 ml of 2 N. the solution of caustic potash. Homogenization should be performed within 15 minutes at a temperature of 25°C. the resulting homogenizate heated at a pressure of 1 ATM. The boiling point of the reaction mixture and incubated for 3 hours. Then the resulting mixture is cooled to a temperature of 40°and filtered. The precipitate on the filter is washed with a solution of caustic potash and hold the 2nd and then the 3rd stage of extraction under the conditions described above. Then the combined extract melanin obtained with 3 speed extraction (82.7 g), add 10%solution of hydrochloric acid in the amount of 61.5 ml of the Solution is maintained at a constant stirring for 3 hours at a temperature of 15°C. Next, the slurry is fed to the filter and the precipitate washed with 10%hydrochloric acid, then with distilled water. The washed crystals melanin dried under vacuum at a temperature of 80°C for 3-4 hours. The result is 2.3 g of melanin.

Example 4.

The culture fluid obtained after separation of the biomass of yeast, in the amount of 450 ml is mixed with 1.2 l of ethanol. The reaction mixture is maintained under stirring for 30 minutes at a temperature of 40°C. Next, the resulting solution of salts of the melanin philtrums is. The precipitate on the filter is washed with ethanol. Then the precipitate salts of melanin diluted in 10 ml of distilled water and kept under stirring for 30 minutes at room temperature. Next to a water solution of salts of melanin add 2.5 ml of 10%hydrochloric acid and the resulting solution maintained at a temperature of 35°C for 30 minutes. Then the obtained solution is filtered, the precipitate washed with 10%hydrochloric acid, then with distilled water. The washed crystals melanin dried under vacuum at a temperature of 80°C for 3-4 hours. The result is 0.7 g of melanin.

Sources of information

1. Borschevskaya M.I., Vasiliev S.M. // Questions of medical chemistry, 1999, No. 1, p.13-23.

2. Moss IB, Kostrov, L.N., Oak B.V., Plotnikova SR, Molofee VP // Radiation biology. Radioecology, 1999, no. 2-3, str-333.

3. RF patent №2215761.

4. Lyakh S. p., Ruban EL // Microbial physiology. M.: Nauka, 1972

5. Prototype - RF patent №2116036.

6. Prototype - RF patent №2069696.

The method of obtaining melanin, which includes the cultivation of yeast Nadsoniella nigra in a nutrient medium containing sources of nitrogen, carbon, phosphorus, trace elements, followed by separation of melanin, characterized in that as a producer of melanin using strain Nadsoniella nigra B-3.



 

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