The method of selection of a strain of the fungus aspergillus niger is the product of citric acid

 

(57) Abstract:

The invention relates to the microbiological industry, in particular to the selection of the active strains - producers of citric acid. Active strains selected in the process of inoculation of conidia of the fungi culture Asp. niger subjected to mutagenesis, and/or hybridization, and/or recombination, on agar nutrient medium with the content transgenically acid number (5,0-9,0)103g/cm3environment. Grow crops for 4-6 days, with 1.5-2 day mark the location of the colonies, 2-3 times smaller in diameter. After completion of the cultivation of these colonies are selected conidia of the fungus and determine its acid activity. The method allows to simplify and accelerate the selection process. table 1.

The invention relates to the microbiological industry, in particular to methods of selection of active strains of the fungus Aspergillus niger - producer of citric acid.

There is a method in which to obtain citric acid by culturing yeast, mold fungus Penicillium or bacteria in the hydrocarbon medium in the presence of mineral salts are selected UV mutants that are sensitive to the inhibitor of growth to monitorstatus or monitorstart. So, h is odny strain only 42 mg/l /1/.

In this way the sensitivity of the mutant strain of microorganism for growth inhibitor - quickly select an active strain - producer of citric acid. In the work there is no information regarding the method of selection of mutant Aspergillus niger - producer of citric acid by fermentation of carbohydrate-containing environments.

Closest to the proposed invention is a method of selection of mutant strains of mold fungus Aspergillus niger is the product of citric acid by fermentation of carbohydrate-containing media in the presence of mineral salts, including planting of UV-irradiated conidia on wort agar in 200 Petri dishes, growing colonies, the selection of conidia of morphologically altered colonies, growing crops, determination of their acid-forming activity by bioconversion of carbohydrate-containing environment /2/. The primary selection process is carried out after each dose of UV irradiation culture of the fungus, repeatedly and requires much time and labor, until it finds an active mutant strain of the fungus.

The technical result of the invention is the reduction of time and labour on the primary selection active acid producing strain of the fungus Aspergillus niger.

Technical , citric acid, including planting conidia culture of the fungus on agar nutrient medium, cultivation on a nutrient medium colonies within 4-6 days, the selection of conidia from the colonies, with the modified structure, the subsequent determination of acid-forming activity of the strain of the fungus by bioconversion of carbohydrate-containing medium, seeded with conidia of the fungi culture is subjected to mutagenesis and/or hybridization and/or recombination, on a nutrient medium in which pre-add TRANS-Aconitum acid number (5,0 - 9,0)10-3g/cm3environment, cultivation say 1.5-2 day location of colonies, 2-3 times smaller in diameter, from which are selected after growing the conidia of the fungus for the subsequent determination of activity of a strain of the fungus.

Information about the possibility of carrying out the invention and the achievement of the technical result is presented in the examples.

In the examples used TRANS-konitova acid, WITH6H6ABOUT6, M. C. 174, in the form of a solution, which is prepared from 0,174 g of the acid by dissolving in 5 cm3of distilled water.

The objects of the proposed method is tested mutants of the fungus Aspergillus niger, already known active producers of citric acid stapledon fungus and natural strain 82.

Active producers of citric acid - strains L-1, VKPM F-326, VKPM F-681, been selected previously known method (the prototype), became the object of control of the proposed method.

Acid-forming activity of strains of the fungus was determined by bioconversion of molasses and saharozameniteley media /3/.

Example 1

0.5 cm3solution of TRANS-amanitowoc acids contribute 10 cm3molten wort-agar 4-6 Petri dishes. Thus, 1 cm3molten nutrient medium contains 9,010-3g TRANS amanitowoc acid.

Conidia of strain L-1 of the fungus Aspergillus niger is filtered through a sterile filter and diluted with sterile water to 1106conidia in 1 cm3that is equivalent to 4 conidia in 1 big square camera Goryaeva. 0.5-1.0 cm3diluted in 104once suspensions of conidia spread over the surface of the nutrient medium in Petri dishes.

Colony culture of the fungus is grown at a temperature of 32oC for 1.5-2 days and then mark the location of the colonies, whose diameter is 2-3 times less than the rest. After 5 days of cultivation of the selected colonies are discarded conidia, which are then germinated on wort agar for definition wide-angle>Data and results of the experiment are shown in the table.

Example 2

In this example we used the strain VKPM F-326 and dose of TRANS-amanitowoc acid 7,010-3g/cm3on the environment of Ĩapek, other conditions similar to example 1.

Example 3

In this example we used the strain VKPM F-681 and the dose of TRANS-amanitowoc acid 6,010-3g/cm3other conditions are similar to example 1.

Examples 4-7

Examples similar to example 1, but distinguished by a strain of the fungus and the dose of TRANS-amanitowoc acid.

The results of the experiments presented in the table.

Analysis of the data presented in the table shows that in comparison with the known, the proposed method reduces the number of subjects cultures to 100 or less, so as to test the activity of acid generating selected culture, sensitive to the action of TRANS-amanitowoc acid, that is, forming colonies of small size and conidia of these colonies have a high acid activity.

If the culture of the fungus is not sensitive to TRANS-amanitowoc acid, that is, forms colonies of normal strains of the fungus diameter, acid-forming activity of these strains is low, as in examples 4, 7.

ri the initial selection of the active strains of the fungus Aspergillus niger - producers of citric acid.

SOURCES OF INFORMATION

1. A. A. imshenetskii, E. I. Shcherbakov "On the correlation that exists between morphological and physiological changes of mutant Aspergillus niger" //Microbiology// - 1964, - 31, - vol. 2, S. 252-256.

2. U.S. patent 3801455, NCI 195/28R, MKI 12 b, 1974.

3. Instruction biological and chemical control of production of citric acid, s-Petersburg, 1997, S. 109-122.

4. USSR author's certificate 407946, MKI With 12 To 3/00, 1974.

5. RF patent 1315472, MKI With 12 N 1/14, 1987.

6. RF patent 2089615, IPC 6 12 P 7/48, 1997.

The method of selection of a strain of the fungus Aspergillus niger - producer of citric acid, including planting conidia culture of the fungus on agar nutrient medium, cultivation on a nutrient medium colonies within 4-6 days, the selection of conidia from the colonies, with the modified structure, the subsequent determination of acid-forming activity of the strain of the fungus by bioconversion of carbohydrate-containing environment, wherein seeded with conidia of the fungi culture is subjected to mutagenesis, and/or hybridization, and/or recombination, on a nutrient medium in which pre-add transcentury acid number (5,0-9,0)10-3g/cm3

 

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SUBSTANCE: invention relates to a method for preparing organic acids, in particular, citric acid. Method involves isolation of calcium citrate and acid stable amylolytic enzymes from the solution cultural fluid after fermentation with fungus Aspergillus niger. The isolation process is carried out at temperature 10-50°C and pH 3.2-5.9. Method provides increasing yield of calcium citrate and complex of acid stable amylolytic enzymes: α-amylase and glucoamylase.

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1 tbl, 6 ex

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