RU2560598C2 - Nutrient medium for growing microorganisms deinococcus radiodurans - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: nutrient medium comprises soybean milk, glucose, MgSO₄×7H₂O, MnCl₂, FeSO₄×7H₂O and distilled water in a predetermined ratio of components.

EFFECT: invention enables to simplify the method of preparing the nutrient medium and to increase the yield of Deinococcus radiodurans.

1 tbl, 10 ex

Description

The present invention relates to microbiology, in particular to the production of culture media for the cultivation of microorganisms, in particular Deinococcus radiodurans.

The non-pathogenic bacterium Deinococcus radiodurans has the ability to utilize petroleum hydrocarbons, and is also resistant to radiation doses that are ten times higher than lethal for the microorganisms studied to date.

One of the characteristic features of this bacterium is the production of deinoxanthin-carotenoid, which gives the colonies a characteristic pink-orange color. This substance has a high antioxidant activity. Deinoxanthin is capable of significantly more effective than β-carotene and α-tocopherol in intercepting singlet oxygen and hydroxyl radical (the most dangerous types of reactive oxygen species). The ability of deinoxanthin to accelerate the regeneration of skin lesions, including in animals with type 1 diabetes mellitus, was discovered (S.V. Demyanenko, V.A. Chistyakov, S.N. Panchenko, M.A. Sazykina, BC Lysenko “Regenerative effect of carotenoids DEINOCOCCUS RADIODURANS in the course of a normal and complicated wound healing process. Proceedings of the IV international scientific-practical conference "Actual problems of biology, nanotechnology and medicine. Rostov-on-Don. P.112. 2011.) (1). The low toxicity of this substance suggests a number of uses:

- in food technology as a dye for a variety of products; or a natural antioxidant that prevents the spoilage of fatty foods (mayonnaise, chocolate paste, etc.) as a result of peroxidation processes;

- in pharmacology as an effective antioxidant with a systemic adaptogenic effect; including as the basis of regeneration stimulants, agents that slow down ultraviolet skin aging.

In connection with the foregoing, the relevance of developing cheap culture media for the cultivation of the bacterium Deinococcus radiodurans is beyond doubt.

The culture medium used should support the metabolism of a particular organism as efficiently as possible, in this case the bacteria Deinococcus radiodurans.

Media suitable for incubation of microorganisms contain a carbon source, for example carbohydrates; a nitrogen source in the form of various nitrogen-containing compounds; as well as various complex additives, for example metal salts, which are necessary for growth.

A nutrient medium is known (Passport for a strain of the microorganism Deinococcus radiodurans VKPM 8209. All-Russian Collection of Industrial Microorganisms. FSUE GosNIIGenetika) (2) used to grow a strain of microorganisms Deinococcus radiodurans VKPM B-8209 containing a nutrient base containing nitrogen compounds as nitrogen source - peptone, yeast extract, as a salt - sodium chloride, as well as agar, with the following quantitative ratios of components, g / l:

yeast extract  5,0 peptone  15.0 NaCl  5,0 agar  15.0 distilled water up to 1 l

A disadvantage of the known nutrient medium is the high cost of the constituent components, because it includes animal products - peptone, yeast extract, agar. Moreover, the productivity of the cultivation of bacteria Deinococcus radiodurans using this medium is low.

A nutrient medium is also known for growing the strain of the microorganism Deinococcus radiodurans (Zhang YM, Wong TY, Chen LY, Lin CS, Liu JK Induction of a futile Embden-Meyerhof-Parnas pathway in Deinococcus radiodurans by Mn: possible role of the pentose phosphate pathway in cell survival. Applied and Environmental Microbiology. 2000. Vol.66. No. 1, P.105-112.) (3) containing a nutritional base, where glucose was used as a source of carbohydrates, yeast extract was used as organic nitrogen-containing compounds, and an important source of amino acids for growth is tryptone, with the following quantitative ratios of components, g / l:

tryptone  5,0 glucose  1,0 yeast extract  3.0 distilled water  up to 1 l

The disadvantage of this medium is the use of expensive ingredients (tryptone - trypsin hydrolyzate of protein substrate of animal origin, yeast extract - autolysate of baker's yeast) with a low biomass yield of cultivated bacteria.

Closest to the proposed invention is a nutrient medium for the cultivation of microorganisms Deinococcus radiodurans (RF Patent No. 2407786 MKI C12N 1/20) (4) containing soy flour, glucose or sucrose and trace elements of salts of magnesium sulfate and manganese chloride, with the following quantitative ratios of components, g / l:

soy flour  40.0-60.0 glucose / or sucrose  8.0-20.0 MgSO ₄ · 7H ₂ O  0.02-0.07 MnCl ₂  0.0004-0.001 distilled water  up to 1 l

In the known medium, soya flour is used as a nutrient base, which is a source of carbon and a nitrogen-containing compound and provides optimal conditions for the development of Deinococcus radiodurans microorganisms, and also significantly reduces the cost of this nutrient medium. As an additional carbon source, glucose or sucrose are used, which, in addition, contribute to better pigmentation. Magnesium sulfate MgSO ₄ · × 7H ₂ O and manganese chloride MnCl _{2 were} added to the nutrient medium as growth components, as well as for the greatest degree of pigment formation.

The disadvantage of this medium is that it is not a solution, but a suspension. The presence in its composition of solid particles of soy flour makes it difficult to separate bacterial cells from the environment. Both during filtration and centrifugation, the bacterial mass is contaminated with solid particles. In addition, when developing this environment, the needs of bacteria for trace elements are not fully taken into account, the most important of which, in addition to manganese and magnesium, is iron.

The objective of the invention is to develop a new suitable for growing microorganisms Deinococcus radiodurans nutrient medium, which would not contain solid suspended particles and provide a higher yield of bacterial biomass Deinococcus radiodurans, not contaminated with solid particles.

This task is achieved in that the nutrient medium for growing microorganisms Deinococcus radiodurans, containing a nutrient base, glucose, trace elements of magnesium sulfate, manganese chloride and distilled water, according to the invention additionally contains a trace element of iron sulfate, and as a nutrient base contains soy milk, with the following quantitative ratios of components, g / l:

soy milk  50-200 glucose  8.0-20.0 MgSO ₄ × 7H ₂ O  0.02-0.07 MnCl ₂  0.0004-0.001 FeSO ₄ × 7H ₂ O 0.001-0.005 distilled water up to 1 l

In the proposed medium, soy milk, which is a protein-fat emulsion, does not contain solid particles, is used as a nutrient base, but, like soy flour, it is a source of carbon and nitrogen-containing compounds and provides optimal conditions for the development of Deinococcus radiodurans microorganisms.

In addition, to achieve a higher density of Deinococcus radiodurans cells, as well as for a higher degree of pigment formation, an additional microelement source, iron (II) sulfate, has been added to the nutrient medium.

The combination of the proposed components for growing microorganisms Deinococcus radiodurans provided an increase in the growth of their biomass compared to the prototype 1.7 times. In this case, the isolated bacterial mass is not contaminated with solid particles.

The combination of the proposed components for the cultivation of microorganisms Deinococcus radiodurans is unknown, which is a hallmark of the proposed nutrient medium.

As a result of the analysis of the prior art, an analogue was not found, characterized by features identical to all the essential features of the claimed invention, and the determination of the prototype from the identified analogues made it possible to identify a set of distinctive features that are significant in relation to the technical result.

Therefore, the claimed invention meets the condition of "novelty."

In an additional search for other solutions related to nutrient media, these distinguishing features were not found. Thus, the claimed invention meets the condition of "inventive step".

The development of a culture medium is illustrated by the following examples.

The nutrient medium is prepared as follows: To 100 g (95 ml) of soy milk is added 500 ml of distilled water, 10 g of glucose, 0.05 g of MgSO ₄ · 7H ₂ O are dissolved in the resulting liquid; 0.0006 g of MnCl ₂ and 0.002 g of FeSO ₄ , adjusted to 1 liter with distilled water, sterilized at 1 atm for 20 minutes. For the preparation used soy milk produced by LLC "RASH" (Aksai), TU 9220-001-48262956-02.

According to the methodological recommendations (MR "Control of the quality of nutrient media" No. 04-3-16 / 1615 dated 06/27/2003.) (5) a qualitative and quantitative control of the biological properties of the proposed medium was carried out using Deinococcus radiodurans VKPM B-8209 as a test strain .

Quality control was carried out by plating a one-day test strain in the proposed nutrient medium using conventional methods.

The Deinococcus radiodurans VKPM B-8209 culture was grown in 250 ml, wide-necked Erlenmeyer flasks with 50 ml of this liquid medium, into which 50 μl of a night culture of bacteria was added. Flasks with crops were incubated on a circular shaker at 30 ° C and 150 rpm for 48 hours.

At the end of the incubation period, the test strain gave a distinct growth with typical distinguishing features: turbidity of the liquid medium with uniform staining in pink-orange color (the strain forms a pink-orange pigment) and the formation of a pink-orange precipitate. The quality control showed the ability of the test strain Deinococcus radiodurans VKPM B-8209 to grow on the proposed medium, while obtaining characteristic differentiating features.

We also conducted a quantitative control of the proposed environment for biological indicators. For this, a suspension of the test strain was prepared using a turbidity standard of 10 units (microbial cell concentration of 1 billion) and ten-fold serial dilutions (8 dilutions inclusive).

To control dilution from 6 and 7 dilutions, 0.1 ml of the suspension was sown directly by plating on plates with culture medium. Three such crops were made from each dilution. The plated cups were incubated in a thermostat at 30 ° C for 24-48 hours. For each series of crops, the average number of colonies grown on three plates was calculated.

The average number of colonies grown during sowing with 0.1 ml of suspension of the test strain of Deinococcus radiodurans VKPM B-8209 from the sixth dilution was 123, and from the seventh - 11. According to methodological recommendations (5), the average number of colonies grown during sowing was 0.1 ml suspension of the test strain from the sixth dilution should be about 100 CFU (colony forming units), and the ratio of the average values when sowing from the sixth to the seventh dilution should be close to 10: 1. Thus, the obtained values indicate the suitability and effectiveness of the proposed environment for growing Deinococcus radiodurans.

The selection of the optimal amount of components (ingredients) in a nutrient medium was experimentally carried out.

Example 1. For the cultivation of the strain Deinococcus radiodurans VKPM B-8209 was prepared nutrient medium having the following composition, g / l:

soy milk  one hundred glucose  10.0 MgSO ₄ × 7H ₂ O  0,07 MnCl ₂  0,0005 FeSO ₄ × 7H ₂ O  0,0005 distilled water  up to 1 l

The Deinococcus radiodurans VKPM B-8209 culture was grown in 250 ml, wide-necked Erlenmeyer flasks with 50 ml of this liquid medium, into which 50 μl of a night culture of bacteria was added. Flasks with crops were incubated on a circular shaker at 30 ° C and 150 rpm for 48 hours. To calculate the number of CFU, the standard method of parallel dilution in physiological saline and deep plating on solid agarized media was used.

The calculated CFU / ml is given in table 1.

Example 2. For the cultivation of the strain Deinococcus radiodurans VKPM B-8209 was prepared nutrient medium having the following composition, g / l:

soy milk  one hundred glucose  10.0 MgSO ₄ × 7H ₂ O  0,07 MnCl ₂  0,0005 FeSO ₄ × 7H ₂ O  0.001 distilled water  up to 1 l

The Deinococcus radiodurans VKPM B-8209 culture was grown in 250 ml, wide-necked Erlenmeyer flasks with 50 ml of this liquid medium, into which 50 μl of a night culture of bacteria was added. Flasks with crops were incubated on a circular shaker at 30 ° C and 150 rpm for 48 hours. To calculate the number of CFU, the standard method of parallel dilution in saline and deep plating on dense agar media was used.

The calculated CFU / ml is given in table 1.

Example 3. For the cultivation of the strain Deinococcus radiodurans VKPM B-8209 was prepared nutrient medium having the following composition, g / l:

soy milk  one hundred glucose  10.0 MgSO ₄ × 7H ₂ O  0,07 MnCl ₂  0,0005 FeSO ₄ × 7H ₂ O  0.002 distilled water  up to 1 l

The Deinococcus radiodurans VKPM B-8209 culture was grown in 250 ml, wide-necked Erlenmeyer flasks with 50 ml of this liquid medium, into which 50 μl of a night culture of bacteria was added. Flasks with crops were incubated on a circular shaker at 30 ° C and 150 rpm for 48 hours. To calculate the number of CFU, we used the standard method of parallel dilutions in saline and deep plating on dense agarized media.

The calculated CFU / ml is given in table 1.

Example 4. For the cultivation of the strain Deinococcus radiodurans VKPM B-8209 was prepared nutrient medium having the following composition, g / l:

soy milk  one hundred glucose  10.0 MgSO ₄ × 7H ₂ O  0,07 MnCl ₂  0,0005 FeSO ₄ × 7H ₂ O  0.005 distilled water  up to 1 l

The Deinococcus radiodurans VKPM B-8209 culture was grown in 250 ml, wide-necked Erlenmeyer flasks with 50 ml of this liquid medium, into which 50 μl of a night culture of bacteria was added. Flasks with crops were incubated on a circular shaker at 30 ° C and 150 rpm for 48 hours. To calculate the number of CFU, the standard method of parallel dilutions in physiological saline and deep plating on solid agarized media was used.

The calculated CFU / ml is given in table 1.

Example 5. For the cultivation of the strain Deinococcus radiodurans VKPM B-8209 was prepared nutrient medium having the following composition, g / l:

soy milk  one hundred glucose  10.0 MgSO ₄ × 7H ₂ O  0,07 MnCl ₂  0,0005 FeSO ₄ × 7H ₂ O  0.01 distilled water  up to 1 l

The Deinococcus radiodurans VKPM B-8209 culture was grown in 250 ml, wide-necked Erlenmeyer flasks with 50 ml of this liquid medium, into which 50 μl of a night culture of bacteria was added. Flasks with crops were incubated on a circular shaker at 30 ° C and 150 rpm for 48 hours. To calculate the number of CFU, the standard method of parallel dilutions in physiological saline and deep plating on solid agarized media was used.

The calculated CFU / ml is given in table 1.

Example 6. Next, we checked the dependence of the growth of the culture of Deinococcus radiodurans on the amount of soy milk in the proposed nutrient medium. For the cultivation of the strain Deinococcus radiodurans VKPM B-8209 was prepared nutrient medium having the following composition, g / l:

soy milk  150 glucose  10.0 MgSO ₄ × 7H ₂ O  0,07 MnCl ₂  0,0005 FeSO ₄ × 7H ₂ O  0.002 distilled water  up to 1 l

The Deinococcus radiodurans VKPM B-8209 culture was grown in 250 ml, wide-necked Erlenmeyer flasks with 50 ml of this liquid medium, into which 50 μl of a night culture of bacteria was added. Flasks with crops were incubated on a circular shaker at 30 ° C and 150 rpm for 48 hours. To calculate the number of CFU, the standard method of parallel dilutions in physiological saline and deep plating on solid agarized media was used.

The calculated CFU / ml is given in table 1.

Example 7. For the cultivation of the strain Deinococcus radiodurans VKPM B-8209 was prepared nutrient medium having the following composition, g / l:

soy milk  200 glucose  10.0 MgSO ₄ × 7H ₂ O  0,07 MnCl ₂  0,0005 FeSO ₄ × 7H ₂ O  0.002 distilled water  up to 1 l

The Deinococcus radiodurans VKPM B-8209 culture was grown in 250 ml, wide-necked Erlenmeyer flasks with 50 ml of this liquid medium, into which 50 μl of a night culture of bacteria was added. Flasks with crops were incubated on a circular shaker at 30 ° C and 150 rpm for 48 hours. To calculate the number of CFU, the standard method of parallel dilutions in physiological saline and deep plating on solid agarized media was used.

The calculated CFU / ml is given in table 1.

Example 8. For the cultivation of the strain Deinococcus radiodurans VKPM B-8209 was prepared nutrient medium having the following composition, g / l:

soy milk  250 glucose  10.0 MgSO ₄ × 7H ₂ O  0,07 MnCl ₂  0,0005 FeSO ₄ × 7H ₂ O  0.002 distilled water  up to 1 l

The Deinococcus radiodurans VKPM B-8209 culture was grown in 250 ml, wide-necked Erlenmeyer flasks with 50 ml of this liquid medium, into which 50 μl of a night culture of bacteria was added. Flasks with crops were incubated on a circular shaker at 30 ° C and 150 rpm for 48 hours. To calculate the number of CFU, the standard method of parallel dilutions in physiological saline and deep plating on solid agarized media was used.

The calculated CFU / ml is given in table 1.

Table 1 Examples The content of components, g / l The number of microorganisms (CFU / ml) soy milk, g Glucose, g MgSO ₄ × 7H ₂ O, g MnCl ₂ , g FeSO ₄ × 7H ₂ O, g one one hundred 10 0,07 0,0005 0 3.8 × 10 ⁹ 2 one hundred 10 0,07 0,0005 0,0005 3.7 × 10 ⁹ 3 one hundred 10 0,07 0,0005 0.001 6.8 × 10 ⁹ four one hundred 10 0,07 0,0005 0.002 6.6 × 10 ⁹ 5 one hundred 10 0,07 0,0005 0.005 6.65 × 10 ⁹ 6 one hundred 10 0,07 0,0005 0.01 1.9 × 10 ⁹ 7 150 10 0,07 0,0005 0.002 6.7 × 10 ⁹ 8 200 10 0,07 0,0005 0.002 6.6 × 10 ⁹ 9 250 10 0,07 0,0005 0.002 6.7 × 10 ⁹

From the data shown in table 1, it is seen that the introduction of iron at a concentration of less than 0.001 g / l does not lead to an increase in the biomass yield of microorganisms in comparison with example 1, where iron was not added. An increase in the concentration of iron from 0.001 to 0.005 increases the yield of biomass of microorganisms to 6.8 × 10 ⁹ CFU / ml. The number of CFU / ml on the media from examples 3-5 and 7-9 is approximately the same. An increase in iron concentration to 0.01 g / l leads to a decrease in the yield of biomass of microorganisms (the toxic effect of iron is manifested). In the examples with the introduction of optimal iron concentrations of 0.002 g / l (examples 4 and 7-9), it is seen that an increase in the content of soy milk in the medium from 100 ml / l to 250 ml / l does not change the yield of Deinococcus radiodurans. Thus, the introduction of iron allows to increase the yield of Deinococcus radiodurans VKPM B-8209 cells by 1.7-1.8 times.

In addition, a comparative characterization of the growth of Deinococcus radiodurans was carried out when grown on different nutrient media.

The growth rate of Deinococcus radiodurans on different media was evaluated by growing the strain and preparing a number of dilutions of the suspension of the strain from each culture medium, plating on agar media and taking into account the number of CFU / ml.

Example 9. For the cultivation of the strain Deinococcus radiodurans VKPM B-8209 was prepared nutrient medium described in the prototype (3), and having the following composition, g / l:

soy flour  50,0 glucose  10.0 MgSO ₄ · 7H ₂ O  0,07 MnCl ₂  0,0005 distilled water  up to 1 l

The culture of Deinococcus radiodurans VKPM B-8209 was grown in 250 ml, wide-necked Erlenmeyer flasks with 50 ml of this medium, into which 50 μl of bacteria night culture was added. Flasks with crops were incubated on a circular shaker at 30 ° C and 150 rpm for 48 hours. To calculate the number of CFU, we used the standard method of parallel dilutions in saline and deep plating on dense agarized media.

Example 10. For the cultivation of the strain Deinococcus radiodurans VKPM B-8209 was prepared, the proposed nutrient medium having the following composition, g / l:

soy milk  one hundred glucose  10.0 MgSO ₄ × 7H ₂ O  0,07 MnCl ₂  0,0005 FeSO ₄ × 7H ₂ O  0.01 g Distilled water  up to 1 l

The culture of Deinococcus radiodurans VKPM B-8209 was grown in 250 ml of wide-necked Erlenmeyer flasks with 50 ml of the proposed soybean medium, into which 50 μl of overnight bacteria culture were added. Flasks with crops were incubated on a circular shaker at 30 ° C and 150 rpm for 48 hours. To calculate the number of CFU, we used the standard method of parallel dilutions in saline and deep plating on dense agarized media.

As can be seen from table 1, the maximum growth of the culture Deinococcus radiodurans VKPM B-8209 was observed when grown in the proposed environment - the number of CFU / ml was 1.7 times greater than CFU / ml in the known medium. In addition, in the proposed environment there are no solid particles of soy, which facilitates the separation of the biomass of bacteria from the environment. In this case, the selected biomass is not contaminated with solid particles of soy.

This allows us to conclude that the obvious advantages of the proposed nutrient medium in comparison with known environments for the cultivation of bacteria Deinococcus radiodurans, including the environment of the prototype.

Thus, the new nutrient medium allows to obtain higher yields of bacterial colonies Deinococcus radiodurans. Moreover, the proposed nutrient medium is technically easy to prepare (without a preliminary stage of preparation of the base), it does not include expensive components.

Information sources

1. S.V. Demyanenko, V.A. Chistyakov, S.N. Panchenko, M.A. Sazykina, B.C. Lysenko Regenerative action of carotenoids DEINOCOCCUS RADIODURANS during normal and complicated wound healing. Materials of the IV international scientific-practical conference “Actual problems of biology, nanotechnology and medicine. Rostov-on-Don. C.112. 2011.

2. Passport for a strain of the microorganism Deinococcus radiodurans VKPM 8209. All-Russian Collection of Industrial Microorganisms. FSUE GosNII Genetics.

3. Zhang Y.M., Wong T.Y., Chen L.Y., Lin C.S., Liu J.K. Induction of a futile Embden-Meyerhof-Parnas pathway in Deinococcus radiodurans by Mn: possible role of the pentose phosphate pathway in cell survival. Applied and Environmental Microbiology. 2000. Vol. 66. No. 1, P.105-112.

4. RF patent No. 2407786, MKI C12N 1/20 (prototype).

5. MR "Quality control of nutrient media" No. 04-3-16 / 1615 from 06/27/2003.

Claims (1)

Nutrient medium for growing microorganisms Deinococcus radiodurans, containing a nutrient base, glucose, trace elements of magnesium sulfate, manganese chloride and distilled water, characterized in that it additionally contains a trace element of iron sulfate, and soy milk as a nutrient base in the following quantitative ratios of components, g / l:
soy milk 50-200 glucose 8.0-20.0 MgSO ₄ × 7H ₂ O 0.02-0.07 MnCl ₂ 0.0004-0.001 FeSO ₄ × 7H ₂ O 0.001-0.005 distilled water up to 1 l