RU2418061C2 - NUTRIENT MEDIUM FOR DEINOCOCCUS RADIODURANS Microorganisms growing - Google Patents

Abstract

FIELD: food industry.

SUBSTANCE: nutrient medium contains soya flour and distilled water at preset quantitative ratios.

EFFECT: Deinococcus radiodurans yield increase.

2 tbl, 8 ex

Description

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

The non-pathogenic bacterium Deinococcus radiodurans is known to be resistant to radiation doses ten times higher than lethal for the microorganisms studied to date.

One of the characteristic features of this bacterium is the production of deinoxanthin, a carotenoid, which gives the colonies a characteristic pink-orange color. This substance has a uniquely 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).

Therefore, this harmless substance can have a number of uses:

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

- in pharmacology as an effective antioxidant; including as the basis of adaptogens, geroprotectors, new generation anesthetics, protectors of near-ultraviolet radiation.

In connection with the foregoing, the development of low-cost nutrient media for growing Deinococcus radiodurans bacteria is currently an urgent task.

Known nutrient broth (TU 42-14-83-78) [1] for the cultivation of a wide range of microorganisms and distilled water, having the following composition, g / l:

Sprat pancreatic hydrolyzate 10.05 Sodium chloride 4.95

pH 7.2 ± 0.2.

A disadvantage of the known nutrient broth is the low productivity of the cultivation of bacteria Deinococcus radiodurans.

A known nutrient medium [2] used for growing a strain of microorganisms Deinococcus radiodurans VKPM B-8209, including, 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 (animal product - peptone, yeast extract, agar). Moreover, the productivity of the cultivation of bacteria Deinococcus radiodurans using this medium is not high enough.

Closest to the proposed invention is selected as a prototype nutrient medium [3] used for growing microorganisms Deinococcus radiodurans, the following composition, g / l:

Tripton 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 for the nutritional basis (tryptone - trypsin hydrolyzate of animal protein substrate, yeast extract - baker's yeast autolysate). Moreover, the productivity of the cultivation of bacteria Deinococcus radiodurans using this medium is not high enough.

The objective of the invention is to reduce the cost of the environment, simplifying its composition, as well as increasing the yield of biomass of bacteria Deinococcus radiodurans.

This goal is achieved in that the nutrient medium containing a nutrient base and distilled water, according to the invention as a nutrient base contains soy flour in the following ratio, g / l:

Soya flour 50,0 Distilled water up to 1 l

Unlike the prototype, the proposed environment is simpler and cheaper and provides optimal conditions for the growth of Deinococcus radiodurans.

It is not known the use of soy flour as a nutrient base for growing Deinococcus radiodurans microorganisms, which is a hallmark of the proposed nutrient medium.

The method is illustrated by the following examples.

Experimentally, the selection of the optimal amount of soy flour in a nutrient medium was carried out.

Example 1. To obtain one liter of nutrient medium, take 10 g of soy flour, add up to 1 liter of distilled water, sterilize at 1 ATM for 20 minutes.

For reliability in all experiments, 3 strains of microorganisms were used: Deinococcus radiodurans VKPM B-8209, Deinococcus radiodurans BKM B-1422, Deinococcus radiodurans BKM B-1467.

Strains of Deinococcus radiodurans were grown in 250 ml wide-necked Erlenmeyer flasks with 50 ml of this liquid medium, into which 50 μl of overnight bacterial 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 / ml, the standard method of parallel dilutions in physiological saline and deep plating on solid agar media was used.

The calculated CFU / ml for all strains of Deinococcus radiodurans microorganisms is given in table 1.

When grown in this medium for all strains of Deinococcus radiodurans, there was a slight uniform staining of the medium in pink-orange color and a slight formation of a pink-orange biomass sediment.

Example 2. According to example 1, a nutrient medium was prepared having the following composition, g / l:

Soya flour 50,0 Distilled water up to 1 l

Cultures of Deinococcus radiodurans were grown in accordance with Example 1. To calculate the number of CFU / ml, a standard method of parallel dilutions in physiological saline and deep plating on solid agar media was also used.

The calculated CFU / ml for all strains of Deinococcus radiodurans microorganisms is given in table 1.

When growing Deinococcus radiodurans strains in this nutrient medium, a significant uniform pink-orange color of the medium and a significant pink-orange biomass sediment were observed for each of them.

Example 3. According to example 1 was prepared a nutrient medium having the following composition, g / l:

Soya flour 100.0 Distilled water up to 1 l

The strains of microorganisms Deinococcus radiodurans were grown in accordance with example 1. Analogously to example 1, the number of CFU / ml was counted.

The calculated CFU / ml for all strains of Deinococcus radiodurans microorganisms is given in table 1.

When different strains of Deinococcus radiodurans microorganisms were grown in this medium, for each of them a significant uniform staining of the medium in pink-orange color and a significant formation of pink-orange biomass sediment were observed.

The table shows that the maximum growth for all strains of microorganisms Deinococcus radiodurans was observed in media with soy flour content of 50 g / l and 100 g / l. The number of CFU / ml for all strains on these media was approximately the same, but 13.6-17.7 and 11.8-15.6 times higher, respectively, than the number of CFU / ml on the medium with these strains containing soy flour 10 g / l.

The results obtained made it possible to determine that the optimal variant of a nutrient medium based on soy flour is 50 g / l of distilled water.

Table 1 The amount of soy flour in 1 liter of culture medium, g The number of colony forming units / ml (CFU / ml) Deinococcus radiodurans VKPM B-8209 Deinococcus radiodurans BKMB-1422 Deinococcus radiodurans BKMB-1467 10 1.1 × 10 ⁸ 1.2 × 10 ⁸ 0.9 × 10 ⁸ fifty 1.5 × 10 ⁹ 1.7 × 10 ⁹ 1.6 × 10 ⁹ one hundred 1.3 × 10 ⁹ 1.3 × 10 ⁹ 1.4 × 10 ⁹

This ratio of ingredients allows you to achieve maximum growth of the Deinococcus radiodurans culture at the lowest consumption of soy flour. In addition, the possibility of culturing various strains of Deinococcus radiodurans on this nutrient medium was experimentally shown.

Example 4. According to the guidelines [4], a qualitative and quantitative control of the biological properties of the proposed medium was carried out using three test strains of Deinococcus radiodurans: VKPM B-8209, VKM B-1422, VKM B-1467. Quality control was carried out by plating a one-day test strain in the proposed nutrient medium using conventional methods.

The nutrient medium was prepared as follows.

To obtain one liter of culture medium, 50 g of soy flour was taken, up to 1 l of distilled water was added and sterilized at 1 atm for 20 min. 50 μl of a night culture of Deinococcus radiodurans VKPM bacteria was added to 250 ml of Erlenmeyer wide-necked flasks. B-8209, Deinococcus radiodurans VKM B-1422, Deinococcus radiodurans VKM B-1467. Flasks with crops were incubated on a circular shaker at 30 ° C, 150 rpm for 48 hours.

At the end of the incubation period, the test strains showed clearly distinguishable 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 strains Deinococcus radiodurans VKPM B-8209, Deinococcus radiodurans VKM B-1422, Deinococcus radiodurans VKM B-1467 to grow on the proposed medium, while obtaining characteristic differentiating characters.

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

To control dilution from the 6th and 7th dilutions, 0.1 ml of the suspension was sown directly by plating on plates with nutrient agar. Three such crops were made from each dilution. Cups with crops were incubated in an incubator 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 by sowing 0.1 ml of a suspension of the test strain Deinococcus radiodurans VKPM B-8209 from the sixth dilution was 93, and from the seventh - 9.

The average number of colonies grown by sowing 0.1 ml of a suspension of the test strain Deinococcus radiodurans BKM B-1422 from the sixth dilution was 97, and from the seventh - 9.

The average number of colonies grown by sowing 0.1 ml of a suspension of the test strain Deinococcus radiodurans BKM B-1467 from the sixth dilution was 88, and from the seventh - 9.

According to the guidelines [4], the average number of colonies grown by sowing 0.1 ml of the 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: one. Thus, the obtained values indicate the suitability and effectiveness of the proposed environment for the cultivation of microorganisms Deinococcus radiodurans.

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

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

Example 5. For the cultivation of strains of Deinococcus radiodurans VKPM B-8209, Deinococcus radiodurans BKM B-1422, Deinococcus radiodurans BKM B-1467, the TGY growth medium described in the prototype [3] and having the following g / l composition was prepared:

Tripton 5,0 Glucose 1,0 Yeast extract 3.0 Distilled water up to 1 l

Cultures Deinococcus radiodurans VKPM B-8209, Deinococcus radiodurans BKM B-1422, Deinococcus radiodurans BKM B-1467 were 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, 150 rpm for 48 hours. To calculate the number of CFU / ml, the standard method of parallel dilutions in physiological saline and deep plating on solid agar media was used.

The calculated CFU / ml for all Deinococcus radiodurans bacteria strains is given in Table 2.

Example 6. For growing strains of Deinococcus radiodurans VKPM B-8209, Deinococcus radiodurans BKM B-1422, Deinococcus radiodurans BKM B-1467, a liquid nutrient medium L was prepared, described in analogue [2] and having the following composition, g / l:

Yeast extract 5,0 Peptone 15.0 NaCl 5,0 Distilled water up to 1 l

Cultures of Deinococcus radiodurans VKPM B-8209, Deinococcus radiodurans BKM B-1422, Deinococcus radiodurans BKM B-1467 were 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, 150 rpm for 48 hours. To calculate the number of CFU / ml, the standard method of parallel dilutions in physiological saline and deep plating on solid agar media was used.

The calculated CFU / ml for all strains of Deinococcus radiodurans are shown in table 2.

Example 7. For growing strains of microorganisms Deinococcus radiodurans VKPM B-8209, Deinococcus radiodurans BKM B-1422, Deinococcus radiodurans BKM B-1467, the nutrient broth (TU 42-14-83-78) described in analogue [1] was prepared by FSUE “ NPO MICROGEN of the Ministry of Health of the Russian Federation of the following composition, g / l:

Sprat pancreatic hydrolyzate 10.05 Sodium chloride 4.95

pH 7.2 ± 0.2.

Cultures of Deinococcus radiodurans VKPM B-8209, Deinococcus radiodurans BKM B-1422, Deinococcus radiodurans BKM B-1467 were 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, 150 rpm for 48 hours. To calculate the number of CFU / ml, the standard method of parallel dilutions in physiological saline and deep plating on solid agar media was used.

The calculated CFU / ml for all strains of Deinococcus radiodurans is given in table 2.

Example 8. For the cultivation of strains of Deinococcus radiodurans VKPM B-8209, Deinococcus radiodurans BKM B-1422, Deinococcus radiodurans BKM B-1467, the proposed nutrient medium was prepared having the following composition, g / l:

Soya flour 50,0 Distilled water up to 1 l

Cultures of Deinococcus radiodurans VKPM B-8209, Deinococcus radiodurans BKM B-1422, Deinococcus radiodurans BKM B-1467 were grown in 250 ml wide-necked Erlenmeyer flasks with 50 ml of the proposed soybean medium, into which 50 μl of bacteria night culture was added. Flasks with crops were incubated on a circular shaker at 30 ° C, 150 rpm for 48 hours. To calculate the number of CFU / ml, the standard method of parallel dilutions in physiological saline and deep plating on solid agar media was used.

The calculated CFU / ml for all strains of Deinococcus radiodurans are shown in table 2.

table 2 Medium name The number of colony forming units / ml (CFU / ml) Deinococcus radiodurans VKPM B-8209 Deinococcus radiodurans BKM B-1422 Deinococcus radiodurans BKM B-1467 Tgy 5.0 × 10 ⁸ 7.7 × 10 ⁸ 7.1 × 10 ⁸ L 1.0 × 10 ⁸ 1.6 × 10 ⁸ 2.1 × 10 ⁸ Nourishing broth 3.5 × 10 ⁷ 5.5 × 10 ⁷ 6.3 × 10 ⁷ Soybean medium 1.3 × 10 ⁹ 1.7 × 10 ⁹ 1.3 × 10 ⁹

As can be seen from table 2, the maximum growth of strains of microorganisms Deinococcus radiodurans VKPM B-8209, Deinococcus radiodurans BKM B-1422, Deinococcus radiodurans BKM B-1467 was observed when grown in the proposed environment. Moreover, the number of CFU / ml on soybean medium for all strains of Deinococcus radiodurans microorganisms was 1.8-2.6 times higher than on TGY medium, 6.2-13.0 times more than the number of CFU / ml on medium L, and 20.6-37.1 times more than on nutrient broth.

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

Thus, a simpler and cheaper nutrient medium allows to obtain a higher yield of the number of bacteria 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. Handbook of microbiological culture media. Edited by Medzhidova M.M. Dagestan book publishing house, Makhachkala, 1989, p.14.

2. Passport for the 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 pathway in cell survival. Applied and Enivironmental Microbiology. 2000. Vol. 66. No. 1, p. 105-112 (prototype).

4. MR "Quality control of nutrient media" No. 04-3-16 / 1615 from 27.06 + .2003.

Claims (1)

Nutrient medium for growing microorganisms Deinococcus radiodurans, containing a nutrient base and distilled water, characterized in that it contains soy flour as a nutrient base in the following quantitative ratios of components, g / l:
soy flour 50,0 distilled water up to 1 l