RU2299904C1 - Preserving agent for microorganism storage - Google Patents

Abstract

FIELD: biotechnology, microbiology.

SUBSTANCE: invention relates to storage of microorganisms. Invention proposes using 5-methyl-3-phenyl-2,4-diethoxycarbonyl-N-(4-methylphenyl)-cyclohexa-1,5-dienylamine as a preserving agent. Invention provides increasing time for storage of bacterial cultures and retention of their viability.

EFFECT: improved and valuable properties of preserving agent.

1 tbl, 6 ex

Description

The invention relates to microbiology, in particular to the storage of bacteria, and can be used in scientific and practical medicine, in biotechnology and the food industry for storage of strains producing various substances.

Known compositions of the media used in the conservation of microorganisms. Mist dissicans medium (Sager R. “Cytoplasmic genes and organelles”, M., Mir, 1975, 423 pp.), Containing 7.5% glucose, 3 parts normal horse serum and 1 part broth.

The disadvantage of this environment should be recognized as the lack of standardization of its preparation due to the presence in it of horse serum and broth prepared from raw materials of various quality,

Sugar-gelatin medium (Danilova MV, Nadirova IM, Kudryavtsev VI “Lyophilization of bacteria”, In: Storage methods for collection cultures of microorganisms.- M .: Nauka, 1967, p. 131), containing 10 % sucrose and 0.1% agar in 1.5% gelatin.

The disadvantage of this environment is the inability to store various bacterial strains for a long time.

Protective media are known (RF patents No. 2088657, IPC C12N 1/04; No. 2045573, IPC C12N 1/04; No. 2041942, IPC C12N 1/04) used for lyophilization of strains of various genus and species. Studies have shown that the high protective properties of these media are mainly due to the high antioxidant activity of their constituent substances. For example, 2,4-diphenyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline (Pat. No. 2045573) has antioxidant activity of 3.06, 7,7-dimethyl-2-phenyl-4- (4 -methoxyphenyl) -5-oxo-1,4,5,6,7,8-hexahydroquinoline (Pat. No. 2088657) - 2.41. Closest to the proposed solution is the invention according to Pat. No. 2041942.

The disadvantage of the above components is the inconvenience of the procedure for their preparation, in particular heating in a sealed ampoule in the synthesis of 7,7-dimethyl-2-phenyl-4- (4-methoxyphenyl) -5-oxo-1,4,5,6,7, 8-hexahydroquinoline and 2,4-diphenyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline.

The objective of the present invention is to use as a preservative substances with high antioxidant activity, expanding the range of antioxidant components for protecting bacteria, simplifying and reducing the cost of their production.

The technical result is to increase the duration of storage of crops while maintaining viability.

The problem is solved in that as a preservative for the storage of bacteria, 5-methyl-3-phenyl-2,4-diethoxycarbonyl-N- (4-methylphenyl) cyclohexa-1,5-dienylamine (dienamine) is used.

The facts of the suitability of dienamine as a component of a protective environment are not known. Compared with the known components for the protection of microorganisms (7,7-dimethyl-2-phenyl-4- (4-methoxyphenyl) -5-oxo-1,4,5,6,7,8-hexahydroquinoline, 2,4- diphenyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline) the proposed compound can be obtained by a simpler method and contains structural fragments that determine high antioxidant properties, in particular N-arylenamine and dihydrobenzene moieties, hydroxyl group . So, the antioxidant activity of dienamine, measured by the chemiluminescent method, is 2.14 (in analogues 2.41 and 3.06), which allows us to offer this compound as a component for protecting bacteria.

On the basis of dienamine, a composition for storing bacteria can be obtained containing an aqueous solution of a protective protein medium, a cryoprotectant and a solution of dienamine, wt.%:

Protective Protein Medium 0.1 ÷ 3.0 Dienamine 0.001 ÷ 0.1 Dienamine solvent 0.1 ÷ 10 Water rest.

Gelatin and agar-agar can be used as a protective protein medium. As a stabilizer of the composition can be added sucrose in an amount of 1 ÷ 10 wt.%.

Dimethyl sulfoxide (DMSO) or ethanol may be used as a dienamine solvent. The composition for storing bacteria may have the following ratio of components, wt.%:

Sucrose 1 ÷ 10; Protective protein components: gelatin 0.5 ÷ 3; agar agar 0.01 ÷ 0.1; Dienamine 0.001 ÷ 0.1; Dienamine solvent 0.1 ÷ 10; Water rest.

In the scientific, patent literature there is no data on such compositions for the storage of bacteria. The facts of the suitability of dienamine as a preservative (a component of the cryoprotective medium) are not known. This is not only an unobvious, moreover, unexpected property of this compound. The increase in the shelf life of bacteria to 2.4 ÷ 18.2 years led to the positive effect of the invention.

Thus, this solution meets the criteria of the invention.

The composition for storing bacteria is prepared as follows (calculation per 1 l of water): 0.1 ÷ 1 g of agar-agar is soaked in 0.7 l of distilled water, put on fire, with constant stirring, pre-soaked swollen gelatin is added (7 ÷ 50 g ) After dissolving the gelatin, sucrose (10 ÷ 100 g) is added, brought to a boil. Remove from heat and bring the volume to 1 liter with distilled water, filter, pour into dishes and sterilize at 110 ° C for 30 minutes. Then, dienamine dissolved in one of the indicated solvents (dimethyl sulfoxide - DMSO, ethanol) is introduced into the resulting mixture.

5-Methyl-3-phenyl-2,4-diethoxycarbonyl-N (4-methylphenyl) cyclohexa-1,5-dienylamine obtained by arylamination of 5-hydroxy-5-methyl-3-phenyl-2,4-diethoxycarbonylcyclohexanone p-toluidine (yield 88%) [Arylamination of 2,4-diacetyl (diethoxycarbonyl) -5-hydroxy-5-methyl-3-phenyl (2-furyl) cyclohexanones / V.V. Sorokin, M.V. Kuzmin, N.S. Smirnova, N.I. Kozhevnikova, A.P. Krivenko // Zh. 1994.V. 30. Issue 4. S.528-530.].

The advantages of the method of obtaining the claimed compound (preservative) include: use for the synthesis of affordable and cheap starting materials (products of basic organic synthesis), simplicity of instrumentation, high yield of the target product, which may be important for obtaining substances in large quantities.

The structure of the obtained dienamine was confirmed by elemental analysis and IR spectroscopy. A sample of the compound used in these studies was identified by the melting temperature of the mixing sample with the sample obtained for the first time by us earlier. [Arylamination of 2,4-diacetyl (diethoxycarbonyl) -5-hydroxy-5-methyl-3-phenyl (2-furyl) cyclohexanones / VV Sorokin, MV Kuzmin, N. S. Smirnova, N. I.Kozhevnikova, A.P. Krivenko // ZhORKh. 1994.V. 30. Issue 4. S.528-530.].

Example 1. The study of the influence of the protective environment with dienamine using DMSO as a solvent was carried out on a model of a representative of the Enterobacterlaceae family, which are widely distributed in nature and are most often used in scientific studies of the microbiological, genetic, immunological and biochemical plan — the vaccine strain of the plague microbe EV NIIEG.

The strain was grown under optimal temperature conditions on Hottinger mowed agar until a stationary growth phase and suspended in a compositionally varying medium consisting of sucrose (1 ÷ 10%), gelatin (0.5 ÷ 3.0%) and agar-agar (0, 01 ÷ 0.1%) with the addition of 0.001 ÷ 0.1% of dienamine dissolved in 0.1% DMSO. The resulting mixture was poured into ampoules and lyophilized. As a result of the experiments conducted in a series of experiments, it was found that dienamine at concentrations of 0.01 ÷ 0.05% increases the duration of the strain in the lyophilized state while maintaining viable cells from 1.8 years (in the control) to 2.4 ÷ 5.6 years in depending on the concentration of dienamine, i.e. 1.3 ÷ 3.1 times.

Example 2. In the composition described in example 1, the same strain was stored under similar conditions, but with a changed concentration of DMSO - 10%. The shelf life in a series of experiments increased from 1.8 to 3.0–6.9 years (1.7–3.8 times) while maintaining viability.

Example 3. In the composition described in example 1, the same strain was stored when using dienamine 0.1% ethanol as a solvent. The shelf life in a series of experiments increased from 1.8 to 3.3–8.2 years (1.8–4.6 times) while maintaining viability.

Example 4. In the composition described in example 1, the strain of Escherichia coli C-600, a member of the Enterobacterlaceae family, was stored. The shelf life increased from 3.5 to 18.2 years (5.2 times).

Conclusion: from the data given in the examples, it follows that the optimal result is obtained when using dienamine at a concentration of 0.001 ÷ 0.01% dissolved in 1 ÷ 10% DMSO (the storage period increases by 1.3 ÷ 4.6 times). The use of ethanol as a solvent, which is also an antioxidant, made it possible to obtain an increase in the shelf life compared to the control sample while maintaining viability by 5.2 times.

In the next step, an additional protective protein medium was used.

Example 5. The study of the influence of the protective environment with dienamine using normal horse serum as an additional component of the protective protein environment was carried out on a representative of the Enterobacteriaceae family - the vaccine strain of the plague microbe EV NIIEG.

The strain was grown on mowing Aging Hottinger pH 7.2 at the optimum temperature until the stationary phase of growth and suspended in a medium consisting of normal horse serum and Hottinger broth pH 7.2 (3: 1) in a ratio of 1.7 ÷ 18 wt.% to the total composition with the addition of dienamine 0.001 ÷ 0.1% dissolved in 0.1% DMSO.

As a result of the experiments, it was found that the protective protein medium using horse serum and Hottinger broth with dienamine in the above concentration increases the duration of storage of the strain in a lyophilized state while maintaining 50% of surviving cells from 3.2 years (in control) to 8.1 years , i.e. 2.5 times.

Example 6. The study of the influence of the protective environment with dienamine on the storage time of widely distributed in nature spore-forming microorganisms of the Bacillaceae family in a lyophilized state was carried out on a vaccine strain of the causative agent of anthrax - "STI".

The strain was grown on Hottinger agar pH 7.2 at a temperature of 37 ° C until the stationary phase of growth (18-24 hours). The suspension was prepared in a medium consisting of 10% sucrose, 1.5% gelatin, 0.1% agar-agar with the addition of 0.001 ÷ 0.1% dienamine, dissolved in 0.1% DMSO. The resulting mixture was poured into ampoules and lyophilized. As a result of the experiments, it was found that dienamine at a concentration of 0.01% increases the shelf life of the anthrax causative agent strain in a lyophilized state with the preservation of viable cells from 6.5 years (in control) to 17.8 years, i.e. 2.7 times.

The given examples confirm the property of dienamine as a preservative for storing bacteria of various genus and species composition when using DMSO and ethanol as a solvent, while storage periods increase to 1.3–5.2 times while maintaining 50% viability of the tested objects.

Table The results of the use of dienamine in the composition of the protective environment for the conservation of bacteria by lyophilization on the model of the plague microbe vaccine strain EV NIIEG The concentration of dienamine% Solvent Duration of storage of culture (years) 0.010 0.1% DMSO 2,4 0,025 -′′- 5,0 0,050 -′′- 5.2 0.010 -′′- 5,6 0,025 -′′- 2,3 0.0010 10% DMSO 6.9 0.0025 -′′- 5.7 0.0050 -′′- 3.0 0.010 -′′- 3.0 0,025 -′′- 3.0 0.0025 0.1% ethanol 5.7 0.0050 chemically pure 8.2 0.010 -′′- 7.0 0,025 -′′- 3.3

Note. Control samples contain 10% sucrose, 1.5% gelatin, 0.1% agar-agar, and a shelf life of 1.8 years. Experienced - the same, with the addition of dienamine in an appropriate solvent.

Claims (1)

The use of 5-methyl-3-phenyl-2,4-diethoxycarbonyl-N (4-methylphenyl) cyclohexa-1,5-dienylamine as a preservative for storing bacteria.