RU2291193C2 - Preserving agent for microorganism storage - Google Patents

Abstract

FIELD: microbiology.

SUBSTANCE: invention relates to agents used in storage of microorganisms. Invention proposes using 5-hydroxy-5-methyl-3-(3-nitrophenyl)-2,4-diethoxycarbonyl-N-(phenyl)-1-cyclohexenylamine (enamine A) as a preserving agent. Invention provides prolongation of bacterial cultures storage and retaining their viability.

EFFECT: valuable biological properties of agent.

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Description

The invention relates to microbiology, in particular to the storage of microorganisms, 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. The medium "Mist, dissicans" (Sager R. Cytoplasmic genes and organelles. - M .: Mir, 1975, 423 pp.), Containing 7.5% glucose, 3 parts of normal horse serum and 1 part of 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 the book: 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 C 12 N 1/04; No. 2045573, IPC C 12 N 1/04; No. 2041942, IPC C 12 N 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 an antioxidant activity of 3.06, 5,5-dimethyl-2- (1,3-diphenyl -3-oxopropyl) - cyclohexanedione-1,3 (Pat. No. 2041942) - 2.77.

Closest to the proposed solution is the invention according to US Pat. RF №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 the protection of microorganisms, simplifying and cheapening the methods for their preparation. The technical result is to increase the duration of storage of crops while maintaining viability.

The problem is solved in that 5-hydroxy-5-methyl-3- (3-nitrophenyl) -2,4-diethoxycarbonyl-N (phenyl) -1-cyclohexenylamine (enamine (A)) is used as a preservative for storing bacteria.

The facts of the suitability of enamine as a component of a protective environment are unknown. 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 predetermine high antioxidant properties, in particular the N-arylenamine fragment, a hydroxyl group. So, the antioxidant activity of enamine, measured by the chemiluminescent method, is 2.57 (in analogues 2.41 and 3.06), which allows us to offer this compound as a component for protecting microorganisms.

In the scientific, patent literature there is no data on such compositions for the storage of bacteria. Unknown facts of the suitability of enamine (A) as a preservative (component of a cryoprotective environment). This is not only non-obvious, moreover, an unexpected property of this compound. The increase in the shelf life of bacteria to 3.85-16 years led to a positive effect of the invention.

Thus, this solution meets the criteria of the invention.

The inventive composition for storing bacteria is prepared as follows (calculation per 1 liter 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 (7-50 g) is added. 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 enamine (A), dissolved in one of these solvents (dimethyl sulfoxide, ethanol), is introduced into the resulting mixture.

Enamine (A) was obtained by arylamination of 5-hydroxy-5-methyl-3- (3-nitrophenyl) -2,4-diethoxycarbonylcyclohexanone under the influence of aniline (product yield is 68%) [Krivenko A.P., Golikov A.G. , Grigoryev A.V., Sorokin V.V. Intramolecular hydrogen bond in a series of substituted cyclohexanolones and their nitrogen-containing derivatives // Zhork. 2000.V. 36. Issue 8. S.1152-1155.]. The composition and structure of the obtained enamine (A) was confirmed by elemental analysis and IR spectroscopy. A sample of the compound used in these studies was identified by the melting temperature of a mixing sample with a sample obtained for the first time by us earlier [Krivenko A.P., Golikov A.G., Grigoryev A.V., Sorokin V.V. Intramolecular hydrogen bond in a series of substituted cyclohexanolones and their nitrogen-containing derivatives // Zhork. 2000.V. 36. Issue 8. C.1152-1155].

The following composition for storing bacteria was prepared, containing an aqueous solution of a protective protein medium and enamine as a preservative, wt.%:

Protective Protein Medium 0.1-3.0 Enamine (A) 0.001-0.1 Enamine Solvent (A) 0.1-10 Water Rest.

Gelatin and agar-agar can be used as a protective protein medium.

As the enamine solvent (A), dimethyl sulfoxide or ethanol can be used.

As a stabilizer of the composition can be added sucrose in an amount of 1-10 wt.%.

Example 1. The study of the influence of a protective environment with enamine 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.

The strain was grown under optimal temperature conditions on Hottinger mowed agar to 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% enamine dissolved in 0.1% dimethyl sulfoxide (DMSO). The resulting mixture was poured into ampoules and lyophilized. As a result of the experiments, it was found that enamine in concentrations of 0.01-0.05% increases the duration of the strain in a lyophilized state while maintaining 50% of viable cells from 2.0 years (in control) to 2.9-6.5 years, depending on the concentration of enamine (A), i.e. 1.45-3.25 times. The determination of shelf life in all experiments was carried out according to an accelerated method (Method for determining the thermal stability of live dry vaccines and predicting their viability in the process, Stavropol, 1985).

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 increased from 2.0 to 3.5-7.7 years (1.75-3.85 times).

Example 3. In the composition described in example 1, the same strain was stored when enamine (A) 0.1% ethanol was used as a solvent. The shelf life increased from 2.0 to 3.5-7.7 years (1.75-3.85 times).

Example 4. In the composition described in example 1, a pseudotuberculosis microbe (strain II a), a member of the Enterobacterlaceae family, was stored. The shelf life increased from 4.1 to 7.2-16.0 years (1.76-3.90 times).

Example 5. The causative agent of intestinal yersiniosis was stored in the composition, wt.%:

Sucrose 10 Gelatin one Agar agar 0.02 Enamine (A) 0.001-0.1 Solvent - DMSO 0.25 Water Rest.

Subject to the storage technology described in example 1, the shelf life is increased from 1.9 to 2.6 years, which exceeds the control period by 1.37 times.

Conclusion: from the data in the examples it follows that the optimal result is obtained by dissolving enamine (A) in 1-10% DMSO with an enamine concentration of 0.001-0.01% (the shelf life is increased by 3.25-3.90 times).

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

Example 6. The study of the influence of the protective environment with enamine using normal horse serum as the main component of the protective protein environment was carried out on a member of the family. Enterobacteriaceae, a plague microbe EV vaccine strain.

The strain was grown on Hottinger mowed agar 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 enamine 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 enamine in the above concentration increases the storage time of the strain in a lyophilized state while maintaining 50% of surviving cells from 3.8 years (in control) to 7.5 years , i.e. 1.97 times.

Example 7. The study of the effect of the protective environment with 5-hydroxy-5-methyl-3- (3-nitrophenyl) -2,4-diethoxycarbonyl-N (phenyl) -1-cyclohexenylamine on the storage time of the naturally occurring spore-forming microorganisms of the Bacillaceae family in 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). A 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% enamine dissolved in 0.1% DMSO. The resulting mixture was poured into ampoules and lyophilized. As a result of the experiments, it was found that enamine at a concentration of 0.01% increases the shelf life of the anthrax causative agent strain in a lyophilized state while maintaining 50% of viable cells from 6.3 years (in control) to 16 years, i.e. 2.54 times.

The given examples confirm the property of enamine as a preservative for storing bacteria, while the shelf life is increased up to 3.9 times while maintaining the viability of the tested objects.

Claims (1)

The use of 5-hydroxy-5-methyl-3- (3-nitrophenyl) -2,4-diethoxycarbonyl-N (phenyl) -1-cyclohexenylamine as a preservative for the storage of bacteria.