RU2400069C1 - Method of protecting materials from microbial destruction - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to methods of inhibiting growth of microorganisms. The invention discloses a method of protecting materials from microbial destruction through introduction of a substance with antimicrobial activity into the protected material. Said substance is a methane oxyphenyl derivative which also contains one furyl or phenyl or trichloromethyl radical.

EFFECT: method widens the range of environmentally safe substances for antimicrobial protection, having prolonged effect and highly reliable protection.

5 ex, 3 tbl

Description

The invention relates to methods for inhibiting the growth of microorganisms and can be used to protect organic and inorganic materials from biodeterioration, as well as in medicine, veterinary medicine, microbiological and medical industries.

A known method of protecting materials, for example, an aqueous or non-aqueous based cutting fluid, is when a composition of anaerobic growth inhibiting compounds is introduced that contains α-tri-1- (p-nitrophenol) -2-aminopropanediol-1,3 and organic sulfonate (GDR Patent No. 266240, class C10M 141/08, 1987).

The disadvantage of this method is the use of antimicrobial compositions that suppress only microorganisms of one type, for example anaerobes, and are not active against other organisms, such as aerobes, as well as inhibiting the development of only vegetative cells, but not resting forms, such as spores.

A known method of protecting materials, for example technical oil, from the effects of micromycetes, comprising the introduction of citral (3,7-dimethyl-octadiene-2,6-al-1) in the amount of 0.5-1.0% (RU 2074250, 02/27/1992).

A known method of combating bacteria and polluting (contaminating) organisms, comprising applying to these bacteria and polluting organisms or their habitat an effective amount of an antibacterial active ingredient corresponding to formula (a):

where n = 1 or 2; R ¹ is hydrogen; R represents (a) phenyl; phenyl substituted with 1 or 2 substituents independently selected from halogen, C _1-12 alkyl, trihalomethyl, C _1-5 alkoxy, C _1-4 alkylcarbonyl or nitro; thienyl when n ≠ 2, furanyl; or R represents a radical of formula (b):

where X is oxygen or sulfur, Y is nitrogen or CH; R '' is hydrogen or C _1-4 alkyl (RU 2143805, 10.01.2000).

The disadvantage of this method is that the biocides of the specified formula used to protect materials have toxic effects and are environmentally hazardous substances.

It is also known to use antimicrobial proteins and peptides to protect objects from microbial destruction. For example, it is known to use a protein component isolated from plant chromatin after decomposition of the latter as an antimicrobial agent. The specified protein component has an apparent molecular weight in the range from 10 to 20 kDa (RU 2303357, 07.27.2007). However, this substance can be destroyed by microorganisms, which reduces the duration of its protective effect.

A common drawback of the methods used to protect materials from biodeterioration is the development of the process of autolysis of microorganisms inhibited by biocides, as a result of which organic substances enter the environment, facilitating the subsequent development of microorganisms of the second wave of contamination. Thus, the protective effect of biocides is reduced.

Closest to the proposed invention is a method of protecting materials from microbiological and oxidative degradation, according to which a sample of a phenolic substance is introduced into the protected material, using a substance of the general formula (c):

(с)

(from)

where R ₁ = H; HO or C ₂ -C ₂₁ alkyl;

R ₂ = H; IT; (CH ₂ ) _n OH,

where n = 2-6; CH ₂ COOR ₄ (R ₄ = H or C ₁ -C ₂₀ alkyl); alkyl C ₆ -C ₂₁ or CH ₂ COR ₅ [R ₅ = CO (CH ₂ ) _n CH ₃ where n = 0-19 or NH (CH ₂ ) _n CH ₃ where n = 0-19]

R ₃ = H or C ₂ -C ₂₁ alkyl,

moreover, R ₁ , R ₂ and R ₃ at the same time cannot be hydrogens (RU 2086610, 08/10/1993).

Antimicrobial substances in the known method have a wide spectrum of action and a prolonged nature of protection.

However, the disadvantage of the known technical solution adopted for the prototype is the high hydrophobicity of most compounds of the general formula, which significantly limits the scope of their application. In addition, the patent describes the use of only a limited range of environmentally friendly substances for the protection of materials; examples of prolonged antimicrobial action and the prevention of autolysis of cells of inhibited microorganisms are not given.

The objective of the present invention is to expand the range of environmentally friendly substances that can be used for antimicrobial protection of materials of different nature and have a prolonged effect, as well as improving the reliability of protection.

The problem is solved by the described method of protecting materials from microbial destruction by introducing a substance with antimicrobial activity into the material to be protected, while an alkyloxyphenyl methane derivative of the general formula is used as a substance with antimicrobial activity:

where: R ₁ selected from oxyphenyl or alkyloxyphenyl,

R ₂ selected from oxyphenyl or alkyloxyphenyl,

R _{3 is} selected from phenyl, furyl or trichloromethyl, while in alkyloxyphenyl, alkyl is selected from C ₂ -C ₂₁ .

We found that the above substances have antimicrobial activity against vegetative and resting cells of a wide range of microorganisms: bacteria, archaea, fungi, and yeast. They prevent their autolysis and the secondary wave of contamination, which ensures a prolonged effect and lack of addiction. The main biological activity of the compounds is to induce the transition of vegetative cells of microorganisms to a state of suspended animation and maintain this state, which is expressed in the inhibition of germination of dormant forms and the growth of microorganisms. In addition, the claimed methane derivatives are low toxicity compounds. According to the molecular mechanism of action, the methane derivatives used are modifiers of the membranes and biopolymers of the cell, which causes the cell to lose metabolic activity, proliferation ability and its death without subsequent autolysis. Used methane derivatives can be obtained by known methods of synthesis or isolated from natural sources.

The maximum effect of protecting materials from microbial destruction is achieved by introducing the claimed substances in a concentration of 25 to 5000 mg / l, depending on the taxonomic group of microorganisms (eubacteria, archaea, fungi) and the physiological group (heterotrophs, autotrophs, hydrocarbon-oxidizing, sulfate-reducing, etc.). The claimed substances when diluted to concentrations below 10 mg / l do not inhibit the development of microorganisms.

The antimicrobial activity of substances is studied by turbidimetric determination of the growth of microorganisms in selective nutrient liquid media when measuring the optical density (OD) of a microbial suspension on a FEK-56 photoelectrocolorimeter, filter No. 8, the layer thickness in the cuvette is 0.5 cm, or a spectrophotometer, wavelength 660 nm, the layer thickness in the cuvette is 1 cm, or by the method of determining the concentration of viable cells (CFU) by seeding ten-fold dilutions of liquid cultures into solid nutrient media. The inoculum is added in an amount giving the number of cells in the medium of 20.0-25.0 million / ml, which is the initial value of OD = 0.2. The cultivation duration is 1-360 days. Cultivation temperatures are optimal for the growth of cultures of microorganisms (20-37 ° C). Aeration is provided by a rocking chair from 140-160 rpm, anaerobic and microaerophilic microorganisms are grown without stirring and without a layer of air above the medium.

The test substances are introduced in the form of a solution in water or in ethanol (96% rectified) or in a special solvent consisting of acetone containing 10% of Tween-80 detergent, in an amount of 0.5-1 vol / vol. Equal amounts of solvent components are added to the control variants. The concentration of introduced substances in the growth medium from a maximum of 5000 mg / L decreases fractionally to a minimum concentration of 1.0 mg / L.

Antimicrobial activity is evaluated by the value of the minimum growth-inhibitory concentrations (MIC), expressed in mg / L.

Table 1 shows the specific substances that correspond to the general formula indicated above, which we tested to protect the materials from the microorganisms listed in table 2.

Table 1 Substances with antimicrobial activity No. Substances, specification of radicals R ₁ , R ₂ , R ₃ one Dioxyphenyl, phenylmethanes, where R ₁ and R ₂ are hydroxyphenyls, R ₃ is phenyl. 2 Dioxyphenyl, furylmethanes, where R ₁ and R ₂ are hydroxyphenyls, R ₃ is furyl. 3 Dialkyloxyphenyl, phenylmethanes, where R ₁ and R ₂ are alkyloxyphenyls with the length of the alkyl radical C ₂ -C ₂₁ , R ₃ is phenyl. four Dialkyloxyphenyl, furylmethanes, where R ₁ and R ₂ are alkyloxyphenyls with the length of the alkyl radical C ₂ -C ₂₁ , R ₃ is furyl. 5 Dialkyloxyphenyl, trichloromethylmethanes, where R ₁ and R ₂ are alkyloxyphenyls with the length of the alkyl radical C ₂ -C ₂₁ , R ₃ is trichloromethyl.

The invention is illustrated by examples.

Example 1. In a series of tubes containing special nutrient media for the growth of bacterial cultures of S. aureus and M. smegmatis, substances 1-5 (Table 1) are added at concentrations of 2-5000 mg / l, after which an inoculum of microorganisms is added in an amount giving the cell concentration (according to CFU) of 20.0-25.0 million / ml. No biocide is added to control tubes. After 3 and 7 days in the control and experimental variants, the concentration of viable cells (CFU) is determined by seeding decimal dilutions on solid media. The values of the concentrations of substances at which the value of the CFU remains at the level of the inoculum, consider the MIC. The MIC values thus obtained for S. aureus and M. smegmatis are presented in Table 2. For S. aureus, these values range from 25 mg / L (for substance No. 3) to 500 mg / L (substance No. 5). For M. smegmatis, MICs range from 50 for substance No. 4 to 1000 for substances No. 1 and 2.

Similarly, the effectiveness of the claimed antimicrobial group against microorganisms of taxonomic groups in which pathogenic or toxigenic strains are present, including the bacteria Bacillus cereus, Pseudomonas fluorescens, Pseudomonas aeruginosa, Streptomyces coelicoloror, Candida utilis yeast, and lower fungi Penicillium brevi-compactum.

The above example shows that oxyphenyl derivatives of methane (substances No. 1-5, Table 1) inhibit the growth of bacteria of medical significance with different taxonomic affiliations and that, when diluted to concentrations below 20 mg / l, substances of the general formula do not inhibit the development of microorganisms, t .e. Do not pose environmental hazard.

table 2 The values of the minimum growth-inhibitory concentrations (MIC) of compounds No. 1-5 (Table 1) (mg / L) for S.aureus and M. smegmatis Connection No.
Microorganism one 2 3 four 5 S. aureus one hundred one hundred 25 70 500 M. smegmatis 1000 1000 70 fifty 750

Example 2. The nutrient medium is inoculated with a mixture of microorganisms, B. subtilis, P. Fluorescens, S. cerevisiae. Then, according to the method described in example 1, drug No. 2 is introduced into the nutrient medium at a concentration of 1000 mg / l. The CFU titer is determined immediately after seeding and after 1, 3, 6 and 12 months. The growth of microorganisms is not observed for 12 months. A similar result was obtained for the remaining substances presented in table 1.

This example shows that microorganisms do not develop adaptive reactions with respect to biocidal substances of the general formula, and therefore the growth-suppressing effect of the tested substances lasts for at least 12 months.

Example 3. Microorganism cultures (Saccharomyces cerevisiae, Micrococcus luteus, Thioalkalivibrio versutus, Staphylococcus aureus, Mycobacterium smegmatis) are added to test tubes with nutrient media and incubated for 1-3 days until the stationary phase is reached, after which substance No. 2 is added (Table 1 ) to a final concentration of 200-1000 mg / l. Within 30-60 minutes, the death of all cells is observed, and cell lysis does not occur, specific morphological changes are observed (high refractoriness of the cells, the appearance of ruptures of the cytoplasmic membrane with its subsequent disintegration, release of phospholipids into the environment while maintaining the integrity of the cell wall, ribosome conglomeration, cytoplasm vitrification ), characteristic of a new type of biocidal action - the formation of micromummies. The remaining substances presented in table 1, act similarly.

This example proves that the use of substances No. 1-5 (Table 1) in addition to the elimination of all viable bacteria prevents their lysis and the concomitant release of nutrients (contents of killed microorganisms) into the environment. As a result, there will be no nutrients for microorganisms entering this (protected) environment and their development will not occur, which increases the reliability of the antimicrobial action of the proposed biocides.

Example 4. The culture of hydrocarbon-oxidizing bacteria Rhodococcus erytropolis and R. maris was introduced into tubes with 10 ml of Raymond medium with 2% oil to a concentration of 3 × 10 ⁸ cells / ml. Substance 1 (Table 1) was added to the tubes to a concentration of 0.005-0.5%. In the control, no biocide was added. The tubes were cultured at 27 ° C for 14 days. The conclusions about the presence of growth and development of cultures were made on the basis of a turbid environment, the formation of a water-oil emulsion and microscopic examination of samples. Cultures of hydrocarbon-oxidizing bacteria were grown as described above and added (in an amount of 10%) to cultures of sulfate-reducing bacteria, which were then grown on Widdel's medium with lactate in Baltsch tubes for 2 weeks. When inoculated, substance No. 1 was added in the above concentrations. The activity of sulfate-reducing bacteria was judged by the appearance in the environment of hydrogen sulfide, determined by Pachmeier.

The results of the analysis of the development of cultures of hydrocarbon-reducing and sulfate-reducing bacteria in the presence of oil are presented in Table 3. Similar results were obtained for other substances listed in table 1.

Table 3 The effect of various concentrations of substance 1 (Table 1) on the growth and development of hydrocarbon-oxidizing bacteria R. erytropolis and R. maris The concentration of the substance 1,% The titer of cells of hydrocarbon-oxidizing bacteria The activity of sulfate-reducing bacteria 0 10 ⁹ one hundred 0.005 10 ⁹ one hundred 0.01 5 × 10 ⁶ 60 0.05 10 ⁶ twenty 0.1 Less than 10 ⁵ 0 0.5 Less than 10 ⁴ 0

This example shows that biocides of the general formula effectively suppress the development of hydrocarbon-reducing and sulfate-reducing bacteria involved in the decomposition of oil and oil products and can be used to prevent their biocorrosion in concentrations of 0.1-0.5%.

Example 5. The effect of the preparation of substance 1 on steel corrosion was carried out according to the method of determining the protective ability of metal corrosion inhibitors in oil-water environments (GOST 9.506-87) using samples of steel STZ. The content of substance 1 was 0.005%, 0.01%, 0.05%, 0.1%, 0.5% (wt.). Effective were concentrations from 0.05% and higher. When the content of substance 1 was from 0.1% to 0.5% (wt.), The corrosion rate decreased by 70-80%.

Similar results were obtained for substances No. 2-5 (table 1).

The data obtained indicate that substances of the general formula are an inhibitor of metal corrosion under the conditions of operation of oil refining equipment at concentrations of 0.05-0.5% (wt.).

Example 6. Alcohol solutions of substance No. 1 (0.1, 0.2, 0.5, 1, 2, 5, and 10%) are applied to the surface of wooden bars at the rate of 0.01-1 mg / cm ² with an impregnation depth of at least 1 mm, the bars are dried and treated with a spore suspension of cellulolytic fungi of the genera Trichoderma lignorum (concentration 10 ⁷ -10 ⁸ CFU / ml). The bars were left in humid chambers at 30 ° C. After 30 days, the fungal damage to the wood was determined - visually and by application of the bars on a dense Hatchinson medium with the addition of filter paper. When using concentrations of 0.1-1%, fungal growth was observed, as in the control on untreated wood. When treated with a solution with a concentration of 2%, the growth was strongly suppressed, when using 5 and 10% growth of fungi was not observed for 6 months.

Similar data on the suppression of the activity of proteolytic fungi using substance No. 1 were obtained for the protection of leathers and leather raw materials.

This example shows that the substance No. 1 can be used to protect materials of different nature, carbohydrates and proteins, from microbial destruction by applying to their surface.

Claims (1)

A method of protecting materials from microbial destruction by introducing a substance with antimicrobial activity into the protected material, characterized in that a methane derivative of the general formula is used as a substance with antimicrobial activity:

where R ₁ and R ₂ are hydroxyphenyl or alkyloxyphenyl, and R ₃ is phenyl, or furyl, or trichloromethyl, while in alkyloxyphenyl, alkyl is selected from C ₂ -C ₂₁ .