RU2446810C2 - Antimicrobial agents - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to antimicrobial agents exhibiting antibacterial activity with respect to test cultures of gram-positive and gram-negative microorganisms which represent copper nanoparticles and copper oxide nanoparticles. The antimicrobial agents according the invention are characterised by specific particle sizes and phase compositions. The nanoparticles have the size of 33.8-103 nm and contain 67-96% of copper and 4-33% of CuO. The copper oxide nanoparticles have the sizes of 77-124 nm and contain 3.3-23% of crystalline copper, 27.1-90% of CuO and 9.05-69.5% of Cu₂O.

EFFECT: invention provides preparing the antimicrobial agents which when introduced in the composition of non-woven linen ensure its antibacterial properties, as well as maintain its aesthetic and service characteristics.

2 cl, 8 dwg, 12 tbl, 6 ex

Description

The invention relates to medicine, in particular to agents with antimicrobial activity.

One of the urgent problems of modern medicine is the search for agents, components or factors that provide antimicrobial action. This is due to the wide spread of microorganisms and their growth in various environments, including drinking water, soil, food, cosmetics, personal care products, plastic, medical products (rubber and synthetic hoses, surgical instruments, plasters, suture materials, dressings surgical masks, respirators, clothing of medical personnel, etc.), causing not only material damage, but, most importantly, threatening human health. The requirements for the environment and medical supplies are getting higher. Therefore, the search for new antimicrobial agents that are safe for humans does not stop, given the constant increase in the resistance of bacteria to the action of sulfa drugs, antibiotics, and antiseptics. Currently, various substances of organic and inorganic nature can act as antimicrobial agents. However, the use of antimicrobial agents of one nature or another in medicine and pharmacy is limited by their safety. Inorganic compounds, for example metals and their compositions, are more preferred in this regard. Antimicrobial agents based on, for example, compounds of cobalt [15], silver [29, 31, 32], complex metal oxides (zinc oxide in CaO), and mixtures of metal compounds have been developed. Researchers' interest in nanoparticles of individual metals, metal compositions with the general formula MnXy, where Mn can be Al, Si, Zn, Ni, Ti; Xy - oxygen, carbon, sulfur; mixed nanoparticles of the type C-H-Ag-Zn, C-Si-Ag-Zn, C-W-Ti-B, monodisperse colloidal metal nanoparticles [16], etc.

There are a number of works devoted to the use of metal nanoparticles with antimicrobial and antiviral properties to improve protection against microbial and viral infections [17, 30]. Therefore, the further search for antimicrobial agents that are safe for human health is relevant.

Our attention was drawn to copper, an essential element that in animals and humans has a strictly coordinated system of regulation, which, in case of an increased load of the metal, maintains its content in biosystems at a level that ensures optimal vital activity. On the other hand, copper has a pronounced antibacterial effect [4, 14, 19, 24, 26]: diphtheria and typhoid bacilli die on metal copper plates [3], highly dispersed copper powders cause the death of E. coli cells [8, 25]. In medical practice, copper citrate is used as an antiseptic, astringent, cauterizing agent for conjunctivitis, tonsillitis, skin infectious diseases, burns, wounds, pressure sores, copper nitrate for trachoma, conjunctivitis, conjunctivitis, laryngitis, urethritis, skin sulfate infections - ; as a bactericidal, cauterizing agent, copper-calcium hydroxide is used, etc. [1, 3, 5].

Currently, several mechanisms are considered by which copper has an antimicrobial effect:

1. Copper inhibits and disrupts the synthesis of protein and nucleic acids. Copper has the ability to interact with amino acids and SH-groups of enzymes actively involved in protein synthesis, which can disrupt this process. It is known that copper selectively binds to N (1) and N (7) adenine, N (7) guanine, N (3) -cytosine. As a result of binding to negative phosphate groups of nucleic acids by electrostatic forces and partially coordination through amino and oxy groups with nitrogen bases, a strong chelate complex is formed [2, 22].

2. Copper reduces the level of reduced thiols and glutathione in cells. Glutathione is one of the metabolites that increase rapidly with osmotic stress. One of the functions of glutathione is associated with the retention of potassium ions in the cytoplasm through the regulation of potassium output channels [11, 23].

3. Copper affects the energy processes occurring on the membrane and disrupts its barrier functions [9, 12, 13, 25, 27, 33].

4. Copper changes the structural and functional properties of the membranes of prokaryotic cells [8, 10, 13].

The development of modern nanotechnology makes it possible to obtain nanostructured metals, including copper with predetermined biological properties, by regulating the physicochemical characteristics of nanoscale materials. The main features of the action of nanoparticles upon their introduction into the eukaryotic organism is their low toxicity, prolonged action, the ability in biotic doses to exert a stimulating effect on metabolic processes, enzyme activity, growth and development of the organism as a whole [7, 20]. At the same time, it was shown that copper nanoparticles have a higher antimicrobial activity compared to silver nanoparticles [8–13, 25].

Among the analogues of the present invention can be called metallic copper powder (as part of homeopathic medicines) as an antimicrobial, immunoprotective effect in inflammatory diseases, in the complex treatment of whooping cough, bronchial asthma and Asian cholera (Russia) [3].

Closest to our invention is the composition described in US patent 534 4636 Anti-microorganism agent and anti-microorganism resin or rubber composition [18], where a complex metal hydroxide [(M ₁ ²⁺ ) ₁ -x is used as an antimicrobial agent (M ₂ ²⁺ ) _x (OH) ₂ ] in the form of particles 0.1 to 2.0 μm in size, in which M ₁ ²⁺ is one of the Mg ²⁺ and / or Ca ²⁺ elements, M ₂ ²⁺ is one of the Cu elements ²⁺ and / or Zn ²⁺ . Such a composition can be incorporated into rubber, rubber, resin, etc., is slightly toxic and has an antibacterial effect in relation to test cultures of E. coli and St.yellow.

The aim of this invention is to establish the antimicrobial effect of copper nanoparticles and copper oxide nanoparticles having different physicochemical characteristics on the growth of test cultures of gram-positive and gram-negative bacterial cells.

The goal is achieved in that, as agents that have an antimicrobial effect, copper nanoparticles and copper oxide nanoparticles are used, having the following physicochemical characteristics:

- copper NPs: particle size 33.8 ÷ 103 nm; oxide film thickness 6 ÷ 10 nm; phase composition: crystalline copper 67 ÷ 96%, copper oxide CuO - 4 ÷ 33%.

- NP copper oxide: particle size 77 ÷ 124 nm; phase composition: crystalline copper 0.5 ÷ 3.3 ÷ 23%, CuO - 27.1 ÷ 90%. and Cu ₂ O - 9.05 ÷ 69.5.

Copper oxide nanoparticles with characteristics: particle size 119.0 ± 1.0; phase composition: crystalline copper ≤0.50 ± 0.02, CuO≈90% and Cu ₂ O≈9.05, taken as an antimicrobial agent and incorporated into the non-woven linen fabric, provide antibacterial properties of flax fiber and preserve its aesthetic and operational properties.

Example 1. Physico-chemical characteristics of copper nanoparticles and copper oxide nanoparticles.

To establish the antimicrobial activity of copper nanoparticles and copper oxide nanoparticles under conditions of controlled synthesis of nanoparticles by high-temperature condensation [6] under the influence of factors: air, water vapor and oxygen, copper nanoparticles and copper oxide nanoparticles were obtained. Only eight samples that were certified according to indicators: particle size, phase composition, oxide film thickness (Table 1).

Transmission electron microscopy of copper nanoparticles showed that the copper nanoparticles of the samples studied are single-crystal structures coated with a translucent film of copper oxide, as shown in (Fig. 1).

However, copper nanoparticles differ in shape. Copper nanoparticles modified with oxygen (samples No. 2, No. 4) and air (samples No. 5, No. 6) have a spherical shape, with copper nanoparticles modified with water vapor (sample No. 1), clear faces are visible on the surface (Fig. 2 ) The average size of copper nanoparticles ranges from 33.8 nm (sample No. 6) to 103 nm (sample No. 2). Copper nanoparticles differ in phase composition. So, the content of crystalline copper in the core of nanoparticles ranges from 67% (sample No. 6) to 96% (sample No. 2), copper oxide CuO - from 4% (sample No. 2) to 33% (sample No. 6). The size of the copper oxide nanoparticles ranges from 77 nm (sample No. 3) to 124 nm (sample No. 8). Moreover, the content of crystalline copper in them is small and ranges from 0.50% (sample No. 7) to 23% (sample No. 8), the content of oxides: Cu (II) varies from 27.1 (sample No. 3) to 90% (sample No. 7) , Cu (I) - from 9.05 (sample No. 7) to 69.5% (sample No. 3). The thickness of the oxide film on the surface of the modified copper nanoparticles varies between 6-10 nm depending on the type of sample (Table 1).

Consequently, the synthesized samples of copper nanoparticles and copper oxide nanoparticles differ in size, shape and structure.

Example 2. Evaluation of the antimicrobial activity of copper nanoparticles and copper oxide nanoparticles by the diffusion-disk method.

Medium suitable for growing test microbes: Staphylococcus albus and Escherichia coli AB1157, is poured into sterile Petri dishes of the same diameter with a flat bottom. The composition of the growth medium of microorganisms (in g): per 1 liter of distilled water: peptone - 10, yeast extract - 5, sodium chloride - 5, agar-agar - 20. After solidification of the agar-agar, a test culture of microorganisms is sown in Petri dishes. Sowing is carried out evenly and equally in all cups from a previously prepared suspension. Sowing density should be sufficient for continuous lawn growth. The cups are ground with a sterile spatula. The studied copper nanoparticles and copper oxide nanoparticles with different physicochemical characteristics were placed in an amount of 5 mg on paper glycerin disks. Disks were laid on the surface of the agar medium in pre-prepared Petri dishes, seeded with test cultures. Ready Petri dishes for a day put in a thermostat at a temperature of 37 ° C. After a day, the cups are removed from the thermostat and photographed. If the test sample affects the growth of the test microbe, a growth inhibition zone is formed. By the area of the growth inhibition zone (mm ² ), one can judge the intensity of the influence of the test sample on test microbes [24]. The reference standard was streptomycin at a dose of 100 μg / disk.

The results are shown in table 2. Our studies showed that all the studied samples of copper nanoparticles and copper oxide nanoparticles have antibacterial activity against both gram-positive and gram-negative microorganism strains. However, there are differences in the effectiveness of nanoparticles. It is seen that the areas of bacterial growth inhibition zones are more pronounced under the action of copper nanoparticles in relation to St.albus cells in comparison with E. coli AB1157 cells. Moreover, the nanoparticles of copper samples have different effects on bacteria: the greatest antimicrobial effect on St.albus is exerted by copper nanoparticles of samples No. 5 and No. 6, and on E. coli AB1157 - copper nanoparticles of sample No. 4. The least pronounced antibacterial effect with respect to St.albus is possessed by copper nanoparticles of samples No. 1 and No. 2, and with respect to E. coli AB1157 - sample No. 1.

Copper oxide nanoparticles of samples No. 3 and No. 7 have the same antimicrobial effect against E. coli AB1157. Copper oxide nanoparticles of sample No. 8 are more active (69%) against St.albus compared with the effect on E. coli AB1157 cells.

A classic antibiotic streptomycin at a concentration of 100 μg / disk was used as a reference standard. The antimicrobial activity of the antibiotic at this concentration is comparable to the antimicrobial effect of copper nanoparticles and copper oxide nanoparticles.

Therefore, copper nanoparticles and copper oxide nanoparticles (8 samples), regardless of their size and structure, have antimicrobial activity, slightly differing from each other in efficiency.

Example 3. Evaluation of the antimicrobial activity of copper oxide nanoparticles by the disk diffusion method depending on the concentration of metal in the disk.

The investigated nanoparticles of copper oxide in the amount of 2, 5 and 10 mg were placed on paper glycerin disks. Disks were laid on the surface of the agar medium in pre-prepared Petri dishes, seeded with test cultures of E. coli AB1157 and St.albus. Ready Petri dishes for a day put in a thermostat at a temperature of 37 ° C. After a day, the cups are removed from the thermostat and photographed. If the test sample affects the growth of the test microbe, a zone of growth inhibition of bacteria is formed around the disk. By the area of the growth inhibition zone (mm ² ), one can judge the intensity of the influence of the test sample on test microbes [24]. The reference standard was streptomycin at doses of 10, 20, 50 and 100 μg / disk. The experimental results were processed statistically (Statistica 6). To evaluate the effectiveness of the studied samples, the following formulas were used:

1) Deviation d _i - random deviation of the i-th version from the average:

2) Standard deviation (or standard deviation) sd - dispersion characteristic variant relative to the mean:

,

,

where f = n-1 is the number of degrees of freedom.

3) Standard deviation of the mean (standard error of the arithmetic mean) se:

4) Confidence interval (confidence interval of the average) - the interval in which with a given confidence probability P is the real value of the determined value (general average):

,

,

- полуширина доверительного интервала.Where

- the half-width of the confidence interval.

5) Confidence probability P - the probability of finding the actual value of the determined quantity a within the confidence interval.

In the analysis, the confidence level was taken equal to 95%, P = 0.95.

:6) The half-width of the confidence interval

:

,

,

where t _{p, f} is the coefficient of normalized deviations (student coefficient, student function, student criterion), which depends on the confidence probability P and the number of degrees of freedom f = n-1, i.e. of the number n of carried out determinations.

The numerical values of t _{p, f} were taken from the directory.

:7) Relative (percentage) error of the average result

:

The research results are presented in table 3, 4, figure 3 and figure 4. Our studies showed that around the discs with copper oxide nanoparticles of sample No. 7 at concentrations of 2, 5, and 10 mg, growth retardation zones of gram-positive (St.albus) and gram-negative bacteria (E. coli AB1157) are formed. So, around the disks with copper oxide nanoparticles in concentrations of 2, 5, 10 mg, zones of growth inhibition are formed with an area of 733.3; 912.8; 1153.6 mm ² St.albus cell culture, respectively, and 604.2; 689.8; 829.2 mm ² cell culture of E. coli AB1157, respectively (table 3). An antibiotic streptomycin was chosen as a reference standard. Streptomycin depending on the concentration also has an inhibitory effect on the surface growth of test cultures. The growth retardation zones of St.albus are formed around the disks with streptomycin at concentrations of 10, 20, 50, 100 μg and around the disks with streptomycin at a concentration of 50, 100 μg, E.coli AB1157 growth retardation zones are formed (Table 4). As a control, a 50% glycerol solution was used to impregnate the discs. It was found that around the disks with glycerol, no growth retardation zone was formed either of a representative of gram-positive (St.albus) or gram-negative (E. coli AB1157) bacteria (Figure 3). Analysis of the changes in the zones of growth inhibition of bacteria by copper oxide nanoparticles depending on the concentration showed that there is a direct relationship between the areas of the zones of growth inhibition of cells and the content of copper oxide nanoparticles in the disk, as shown in Fig. 4.

Example 4. Evaluation of the effect of various concentrations of copper nanoparticles on the growth of cells of the test culture of E. coli K12 depending on the phase of growth of bacteria in a nutrient medium (M-9).

The effect of copper nanoparticles on the growth phases of the test culture in a liquid nutrient medium was judged by the change in cell density with respect to the control nutrient medium without adding copper nanoparticles by light scattering at a wavelength of λ = 450 nm (M-9 medium) on a Sp200 spectrophotometer - 20 company "Hitachi" (Japan) [24].

The research results are shown in table 5. It was found that when adding to the medium copper nanoparticles of sample No. 2 in the concentration range from 0.1 to 10 μg / ml during the first 3 hours (incubation growth phase), differences in the biomass of bacterial cells of E. coli K12 are not occurs compared to control. The logarithmic growth phase follows, which is characterized by an active increase in bacterial biomass in the control and the absence of significant changes in the experimental samples, regardless of the concentration of copper nanoparticles. After 24 hours (stationary growth phase), differences in the effectiveness of different concentrations of copper nanoparticles on the growth of the E. coli K12 microorganism are visible. In samples containing copper nanoparticles at a concentration of 0.1 μg / ml and 0.2 μg / ml, stimulation of the growth of the test microbe is observed, leading to an increase in bacterial biomass by 6.6 ± 3% and 29.6 ± 5%, respectively, compared with the control. In samples containing copper nanoparticles in higher concentrations in the range from 1 to 10 μg / ml, the growth of microorganisms does not occur.

Example 5. Evaluation of the effect of various concentrations of copper nanoparticles and copper oxide nanoparticles on the growth of test microbes in different nutrient media (M9 and MPB).

The effect of copper nanoparticles on the growth phases of test cultures in a nutrient medium (as compared with the control) was judged by the change in biomass, which was recorded by the light scattering at a wavelength of λ = 450 nm (M-9 medium) and 630 nm (MPB) on a spectrophotometer Sp200 - 20 firms "Hitachi" (Japan) [24]. Evaluation of the effect of copper nanoparticles, copper oxide nanoparticles, and the reference standard streptomycin was evaluated by the metrological parameters described in Example 3 (Statistica 6).

An assessment of the effect of copper nanoparticles (samples No. 4, No. 5, No. 6) depending on the concentration on the growth of the test culture of E. coli K12 in a nutrient medium (M9) is presented in Table 6, copper oxide nanoparticles (sample No. 7) depending concentration on the test culture of E. coli AB1157 in a nutrient medium (BCH) in table 7, on the test culture of E. coli K12 in a nutrient medium (BCH) in table 9, on the test culture of St.albus in a nutrient medium (BCH) in table 11, streptomycin (reference standard) for test cultures of E. coli AB1157, E. coli K12 and St.albus in a nutrient medium (BCH) depending on the concentration in tables 8, 10 and 12.

From the data of table 6 it is seen that copper nanoparticles of samples No. 4, No. 5 and No. 6 inhibit the growth of E. coli K12. At the lowest concentration (0.2 μg / ml), copper nanoparticles on average inhibit cell growth by 65-80%, and at a concentration of 0.5 μg / ml by 90-95%. A further increase in the concentration of copper nanoparticles in the medium does not lead to an increase in the effect. There are differences in the effect of copper nanoparticles of different samples. At a concentration of 0.2 μg / ml, nanoparticles of sample No. 4 inhibit cell growth by 65%, No. 5 by 45%, and No. 6 by 79%. Such differences in the action of copper nanoparticles of different samples are smoothed out when large concentrations of metal are added to the medium. This may be due to the fact that at concentrations exceeding 0.5 μg / ml, there are more than 250 particles per cell, which is not compatible with the normal functioning of its vital systems. Therefore, the most inhibitory effect on the growth of the test microbe is possessed by copper nanoparticles of sample No. 6, suppressing the growth of E. coli K12 2.5 times more efficiently than copper nanoparticles of sample No. 5.

When copper oxide nanoparticles of sample No. 7 were added to the growth medium of the E. coli AB 1157 culture at concentrations from 0.5 to 20 μg / ml in the medium of MPB, inhibition of microbial cell growth was observed, which had a direct dependence on concentration (Table 7). At a concentration of 20 μg / ml, a maximum growth inhibition of 35% occurs. The reference standard - streptomycin in concentrations from 0.01 to 20 μg / ml also inhibits the growth of the culture. The maximum inhibition is observed at a concentration of 20 μg / ml and is 45%, i.e. the inhibitory effect of the antibiotic is commensurate with the action of copper nanoparticles (table 8, Fig. 5).

When copper oxide nanoparticles (sample No. 7) are added to the growth medium of the E. coli K-12 culture at concentrations from 0.05 to 20 μg / ml, microbial cell growth is also inhibited. There is a direct correlation between the degree of inhibition and the concentration of copper oxide nanoparticles (Table 9). Growth inhibition by 62.9% is already observed at a nanoparticle concentration of 0.5 μg / ml, while streptomycin at the same concentration inhibits growth by only 28.4% (Table 10). Therefore, the inhibitory effect of copper oxide nanoparticles is comparable with the effect of streptomycin on the growth of this culture, reaching 98% inhibition at a concentration of 20 μg / ml.

When copper oxide nanoparticles (sample No. 7) were added to the growth medium of the St.albus culture at concentrations from 1 to 20 μg / ml, inhibition of microbial cell growth was observed (Table 11). At a concentration of 5 μg / ml, growth inhibition is 59%. Streptomycin in concentrations from 0.5 to 20 μg / ml sharply inhibits the growth of culture up to 100% (table 12).

Therefore, the studied samples of copper nanoparticles of samples No. 4, No. 5, No. 6 and copper oxide nanoparticles of sample No. 7 have an antimicrobial effect on different strains of gram-negative bacteria E.coli K-12 and E. coli AB1157 and the test culture of gram-positive bacteria St.albus in a wide range of concentrations, starting from 0.05 μg / ml. Moreover, the effect of copper nanoparticles on E. coli AB1157 is comparable with the action of streptomycin in the same concentration range.

Example 6. Evaluation of the effect of copper oxide nanoparticles as an antimicrobial agent on the state of non-woven linen.

The above examples (2-5) prove that copper nanoparticles and copper oxide nanoparticles have antimicrobial activity against gram-positive and gram-negative bacteria. Given the high need for effective and non-toxic antimicrobial agents, these properties of copper nanoparticles and copper oxide nanoparticles can be used to create bacteria-resistant materials for technical and medical purposes. In this regard, the non-woven linen cloth was treated with copper oxide nanoparticles.

To assess the effect of copper oxide nanoparticles as an antimicrobial agent on the fabric property, copper oxide nanoparticles of sample No. 7 and a nonwoven material with a surface density of 70 g / m ² based on bleached flax fibers were used. Liquid treatment of flax fibers was carried out after ultrasonic dispersion of aqueous systems containing copper nanoparticles at a concentration of 0.1-0.5 mg / ml. The antibacterial activity of copper nanoparticles was evaluated by diffusion into agar on a solid medium. Pieces of non-woven linen flax 10 × 10 mm in size treated with copper oxide nanoparticles were placed on the prepared bacterial lawn, seeded with test cultures of E. coli AB1157 and St.albus. A non-woven linen cloth without copper nanoparticles was used as a control. Ready Petri dishes for a day were introduced into the thermostat at a temperature of 37 ° C. After a day, the cups were removed from the thermostat and photographed (Fig.6).

In FIG. 7 shows electron microscopic images of copper oxide nanoparticles sorbed on microfibres of a nonwoven fabric. Particles are located in the microroughnesses of the surface of the fibers both in the form of small aggregates with sizes up to several tens of particles and individual particles. Particles are observed both on the fibers of the outer layers and on the fibers of the inner layers of the tissue.

On Fig presents the results of the experiment. It can be seen that under the non-woven linen cloth not treated with copper oxide nanoparticles, the growth of the studied test cultures is observed. At the same time, pronounced stimulation of the growth of microorganisms is observed along the edge of tissue samples. A study of the growth of test cultures under a non-woven linen cloth with copper oxide nanoparticles showed the presence of separate colonies of microorganisms under the tissue and the absence of stimulation of the growth of microorganisms along the edge of the flax.

In addition, an analysis was carried out of the transformations occurring in the linen matrix with the creation of optimal conditions for the development of the natural microflora complex (growth of fungi, bacteria, actinomycetes) without copper oxide nanoparticles and with copper oxide nanoparticles. The external manifestation of the vital activity of microbial cultures, their adaptation to existence on flax fiber material is the “colonization” of its surface, the appearance of cobweb-like films, pigmentation, and violation of the integrity of the structure. We found that after a long (10-14 days) aging of the nonwoven material in the TC-80 thermostat at 29 ± 0.2 ° C and 100% humidity, the nonwoven fabric treated with copper oxide nanoparticles showed no visible damage to the flax nanocomposites. Under the same conditions, a non-woven fabric not treated with copper oxide nanoparticles, aesthetic and operational properties deteriorated to a large extent. It was shown that when copper oxide nanoparticles were introduced into the flax matrix at a concentration of 0.3-0.5 mg / g fiber, the coefficient of microbial destruction resistance of the nonwoven material was 60-90%, and the nonwoven material without copper oxide nanoparticles was only 2-5%.

Therefore, the non-woven linen web treated with nanoparticles of copper oxide has an antimicrobial effect in the contact interaction between the linen and bacterial cells. In addition, treating the fabric with copper oxide nanoparticles significantly enhances the aesthetic and operational characteristics of flax fiber.

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table 2 The area of growth retardation zones (mm ² ) of test cultures of St.albus and E. coli AB1157 under the action of copper nanoparticles and copper oxide nanoparticles at a concentration of 5 mg / disk, streptomycin 100 μg / disk Copper LF Sample St.albus growth retardation zone, mm ² The growth retardation zone of E. coli AB1157, mm ² Sample No. 1 1004 + 42 805 ± 57 Sample No. 2 1014 ± 72 857 ± 15 Sample No. 3 1001 ± 88 868 ± 33 Sample No. 4 1054 ± 79 940 ± 44 Sample No. 5 1210 ± 32 839 ± 10 Sample No. 6 1207 ± 40 835 ± 60 Sample No. 7 1103 ± 59 868 ± 25 Sample No. 8 1168 ± 17 689 ± 19 Reference standard - streptomycin 794 ± 16 361 ± 14

Table 3 Metrological parameters of the results of measuring the areas of growth inhibition zones when applying copper oxide nanoparticles to a paper disk (sample No. 7) Metrological parameters Concentration, mg / disk St.albus E.coli AB1157 2 5 10 2 5 10 n 3 3 3 3 3 3 Square areas of growth delay πd ^2/4 716.0 918.2 1151.5 576.5 651.1 824.1 865.3 896.8 1175.7 585.1 735.0 829.2 798.3 923.5 1133.5 651.1 683.1 834.3

733.3 912.8 1153.6 604.2 689.8 829.2 sd 74.8 14.1 21.1 48.0 42,4 5.1

185.7 35.0 52,4 119.2 105.3 12.7

% 25.3 3.8 4,5 19.7 15.3 1,5

Table 5 The influence of copper nanoparticles of sample No. 2 in different concentrations on the growth (A ₄₅₀ , O / K,%) of the E. coli K12 test culture in a nutrient medium (M-9) by the growth phases Time hour The change in the biomass of bacteria during growth (A ₄₅₀ , O / K,%) The concentration of copper nanoparticles, μg / ml 10 four 2 one 0.5 0.2 0.1 the control one 10.9 11.1 8.9 8.9 9.5 10,2 10.7 11,4 1,5 11.3 11,2 10.8 12.1 13,2 14.0 14,4 15.9 2 11,2 10.6 10,2 12.7 14.2 16.8 18.6 19.5 2.5 12.0 10.9 11.8 17,2 16.5 20.5 21.0 26.0 3 13.7 12.5 12.0 16.5 16.6 20.1 21,2 32,7 3,5 13.5 12,4 12.1 16.6 17.3 21,2 20.8 41,4 four 13.5 12.6 12.9 17.3 18.1 22.1 21.1 50.9 4,5 13.6 12,4 12.0 18.1 19.1 23.1 22.2 63.8 5 14.0 12.0 11.9 17.7 21.0 26.1 23.6 72.3 24 13.9 12.3 11.9 14.6 17.0 129.6 106.6 one hundred

Table 6 The effect of copper nanoparticles of samples No. 4, No. 5 and No. 6 depending on the concentration on the growth of the test culture of E. coli K12 (A ₄₅₀ , O / K,%) in a nutrient medium (M-9) after 48 hours of cultivation Samples of copper nanoparticles No. The concentration of copper NPs in the nutrient medium (M-9), μg / ml 0 0.2 0.5 1.0 2.0 four one hundred 35.0 ± 1.47 9.0 ± 0.15 9.4 ± 0.15 8.4 ± 1.19 5 one hundred 55.1 ± 0.96 9.2 ± 0.15 8.5 ± 0.45 8.2 ± 0.10 6 one hundred 21.1 ± 0.47 9.0 ± 0.15 8.6 ± 0.30 11.1 ± 0.45

Table 7 Metrological parameters of the results (α,%) of the study of the effect of copper oxide nanoparticles (sample No. 7) in various concentrations on the growth of the E. coli AB1157 test culture in a nutrient medium (MPB), with n = 3 Metrological parameters Concentration, mcg / ml 0 0.5 5 10 twenty sd 0 0.88 2.43 1,64 1.36

one hundred 97.49 85.71 76.9 65.57

0 2.19 6.05 1,64 3.39

% 0 2.25 7.06 5.3 5.17

Table 11 Metrological parameters of the results of the study of the influence of copper oxide nanoparticles (sample No. 7) in various concentrations on the growth of the St.albus test culture in a nutrient medium (MPB), at n = 3 Metrological parameters Concentration, mcg / ml 0 one 5 10 twenty sd 0 0.75 5.37 7.95 2,36

one hundred 43.36 41.00 62.41 58.92

0 1.87 13.37 19.8 5.88

% 0 4.31 32.6 31.7 9.97

Claims (2)

1. Antimicrobial agent with a high antibacterial effect against test cultures of gram-positive and gram-negative microorganisms, characterized in that copper nanoparticles having the following characteristics are used as the active substance: particle size 33.8 ÷ 103 nm; oxide film thickness 6 ÷ 10 nm; phase composition: crystalline copper 67–96%, copper oxide CuO 4–33%. 2. An antimicrobial agent with a high antibacterial effect against test cultures of gram-positive and gram-negative microorganisms, characterized in that copper nanoparticles having the following physicochemical characteristics are used as the active substance: particle size 77 ÷ 124 nm; phase composition: crystalline copper 3.3–23%, CuO 27.1 ÷ 90% and Cu ₂ O 9.05 ÷ 69.5% or particle size 119.0 ± 1.0 nm, phase composition: crystalline copper 0, 50 ± 0.02%, Cu ₂ O 9.05%, CuO the rest.

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