RU2460797C2 - Method for preparing composition containing colloidal nanosilver or nanogold (versions) - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method for preparing a composition containing colloidal nanosilver or nanogold involves incubation of probiotic bacteria specified in Lactobacillus fermentum species with an aqueous solution containing at least 4 mM of silver nitrate or auric chloride. The composition containing colloidal nanosilver is prepared by contact of said bacteria at 5-45°C and the aqueous solution containing mixed silver nitrate, ammonium and/or ammonia salt, as well as alkali metal hydroxide. The composition containing colloidal nanogold is prepared by contact of said bacteria at 5-45°C and the aqueous solution containing mixed auric chloride and alkali metal hydroxide.

EFFECT: prepared compositions are used as an antimicrobial agent or an algicidal agent.

17 cl, 4 dwg, 5 tbl, 16 ex

Description

Technical field

The present invention relates to the production of colloidal metal compounds on a bacterial membrane. The present invention also relates to methods for producing silver or gold nanoparticles using a biological process. In particular, the invention relates to the use of probiotic bacteria, such as, but not limited to, Lactobacillus, under specific conditions in the preparation of metal nanoparticles, in particular silver or gold nanoparticles, in order to improve their antimicrobial effect. The present invention relates to disinfectant products, including a carrier, impregnated with a composition containing colloidal nanosilver or nanogold obtained by this method.

State of the art

Effective disinfection methods are necessary for the treatment of large quantities of contaminated materials, such as water, especially domestic and industrial circulating water, as well as water streams (for example, used for food processing) containing microorganisms that cannot be drained or reused untreated for hygienic, technological or environmental reasons. Effective disinfection methods are also necessary for treating surfaces such as surfaces of rooms, equipment, containers, air conditioning systems, etc. Environmental compatible disinfection methods are most often based on the use of active oxygen compounds such as hydrogen peroxide or monomeric quaternary ammonium compounds.

Hydrogen peroxide is a moderately active disinfectant with bactericidal properties. It is known that hydrogen peroxide, having a concentration of 25 mg / l, inhibits the growth of some bacteria, however, an effective reduction in the number of microbes even with a significantly higher concentration of hydrogen peroxide takes many hours or requires additional ultraviolet radiation. However, the use of the latter requires both expensive equipment and significant electricity costs. Therefore, when disinfecting large quantities of contaminated materials, such as water, for example, for treating water at wastewater treatment plants and their products, such measures are practically inadequate and / or uneconomical. Therefore, various attempts have been made in the art to overcome these disadvantages.

It is well known in the art that silver ions and silver-based compounds are highly toxic to microorganisms and therefore have a strong bactericidal effect on many common bacterial species, including Escherichia coli. It was also proved that hybrids of silver nanoparticles with amphiphilic hyperbranched macromolecules provide effective antimicrobial surface coatings. It was found that stable aqueous dispersions of silver nanoparticles in the form of non-toxic elementary silver hydrosols have a strong bactericidal effect on E. coli, while a concentration of 50 μg / cm ³ causes 100% inhibition of bacterial growth. It was found that silver nanoparticles accumulate in bacterial membranes, somehow interacting with certain building elements of the bacterial membrane, thereby causing structural changes, destruction and, finally, cell death. It is noted that the surface of bacteria is fully charged negatively at the level of biological pH values due to the dissociation of an excessive amount of carboxyl and other groups in the membrane. It has been suggested that silver nanoparticles embedded in the carbon matrix of a membrane produce a surface charge due to their movement and friction inside the matrix, so electrostatic forces can cause the nanoparticles to interact with bacteria. Moreover, silver tends to have a closer affinity for interaction with phosphorus and silver compounds contained in the membrane, as well as in DNA. The third possible type of interaction is the release of silver ions, which can further contribute to the bactericidal action of silver nanoparticles.

It was found that some types of microorganisms, for example Lactobacillus sp. and the fungus Fusarium oxysporum, biosorb Ag (I) on the surface of their cells and neutralize this ion by reduction to Ag (0) either by the action of reductase, or by shuttle electron quinones, or both.

A non-cytotoxic antimicrobial composition is already known in the art, including biologically stabilized silver nanoparticles having a size in the range from 1 to 100 nm, and a carrier in which the concentration of said biologically stabilized silver nanoparticles is from 1 to 6 ppm.

Also known is a method of producing a colloidal complex of silver-biomolecules, including:

- obtaining a mixture of a biomolecule, a silver salt and a source of halogen ions in one solution; and

- irradiation of the mixture with light having a wavelength in the visible range, while the silver salt and the source of halogen ions are soluble in water; the amounts of the biomolecule, the silver salt and the source of the halogen ions are such that the irradiation stage leads to the formation of colloidal complexes of silver-biomolecules.

A method for producing colloidal metal nanoparticles has also been described, comprising treating wet fungi or a fungal extract with a solution of metal ions at a temperature in the range from 15 to 40 ° C. for a period of time from 2 to 120 hours and separating the biomass to obtain colloidal metal nanoparticles.

Traditional methods for producing silver nanoparticles have several disadvantages, such as high production cost, the formation of a significant amount of by-products, or the presence of an upper concentration limit for the resulting nanoparticles. For example, the latter production method is time consuming and is based on the use of fungi that may be pathogenic. Therefore, in the art there is a need to develop a reliable and inexpensive method for producing silver nanoparticles, reducing or preventing the formation of by-products.

We also studied the biosorption process of Ag (I) using Lactobacillus, its pH dependence in the pH range from 2 to 6, and the temperature dependence in the range from 10 to 60 ° C, as well as the mechanism of reduction of Ag ⁺ to Ag ⁰ using Lactobacillus.

A method for producing silver nanoparticles by bioreduction using Aeromonas sp. Is also known in the art. mixed with silver ions, ammonia and sodium hydroxide at 60 ° C for several hours.

The above methods have disadvantages, such as the need to use an elevated temperature, acid pH or a long incubation period, or insufficient bactericidal activity of the resulting silver nanoparticles.

Therefore, in the art there is a need to obtain silver or gold nanoparticles in a manner free of the above disadvantages.

There is also a need in the art to develop a simple, environmentally friendly and reproducible process for producing silver or gold nanoparticles with good antimicrobial properties.

There is also a need in the art to develop an appropriate method for producing silver or gold nanoparticles, applicable for some medical purposes.

It is also known in the art that colloidal forms of metals other than gold or silver and compounds of such metals have valuable properties and uses. For example, water-soluble colloidal bismuth subcitrate, especially at a pH of about 3 to 8, has been used for decades along with antibiotics to treat stomach and duodenal ulcers, as well as Helicobacter pylori infection. Colloidal forms of mercury, inorganic compounds of mercury and ointments with metallic mercury were used locally for various therapeutic purposes, including the treatment of infectious eczema or impetigo (salt of mercury), the treatment of syphilis (calomel), the treatment of psoriasis (mercury oxide or mercuric chloride). Colloidal forms of palladium and platinum were used as catalysts for various chemical reactions, including organic reduction, hydrogenolysis, etc. Colloidal platinum nanoparticles are also known as anti-cancer agents. Colloidal copper, optionally chelated with salicylic acid, is a powerful anti-inflammatory agent, and it is known that sublingual forms of colloidal copper or colloidal zinc are active against colds and flu. Colloidal zinc is also able to have a particularly effective antiviral effect. In all these various fields, there is a continuing need for alternative physical forms of colloidal metals or colloidal metal compounds in order to increase their effectiveness in their respective fields of application.

SUMMARY OF THE INVENTION

In the broadest sense, the present invention relates to the use of bacteria for producing colloidal metal compounds on a bacterial membrane and the subsequent use of coated bacteria as an antimicrobial agent. In particular, the invention relates to:

- the use of bacteria to obtain colloidal metal compounds by contacting these bacteria with a mixture of metal salts and other salts at a controlled pH, which ensures that bacteria produce colloidal metal compounds on their membrane; and

- the use of these bacteria coated with metal compounds on the membrane as an antimicrobial agent.

In one embodiment, the invention relates to the production of metal nanoparticles by probiotic and other bacteria that can be used as an antimicrobial agent in drinking water, in surface coatings and other materials.

More specifically, some bacteria are able to reduce salts of Ag (I) to colloidal Ag (0), which is deposited in the form of nano-Ag particles on the surface of the cell. The biomass coated with colloidal silver or another metal nano-precipitate can be easily separated from the aqueous phase by filtration or centrifugation, can be washed and rinsed and subjected to further processing to obtain a colloidal product with strong antimicrobial properties both in the form of (diluted) suspension and during processing in coatings.

Interestingly, the group of probiotic bacteria, i.e. bacteria obtained industrially, due to the healing effect on human health, if they are present in the human digestive tract, demonstrates this ability to form nano-deposits on the surface of their cells. Such bacteria include, but are not limited to, probiotic strains of Lactobacillus fermentum.

Adding a specific combination of salts (AgNO ₃ , NH ₄ Cl, NaOH and others) to a concentrated cell culture of bacteria and controlling the pH, a colloidal silver product with strong antimicrobial properties is obtained. Salts of other metals in combination with certain bacterial strains also cause the formation of nano-deposits with similar properties, which is also part of this invention.

By adjusting the ratio of “mass of silver” to “mass of biological cells” (Ag: CDW, where CDW = mass of cells in the dry state), one can change the reactivity and properties of the final product of colloidal silver regarding the size of colloidal particles, the distribution of colloidal particles and their other properties .

Colloidal silver compounds obtained on the surface of bacteria have a very wide range of uses, including, but not limited to: disinfection of water, use as a disinfecting agent in cleaning products, as a cleaning agent, composition in antimicrobial coatings, for medical purposes, medicinal preparations for people, use in textiles, in ointments and lubricants, as a catalyst, etc.

The production process is direct, cost-effective, has a high yield and can be easily updated, particle size and distribution can be controlled, and the antimicrobial reactivity of the resulting nanosilver surpasses other colloidal silver products at very low (ppm) concentrations. Moreover, such a product can be processed in various forms: dry, suspended or in the form of “wet” pellets, it can be part of various compositions. The final product does not contain chemical residues, since it can be washed with clean water without loss of activity.

Probiotic bacteria can be used for a wide variety of purposes in the healthcare and food industries. Ag coated bacterial product is especially suitable for the following applications:

- a component of disinfectant cleaners (hospitals, laboratories, animal breeding grounds, etc.);

- use in ceramic filters or other filters for the disinfection of water, both drinking water and water in swimming pools, water for raising animals, water for growing aquatic cultures, etc .;

- use in disinfecting coatings: polymers, textile fibers, metals;

- Suitable for use as part of skin disinfecting ointments, lubricants, etc .;

- use of drinking water for disinfection: developing countries, satchels, airplanes and much more (simple addition in the form of drops), and

- fight against pathogenic microorganisms: Legionella, Cryprosporidium, Hepatitis, Herpes, Pseudomonas, Staphylococcus, various types of bacteria, fungi and viruses.

Another objective of the present invention is to obtain high quality gold and silver nanoparticles. A first aspect of the present invention relates to the development of an improved biological method for preparing a composition comprising colloidal gold and silver nanoparticles, said method comprising using probiotic bacteria, in particular Lactobacillus species such as Lactobacillus fermentum, and contacting said biomass with an aqueous solution of silver salt (I ) or gold salt (III). The present invention is based on the unexpected discovery that some specific parameters of the method for producing gold or silver nanoparticles by bioremediation have a strong influence on the efficiency of their preparation and the characteristics of the resulting nanoparticles. In particular, the specific methods of the present invention have a strong effect on the antimicrobial activity of the resulting composition, including silver nanoparticles.

Another aspect of the present invention is that the composition of gold and silver nanoparticles obtained by bioremediation under such specific conditions can be further processed, for example, separated from the biomass while maintaining or even enhancing its activity or other relevant properties, such as storage stability . Alternatively, subsequent chemical treatment, for example with oxidizing agents such as peroxide or per-salt, of a gold and silver nanoparticle composition obtained by bioreduction under such specific conditions, can even enhance the properties of the resulting nanoparticle composition.

An advantage of the method according to the present invention also lies in the fact that the size and distribution of the resulting gold and silver nanoparticles can be adjusted in a reproducible manner.

An advantage of the present invention also lies in the fact that this method provides a result with a high degree of reliability in a significantly shorter time at low cost and without harm to the environment by reducing the need for potentially toxic and / or expensive chemicals. In the composition obtained by the method according to the invention, no harmful residues of chemical reagents remain, largely due to the fact that the biomass used is obtained from harmless, for example probiotic, microorganisms. Therefore, an advantage of the present invention is that the method provides a composition which, when used together with eukaryotic organisms, does not significantly affect such organisms. According to a specific embodiment, the invention provides a composition with high antimicrobial activity that also acts against marine microorganisms without significantly affecting eukaryotic organisms. An additional advantage of the present invention is that the invention allows to obtain a composition having a high concentration of nanosilver or nano-gold as such, and also includes nano-silver or nano-gold, essentially consisting of silver or gold in their metallic state, respectively, for example, includes more than about 95% Ag ⁰ of the total silver content or more than about 95% Au ⁰ of the total gold content, respectively.

A further advantage of the present invention is that the resulting product or composition can be easily and safely processed while maintaining or even enhancing their activity. The composition can be dried or in the form of a suspension or wet pellets, it can also take various forms, such as aerosol or impregnation of a carrier, without impairing antimicrobial activity due to the stability of nanosilver particles.

According to yet another embodiment, the present invention relates to the use of a colloidal silver composition obtained in accordance with the above method as an algicidal or herbicidal agent.

Definitions

As used herein to describe the present invention, the terms “nanosilver” or “nano-Ag” refer to metallic silver nanoparticles (Ag ⁰ ). According to the present invention, said nanoparticles may or may not be precipitated onto biomass. The size of such nanoparticles can vary from about 0.1 nm to 100 nm, for example in the range from about 0.5 nm to 5 nm. The particle size distribution of such nanoparticles can also vary around their average size.

As used herein to describe the present invention, the terms “nano-gold” or “nano-Au” refer to metal gold nanoparticles (Au ⁰ ). According to the present invention, said nanoparticles may or may not be precipitated onto biomass. The size of such nanoparticles can vary from about 0.1 nm to 100 nm, for example in the range from about 0.5 nm to 5 nm. The particle size distribution of such nanoparticles can also vary around their average size.

As used herein to describe the present invention, the term “biomass” refers to organic material consisting of or derived from bacterial species used to produce “nanosilver” or “nanogold”.

As used herein to describe the present invention, the term “probiotic bacteria” refers to bacteria that, when administered in appropriate amounts to a host, such as a mammal, marine species (eg, fish), or humans, have a beneficial effect on the health of said host.

As used herein to describe the present invention, the term “silver (I)” or “Ag (I)” refers to monovalent positively charged silver or Ag ⁺ ions.

As used herein to describe the present invention, the terms “gold (I)” and “gold (III)” refer to monovalent and trivalent positively charged gold ions, respectively.

Brief Description of the Drawings

FIG. 1 illustrates the antimicrobial effect of treatment with nanosilver particles according to one embodiment of the invention at various concentrations of the total number of cells and the survival of E. coli.

FIG. 2 illustrates an X-ray diffraction spectrum of nanosilver particles according to one embodiment of the invention.

FIG. 3 illustrates the effect of the ratio of silver to the mass of cells in the dry state during the production of nanosilver particles according to one embodiment of the invention on the antimicrobial effect of these particles against Salmonella typhimurium.

FIG. 4 illustrates an X-ray diffraction spectrum of nanosilver particles according to another embodiment of the invention.

DETAILED DESCRIPTION OF THE INVENTION

A first aspect of the present invention relates to the development of a simple method for producing a composition containing colloidal nanosilver or nanogold, comprising the step of incubating probiotic bacteria with an aqueous solution containing at least 4 mM silver or gold salt.

Suitable probiotic bacteria according to the present invention belong to genera, such as, but not limited to, Lactobacillus, Bifidobacterium, Escherichia, Enterococcus, Saccharomyces and Bacillus. Probiotic bacteria are free to belong to one or more of the following species: Lactobacillus sakei, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus cripatus, Lactobacillus delbrueckii subspecies bulgaricus, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus rhamnosus , Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium lactis, Bifidobacterium adolescentis, Escherichia coli Nissle, Saccharomyces boulardii, Streptococcus thermophilus, Enterococcus faecium, Bacillus licheniformis, Bacillus cereus, Bacillus subtilis, Bacillus megaterium, Bacillus acidophilus, Bacillus pumilus, Bacillus polyfermenticus, Bacillus clausii, Bacillus laterosporus, Bacillus sporogenes, Bacillus coagulas and Bacillus polymyxa.

Any water-soluble silver salt can be used to carry out various variations of the process of the present invention. In the description, the term “silver salt” also includes hydrates and other solvates of such silver salts. A water-soluble silver salt can be defined here as a silver salt with a solubility in water of at least 0.1 g / l at the temperature of the method according to the invention, for example at room temperature. The silver salt may, without limitation, be an inorganic silver salt or an organic silver salt, such as, but not limited to, silver acetate, silver chloride, silver perchlorate, silver chlorate, silver bromide, silver fluoride, silver lactate, silver nitrate, silver sulfate or silver tartrate.

Any water soluble gold salt may be used to implement the various process variants of the present invention. In the description, the term “gold salt” also includes hydrates and other solvates of such gold salts. A water soluble gold salt can be defined here as a gold salt with a solubility in water of at least 0.1 g / l at the temperature of the process according to the invention, for example at room temperature. A gold salt may, without limitation, be monovalent or trivalent. The gold salt may, without limitation, be an inorganic gold salt or an organic gold salt, or a mixed gold salt, such as, but not limited to, gold (III) chloride, gold-sodium thiomalate monohydrate, gold (III) bromide, gold (III) iodide ) and gold (III) nitrate.

According to one embodiment of the present invention, the initial concentration of the silver or gold salt in the incubated aqueous solution should be at least 4 mM, for example at least 10 mM or, as a specific example, at least 50 mM.

According to another embodiment of the present invention, said aqueous solution may also include additional components capable of influencing behavior, in particular improving the properties of the resulting composition. In this regard, in an embodiment of the present invention to produce nanosilver, the described method may include the step of incubating the probiotic bacteria (described above) with an aqueous solution comprising at least 4 mM silver salt and further comprising ammonia and / or ammonium salt. Ammonium salts useful in this embodiment include, but are not limited to, ammonium chloride, ammonium nitrate, ammonium phosphate, ammonium sulfate, ammonium carbonate, ammonium formate and ammonium bromide. The amount of ammonia and / or ammonium salt used in this embodiment of the present invention should preferably be sufficient to allow the formation of a significant amount of a silver-ammonia or silver-ammonium complex, such as, but not limited to, a silver (I) complex -ammonia in the form of Ag (NH ₂ ) ⁺ and / or {Ag (NH ₃ ) ₂ } ⁺ . According to another aspect of an embodiment of the present invention, the incubated aqueous solution may also include an appropriate amount of an alkali metal hydroxide, such as, but not limited to, sodium hydroxide or potassium hydroxide. In accordance with the description below, such an appropriate amount can be determined taking into account the set suitable pH range.

According to another embodiment of the present invention, the described method includes the step of incubating the probiotic bacteria (described above) with an aqueous solution comprising at least 4 mm gold salt and further including an appropriate amount of alkali metal hydroxide, such as, but not limited to, sodium hydroxide or hydroxide potassium, in the absence of ammonia and / or ammonium salt.

Suitable alkali metal hydroxides, such as, but not limited to, sodium hydroxide or potassium hydroxide, may be added to the incubation aqueous solution at a concentration of up to about 1M. The incubation is preferably carried out at a pH of at least 8, for example in the range of from about 8 to 12, or, as a more specific embodiment, in the range of from about 8 to 11.

According to one embodiment of the present invention, the ratio of the mass of silver or mass of gold to dry cell mass (hereinafter abbreviated as CWD) of probiotic bacteria is at least about 0.01, for example at least about 0.05 or at least about 0, one. According to another embodiment of the present invention, the weight ratio of Ag: CDW or Au / CDW is not more than about 20, preferably less than about 10, for example less than 5.

According to one embodiment of the present invention, the incubation step in this method is carried out at a temperature of from about 5 ° C to 45 ° C, preferably at a temperature of from about 15 ° C to 35 ° C, for example at room temperature.

According to another embodiment of the present invention, the incubation step in this method can be carried out for a period of time of from about 1 second to 30 minutes, for example from about 5 seconds to 20 minutes. The person skilled in the art is able to determine, by minimal experiments, the most suitable time period for incubation, depending on other process parameters, such as, but not limited to, silver or gold salt concentration, incubation temperature, Ag: CDW or Au / CDW weight ratio, the presence or absence of ammonia or an ammonium salt and the like. As is customary in the art, incubation can be carried out with stirring for at least part of the incubation period.

The method according to the present invention may also include the step of further processing the resulting composition containing colloidal silver or gold nanoparticles. Such further processing may include one or more steps, such as, but not limited to, separating at least a portion of the biomass from silver or gold nanoparticles or fractionating the biomass by mechanical, enzymatic and / or physicochemical treatment, for example, by means of ultrasound. All of these methods for separating or fractionating biomass are well known to those skilled in the art. Alternatively or in addition to these methods, such a treatment may include a chemical treatment step to stabilize or even improve certain desired properties of the resulting composition, including colloidal silver or gold nanoparticles. According to a specific embodiment of such a chemical treatment, the silver or gold nanoparticle composition of the present invention can be treated after incubation and optionally removing the biomass with an oxidizing agent such as peroxide or per-salt to form a precipitate of silver or gold nanoparticles with improved stability and / or (relative to nanosilver) with higher antimicrobial activity. According to this embodiment of the present invention, suitable organic and inorganic peroxides include, but are not limited to, hydrogen peroxide, peracetic acid, and the like. Suitable per-salts applicable in this embodiment of the present invention include, but are not limited to, alkaline water-soluble salts capable of forming hydrogen peroxide upon dissociation, for example, peroxide ion is released when such salts are dissolved in water. Suitable examples of such salts include percarbonates, perborates, persilicates and perphosphates associated with a cation, such as an alkali metal. Particularly preferred is sodium percarbonate having the empirical formula 2Na ₂ CO ₃ · 3H ₂ O ₂ . In support of this embodiment of the present invention, a person skilled in the art knows that:

- such per-salts may be superior in disinfection ability to hydrogen peroxide;

- hydrogen peroxide is a weak disinfectant and penetrates poorly into bacteria;

by dissolving per-salt in water and releasing peroxide

hydrogen, the alkaline salt extracts the proton from the liberated hydrogen peroxide, forming a hydroperoxide ion, which, unlike hydrogen peroxide, is a powerful disinfectant and easily penetrates bacteria.

At any point in the process according to the invention, the solid part, including colloidal nanosilver or nanogold, can be separated from the liquid part by any method well known to a person skilled in the art. For example, it can be separated by centrifugation and subsequent decantation of the liquid fraction or by filtration.

In the method according to the present invention, the silver or gold salt can be at least partially replaced by a copper salt while carefully regulating one or more reaction operating conditions, such as, but not limited to, pH, incubation temperature, type of salt, and salt concentration. In the method according to the present invention, the types of probiotic bacteria can also be at least partially replaced by alternative organisms or bacteria, while carefully regulating one or more reaction working conditions, such as, but not limited to, pH, incubation temperature, type of salt, and salt concentration ( gold or silver). Such alternative bacteria can be selected from the group consisting of bacteria that are generally considered environmentally friendly, more specifically, bacteria that are known to have bio-recovery potential.

Although the method according to the present invention is mainly described here with respect to silver and gold, it is not limited to these metals, but, as indicated in its broadest expression, is also applicable to other metals or metal compounds, provided that one or more reaction workers conditions, such as, but not limited to, pH, incubation temperature, type of salt and salt concentration, are adapted accordingly. Such adaptation is included in the scope of routine experiments for a specialist, taking into account the general provisions set forth here. Metals of particular interest in the framework of this invention include zinc, mercury, copper, palladium, platinum and bismuth.

The second aspect of the present invention relates to the antimicrobial use of a nanosilver composition obtained by the above method, based on an unexpected discovery, namely that the effective concentration of such a nanosilver composition during antimicrobial treatment can be extremely low, depending on the target bacteria, for example, about 0.5 m .d. or even less, for example about 0.05 ppm. or even less, and also at the discovery that a significant reduction in the number of unwanted bacteria can occur within a limited period of time, for example, not more than about 5 hours. Suitable target bacteria for this aspect of the present invention include a wide range of gram-positive and gram-negative microbes, such as, but not limited to, Pseudomonas aeruginosa (e.g. strain CMCM-2-22), Pseudomonas cepacia, Enterobacter cloacae, Enterobacter agglomerans, Klebsiella pneumonia e.g. strain ATCC-10031), Eschericia coli, Streptococcua faecalis (e.g. strain ATCC-10541), Staphylococcus cohnii, Staphylococcus aureus (e.g. strain IP 52154 or ATCC-6538), Bacillus subtilis (strain ATCC-19659) (which are common hospital bacterial strains), Enterococcus facium, Enterococcus hirae, Thibacillus ferrooxidans (e.g. strain ATCC-13661), Lactobacilli, Therm ophilic bacilli, Trychophyton interdigitale (e.g., ATCC-640 strain), Clostridium sporogenes (ATCC-3584 strain), Clostridium perfringens (ATCC-13124 strain), Salmonella typhimurium, Listeria monocytogenes, etc. The nanosilver composition according to the invention is also capable of showing activity against fungi, including, for example, Candida albicans (e.g., strain APCC-2091), Mycobacterium smegmatis (e.g., strain IP 7326), Aspergillus niger (e.g., strain 218 IP), Penicillium verrucosum and t .p., and is also capable of exhibiting antiparasitic activity against, for example, Schistosoma haematobium, Schistosoma mansoni and the like.

According to a specific embodiment of the present invention, the antimicrobial (or antifungal, or antiparasitic, or antiviral) use may be in the form of a liquid disinfectant composition in which the nanosilver composition obtained by the above methods can be combined with a second antimicrobial agent or a mixture of such agents. Suitable examples of the second antimicrobial agent include, but are not limited to, hydrogen peroxide, quaternary ammonium salts, peracetic acid, per-salts (mentioned above in the description of the first aspect of the present invention), as well as mixtures thereof in any known proportions. In particular, this combination is capable of providing a synergistic effect of antimicrobial activity. In a particular embodiment, said second antimicrobial agent may be an oxidizing antimicrobial agent, such as, but not limited to, chlorine dioxide, monochloramine, hypochlorite, potassium permanganate, iodine or chlorine. The liquid disinfectant compositions according to this embodiment of the invention may further include one or more stabilizers, such as, for example, phosphoric acid, nitric acid, sulfuric acid, hydrobromic acid or boric acid, or mixtures thereof, the purpose of which is to adjust the pH of the composition within a range suitable for its transportation and use. Among inorganic acid stabilizers, phosphoric acid is particularly preferred. In practice, a suitable amount of said acidic stabilizer can usually be added to commercially available hydrogen peroxide grades. The stabilizer optionally used in the invention may also be an organic carboxylic acid, such as tartaric acid, citric acid (or its hydrate), benzoic acid, picolinic acid, nicotinic acid, and isonicotinic acid. Mixtures of organic and inorganic acids can also be used for this purpose. The specified stabilizer (s), if any, is preferably present in an amount effective to control pH and / or long-term storage stability of the liquid disinfectant composition. Such disinfectant liquid compositions according to the invention may also include at least one component selected from the group consisting of surfactants, corrosion inhibitors and fragrances.

Suitable surfactants that can be used in the disinfectant compositions of the present invention include, for example, but not limited to, cationic, nonionic, amphoteric, and zwitterionic surfactants, preferably surfactants, suitable for contact with food or drinking water , in appropriate doses, as well as mixtures of such compounds. A wide range of nonionic surfactants is potentially applicable for this purpose. Non-limiting examples of anionic surfactants include, for example, surfactants selected from the group consisting of polyethoxylated and / or polypropoxylated glycols, C ₈ -C ₂₀ fatty acid monoesters, sorbitan monopalmitate, and the like. Specific examples of suitable amphoteric surfactants include sodium 3-dodecylaminopropionate, sodium 3-dodecylaminopropanesulfonate, N-alkyl taurines and betaines.

A disinfectant composition comprising nanosilver obtained by the method according to the present invention can be stabilized in the biomatrix and can be used directly or after further processing as described above to treat, clean or disinfect the medium. For example, nanosilver particles can be dispersed at or where bacteria should be removed, by any suitable means or application methods. The nanosilver component of the composition is able to interact with the components of the bacterial cells, thereby effectively destroying them and reducing the total number of bacterial cells to an acceptable level.

The nanosilver-containing liquid composition described above can be used to clean, disinfect, disinfect, or sterilize a solid surface or any volume of gas or liquid. In the event that the specified liquid composition according to the present invention is used as a disinfecting or sterilizing composition (for example, dispersing it into a liquid or gas), it is usually used under appropriate conditions, including its concentration and duration of use, which can be easily determined by a specialist on based on standard knowledge in the field of disinfection and sterilization.

When applying a disinfectant liquid containing a nanosilver composition according to the invention to a solid surface, it is preferable to apply, for safety, a diluted composition prepared by mixing a suitable amount of the concentrated composition with water, and then wetting said solid surface with a diluted composition obtained for the time during which complete wetting of the solid surface occurs (which, as is known to a specialist, may depend on pores surface truths).

As is known to those skilled in the art, the preferred amount of a disinfectant, liquid, nanosilver-containing composition to be used varies widely depending on the type and number of microorganisms present on a solid surface or present in the liquid or gas being treated.

With regard to the above use of liquid containing nanosilver compositions according to the invention as a disinfectant, more specifically, the following application methods may be recommended:

- immersion of the processed product in the specified containing nanosilver composition;

- spraying the disinfectant composition onto a hard surface to be treated; and

- the introduction of a disinfectant (diluted or concentrated) composition in the treated water (in particular, in the water of a swimming pool, water from industrial processes, waste water, etc.).

Therefore, liquid disinfectant containing nanosilver compositions according to the present invention are particularly applicable to:

(a) the disinfection and sanitization of hospital and laboratory facilities, industrial premises (such as dairy farms, cheese factories, malt houses, breweries, plants for the production of mineral water, wine, spirits, fruit and vegetable juices; greenhouses; cowsheds, poultry houses and stables ; lines for packaging food products, beverages or pharmaceuticals; interior spaces of aircraft and ships) and the contents of such spaces, especially equipment or tools in such spaces;

(b) sterilizing aseptic enclosed spaces, such as incubators for nursing prematurely born animals or growing axenic animals;

(c) combating Legionella in air conditioning systems;

(d) the disinfection and sanitization of storage containers (especially silos) and lines for supplying liquid or solid products, such as food products (sugar, tea, coffee, grains, drinks) and animal feed;

(e) disinfection and sanitization of swimming pools and other balneological equipment (in which case, the composition preferably does not contain surfactants);

(f) disinfecting systems for receiving, transporting and storing drinking water (for example, in wells or storage containers), wherein the composition preferably does not contain surfactants; and

(g) the protection of outdoor crops (such as crops, tomatoes, banana plantations, hydroponic crops, including witloof, seeds, small tubers, etc.) due to their bactericidal, fungicidal, antiviral and antiparasitic properties.

The high and selective antimicrobial activity of the nanosilver composition obtained by the method according to the present invention is widely used both in the home and in industry, including, but not limited to, disinfecting water, preventing the growth of algae in water, using as a cleaning agent and forming antimicrobial coatings, for example, for medical purposes or processing nutritious or other products for humans or animals (especially in the absence of minimal eukaryotic effects lettuce or organisms), for the antimicrobial protection of textile products, as an ingredient in medicines intended to prevent infectious or microbial contamination of exposed tissues, such as, but not limited to creams, ointments or lotions, or as catalysts in chemical or other processes transformations. Each of the above applications can be carried out using nanosilver in suspension, as well as after introducing nanosilver into polymers and / or other types of coatings.

A third aspect of the present invention relates to the production of metal nanoprecipitation by probiotic and other bacteria, which, as it turned out unexpectedly, can be used as an algicidal agent (for example, but not limited to Chlorella vulgaris) in drinking water, water for aquariums, ponds or swimming pools or in other tanks with fresh or salt water, in polymers and paints, in surface coatings and other materials to protect against light pollution (aesthetic aspect) or heavy pollution ia (pollution of materials). A fourth aspect of the present invention relates to the production of metal nanoprecipitation by probiotic and other bacteria, which, as it turned out unexpectedly, can have a herbicidal effect against some dicotyledonous or monocotyledonous plants or an action against lower plants, such as moss, both in dilute form in water and when further mechanical, enzymatic and / or physico-chemical treatment. The selection of appropriate plants for this purpose is not a critical parameter of the present invention. Suitable plants for this purpose include, among others, dicotyledonous plants such as tobacco (Nicotiana tabacum), duckweed (Lamna sp.), Soybean (Glycine max), apple tree, sugar beets, Arabidopsis thaliana, alfalfa, petunia, cotton, carrots, celery, cabbage, cucumber, pepper, canola, tomato, potato, lentil, flax, broccoli, bean, lettuce, colza, cauliflower, spinach, Brussels sprouts, artichoke, peas, okra, pumpkin, forage cabbage, leaf cabbage, tea, coffee and Selaginella lepidophylla. They also include monocotyledonous plants such as Oryza sativa rice, corn, barley, maize, sunflowers (Helianthus annuus), wheat, oats, millet, sorghum, amaranth, onions, asparagus and sugarcane.

The above aspects of the present invention are particularly applicable in the following areas:

- inhibition of algae growth in aquarium water, in drinking water distribution systems for animals and people, in water distribution systems for gardening, in ponds, in swimming pools, in filtration systems for treating water from ponds or swimming pools, as well as in irrigation systems of various species;

- inhibition of algae growth on surfaces, including surfaces in contact with water, such as ship hulls;

- use in paints, polymers or coatings to prevent the growth of algae on surfaces, including the growth of algae on the surfaces of more highly organized organisms, such as plants;

- inhibiting the growth of moss or other undesirable plants, both monocotyledonous and dicotyledonous, by applying to the leaves, trunks, flowers or root systems of colloidal silver, for example colloidal silver, obtained using probiotic bacteria and deposited on them according to the above production method; and

- inhibiting the growth of certain plants or weeds on surfaces, including coating or any other effect on such surfaces of colloidal silver, for example colloidal silver, obtained using probiotic bacteria and deposited on them according to the above production method.

The following examples are provided to illustrate certain process variations and the disinfectant compositions of the present invention, without limiting them in any way.

Example 1 - obtaining nanosilver

A culture of Lactobacillus fermentum Beijerinck 1901 AL (ATCC 11976, LMG 8900, from the intestines of an 8-day-old breast-feeding baby) is propagated in MRS broth (manufactured for commercial purposes by Oxoid, Basingstoke, United Kingdom) under microaerobic conditions at 37 ° C for 15 hours. Cells were harvested from MRS by centrifugation at 3000 g for 10 minutes at 15 ° C and washed 2 times with milliQ water, then resuspended in milliQ water to a final optical density of 1.5 at 600 nm (OD ₆₀₀ ). Sodium hydroxide from 1 N NaOH stock solution was added to the cell suspension so that the final concentration was 0.05 N NaOH and 0.10 N NaOH, respectively.

A mother liquor of Ag (I) is prepared from 425 mg of AgNO ₃ and 225 mg of NH ₄ Cl in 50 ml milliQ of water. One volume of the prepared stock solution of Ag (I) is added to ten volumes of cell suspensions with 0.05 and 0.10 N NaOH, respectively. The resulting mixtures are allowed to incubate under visible light and a temperature of 25 ° C under gentle mixing (100 revolutions of the vibrator per minute) for 30 minutes. Obtain 5.0 mm final solution of Ag (0) (535 mg Ag (0) / l), precipitated on the biomass of Lactobacillus fermentum, referred to in this description, “nanosilver” or “nano-Ag”. Coated Lactobacillus fermentum cells are centrifuged and washed three times with milliQ water to remove residual growth medium and other additives by centrifuging, decanting and resuspending the composition in fresh milliq water. After that, the concentration of nano-Ag is brought to a final value. The composition is then either diluted with milliQ water or concentrated by centrifugation at 3000 g and resuspended in milliQ water according to the needs of the end user.

Example 2 - XRD analysis of nanosilver

X-ray diffraction (XRD) analysis of biomass with silver particles obtained in Example 1 and then dried at 30 ° C is carried out on a Siemens D5000 diffractometer using Bragg-Brentano optical instruments (commercially available from Siemens, Munich, Germany). X-rays emanate from a 1.6 kW copper x-ray tube (40 kV, 40 mA). Measurements are carried out between 25 and 90 degrees 2-theta with a step duration of 1.6 seconds and a step size of 0.05 degrees. The resulting spectrum (not shown) shows the presence of x-rays of metallic silver and sodium oxide. The latter is the residue of sodium hydroxide used in the preparation of nanosilver.

Example 3 - EDX analysis of nanosilver

X-ray spectral analysis based on the energy dispersion (EDX) method of dry biomass with nanosilver obtained in Example 1 and additionally dried at 30 ° C is carried out using a JSM6100 scanning electron microscope with an EDX detector (manufactured by JEOL USA, Inc.) with a resolution corresponding to incident an energy component of 20.0 keV. The results of the analysis are presented in table 1 (both weight% and atomic%) and clearly show the presence of mainly organic matter (due to the high content of carbon and oxygen) and silver, the total content of which is about 91 wt.% Of dry matter. The rest of the dried product consists of trace elements of Ca, Mg, Si, P, S and Cl, mainly due to the mineral residues of the dried biological matrix.

Table 1 Element Weight% Atomic% C 55.90 ± 0.28 69.18 ± 0.16 O 26.21 ± 0.08 24.35 ± 0.15 Na 4.97 ± 0.02 3.21 ± 0.00 Mg 0.85 ± 0.06 0.52 ± 0.04 Si 0.19 ± 0.06 0.10 ± 0.03 P 1.64 ± 0.04 0.79 ± 0.02 S 0.22 ± 0.01 0.11 ± 0.01 Cl 0.31 ± 0.03 0.13 ± 0.014 Ag 8.51 ± 0.20 1.17 ± 0.03 Ca 1.22 ± 0.18 0.45 ± 0.07

Example 4 - antimicrobial activity of nanosilver on solid medium for growing Escherichia coli

100 ml of a suspension of silver with an Ag concentration of 5 mM precipitated on the Lactobacillus fermentum biomass obtained in Example 1 is plated on a cured growth medium in order to cultivate Escherichia coli (Luria Bertani agar). As a control, 100 ml of a solution of 5 mM AgNO ₃ in sterile milliQ water with 0.1 N NaOH was plated on the same growth medium. This technique is consistent with a total amount of 0.05 mg Ag per cup of agar or 11 mg Ag per m ^{2 of} total surface area. This experiment is repeated with the last concentration doubled, i.e. with 0.11 mg Ag per cup of agar or 22 mg Ag per m ^{2 of} total surface area.

By plating such silver suspensions, a homogeneous layer of Ag (I) NO ₃ or nano-Ag, respectively, is applied to the cured growth medium.

After such pretreatment of the cured growth medium, 100 μl of 2x10 ⁶ CFU / ml of a suspension of Escherichia coli in physiological saline solution (8.5 g of NaCl / L in sterile water) are seeded in pre-treated agar plates. Then the plates are incubated for 24 hours at 30 ° C and the number of colonies is counted. The results obtained are illustrated in figure 1. At nano-Ag concentrations of 11 mg Ag / m ² and 22 mg Ag / m ² , no viable E. coli cells were detected on the treated solid growth medium (<detection limit (DL) = 1x10 ¹ CFU / ml). Thus, treatment with nano-Ag at the indicated concentrations leads to a decrease in the number of E. coli cells from 2x10 ⁶ CFU / ml to less than 1x10 ¹ CFU / ml (DL). At concentrations of Ag (I) NO _{3 of} 11 mg Ag / m ² and 22 mg Ag / m ² , the number of E. coli cells decreases significantly from 2x10 ⁶ CFU / ml to 4x10 ² CFU / ml and 1x10 ² CFU / ml respectively.

As a control, E. coli suspensions with a concentration of 2x10 ⁶ CFU / ml are seeded onto untreated growth medium, i.e. without Ag, and to the same growth medium treated with 100 μl of sterile mQ water only with Lactobacillus fermentum ATCC 11976, at the same concentration as when processing Ag, but without nano-Ag. With a total count of the number of cells, an inhibitory effect is not observed. Therefore, the inhibitory effect on nano-Ag and Ag (I) is explained by the treatment of Ag, and not by the treatment procedure or the Lactobacillus strains used in this experiment.

Example 5 - antimicrobial activity in suspension relative to various pathogenic bacteria

The survival of pathogenic cultures of Escherichia coli, Salmonella typhimurium, Staphylococcus aureus and Listeria monocytogenes diluted in physiological solutions having different concentrations (0 mg / L, 0.10 mg / L, 1.0 mg / L, 10 mg / L and 50 mg / l) of the composition of nano-Ag obtained in example 1. Nano-Ag is applied in saline containing a live culture of one of the above pathogenic bacteria. A physiological solution containing 8.5 g of NaCl per 1 liter of water is obtained in such a way that its osmotic potential is neutral with respect to bacterial cells, thus not killing them due to the osmotic effect. Control treatments include the use of a bacterial culture in saline in the absence of nano-Ag.

A mother solution of 100 mg of nano-Ag / L in mQ water is prepared and added to bacterial cultures diluted in physiological saline in amounts suitable to obtain the final concentrations of nano-Ag shown in Table 2.

The treatment is repeated separately for each type of the above pathogenic bacteria, while the “bacterial culture” is a diluted liquid broth with bacterial species in the exponential growth phase, diluted in saline to a final cell concentration of 10 ⁴ -10 ⁵ CFU / ml. Each treatment is carried out twice. Incubation is carried out in sterile, cork-closed tubes, which are incubated with shaking at 37 ° C for 72 hours. After incubation, 100 μl from each tube was plated on solid growth medium from tryptase-soy agar (TSA) and the number of colonies was counted. The results are presented in table 2 for various investigated pathogenic microorganisms.

table 2 Nano Ag concentration
(mg Ag / L) Pathogen (CFU / ml) E.coli S.aureus Salmonella Listeria 0 1,1x10 ² 1.3x10 ² 1,0x10 ³ 1,5x10 ¹ 0.10 4.6x10 ¹ one 5 1,0x10 ¹ 1,0 0 0 0 2 10 2 0 0 0 fifty 0 0 0 0

From table 2 it is obvious that a concentration of 1 mg / l of nano-Ag obtained in example 1 is sufficient to reduce the number of viable cells of E. coli, S.aureus and S. typhimurium within 72 hours to a cell concentration of <10 CFU / ml (i.e. below the detection limit). Significant cell death is already observed at a concentration of 0.10 mg / L. For Listeria, a decrease in the concentration of viable cells below the detection limit occurs at 10 mg / L nano-Ag. Thus, it can be concluded that the nano-Ag obtained in example 1, at concentrations equal to 1.0 mg / l or less in liquid cell suspensions, acts as a strong antimicrobial agent, significantly and effectively killing viable pathogenic bacteria in the liquid.

Example 6 - antimicrobial activity of nano-Ag in suspension in combination with Artemia franciscana

Sterile artificial seawater (Instant Ocean ^R manufactured by Aquarium Systems USA) is obtained in milliQ water by autoclaving. 20 ml aliquots of sterile artificial seawater in 50 ml Falcon tubes are treated. Each treatment (in triplicate) involves the use of 20 axenic Artemia nauplii in 20 ml of artificial sea water in combination with 10 ⁵ CFU / ml (colony forming units) of Vibrio campbellii LMG21363 and / or nano-Ag obtained in example 1, with a final concentration, presented in table 3. Thus, the pathogenic bacterium V. campbellii is incubated together with its host organism Artemia franciscana.

The following tests are carried out:

- Artemia franciscana + 10 ⁵ CFU / ml Vibrio campbellii

- Artemia franciscana + 10 ⁵ CFU / ml Vibrio campbellii + 100 mg nano-Ag / L

- Artemia franciscana + 10 ⁵ CFU / ml Vibrio campbellii + 10 mg nano-Ag / L

- Artemia franciscana + 10 ⁵ CFU / ml Vibrio campbellii + 1.0 mg nano-Ag / L

- Artemia franciscana + 10 ⁵ CFU / ml Vibrio campbellii + 0.1 mg nano-Ag / L

- Artemia franciscana + 0.10 mg nano-Ag / L

- Artemia franciscana + 100 mg nano-Ag / L

- Artemia franciscana + 10 mg nano-Ag / L

- Artemia franciscana + 1.0 mg nano-Ag / L and

- Artemia franciscana + 0.10 mg nano-Ag / L.

After 48 hours of incubation, the concentration of V. campbellii in sterile artificial seawater with Artemia franciscana was determined by cup method on a specific Vibrio growth medium. The average processing results are presented below in table 3 (where D.L. means detection limit).

Table 3 Concentration
nano-Ag (mg Ag / L) Survival of pathogenic bacteria Vibrio campbellii 0 1,0x10 ⁵ CFU / ml 0.10 3.0x10 ² CFU / ml 1,0 <10 ¹ CFU / ml (DL) 10 <10 ¹ CFU / ml (DL) one hundred <10 ¹ CFU / ml (DL)

In addition, it should be noted that at concentrations of 0.10 and 1.0 mg / L nano-Ag, there is no significant effect on the survival rate (80%) of Artemia franciscana compared to untreated control samples. This means that at these concentrations nano-Ag obtained according to example 1 does not have a toxic or inhibitory effect on more highly organized organisms.

Example 7 - determination of the effective duration of contact for the manifestation of antimicrobial activity

The purpose of this test is to determine the appropriate duration of contact of the composition of nano-Ag according to example 1 with pathogenic cultures of bacteria Escherichia coli, Salmonella typhimurium, Staphylococcus aureus and Listeria monocytogenes, diluted in physiological solutions, to obtain effective antimicrobial activity at concentrations of 0.1 and 1.0 mg / L Ag, respectively.

These concentrations of nano-Ag act on bacterial cultures in physiological saline (8.5 g of NaCl in 1 liter of water), obtained in such a way that its osmotic potential is neutral with respect to bacterial cells, thus not killing them due to the osmotic effect. Control treatment involves the use of a bacterial culture in physiological saline without a composition of nano-Ag.

A mother solution of 100 mg of nano-Ag / L in mQ water is prepared and added to bacterial cultures (in the same manner as in Example 5) in physiological saline in amounts suitable to obtain the desired final concentrations of nano-Ag.

Incubation (in duplicate) is carried out in sterile, corked tubes by shaking at 37 ° C, after which the number of cells at different contact times is counted (taking measurements). For each measurement, 100 μl from each tube is plated on solid growth medium from tryptase-soy agar (TSA) and the number of colonies is counted. The results obtained after 15 hours, 16 hours, 17 hours, 18 hours and 40 hours, respectively, are presented below in table 4 (where ND means “undetectable”, i.e. below the detection limit).

Example 8 - obtaining compositions of nanosilver at various weight ratios of silver to biomass of cells in a dry state

Prepare a mother liquor of silver (I) in liquid ammonia (28 vol.% NHO ₃ in water) with a final concentration of 425 g AgNO ₃ / l (= 50 mm AgNO ₃ ). A culture of Lactobacillus fermentum is obtained in the same manner as in example 1.

2.8 g (wet) of centrifuged pellet cells are resuspended in 3 different amounts (50 ml, 100 ml and 1 l) milliQ of water to obtain reaction mixtures indicated by letters A, B and C, respectively.

Then, NaOH from 1 N stock solution of NaOH in milliQ water is added to each tube to obtain a normality of 0.10 N NaOH in the above suspension.

After this, the mother liquor of silver (I) is added as follows:

- Reaction mixture A: 0.24 ml of a silver (I) mother liquor is added to obtain a final concentration of 1.30 g Ag / L (or 12 mM) Ag. Almost immediately, a precipitation reaction (brownish-reddish sediment) occurs at 56 g / l biomass (in the wet state). When the average ratio of the centrifuged mass in the dry state, comprising from 10 to 30%, get the ratio of the mass of silver to the mass of cells in the dry state, comprising from 1: 4 to 1:12.

- Reaction mixture B: 2.4 ml of a silver (I) mother liquor are added to give a final concentration of 5.78 g Ag / L (55 mM) Ag. Almost immediately, a precipitation reaction (brownish-reddish sediment) occurs on 28 g / l of biomass (in the wet state). Since the ratio of the centrifuged mass in the dry state averages 10-30%, a ratio of the mass of silver to the mass of cells in the dry state is obtained, which is from 2: 1 to 0.7: 1. The pH during this reaction is 11.6.

- Reaction mixture C: 2.4 ml of a silver (I) mother liquor are added to obtain a final concentration of 5.78 g Ag / L (55 mM) Ag. Almost immediately, a precipitation reaction occurs on 2.8 g / l of biomass (in the wet state). Since the ratio of the centrifuged mass in the dry state is on average 10-30%, a ratio of the mass of silver to the mass of cells in the dry state is obtained, comprising from 20: 1 to 7: 1.

The reaction mixture was left for 30 minutes, and then the resulting composition was collected from nano-Ag.

The resulting precipitate from nano-Ag is centrifuged along with biomass at 3000 g for 10 minutes at 15 ° C, and then washed twice with milliQ water, removing all residual ammonia and other water-soluble components used in the process. After that, the purified product from nano-Ag pellets is subjected to analysis (Example 9) or diluted even further in milliQ water to the corresponding concentrations of nano-Ag for further investigation.

Example 9 - XRD analysis of nano-Ag obtained when the ratio of the mass of silver to the biomass of cells in the dry state, equal to 0.7: 1

XRD analysis of biomass with silver particles obtained at a ratio of the mass of silver to the dry cell biomass of 0.7: 1 according to Example 8, and then dried in an oven at 100 ° C for 24 hours, is carried out in accordance with Example 2. In this XRD spectrum, only an X-ray of metallic silver can be determined. Since it can be safely stated that crystalline trace elements contained in the dried product in an amount of less than 5 wt.% Cannot be determined using XRD, it can be estimated that at least 95% of silver determined using XRD, is in state Ag (0).

Example 10 - subsequent processing of nanosilver N ₂ ABOUT ₂

The washed nano-Ag pellets obtained according to example 1 or example 8 are subjected to subsequent processing of 30% (vol.) H ₂ O ₂ in water. For this, the pellets are suspended in H ₂ O ₂ in an amount up to 6 g of Ag / L H ₂ O ₂ (30%). Get more stable precipitation. Then, the suspension of the resulting precipitates is further diluted in milliQ water to obtain the corresponding concentrations of nano-Ag for further studies.

Example 11 - the antimicrobial properties of nano-Ag after treatment N ₂ ABOUT ₂  or without her

Compositions of nano-Ag are obtained according to example 8 at various weight ratios of silver to biomass of cells in the dry state, comprising 7: 1, 1:10 and 0.7: 1, respectively (samples are indicated by the letters A, B and C, respectively). In addition, nano-Ag preparations obtained with a weight ratio of silver to biomass of cells in the dry state equal to 7: 1 were additionally treated with H ₂ O ₂ , as in Example 10, to obtain a fourth sample designated by the letter D.

In order to evaluate the effect of this weight ratio of silver to dry biomass of cells on the antimicrobial activity of nano-Ag products, a cell suspension containing 1x10 ⁴ CFU / ml Salmonella typhimurium is prepared in sterile saline and distributed into various tubes. Samples A, B, C and D are added to the same tubes until a final concentration of 0.05 mg / L (or 50 ppm) of nano-Ag is obtained in each tube.

As control samples, bacterial cultures were incubated with AgNO ₃ in an amount of 0.05 ppm, and also without silver. The tubes are capped and incubated with shaking at 37 ° C in duplicate. After 4.5 hours of incubation, samples were taken, a series of dilutions were performed in saline, and after plating on TSA, plates were incubated at 37 ° C. overnight in order to determine the total amount of Salmonella. The results of such calculations are presented in figure 3 in the form of the average number of cells and the standard deviation of two independent takes. After 4.5 hours of incubation, a significant decrease in the number of bacteria in the presence of 0.05 ppm is observed. nano-Ag obtained as described in example 8. The results presented in FIG. 3 show that the higher the weight ratio of silver to biomass of cells in the dry state, the lower the antimicrobial activity of the obtained nano-Ag. Processing the product with nano-Ag H ₂ O ₂ also significantly increases its antimicrobial activity.

Example 12 - determination of the particle size of nanosilver using transmission electron microscopy (TEM)

In order to obtain thin sections for analysis using TEM, bacterial pellets are fixed in 0.1 M cacodilate buffer (pH 7.4) containing 2.5% glutaraldehyde and 2% formaldehyde, and they are enclosed in 3% low melting agarose (from Difco Laboratories, Detroit, Michigan, USA). Then, the obtained samples are fixed in 1% osmium tetroxide. Between and after the fixation steps, the samples are washed with distilled water. After that, the samples are dehydrated in ethanol with increasing concentration and, finally, in anhydrous propylene oxide. Once embedded in an Epon-Spurr medium, sample blocks are cut with a TM60 trimmer (from Reichert-Jung AG, Vienna, Austria) to obtain a front surface of 0.5x1 mm ² - 1x2 mm ^{2 in} size, and ultrathin sections are cut in gold so that to color silver interference in the color range using an Ultracut microtome (from Reichert-Jung AG, Vienna, Austria). Sections were transferred to pioloform and carbon-coated copper grids (200 mesh). Thin sections were then stained with 2% uranyl acetate and then lead citrate in order to determine the ultrastructure of the cells. The chemicals and grates used are manufactured by Agar Scientific (Stansed, United Kingdom). Images were obtained using an EM208S transmission electron microscope (from FEI, Eindhoven, the Netherlands) operating at 80 kV.

TEM images were obtained (not shown) of nanosilver particles obtained from reaction mixtures A, B and C described in Example 8. The obtained images confirm that spherical nanosilver particles were obtained in the composition in the form of sediments on the surface of bacterial cells and in suspension between biomass.

- The particle sizes of nano-Ag obtained from reaction mixture A (ratio 1:10): the diameter of 35 measured nanoparticles varies from 3.3 nm to 72 nm, with an average diameter of 14 nm; the surface area of the particles varies from 6.4 to 2.996 nm ² , and therefore the sphericity varies from 0.14 to 0.97.

- The particle sizes of nano-Ag obtained from reaction mixture B (1: 1 ratio): the diameter of 202 measured nanoparticles varies from 3 nm to 116 nm, with an average diameter of 15 nm; the surface area of the particles varies from 6 to 4.805 nm ² , and therefore the sphericity varies from 0.12 to 0.96.

- The particle sizes of nano-Ag obtained from reaction mixture C (ratio 10: 1): the diameter of 56 measured nanoparticles varies from 3.3 nm to 56 nm, with an average diameter of 16 nm; the surface area of the particles varies from 6.4 to 1.841 nm ² , and therefore the sphericity varies from 0.15 to 0.95.

Example 13 - obtaining a colloidal nanogold

A mother solution of gold (III) is prepared in milliQ water with a final concentration of 7.5 g AuCl ₃ / L. A culture of Lactobacillus fermentum is obtained in the same manner as in example 1.

Centrifuged wet pellets weighing 2.5 g in the wet state are added to 100 ml milliQ of water.

NaOH is added from 1 N stock solution of NaOH in milliQ water to obtain the normality of the above suspension of 0.10 N NaOH.

After that, 10 ml of the mother solution of gold (III) was added to the resulting suspension to obtain a final concentration of 75 mg Au (III) / 100 ml, in the form of AuCl ₃ -Au (3.8 mm Au). The deposition of Au (0) on 2.5 g / 100 ml of biomass (in the wet state) is completed within 4 hours. Since the ratio of the centrifuged mass in the dry state is on average 10-30%, a ratio of the mass of gold to the mass of cells in the dry state is obtained, ranging from 1: 3 to 1:10.

Reactions make it possible to continue for 4 hours, after which nanogold particles are collected. The resulting purple precipitate was centrifuged at 3000 g for 10 minutes at 15 ° C and washed 2 times with milliQ water, removing water-soluble components from the process.

Example 14 - XRD analysis of nanogold

XRD analysis of biomass with gold particles from example 13, dried in an oven at 100 ° C for 24 hours, is carried out on a Siemens D5000 diffractometer using optical Bragg-Brentano instruments in accordance with example 2. The resulting spectrum, shown in figure 4, shows the presence of x-ray diffraction peaks is only Au ⁰ .

Example 15 - the effectiveness of bio-deposition and recovery on biomass with different ratios of the mass of silver to the biomass of cells in the dry state

In order to evaluate the effect of biomass on the bioreduction of Ag (I) particles to Ag (0), the recovery on biomass and in solution is determined for various Ag: CDW ratios.

Silver compositions are obtained at various Ag: CDW ratios as described in Example 8. After 4 hours of incubation of the Ag (I) mass and after its fractionation into the soluble phase (in solution) and the precipitated phase (biomass) by centrifugation at 7000 g within 10 minutes, determine the percentage recovery of silver. The results are presented below in table 5.

Table 5 Sample
(Ag: CDW ratio) Mg Ag / L in solution Mg Ag / L in biomass General recovery A (1:10) 76 1295 one hundred% In (1: 1) 819 5308 one hundred% C: Ag: CDW = 10: 1 1612 4844 one hundred%

From the above results, it is clear that the reduction of silver in the form of particles associated with biomass is higher at lower Ag: CDW ratios. For example, an Ag: CDW ratio of 1:10 provides an acceptable reduction of Ag (I) (about 95%) from Ag ⁰ in solution during bioremediation.

Example 16 - Algicidal properties of the composition of nanosilver without subsequent treatment with hydrogen peroxide

The composition of nanosilver obtained in accordance with the description given in example 8, with the ratio of the mass of silver to the biomass of cells in the dry state, comprising 1: 4.

In order to evaluate the algicidal effect of this formulation, tubes containing 10 ml of BG11 medium (described by Stanier et al. In Bacteriol. Rev. (1971) 35: 171-205) were inoculated with 0.5 ml of the actively growing liquid culture of BG11 Chlorella vulagris and incubated at 20 ° C, 65% relative humidity and 1000 lux (16 hours / day). Growth was evaluated after 2 weeks using spectrophotometric measurement. Different concentrations of silver, ranging from 20 mg Ag / L to 0.01 mg Ag / L, are examined in test tubes per dose. The MIC value represents the lowest studied concentration at which complete inhibition of the growth of organisms is observed. It was found that the MIC value of this nanosilver composition inhibiting Chlorella vulgaris is 0.125 mg Ag / L.

Claims (17)

1. A method of obtaining a composition containing colloidal nanosilver or nano-gold, comprising the step of incubating probiotic bacteria selected from Lactobacillus fermentum species with an aqueous solution containing at least 4 mM silver nitrate or gold chloride. 2. The method according to claim 1, wherein said aqueous solution contains at least 4 mM silver nitrate, and also contains ammonia and / or an ammonium salt and an alkali metal hydroxide. 3. The method according to claim 2, in which ammonia and / or ammonium salt is present in an amount sufficient to form the complex Ag (NH ₃ ) ⁺ or {Ag (NH ₃ ) ₂ } ⁺ . 4. The method according to claim 2, wherein said alkali metal hydroxide is sodium hydroxide. 5. The method according to claim 1, in which the ratio of the mass of silver or gold to the mass of bacterial cells in the dry state is from 0.05 to 20. 6. The method according to claim 1, in which the duration of incubation or contact is from 1 s to 30 minutes 7. The method according to claim 1, in which the incubation is carried out at a pH of from 8 to 12. 8. The method according to claim 1, further comprising the step of removing at least a portion of said probiotic bacteria selected from Lactobacillus fermentum species from the incubation mixture by mechanical treatment. 9. The method according to claim 1, further comprising the following steps:
- centrifuging the incubating mixture to a solid phase, including a composition containing colloidal nanosilver or nanogold and the liquid part, and
- separation of the specified solid part from the liquid part. 10. The method according to claim 1, further comprising the step of treating said composition containing colloidal nanosilver or nanogold with peroxide or per-salt. 11. The method of claim 10, wherein said peroxide is hydrogen peroxide. 12. The method according to claim 1, wherein said colloidal nanosilver is administered as a component of an antimicrobial agent. 13. The method of claim 12, wherein said antimicrobial agent is active against pathogenic bacteria selected from the group consisting of Escherichia, Salmonella, Staphylococcus, Listeria and Vibrio. 14. The method according to claim 1, wherein said colloidal nanosilver is administered as a component of an algicidal agent. 15. A method of obtaining a composition containing colloidal nanosilver, comprising the step of contacting at a temperature of from 5 ° C to 45 ° C of probiotic bacteria selected from Lactobacillus fermentum species, with an aqueous solution containing a mixture of silver nitrate, ammonia and / or ammonium salt, and alkali metal hydroxide. 16. The method according to clause 15, in which ammonia and / or ammonium salt is present in an amount sufficient to form the complex Ag (NH ₃ ) ⁺ or {Ag (NH ₃ ) ₂ } ⁺ . 17. A method of obtaining a composition containing colloidal nanogold, comprising the step of contacting at a temperature of from 5 ° C to 45 ° C of probiotic bacteria selected from Lactobacillus fermentum species with an aqueous solution containing a mixture of gold chloride and an alkali metal hydroxide in the absence of ammonia or ammonium salt .

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