WO2006049479A1

WO2006049479A1 - Antimicrobial spray compositions

Info

Publication number: WO2006049479A1
Application number: PCT/KR2005/003774
Authority: WO
Inventors: Hae-Jun Park; Sung Ho Kim; Hwa Jung Kim; Hae Jin Park
Original assignee: Bio Dreams Co. Ltd.
Priority date: 2004-11-08
Filing date: 2005-11-08
Publication date: 2006-05-11
Also published as: WO2006049478A1

Abstract

Disclosed is an antimicrobial spray composition comprising nanosized silica-silver particles, in which nano-silver is bound to silica molecules and a water- soluble polymer, the nanosized silica-silver particles prepared by irradiating a solution comprising a silver salt, silicate and the water-soluble polymer with radiation rays. Also disclosed is a spray formulation formulated from the composition.

Description

ANTIMICROBIAL SPRAY COMPOSITIONS

Technical Field

The present invention relates to an antimicrobial spray composition. More particularly, the present invention relates to an antimicrobial spray composition comprising nanosized silica-silver and a spray formulation.

Background Art

Microorganisms are always present in the environment. Some microorganisms are beneficial to humans, and others are harmful because they cause problems including diseases, offensive odors and unpleasant appearances. In particular, fungi typically inhabit humid basements or enclosed spaces (e.g., clothes chests, shoe chests, etc.) and generate offensive odors therein, modify articles, have unpleasant appearances, and cause diseases. In the rainy season, which is characterized by high temperature and high humidity, fungi inhabit, for example wallpaper in indoor environments, and cause the aforementioned problems.

Many efforts have been made to improve the unsanitary environments formed by such microorganisms, and a large number of antimicrobial products have been developed and are commercially available. However, antimicrobial products generally contain chemical substances as antimicrobial agents. In many cases, the chemical substances have significant problems in safety and duration of action because they have low molecular weights. Low-molecular weight synthetic compounds have a shorter duration of antimicrobial activity due to their volatile properties. In addition, synthetic compounds or inorganic substances have antimicrobial activity, but have disadvantages m terms of being inconvenient to use m spray forms because they need to dissolve or disperse m organic solvents to yield aerosol sprays and generating unpleasant odors in indoor environments. Antimicrobial compositions meet the following requirements to be provided in spray forms: 1) high antimicrobial activity in even small amounts of antimicrobial agents; 2) no strong irritation to the skin and eyes; and 3) strong residual activity.

Silver (Ag) , which is known as a strong bactericidal agent, destroys unicellular microorganisms through its antimicrobial activity against enzymes performing metabolic functions m microbes (T. N. Kim, Q. L. Feng, et al. , J. Mater. Sci. Mater. Med., 9, 129 (1998)) . Heavy metals such as copper and zinc also have the same function as silver. However, silver has the strongest bactericidal effect and also has excellent effects on algae. Silver has been studied as a substitute for chloride or other toxic microbicides. To date, a variety of inorganic antimicrobial agents using silver have been developed. Silver-based inorganic antimicrobial agents in current use are commercially available in the form of silver-supported inorganic powder, silver colloids, metal silver powder, and the like. Of them, the silver-supported inorganic powder form makes up the largest part of this demand, and this form is generally referred to as an inorganic antimicrobial agent.

When silver exists in an ion state, it has good antimicrobial activity. However, silver is unstable due to its high reactivity and is easily oxidized or reduced to a metal according to the surrounding atmosphere, thereby spontaneously changing in color or causing other materials to be changed in color. These phenomena lead to a reduction in the duration of the antimicrobial action of silver. When present in a metal or oxidized form, silver is stable in the environment but must be used in relatively large amounts due to its low antimicrobial activity.

Silver, having the advantages and drawbacks as noted above, is spotlighted in the form of nanoparticles. Nanoparitcles are synthesized by a variety of methods including mechanical grinding, coprecipitation, spraying, sol-gel processing, electrolysis and reverse-phase microemulsion processing. However, these methods are problematic in terms of being difficult to control the size of formed particles or requiring high production costs for micro metal particles. For example, the coprecipitation method is incapable of controlling the size, shape and size distribution of particles because it is based on forming particles m an aqueous solution The electrolysis and sol gel techniques require high production costs and have difficulty producing nanoparticels in a large scale The reverse-phase microemulsion processing easily controls the size, shape and size distribution of particles, but provides a very complicated manufacturing process and thus does not have practical use

On the other hand, a method of preparing nanometer- sized particles by irradiation with radiation rays has the following advantages it easily controls the size, shape and size distribution of particles it can form nanoparticles at room temperature, and it provides a simple manufacturing process and thus makes mass production thereof possible with low costs

Korean Pat Registration No 0425976 discloses a method of preparing nanometer sized silver colloids by irradiation with radiation rays and nanometer-sized silver colloids The method of preparing silver colloids comprises dissolving a silver salt in triple distilled water, adding sodium dodecyl sulfate (SDS) , polyvinyl alcohol (PVA) , polyvinylpyrrolidone (PVP) and others as colloid stabilizers to the solution, carrying out nitrogen purging, and irradiating the resulting solution with radiation rays However, since this method produces silver colloids having a particle size greater than 100 nm, high concentrations of silver colloids should be produced for use as an antimicrobial agent against microbes, especially fungi

In addition to the aforementioned methods, many efforts have been made to provide nano-sxlver applicable to a broad range of fields with purposes including anti- bacteria, cleaning and deodorization. Despite these efforts, there is still a need for the development of a more simple process for preparing cheaper and more stable nano silver

Silicon (Si) , which is the second most abundant material m the earth, is taken up by plants and enhances resistance to diseases and stress therein (Role of Root hairs and Lateral Roots in Silicon Uptake by Rice J F. Ma et al. Ichii Plant Physiology (2001) 127: 1773-1780, etc ) . In particular, when plants are treated with an aqueous solution of silicate, silicate displays excellent preventive effects on major plant diseases including powdery mildew and downy mildew Also, silicate promotes physiological activity of plants and improves plant growth while providing resistance to diseases and stress (Suppressive effect of potassium silicate on powdery mildew of strawberry in hydroponics T Kanto et al . J GenPlant Pathol (2004) 70: 207-211), etc.) . However, since silica does not have direct disinfecting effects on plant pathogens, it does not exhibit positive effects when diseases develop m plants Based on this background, the present inventors prepared nanosized silica-silver particles, in which nano silver is bound to silica molecules and a water-soluble polymer, by mixing a silver salt, silicate and a water soluble polymer and irradiating the resulting mixture with radiation rays, and found that the nanosized silica silver particles thus prepared are uniform in size, are stable, and have excellent antimicrobial effects even m very low concentrations The present inventors further found that, when such particles are formulated into a spray form and sprayed, the particles are able to effectively disinfect harmful microorganisms including pathogens, thus leading to the present invention

Disclosure of the Invention

It is therefore an object of the present invention to provide an antimicrobial spray composition comprising nanosized silica-silver particles, in which nano-silver is bound to silica molecules and a water-soluble polymer, the nanosized silica-silver particles prepared by irradiating a solution comprising a silver salt, silicate and the water soluble polymer with radiation rays, and a method of preparing the composition It is another object of the present invention to provide a spray formulation formulated from the antimicrobial spray composition

It is a further object of the present invention to provide a method of antimicrobially treating an article or area needing to be antimicrobially treated by spraying the spray formulation onto the article or are

Brief Description of the Drawings

The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken m conjunction with the accompanying drawings, in which:

Fig Ia is a flowchart of a process of preparing nanosized silica-silver, and Fig. Ib shows TEM images of nanosized silica-silver formed after irradiation with gamma rays,

Fig 2 shows the colloidal stability of nanosized silica-silver m water,-

Fig. 3 shows the absorption spectrum of nanosized silica-silver at 403 nm compared with the absorption spectra of water and silver ions;

Fig. 4 shows the change of nanosized silica-silver m absorbance at 403 nm according to concentrations of sodium silicate (Na₂SiO₃) ;

Fig. 5 shows the absorption spectra at 403 nm of nanosized silica-silver prepared with varying concentrations of polyvinylpyrrolidone (PVP) ,-

Figs. 6a and 6b show the absorption spectra at 403 nm of nanosized silica-silver prepared with other water soluble polymers (high levan and corn starch, respectively) , Fig 7 shows the absorption spectra at 403 nra of nanosized silica silver according to radiation doses,

Fig. 8 shows the antibacterial effects of nanosized silica-silver on Escherichia coll, Bacillus subtilis and Pseudomonas syringae subsp Syrmgae according to concentrations,

Fig. 9 shows the antimicrobial effects of silica, nanosized silica-silver, 20 nm silver and 100 run silver versus Rh±zoctonia solani, Fig 10 shows the antimicrobial effects of silica, nanosized silica silver, 20 nm silver and 100 nm silver versus Botrytis cmerea, and

Fig 11 shows the results of comparison of nanosized silica-silver of varying concentrations and commercially available products for antimicrobial effects.

Best Mode for Carrying Out the Invention

In one aspect, the present invention relates to an antimicrobial spray composition comprising nanosized silica-silver particles, in which nano silver is bound to silica molecules and a water-soluble polymer, the nanosized silica-silver particles prepared by irradiating a solution comprising a silver salt, silicate and the water-soluble polymer with radiation rays, and a method of preparing the composition

The term "nanosized silica silver", as used herein, refers to a composite in which nano sized silver particles and silica molecules are bound to a water soluble polymer According to a detailed aspect, the nanosized silica-silver may be prepared by irradiating a solution containing a silver salt, silicate and a water soluble polymer with radiation rays A form of the composite is exemplified by a structure in which nano-sized silver particles, formed from silver ions, and silica molecules, formed from silicate, are individually or together surrounded by a water-soluble polymer by irradiation with radiation rays The nanosized silica-silver thus prepared is present in a form m which nanoparticles are dissociated from each other at a colloidal state or assembled into loose spherical aggregates (Fig Ib) The aggregates are disassembled into dispersed nanoparticles when temperature increases Nano silver particles in which nano-silver is coated with silica particles were conventionally developed However, these particles, unlike the nanosized silica silver particles contained in the antifungal pharmaceutical composition of the present invention, do not include a water-soluble polymer m the particle composition Also, a water-soluble polymer was conventionally used to form nano-silver particles However, m this case, the water-soluble polymer was used not as a component of nano-silver particles but as a dispersing agent for forming a colloidal solution

The nanosized silica-silver contained in the present composition, as demonstrated from the absorption spectrum of Fig 3, absorbs lxght at 403 nm, characteristic for nano-silver, and as shown m Fig Ib has a uniform nanoparticle size The nanosized silica silver has a particle size of preferably 0 5 to 30 nm, more preferably 1 to 20 nm, and most preferably 1 to 5 nm The nanosized silica silver is prepared by preparing a solution containing a silver salt, silicate and a water soluble polymer and irradiating the solution with radiation rays This method may further include bubbling (or purging) with inert gas before, after, or before and after irradiation with radiation rays The inert gas is exemplified by nitrogen and argon, and nitrogen gas is preferred The bubbling is preferably carried out for 10 mm to 30 mm In the method, the solution containing a silver salt, silicate and a water-soluble polymer may further include a radical scavenger for scavenging radicals generated by irradiation with radiation rays The radical scavenger is exemplified by alcohols, glutathione, vitamin E, flavonoid and ascorbic acid Available alcohols may include methanol, ethanol, nor-propanol, isopropanol (IPA) and butanol Of them, isopropanol is preferred The alcohol may be used in an amount of 0 1 to 20%, and preferably 3 to 10% based on the total amount of the solution containing a silver salt, silicate and a water soluble polymer The silver salt contained in the present composition may be exemplified by siver nitrate (AgNO₃) , silver perchlorate (AgClO₄) , silver chlorate (AgClO₃) , silver chloride (AgCl) , silver iodide (AgI) , silver fluoride (AgF) , and silver acetate (CH₃COOAg) . A highly water- soluble silver salt (e g., silver nitrate) xs preferred The water-soluble polymer used m the preparation of nanosized silica-silver may be exemplified by polyvinylpyrrolidone (PVP) , polyvinyl alcohol (PVA) , polyacrylic acid and derivatives thereof, levan, pullulan, gellan, water soluble cellulose, glucan, xanthan, water- soluble starch, and corn starch. Of them, polyvinylpyrrolidone (PVP) is preferred. The silicate used xn the preparation of nanosized silica-silver may be exemplified by sodium silicate, potassium silicate, calcium silicate, and magnesium silicate. Of them, sodium silicate is preferred The use of silicate for preparing nano-silver was not reported prior to the present invention. The present inventors are the first to describe the use of silicate, not a silica form, in the reaction with a silver salt in order to provide nanosized silica silver having excellent antibacterial effects, in which silica molecules and a water-soluble polymer are bound to nano-silver. In the preparation of nanosized silica-silver, the silver salt and silicate are reacted m a weight ratio of 1:0.5 to 1.3 (silver salt: silicate) . Preferably, the reaction is carried out m a weight ratio of 1:1. The particle size of nanosized silica silver may be controlled according to the amount of silicate. The use of silicate in a small amount results in increased size of particles. In contrast, when an excess amount of silicate compared to the silver salt is used, particles do not form In the nanosized silica silver preparation, the silver salt and water-soluble polymer are reacted in a weight ratio of 1 0 5 to 2 5 (silver salt water-soluble polymer) Preferably, the reaction is carried out m a weight ratio of 1 1 For the preparation of nanosized silica-silver, radiation rays may be used, which include beta rays, gamma rays, X-rays, ultraviolet and electron rays A gamma ray dose of 10 to 30 kGy is preferred

Generally, nano sized particles are able to penetrate the plasma membrane, and silica is well taken up by fungi The nanosized silica-silver is taken up by fungal cells, in which the nanosized silica-silver exhibits increased antimicrobial activity mediated by silver nanoparticles, and forms a physical barrier against pathogenic fungi due to the property of silica to increase resistance by inducing dynamic resistance to diseases, thereby preventing recurrence of diseases for a considerable period of time after pathogens are disinfected

The antimicrobial spray composition of the present invention may be used m the form of a colloidal solution m which the aforementioned nanosized silica-silver is dispersed/suspended m a solvent (e g , water, alcohol, or combinations thereof, etc ) As used herein, the term "weight percentage (wt%) " is based on the total weight of a composition containing a solvent The nanosxzed silica-silver contained in the antimicrobial spray composxtion of the present invention has a particle sized of 0 5 to 30 nm, preferably 1 to 20 nm, and more preferably 1 to 5 nm

5 The nanosized silica-silver is contained in the antimicrobial spray composition of the present invention m an amount of 0 1 to 100 ppm, preferably 0 1 to 50 ppm, and more preferably 1 to 15 ppm

In addition to the nanosized silica-silver, the

10 antimicrobial spray composition of the present invention may further include a surfactant The surfactant useful in the present invention may include nonionic anionic, cationic and/or amphoteric forms Also, any surfactants known to those skilled m the art are available Available

Ib nonionic suifactants may include polyoxyethylene- polyoxypropylene copolymers, sorbitan ester, polyoxyethylene sorbitan, polyethylene glycol and polyoxyethylene ether Available anionic surfactants may include alkyl sulfate, alkyl ether sulfate, alkaryl

2C sulfonate, alkanoyl lsethionate, alkyl succinate, alkyl sulfosuccmate, N alkyl sarcosmate, alkyl phosphate, alkyl ether phosphate, alkyl ether carboxylate and alpha-olefin sulfonate Available cationic surfactants may include 1,2- dioleoyl-3-trimethylammonium propane (DOTAP) , dimethyl 5 dioctadecyl ammonium chloride (DDAC), N [1-(1,2- dioleoyloxy)propyl] -N,N,N-trimethylammomum chloride (DOTMA), l,2-dioleoyl-3-ethylphosphocholme (DOEPC), and 3β- [N- [ (N,N¹ -dimethylammo)ethane] carbamoyl] cholesterol (DC-Choi) . Available amphoteric surfactants may include cocodimethylcarboxymethylbetame, coca idopropylbetame, cocobetame, laurylbetame, laurylamidopropylbetame, and oleylbetame

The present composition preferably includes a nonionic surfactant, and according to the intended use, may further include, in addition to the nonionic surfactant, other types of surfactants Surfactants more suitable for use in the present composition include Tween 20, Tween 80, sorbitan monooleate and polyethylene glycol.

The present composition comprising a surfactant may appear colorless or colored For this use, a surfactant suitable for the colorless or colored appearance may be selected taking into consideration precipitation, turbidity and other factors. In a preferred aspect, sorbitan monooleate and polyethylene glycol may be used as surfactants

The present composition may include nanosized silica- silver and a surfactant in a weight ratio of 1:0.2 to 20 (nanosized silica-silver: surfactant) , and more preferably 1:1 to 10. The surfactant may be contained in an amount of less than 30 wt%, preferably 0 1 to 20 wt%, and more preferably 0.5 to 10 wt%, based on the total weight of the composition.

The antimicrobial spray composition of the present invention may further include an alcohol. Alcohols suitable for use in the present invention preferably have a carbon number of 5 or less, and are more preferably ethanol, methanol and isopropanol The alcohol may be contained m an amount of less than 15 wt%, preferably 1 to 10 wt%, and more preferably 3 to 5 wt%, based on the total weight of the composition

The antimicrobial spray composition of the present invention may further include an aromatic agent Any aromatic agents that are known in the art are suitable for use in the present invention The aromatic agents may be contained in an amount of less than 10 wt%, preferably 0 05 to 5 wt%, and more preferably 0 125 to 1 25 wt%, based on the total weight of the composition

More particularly, the antimicrobial spray composition of the present invention may include nanosized silica-silver, a surfactant selected from among sorbitan monooleate and polyethylene glycol, an alcohol selected from among ethanol, methanol and isopropanol, and an aromatic agent The components of the composition may be contained in a nanosized silica silver surfactant alcohol aromatic agent weight ratio of 1 0 1 to 30 0 1 to 50 0 125 to 10, preferably 1 0 5 to 20 0 5 to 30 0 125 to 5, and more preferably 1 1 25 to 10 7 5 to 25 1 25 to 2 5 According to the intended use, the antimicrobial spray composition of the present invention may include a deordorizmg agent (e g , flavonoid, phytoncide, wood vinegar liquor, plant extracts, cyclodextrin, metal ions, titanium dioxide) , a precipitation inhibitor (e g , polyvmylalcohol (PVA) , pullulan, gellan, water-soluble cellulose, glucan, xanthan, water-soluble starch, levan) , 5 and a propellant Also the present composition may further include a widely known disinfecting agent, for example, antimicrobial plant extracts and an organic synthetic product

The term "antimicrobial" , as used herein, includes

10 both growth inhibition of pathogenic microbes, including bacteria and fungi, and disinfection through survival inhibition

The nanosized silica-silver, contained m the present composition used in antimicrobial fiber coating, exhibits

±5 excellent antimicrobial effects on fungi, which are exemplified by Candida, Cryptococcus, Aspergillus, Trichophyton, Trichomonas, Chaetomium, Gliocladium, Aureobasidium, PeniciIlium, Rhizopus, Cladosporium, Mucor, Pullularia, Trichoderma, Fusaπum, Myrothecium and

20 Memnoniella, and bacteria, which are exemplified by Escherichia, Bacillus, Pseudomonas, Chetonium, Staphylococcus, Klebsiella, Legionella, Salmonella, Vibrio and Rickettsia

In another aspect, the present invention relates to a

2b spray formulation formulated from the antimicrobial spray composition comprising nanosized silica-silver

The present composition may be formulated into varxous suitable spray forms known to those skilled in the art For example, the present composition may be packaged usxng a manual spray distributor manufactured with a synthetic organic polymeric plastic material, specifically a trigger spray distributor or a pump-type spray distributor

Spray distributors evenly apply the present composition onto a relatively wide area of a surface to be disinfected, and thus contribute to the antimicrobial property of the present composition Such spray distributors are especially suitable for disinfection of perpendicular surfaces Spray distributors suitable for general use are manually operated effervescent trigger-type distributors, m which a liquid composition is divided into droplets and directly sprayed onto a surface to be treated In fact, when a user operates a pump device, a composition contained in the body of such a spray distributor moves toward the head of the spray distributor by energy transmitted to a pumping unit In the head of the distributor, the composition is pressurized by an obstacle (e g , lattice, cone, etc ) and is sprayed from the distributor, forming droplets

In a further aspect, the present invention relates to a antimicrobial treatment method using the spray formulation formulated from the antimicrobial spray composition comprising nanosized silica silver

The spray formulation according to the present mventxon may be sprayed onto a surface needing to be antimicrobialIy treated to disinfect the surface

The term "surface" , as used herein, refers to a certain surface including biological and nonbiological surfaces Nonbiological surfaces include all hard and soft surfaces, for example, tiles, walls, floor materials, chrome, glass, vinyl products, plastics, plastic woods, tables, sewers, cookware, dishes, sanitary facilities (e.g., sewers, showers, show curtains, wash basins, bathrooms, etc.), fabrics (e.g., clothing, curtains, drapes, bedcovers, bathroom linens, table covers, sleeping bags, tents, furniture having covers, carpets, etc.), and household electric applicances (e.g., refrigerators, freezers, washing machines, automatic dryers, ovens, microwave ovens, dishwashers, etc.), but are not limited to the above articles.

A better understanding of the present invention may be obtained through the following examples which are set forth to illustrate, but are not to be construed as the limit of the present invention.

EXAMPLE 1: Preparation of nanosized silica-silver bound to silica molecules and water-soluble polymer

1 g of sodium silicate (Na₂SiO₃) 1 g of silver nitrate (AgNO₃) , 1 g of polyvinylpyrrolidone (PVP) and 12 ml of isopropylalcohol (IPA) were dissolved in distilled water at a total volume of 200 ml Nitrogen gas was injected into the resulting solution for 20 mm After bubbling, the solution was irradiated with gamma rays of 25 kGy, thereby preparing nanosized silica-silver Fig Ia is a flowchart of a method of preparing nanosized silica silver bound to silica molecules and a water soluble polymer according to one embodiment of the present invention After irradiation with gamma rays, the solution appeared yellow, characteristic for nano-silver particles This result indicates the formation of stable nano sized silica silver particles through linkage of silica molecules, the water soluble polymer and silver particles by the above reactions

The particles formed by the above reactions were examined to determine if they were nano silver particles Test samples were prepared according to the compositions described in Table 1, below, and were allowed to stand for 24 hrs at room temperature Thereafter, test samples were examined for color change

TABLE 1

Solution prepared m this example Test samples A and B were the prepared solutions irradiated with radiation rays, and test samples C and D were the prepared solutions containing Ag^* ions but not irradiated with radiation rays Test samples SW and DW were used as controls, not containing silver ions or silver particles

Silver is easily oxidized in an ionic state In the presence of Cl ions, silver ions are precipitated as a brown precipitate, AgCl, wherein they turn brown Based on this fact, the state of silver was investigated using tap water containing Cl ions Silver forms precipitates in an ionic state (Ag⁺) , and appears yellow when present as stable nano silver particles The results are given in Table 2, below

TABLE 2

As shown in Table 2, test samples SW, D and DW were colorless with no change in color after incubation for 24 hrs indicating that silver ions, chloride ions, or neither silver ions nor chloride ions were in existence In contrast, test sample C changed from colorless to reddish brown This is because silver ions bonded to chloride ions contained m tap water to form a precipitate of AgCl Test samples A and B appeared yellow with no change ±n color, indicating that the irradiation with radiation rays formed stable nano-silver particles, bound to silica molecules and a water-soluble polymer, with no formation of AgCl precipitates even in the presence of chloride ions The color changes are also photographically shown in Fig 2

Fig 3 shows the absorption spectrum of the nanosized silica-silver of the present invention, prepared as described above The absorption spectrum of the nanosized silica silver was compared with absorption spectra of test samples DW, B and D, described m Table 2 Only test sample B absorbed light at 403 nm, characteristic for nano-silver Test samples DW and D did not absorb light at the same wavelength As revealed from the results obtained after incubation for 24 hrs and the absorption spectra, the irradiation of a solution containing sodium silicate, silver nitrate and PVP with radiation rays forms stable nanosized silica silver bound to silica molecules and a water soluble polymer

Fig Ib shows TEM (Transmission Electron Microscope) images of the nanosized silica-silver prepared as described above As shown m Fig Ib, nanosized silica-silver particles have a uniform particle size distribution with a particle size less than 20 nm, specially, ranging from 1 nm to 5 nm The nanosized silica silver particles are dissociated from each other or assemble into loose spherical aggregates by intermolecular attractive forces. The aggregates are easily disassembled by heating.

EXAMPLE 2: Preparation of nanosized silica-silver bound to silica molecules and water-soluble polymer

Nanosized silica-silver was prepared according to the same method as in Example 1, except that sodium silicate (Na₂SiO₃) was used in varying amounts of 0.5 to 2 g. Various test samples, described in Table 3, below, were prepared with varying amounts of sodium silicate and examined.

TABLE 3

Fig. 4 shows the changes in absorbance and color of nanosized silica-silver according to varying concentrations of sodium silicate, described in Table 3. As shown in Fig. 4, the highest absorbance was observed in a sodium silicate to silver nitrate ratio of 1:1. The absorbance decreased when sodium silicate was used in a 1.5-fold higher amount than silver nitrate. Also, when sodium silicate was used in a 0.5-fold lower amount than silver nitrate, orange-gold color was observed, indicating that silver particles increased in size

The above results indicate that the added amount of sodium silicate is an important factor upon prepaiation of nanosized silica silver, and that the particle size of nanosized silica-silver can be controlled by varying the amount of sodium silicate

EXAMPLE 3 Preparation of nanosized silica-silver bound to silica molecules and water-soluble polymer

Nanosized silica-silver was prepared according to the same method as in Example 1, except that polyvinylpyrrolidone (PVP) was used in varying amounts of 0 5 to 2 g.

The changes in absorbance and color of nanosized silica-silver according to varying concentrations of polyvinylpyrrolidone (PVP) are given m Table 4, below, and Fig 5

TABLE 4

As shown m Table 4 and Fig. 5, when sodium silicate was used in an equal ratio to silver nitrate, polyvinylpyrrolidone (PVP) can be used m a concentration

>3 0 5 to 2-fold higher than sodium silicate (or silver nitrate)

EXAMPLE 4: Preparation of nanosized silica-silver bound to silica molecules and water-soluble polymer

Nanosized silica-silver was prepared according to the same method as in Example 1, except that high levan or corn starch was used instead of polyvinylpyrrolidone (PVP) .

The absorbance and absorption spectra of the prepared nanosized silica-silver are given m Table 5, below, and Figs. 6a and 6b.

TABLE b

Test samples Ab at 403 nm

High levan 0 208

Corn starch 0 211

As shown m Table 5 and Figs 6a and 6b, polysaccharides such as levan or corn starch are available for preparation of nanosized silica-silver although the use of levan or corn starch resulted in decreased absorbance at

403 nm.

EXAMPLE 5: Preparation of nanosized silica-silver bound to silica molecules and water-soluble polymer

Nanosized silica-silver was prepared according to the same method as m Example 1, except that varying radiation doses were used

The absorbance and absorption spectra of the prepared nanosized sxlica-ailver are given m Table 6, below, and Fig 7

TABLE 6

As shown xn Table 6 and Fig 7, the absorbance at 403 nm occurred even in a gamma-ray dose of 10 kGy, and increased with increasing gamma-ray doses These results indicate that nanosized silica-silver can be prepared using a radiation dose higher than 10 kGy

EXAMPLE 6 Evaluation of antibacterial effects of nanosized silica-silver

Nanosized silica-silver bound to silica molecules and a water-soluble polymer was examined for growth inhibitory effects versus bacteria, Escherichia coli, Bacillus subtilis KCTC 1021, and Pseudomonas synngae subsp syrmgae KCTC 2440, according to concentrations Bacteria were incubated in 500-ml Erlenmeyer flasks containing 100 ml LB medium under aerobic conditions with shaking at 190 rptn for 15 to 16 hrs at 37^°C for Escherichia coli and at 30 C for other bacteria Thereafter, 20 id of each culture was inoculated onto LB agar plates containing nano silver bound to silica molecules and a water-soluble polymer m concentrations of 0, 1, 10, 100 and 1000 ppm Incubation was conducted for 6 to 7 days at 37^°C for E. coli and at 30^°C for other bacteria

Fig 8 shows the growth inhibitory effects of nanosized silica-silver versus Escherichia coli, Bacillus subtilis 1021 and Pseudomonas syringae subsp Syπngae 2440 The gram-positive bacterium, Bacillus subtilis, exhibited decreased growth at 100 ppm of nanosized silica silver compared to a control (LB agar plate) The gram- negative bacteria, Escherichia coli (Probe, PR2) and Pseudomonas syringae, showed similar growth rates at 10 ppm of nanosized silica-silver to those of controls (LB agar plates) , and their growth was completely inhibited at 100 ppm of nanosized silica silver

EXAMPLE 7 Evaluation of antifungal effects of nanosized silica silver

TEST EXAMPLE 1 Antifungal activity of nanosized silica silver against Rhizoctonia and Botrytis

A culture medium for the growth of microorganisms, PDA medium (Difco) , was autoclaved and aliquotted in 25 ml into petri dishes Before the medium was hardened (at about 40X ) , it was mixed with silica molecules for test sample A, the nanosized sxlxca-silver prepared in Example 1 for test sample B, sxlver particles 20 nm in size for test sample C, and silver particles 100 nm in size for test sample D Then, the medium was allowed to cool to give PDA plates PDA plates were inoculated with fungi, Rhizoctoπia solani (Agribiology Department, Chungnam National University) and Botrytis cinerea (Agribiology Department, Chungnam National University) , each sufficxently grown on solid media and excised as a circle 5 mm m diameter Thereafter, each plate was incubated for 2 days at room temperature to examine whether the growth of R solani and B cinerea would be inhibited The additionally supplemented materials m the test samples were used in concentrations of 6 pptn and 0 3 ppm

As shown in Fxg 9, test sample A mixed with silica molecules exhibited the same results as in a control m both concentrations of silica molecules Test samples C and D, mixed with 20 nm silver and 100 nm silver, respectively, displayed the same results as m the control at 0 3 ppm of silver In contrast, test sample B, mixed with the nanosized silica-silver of the present invention, exhibited remarkably inhibited growth of Rhizoctoma solani even in a very low concentration of 0 3 ppm of nanosized silica- silver

As shown m Fig 10, test sample A mixed with sxlica molecules exhxbxted the same results as xn a control in all concentrations of silica molecules Test samples B and D, mixed with 20 nm silver and 100 nm silver, respectively, displayed the same results as in the control at 0 3 ppm of silver In contrast, test sample C, mixed with the nanosized silica silver of the present invention, exhibited remarkably inhibited growth of Botrytis cmerea even in a very low concentration of 0 3 ppm of nanosized silica- sliver in comparison with the treatment of S cmerea with 20 nm silver and 100 nm silver

TEST EXAMPLE 2 Antifungal activity of nanosized silica- sliver against pathogenic fungi

Minimal inhibitory concentrations (MICs) of nanosized silica silver, tolnaftate, amphotericin B and itraconazole against various human pathogenic fungi were measured The pathogenic fungi included Candida lusitaniae, Candida tropicalis, Candida albicans, Candida krusei, Candida glabrata, Candida parapsilosis, Cryptococcus neoformans, Mucor ramosissmus, Aspergillus fumigatus, Aspergillus flavus, and Aspergillus terreus The MICs were measured using a standard procedure proposed by the AFST EUCAST (Anitifungal Susceptibility Testing Subcommittee of the European Committee on Antibiotic Susceptibility Testing, Rodriguez-Tudela et al , (2003) Method for the determination of minimum inhibitory concentration by broth dilution of fermentative yeasts, Clinical Microbiology and Infection, 9, I-VIII) This standard is based on the reference procedure of the National Committee for Clinical Laboratory Standards (NCCLS) , which is described in the literature (National Committee for Clinical Laboratory Standards (2002) Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeast Second Edition- Approved Standard M27-A2. NCCLS, Wayne, PA, USA)

In detail, of the pathogenic fungi, Candida species, Cryptococcus neoformans and Mucor ramosissmus were cultured using SDA (Sabouraud Dextrose Agar) medium at 35^°C for 24 hrs for Candida species and for 48 hrs for C. neoformans and M ramosissmus About five colonies of less than 1 mm were picked, suspended in 5 ml of 0 85% saline (8 5 g/L NaCl), and adjusted to a final density of 2XlO³ cells/ml with RPMI 1640 medium to give an inoculum Also, Aspergillus species were sufficiently cultured at 35^°C for 7 days using PDA (Potato Dextrose Agar) medium. After 5 ml of sterile distilled water and one drop of Tween 20 were poured onto the PDA plate, spores were scratched with a sterile microprpette tip and placed into a test tube. After the test tube was allowed to stand for 3 to 5 mm, the supernatant was recovered and adjusted to a density of 2XlO⁴ CFU/ml to give an inoculum. Of the nanosized silica- silver prepared in the above Examples and demonstrated to have antifungal activity, the nanosized silica-silver prepared in Example 1 was used m this test and w?as two¬ fold serially diluted with RPMI 1640 medium Also, as controls, tolnaftate, amphotericin B and itraconazole were dissolved in DMSO (dimethyl sulfoxide) and two-fold serially diluted with RPMI 1640 medium. The final concentration of DMSO was 2.5%. 100 μΛ of each dilution and 100 β( of each inoculum were aliquotted into 96-well plates, thereby giving a final concentration of 128 μg/mC to 0.0313 μg/iti-P for antifungal agents contained in the two-fold serial dilutions. 96-well plates seeded with Candida, species and Aspergilles fυmigatus were incubated at 35^°C for 48 hrs. Cryptococcus neoformans and Mucor ramosissmus were cultured at 35^°C for 72 hrs. After cultivation, the culture was observed with the naked eye, and the lowest concentration inhibiting visible fungal growth was considered a minimum growth inhibitory concentration (MIC; l≥μg/m-d) . The results are given in Table 7, below.

TABLE 7

(Unit: μg/ml)

As shown xn Table 7, the nanosxzed silica-silver exhibited antifungal activity against pathogenic fungi, Candida, Cryptococcus, Mucor and Aspergillus

TEST EXAMPLE 3 Antimicrobial activity of nanosized silica- silver against indoor microorganisms

In order to determine whether nanosized silica-silver has antimicrobial effects versus indoor microorganisms according to concentrations, the nanosized silica-silver was examined for growth inhibitory effects versus an indoor fungus Chaetomium globosum KCTC 6988, m concentrations of 0 3, 3, 10 and 100 ppm

In detail, MSA (mineral salt agar) plates, containing nanosized silica silver at 0 3, 3, 10 and 100 ppm, were inoculated with the indoor fungi, Chaetomium globosum KCTC 6988 with a disk cut with a 6-mm-diameter cork borer, followed by incubation in an incubator for 7 days at 25 C On Day 7, the cultures were compared with a culture not containing nanosized silica-silver in order to determine whether the nanosized silica-silver has an antimicrobial effect on C globosum

Chaetomium globosum was found not to grow even at 0 3 ppm of nanosized silica-silver These results indicate that the nanosized silica-silver has excellent antimicrobial effects even in small concentrations EXAMPLE 8 • Antifungal activity of nanosized silica-silver according to surfactant addition

The nanosized silica-silver solution prepared in Example 1 was supplemented with a surfactant and was evaluated for antifungal activity. The nanosized silica silver was supplemented with a surfactant, PEG-400 (Polyethylene glycol, CELL CHEMICAL) or CELNON TW80 (Sorbitan monoolate, CELL CHEMICAL) The surfactant- containing nanosized silica-silver solutions (PEG-400 and CELNON-80TW samples) , a positive control and a negative control were inoculated with Aspergillus niger KCTC 6960, diluted with 0.05% Tween 20 to a density of 3.25χlO⁴ spores/ml, in a ratio of 9.1 (surfactant-containing solution: moculaum) After the inoculated samples were allowed to stand for 60 min, 200 μg of each sample was smeared onto PDA and MEA plates. After cultivation, the nanosized silica-silver supplemented with a surfactant was assessed for antifungal activity. The results are given in Table 8, below

TABLE 8

As shown in Table 8, except for the negative control not containing nanosized silica-silver, the nanosized silica silver solutions, supplemented with surfactants or not, were found to have antifungal effects

EXAMPLE 9 Color intensity and clarity of nanosized silica- silver containing solutions according to mixing ratios of nanosized silica-silver and surfactant

In order to investigate the color intensity and clarity of a nanosized silica silver-containing solution according to mixing ratios of nanosized silica-silver and a surfactant, nanosized silica-silver (NSS) and a surfactant, CELNON-8OTW, were mixed m various ratios and then mixed with water, enough to give a total volume of 100 ml The resulting solutions and a control were examined for color intensity and clarity The results are given m Table 9, below

TABLE 9

(no precipitation) 0 >)>>>> 3 (formation of precipitates greater than 5 mm m diametei)

(not turbid) 0 )>)>)> 3 (completely turbid) ^c (colorless (white)) 0 nn)) 5 (no color change, color appears when NSS is added to distilled water)

' changed to grayish brown when NSS is precipated in)

As shown in Table 9, no precipitation was observed xn S-I to S-Il samples, and these samples were not turbid. In contrast, S-12 to S-15 samples were colorless immediately after being prepared, and after 3 days, became turbid and changed color when precipitates were formed xn these samples. The precxpxtatxon and color changes decreased with increasing content of CELNON-8OTW. Samples containing CELNON-80TW in 2- to 7.5-fold higher amounts than nanosized silica-silver were colorless.

EXAMPLE 10: Discoloration test for cloth sprayed with antimicrobial spray compositions

An antimicrobial spray composition according to the present invention was evaluated for causing discoloration when sprayed onto cloth or fabrics. Nanosized silica-silver (NSS) containing solutions having low turbidity and low color intensity were selected and sprayed onto 100% natural cotton fabrics (white) either from a short distance (20 cm) away or from a long distance (40 cm) away. The cotton fabrics were then dried under sunshine or in a drying oven at 80V, and was visually examined for discoloration. The results are given in Table 10, below.

TABLE 10

Std. : Standard * Not studied

" (not turbid) 0 >>>»>> 3 (completely turbid) ¹ (colorless (white) ) 0 >>>;-> 5 (a score of 5 means no color change

When antimicrobial spray compositions were prepared according to the compositions described in Table 10, no precipitation was observed in the compositions. Also, the compositions were not turbid and were colorless When the compositions thus prepared were sprayed onto white cotton fabrics from a short distance (20 cm) away, stains were formed in all cases. In contrast, upon spraying from a long distance (40 cm) away, SP-I, SP-2 and SP-6 samples did not cause any stains. These samples also did not cause any stains even after drying under sunshine and at 80^°C.

EXAMPLE 11: Evaluation of antifungal effects of antimicrobial spray compositions upon spraying

Antimicrobial spray compositions comprising nanosized silica-silver were evaluated for antifungal effects when sprayed onto fungi Antimicrobial spray compositions were prepared with varying mixing ratios of components as described in example 10 and sprayed onto fungi to examine their antifungal effects In detail, 600 ml of MEA (malt extract agar) medium was autoclaved, cooled to 50 to 55^°C, mixed with 0 6 ml of a dilution of spores of Aspergillus niger KCTC 6960, and dried, thus yielding spore containing plates A filter paper 15 mm in diameter was sprayed with the prepared antimicrobial spray composition and placed onto the middle of the plates The plates were incubated for 2 days and examined for the formation of clear zones around the fungal growth The formation of clear zones was indicative of antifungal activity A negative control was prepared by dissolving 0 25 ml of a surfactant, CELNON 80TW, 5 ml of ethanol and 0 5 ml of marine flavor m a solvent, water, to give a total volume of 100 ml The results are given m Table 11, below

TABLE 11

Samples Antifungal activity (+ antifungal - not antifungal;

As shown xn Table 11, all test samples excluding the negatxve control and ethanol samples were found to have excellent antifungal effects

EXAMPLE 12 Evaluation of antimicrobial effects of nanosized silica silver on wallpaper

In order to determine whether nanosized silica silver has an ability to prevent microbial contamination of highly humid indoor wallpaper, an antimicrobial activity assay was carried out using a filter paper The antimicrobial effects of nanosized silica-silver were compared to those of commercially available antimicrobial products

A filter paper 70 mm in diameter was sprayed with a PD (potato dextrose) broth containing numerous microorganisms isolated from a dust filter of an indoor air conditioner The filter paper was placed into a petri dish and then dried at room temperature in an open state to affix microorganisms thereto The nanosized silica-silver (NSS) solutions, prepared according to the same method as in Example 1 were diluted with a high levan solution by 100, 500 and 1000 times Each dilution was sprayed onto the filter paper inoculated with microorganisms The filter paper was then semi dried at room temperature Commercial comparative products, A (nano silver photocatalytic capsule perfume in pure water) and B (pure nano silver solution) , were directly sprayed with no dilution, followed by semi-drying at room temperature

Each Petri dish containing the semi dried filter paper was placed into a rectangular tray (280x250x50 mm) in which a wet paper was placed on the bottom, and was incubated at room temperature under humid conditions On Day 7, the filter paper was captured by a camera, and emerged colonies were counted Also, because colonies were poorly visible on the wet filter paper, the filter paper was completely dried for 3 days m order to make an exact count of the colonies A disk was cut from the middle of the 70 mm-diameter filter paper using a cork borer 14 2 mm m diameter, and was subjected to colony counting The results are given m Table 12, below and Fig 11

TABLE 12

When the inoculated filter paper was treated with sample A containing a natural perfume, no microbial colonies were observed for an initial period of 3 to 4 days when the perfume was released, but microbial contamination rapidly increased when the perfume disappeared. Sample B started to increase microbial contamination at the similar time point as the control, and its antimicrobial activity lasted for a period similar to sample A. In contrast, the nanosized silica-silver exhibited antimicrobial activity greater than 90% even when diluted by 100 and 500 times. Since filter papers have an uneven surface and hygroscopic properties, the nanosized silica-silver contacts microorganisms for a short period of time in the end of fibers and thus has low antimicrobial effects. However, since wallpaper actually is lower hygroscopically than filter paper, the nanosized silica silver may display much higher antimicrobial effects when applied to wallpaper.

Industrial Applicability

As described hereinbefore, the antifungal spray composition comprising nanosized silica-silver particles, in which nano-silver is bound to silica molecules and a water-soluble polymer, the nanosized silica-silver particles prepared by irradiating a solution comprising a silver salt, silicate and the water-soluble polymer with radiation rays, has antimicrobial effects when sprayed onto a surface contaminated wxth microorganisms, and thus can provide non-contaminated clean environment.

Claims

1 An antimicrobial spray composition comprising nanosized silica-silver particles 0 5 to 30 nm m size, m which nano-silver is bound to silica molecules and a water- soluble polymer, the nanosized silica silver particles prepared by irradiating a solution comprising a silver salt, silicate and the water-soluble polymer with radiation rays

2 The antimicrobial spray composition as set forth in claim 1, wherein the nanosized silica-silver is contained m a concentration of 0 1 to 100 ppm

3 The antimicrobial spray composition as set forth m claim 1, further comprising a surfactant

4 The antimicrobial spray composition as set forth in claim 3, wherein the surfactant is contained in a surfactant nanosized silica silver weight ratio of 0 2 to 20 1

5 The antimicrobial spray composition as set forth in claim 3, further comprising an aromatic agent and an alcohol

6 The antimicrobial spray composition as set forth in claim 5, which comprises nanosized silica silver, a surfactant selected from among sorbitan monooleate and polyethylene glycol, an alcohol selected from among ethanol, methanol and isopropanol, and an aromatic agent

7 The antimicrobial spray composition as set forth m claim 6 wherein the nanosized silica silver, surfactant alcohol and aromatic agent are contained in a weight ratio of 1 0 5 to 20 0 5 to 30 0 125 to 5

8 A spray formulation formulated from the antimicrobial spray composition of claim 1

9 A method of preparing an antimicrobial spray composition comprising nanosized silica-silver particles 0 5 to 30 nm in size, comprising

(A) preparing a solution containing a silver salt silicate and a water soluble polymer, and

(B) irradiating the solution with radiation rays

10 A method of antimicrobially treating a surface needing to be antimicrobially treated, which comprises spraying the spray formulation of claim 8 onto the surface