WO2016122995A1

WO2016122995A1 - Antibacterial composition of silver nanoparticles bonded to a dispersing agent

Info

Publication number: WO2016122995A1
Application number: PCT/US2016/014665
Authority: WO
Inventors: Zoe Vineth Quinones JURADO; Miguel Angel Waldo MENDOZA; Luis Manuel Cespedes COVARRUBIAS; Hugo Marcelo Aguilera BANDIN; Jose Elias Perez LOPEZ
Original assignee: A Schulman Inc
Current assignee: Lyondellbasell Advanced Polymers Inc
Priority date: 2015-01-26
Filing date: 2016-01-25
Publication date: 2016-08-04
Anticipated expiration: 2017-07-26
Also published as: CL2017001911A1; MX2015001206A; CO2017008550A2; GT201700165A; PE20180159A1

Abstract

The present invention refers to an antibacterial composition against Gram positive and Gram negative bacteria, which contains silver nanoparticles (Ag NPs) with average diameter of approximately less than 15 nm that have a photocatalytically inactive surface, present in a range between 8.5 and to 70% in weight regarding the total composition, in which Ag NPs are the antibacterial agent that remains homogeneously exposed by a ceramic dispersing agent like TiO₂ and its bactericidal activity is generated in presence of UV-visible light and/or in the dark.

Description

ANTIBACTERIAL COMPOSITION OF SILVER NANOPARTICLES BONDED TO A DISPERSING AGENT

TECHNICAL FIELD OF THE INVENTION

The present invention relates to an antibacterial composition containing silver nanoparticles (Ag NPs) bonded to the surface of a ceramic substrate dispersing agent, in which Ag NPs have a photocatalytically inactive surface, being bactericidal in the dark as well as also under Ultraviolet and visible light (UV-vis).

BACKGROUND

It is known in the state of the technique that nanoparticles of noble metals have more reactivity than such metals in massive state; this is due to their large surface and to the increment in electrical properties.

Silver compositions commonly used as antimicrobial are silver sulphadiazine (CioH9N402SAg), silver acetate (AgCH3COO), silver nitrate (AgN03), other silver salts, or other compositions based on silver ions caught in vitreous structures, of zeolites, clays, etc. According to that reported in the state-of-the-art, metallic silver as nanometric size particles (Ag NPs) has shown better antimicrobial capacity, and even authors like S. Pal et al. [Applied and Environmental Microbiology, 73 (2007) 1712-1720] and N. Ayala et al. [Nanobiotechnology, 5 (2009) 2-9], have referred that at a smaller size of the particle, the superficial area increases and consequently, a greater antimicrobial effect is developed.

In documents MX NL a/2006/000107 and EP 2103364 (R. Benavides et al.), reference is made to the process of obtaining Ag NPs by means of the reduction of silver ions in humid medium in order to manufacture nanoparticles with average sizes from 10 to 20 nm, process in which the particle size is controlled by adjusting the pH to alkaline

values of up to 11.5. If the alkaline control^" is not achieved during the process, particles with average size over 20 nm would be obtained, which is considered as non-desirable because the bactericidal function would be reduced due to a bigger particle size. The presentation of the Ag NPs consists of a humid paste of flocculated particles and their re-dispersion with another material will depend on the mixing by means of mechanical work.

Additional to the effect of the small size of Ag NPs, it is important to avoid their agglomeration, otherwise the superficial area and its bactericidal effect is reduced; for this can be used surfactants and stabilizers.

In order to not affect the high superficial area of Ag NPs due to their tendency to agglomerate, our invention gives outstanding attention to the Ag NPs pre-dispersion by means of their formation on a ceramic substrate (composition where silver is "supported" on the ceramic surface) and this avoids the use of surfactants or additional mechanic mixing to maintain their dispersion. A composition is known as "supported, decorated or core-shell" when atoms of a constituent are adhered to the exterior of a support substrate, either in form of coating or of particles with nanometric dimension homogeneously distributed on the support.

At present time, materials consisting of nanoparticles of noble metals "supported" on the surface of semiconductor materials like titanium dioxide (TiO.) have attracted the attention in the fields of photocatalysis and microbiology; . this is because when both components are bonded, these increase their reaction capacity by means of a greater mobility of electrons, which may be excited with Ultraviolet and visible light (UV-vis) [J Keleher et al., J Microbiol Biotechnol (2002) 133-139]; A. Ashkarran et al. Curr Appl Phys. (2011) 1048-1055], [A. Ashkarran et al. Curr Appl Phys. 11 (2011) 1048-1055]. Excitation can be understood as an elevation in the energy level of a physical system having as consequence an increase of reactivity, mobility, etc. For example; an atom or

any other system can get excited by absorption of photons with a characteristic frequency, or also by means of heat or electricity.

So far, antibacterial compositions of noble metals supported to a semiconductor like Ag/Ti02, require photo-excitation to perform efficiently as bactericidal, however, these are not effective in the dark and require to be supplemented with other antibacterial substances. For example, in the document of patent KR20040001410, the Ag/Ti02 photocatalytic compound is combined with zinc chloride plus hydroxyapatite and in the patent JPH11192290 other metallic oxides such as silica are combinedr

Following are mentioned some documents of patents that protect Ag NPs compositions bonded to a ceramic material with bactericidal capacity, where their antibacterial effect is limited to applications where Ultraviolet and visible light (UV-vis) require to be irradiated to activate reactivity.

• The patent application MX MX/a/2010/010434 refers to a Ag/Ti02 antibacterial compound "supported" by way of aqueous suspension and whose silver nanoparticle size is controlled through the use of Gallic acid in dimensions between 20-60 nm, in which the Ag NPs content that is supported, is only of approximately 5% of the weight in order to maintain the stability of the suspension of the Ag/Ti02 compound, and therefore, given the low Ag NPs content, it may not perform as bactericidal under non-photocatalytic conditions.

• Document KR20090035812 describes an antimicrobial that consists of the combination of metallic nanoparticles or metallic oxides encapsulated with ΤΊΟ2 for photocatalytic use. This composition could only develop bactericidal activity by the generation of reactive species of (ROS) oxygen when there is excitation with UV/visible light.

• Document R0126368 refers to silver nanostructured compounds supported on oxides like ZnO and ΤΊΟ2, with a silver weight content of less than 6% and with antimicrobial activity based on the photocatalytic effect.

• Document KR20010057595 refers to a silver-coated T1O2 antibacterial photocatalyst, with a content of silver less than 5% in weight regarding the total composition, which is a low content to perform efficiently as bactericidal in the dark.

The present invention refers to an antibacterial composition based on Ag nanoparticles "supported" to a ceramic substrate, photocatalytic (semiconductor material) or non photocatalytic, in which Ag NPs have a photocatalytically inactive surface being bactericidal in the dark, as well as under Ultraviolet and visible light (UV- vis).

OBJECTS OF THE INVENTION

In this invention, the use of an adherent substrate of the silver nanoparticles (Ag NPs) helps to prevent their agglomeration, besides optimizing the exposition of the surface area of such Ag NPs, giving as a result that the substrate used in the present invention performs as dispersing agent to ensure greater contact of the antimicrobial active (Ag NPs) with the possible bacteria during its application. The substrate or dispersing agent comprises one or more of the ceramic particles selected among the semiconductors such as Titanium dioxide (T1O2), Zinc oxide (ZnO), Silicon dioxide (S1O2) or even particles of non-semiconductor ceramics such as; Zirconium dioxide (Zr02), Aluminum trioxide (AI2O3), aluminosilicates (S1O2.AI2O3), Tin dioxide (Sn02), Tin oxide (SnO), Copper oxides (CuO, CU2O), Antimony oxide (Sb,Os), Beryllium oxide (BeO), Tellurium oxide (Te02), Indium oxide (Ιη2θ3), Gallium oxide (Ga203), and their respective hydroxides.

The present invention refers to an antibacterial composition containing silver nanoparticles (Ag NPs) "supported" to the surface of a dispersing agent having bactericidal action against Gram negative and Gram positive bacteria, which does not require photo excitation to destroy bacteria, since neither the silver nanoparticles (Ag NPs) or the dispersing agent are of interest by their photocatalytic activity, because their performance is required without the condition of photo excitation. The antibacterial agent (Ag NPs) is characterized to have a photocatalytically inactive surface under visible light.

BRIEF DESCRIPTION OF THE INVENTION

The medical, hygienic industry and of materials destined for auto-cleaning applications have started to be interested in antibacterial compositions of lingering activity and based on products that have not created resistance in bacteria.

The synergy achieved with the bond of Ag NPs with semiconducting materials such as T1O2 is very promissory due to its photocatalytic contribution; however, the technical problem of these compounds is that their bactericidal activity is limited to applications that receive Ultraviolet and visible light (UV-vis). Added to this, the present invention demonstrates that visible light (vis) does not stimulate photocatalytically the different types of silver "supported" compounds, neither to the Ag NPs with an average diameter of approximately less than 15nm, since in so small Ag NPs diameters, the electronic reactivity is confined on the surface making them passive and not reactive, and therefore photocatalytically inactive under visible light (Figure 4).

The present invention is related to an antibacterial composition containing silver nanoparticles (Ag NPs) bonded to the surface of a dispersing agent, characterized because Ag NPs have a high superficial area (Figure 3), but not a photocatalytically surface (Figure 4).

This last characteristic differs to that previously quoted in the state-of-the-art, on generalizing that the bactericidal effect of the nanoparticles of noble metals increases at a higher size reduction, because although the superficial area of the particle increases to ensure greater contact of the surface with the bacteria, the chemical reactivity derived from the electric properties on the surface gets lost in nanometric size particles with average diameter of approximately less than 15 nm.

The antibacterial composition of the present invention mainly acts through the mechanism of permeability and breaking of the cellular membrane at the contact with the antibacterial agent (Ag NPs), in which the surface of silver nanoparticles (Ag NPs) is characterized to have a photocatalytically inactive surface under visible light.

In this invention it is also demonstrated that the antibacterial composition constituted by Ag NPs of passive character must be constituted with a content of 8.5% or of up to 70% in weight regarding the total composition to ensure the bactericidal character in the dark, as well as also under Ultraviolet and visible light (UV-vis).

When photocatalytically inactive Ag NPs are exposed and homogeneously distributed on the ceramic substrate generate bactericidal activity against Gram negative and Gram positive bacteria, whether under the dark and/or with Ultraviolet and visible light (UV-vis). The antibacterial composition is compatible to be mixed with polymer, adhesive, metal and ceramic materials, for applications as for example: materials suitable for medical products, surfaces of appliances, in ventilation systems or air purification, carpets, synthetic grass, paper, fibers for textiles, non-woven fabric, articles in contact with food, kitchen utensils, plastic films, toilet articles, etc.

BRIEF DESCRIPTION OF THE FIGURES

FIGURE 1. It shows a qualitative spectrum of the elemental chemical analysis of the Ag Ti02 "supported" compound corresponding to the formulation 14, obtained by energy-dispersive X-ray spectroscopy (EDX). In axis "X" is shown the energy of the photon emission (keV) and in T the counting of each emitted radiations.

FIGURE 2. It shows a micrograph of the silver nanoparticles compound (Ag NPs) deposited on the surface of the titanium dioxide (T1O2) dispersing agent, corresponding to the Ag/Ti02 "supported" compound obtained from formulation 1 , which does not show bactericidal activity in the dark. Obtained through transmission electron microscopy (TEM), seen at 100000x magnification.

FIGURE 3. It shows a micrograph of the silver nanoparticles compound- (Ag NPs) deposited on the surface of the titanium dioxide (T1O2) dispersing agent, which shows bactericidal activity in the dark. The Ag/Ti02 "supported" compound corresponds to formulation 14. The micrograph was obtained through transmission electron microscopy (TEM), seen at 100000x magnification.

FIGURE 4. It shows the absorption spectra of Ultraviolet and visible light (UV-vis) of Ag/Ti02 "supported" compound, with different content in weight percentage of the Ag NPs in a range approximately between 1.5-5%, obtained from formulation 3, formulation 6 and formulation 7, that correspond to the compound; (1.5Ag/Ti02), (4Ag/Ti02) and (5Ag Ti02), respectively, using as controls both the pure titanium dioxide T1O2 spectrum and the spectrum of the pure silver nanoparticles Ag NPs. In axis "X" is shown the excitation energy of the different materials (nm) and in axis Ύ" is shown the intensity of absorbed energy (L mo crrr¹).

DETAILED DESCRIPTION OF THE INVENTION

In the following description of the invention this terminology will be mentioned;

• Antibacterial composition: Substance used in the control of the growth and/or destruction of bacteria.

• Bactericidal: Antibacterial substance able to kill bacteria

• "Supported" compound. Material consisting of a substrate to which another compound is externally adhered in form of coating or particles with nanometric dimension.

• Substrate or dispersing agent: Surface of the ceramic particles used as dispersing agent of the silver nanoparticles.

• Silver nanoparticles (Ag NPs): Metallic silver particles of nanometric size.

• Photocatalytic activity / photocatalysis: Effect on the electrons mobility in certain substances when exposed to the light, as for example; when titanium dioxide (T1O2) is irradiated with Ultraviolet light (UV), it increases the electrons mobility.

• Plasmon Effect: Is the phenomenon of the collective oscillation of electrons maintained on the surface of a metal; it originates by the incidence of photons that remain caught by free electrons and generates more reactivity on the metal surface.

The present invention is related to an antibacterial composition that comprises an antibacterial agent consisting of silver nanoparticles (Ag NPs) which show a quasi- spherical structure and are bonded to the surface of a dispersing agent that prevents

their agglomeration. In particular, the present invention refers to antibacterial composition in which silver nanoparticles (Ag NPs) have an average diameter approximately smaller than 15 nm; which makes them photocatalytically inactive due to a confinement of electrons on the surface, related to the small size of the Ag NPs; likewise, Ag NPs do not form agglomeration, that is to say, these are dispersed and homogeneously distributed on the on the surface of the dispersing agent.

In particular, in this invention, the ceramic substrates is are not used by the photocatalytic activity that it may develop under UV light, but these are interest as dispersing agent of the silver nanoparticles (Ag NPs), since they behaves as a substrate with capacity to adhere Ag NPs in a massive and homogeneous way. The route through which Ag NPs is formed is by means of the attraction of positive silver ions in the places with Oxygen vacancies (O-) of the ceramic substrate, followed by the size growth of the Ag NPs due to the accumulation of silver atoms through a chemical reduction reaction. In such a way that the antibacterial agent is homogeneously supported on the surface of the ceramic substrate to favor the contact of the Ag NPs with the bacteria, in which such particles are characterized to have an average diameter of approximately less than 15 nm, as shown in figure 3.

More specifically, the invention is defined to be an antibacterial composition against Gram positive bacteria such as Staphylococcus aureus, Staphylococcus epideimidis, Staphylococcus saprophyticus, Enterococcus, pyogenes, Enterococcus faecalis, Lactobacillus sp. and Gram negative, such as Escherichia coli, S., Salmonella, Pseudomonas aeruginosa, among others. For being an antibacterial composition with bactericidal effect independent of the luminous excitation of Ultraviolet-visible light. '

The invention of the antibacterial composition has preferably a concentration of Ag NPs with average diameter approximately lower than 15 nm in the range between approximately from 8.5 to 70% in weight regarding the total composition.

The antibacterial composition of the present invention provides a bactericidal effect through the permeation in the cellular membrane of bacteria, this caused by the effect of the high exposition of the superficial area of the silver nanoparticles (Ag NPs) and, additionally by the electrostatic destabilization of the cellular membrane to the Gram negative bacteria, caused by the presence of ionized silver.

With the aim of having a deeper comprehension of the present invention, some examples are detailed, without being limitative to the invention.

[EXAMPLES

Example lObtention of the antibacterial composition using a T1O2 substrate

The antibacterial composition is constituted with the Ag Ti02 "supported" compound, which is elaborated in an aqueous medium by means of the adhesion of Ag NPs to the titanium dioxide (T1O2) surface, this upon the Oxygen vacancies (0-) in the interface of the T1O2 substrate. Without being limitative of the invention, Table 1 shows different formulations used to obtain the Ag/Ti02 "supported" compound.

Table 1 shows examples of formulations used to obtain the Ag/TiC "supported" compound, in which the numeric value preceding the nomenclature Ag/Ti02, refers to the maximum percentage in weight of the silver to be supported. starting from the AgN03 precursor and, for some compounds is compared with the real content of Ag NPs that could be supported.

Identification Ag NPs

Content of the Content of the of obtained Real silver precursor dispersing agent

Formulation compositions content

(AgNC ) (TiOj )

AgfTi02

[%weight] [%weight] [% weight]

1 0.8 99.2 0.5Ag TiO2 0.3

2 1.6 98.4 1 Ag/Ti02 -

3 2.3 97.7 1.5 Ag/Ti02 1

5 4.6 95.4 3 Ag/TiCte -

6 6.2 93.8 4 Ag Ti02 2.9

9 10.6 89.4 7 Ag/Ti02 -

10 12.0 88.0 8 Ag Ti02 -

11 13.5 86.5 9 Ag/TiCte -

12 14.9 85.1 10 Ag Ti02 8.5

13 21.7 78.3 15Ag/Ti02 -

14 34.4 65.6 25Ag/Ti02 14.1

15 45.9 54.1 35Ag/Ti02 16.9

16 56.3 43.7 45Ag/Ti02 18.5

17 65.8 34.2 55Ag/Ti02 18.1

Example 2. Qualitative spectrum of the elemental chemical analysis of the Ag Ti02 "supported" compound, obtained by energy-dispersive X-ray spectroscopy (EDX).

Figure 1 shows a qualitative spectrum of the elementary chemical analysis acquired by energy-dispersive X-ray spectroscopy (EDX), corresponding to the 25Ag/Ti02 compound, obtained from the formulation 14 identified as one of the "supported" compounds that showed bactericidal activity without the photo-excitation of (UV - vis) light. In axis "X" is shown the energy of the photon emission (keV) that characterizes the chemical elements and in "Y" the counting of each emitted radiation. This X-ray emission spectrum indicates the X-ray energy of each one of the elements

present in the sample, demonstrating with it that the antibacterial composition is formed from the element of Silver (Ag) and Titanium (Ti). The Copper (Cu) sign is due to the grate in which the compound was supported for its analysis.

Example 3. Comparative of the structure of Ag/Ti02 "supported" compound with and without bactericidal activity in the dark, seen at 100,000x magnification.

Figures 2 and 3 show the images of the Ag/Ti02 "supported" compound structure, which is formed from quasi-spherical silver nanoparticles (Ag NPs) with an average diameter approximately smaller than 5 nm, bonded on the titanium dioxide (T1O2) surface, where T1O2 is present as a substrate or dispersing agent that supports Ag NPs keeping them separate, thus achieving the exposition of the superficial area of the silver nanoparticles (Ag NPs), and therefore providing more contact of the antibacterial agent consisting of (Ag NPs) with the bacteria during its application.

Without being limitative of the invention, by way of example, Figure 2 presents the structure of the 0.5Ag TiO2 compound obtained with formulation 1 , which does not have bactericidal activity in the dark due to the low content of Ag NPs on the T1O2 dispersing agent, and it is compared with the structure of the 25Ag/Ti02 compound obtained from formulation 14 (Figure 3), in which occurred bactericidal activity in the dark; this last one is formed from the T1O2 dispersing agent with 14.1 % in weight of the Ag NPs homogeneously distributed.

Example 4. Photocatalytic activity of the Ag/Ti02 "supported" compound in conditions of Ultraviolet radiation and visible light.

Figure 4 shows the absorption spectra of Ultraviolet radiation and visible light (UV-vis) of the Ag Ti02 "supported" compounds, obtained from formulations 3, 6 and 7 identified as 1.5 Ag Ti02, 4 Ag/TiCte and 5 Ag Ti02, using as controls both the pure titanium dioxide T1O2 spectrum and the silver nanoparticles Ag NPs spectrum. In the

axis "X" is shown the wavelength corresponding to the excitation energy (nm) and in the axis "Y" is shown the intensity of absorbed energy (L mol-'cnr¹).

Following is explained how ultraviolet and visible light are related in the electromagnetic spectrum with the photocatalytic activity of the Ag/TiCte "supported" compound, as well as of the pure ΤΊΟ2 used as dispersing agent and of the Ag NPs used as antibacterial agent. The electromagnetic spectrum is the set of wavelengths of the whole electromagnetic radiation; it is formed by gamma rays, X-rays, ultraviolet radiation, visible light and infrared radiation, and others of greater wavelength. The wavelength is defined as the distance that energy travels in a time equivalent to a period. An electromagnetic wave is formed by photons. The energy of each photon is directly proportional to the frequency of the wave. The higher the frequency is the larger is the quantity of energy contained in each photon. It happens that the frequency increases as the wavelength decreases, and vice versa.

The absorption spectra of UV-vis light that form the image of Figure 4 show that in pure state, T1O2 presents photocatalytic activity in the Ultraviolet range and the Ag NPs in the range of visible light. The bond of Ag NPs with average diameter approximately smaller than 15 nm to the T1O2 surface in the Ag/Ti02 "supported" compound modifies the energy of the frequency so that T1O2 photocatalysis may occur. The T1O2 dispersing agent changes the absorption of energy of a 283.5 nm wavelength to a value of 334 nm, reason why it is required the absorption of a frequency of less energy to be photocatalytic. This wavelength sliding indicates the formation of an interface between Ag NPs and T1O2, where there is an increment in the mobility of the electrons, just as it has been reported in the literature and as it is shown in the absorption spectra of UV-vis light of the compounds; 1.5 Ag/Ti02, 4 Ag/Ti02 and 5 Ag/Ti02 as shown in Figure 4.

The type of Ag Ti02 antimicrobials based on the photocatalytic technology presents an electromagnetic phenomenon characteristic of Ag NPs, in which, when being irradiated in the range of the visible spectrum, the reactivity by the mobility of

electrons on the surface increases (Plasmon effect), just as it can be seen in Figure 4 for the spectrum of pure Ag NPs silver nanoparticles. However, when comparing the photocatalysis of Ag NPs with average diameter of approximately less than 15 nm "supported" in the Ag/Ti02 compound, it is shown that there is a confinement in the photocatalytic activity of the Ag NPs, that is to say, the photo-activity gets lost with the visible light radiation, because these did not show to have enough electrons mobility to generate plasmon resonance in the range of the visible light at the 400 nm wavelength.

Example 5. Microbiological analysis to determine the minimum bactericidal concentration (MBC) under the dark of the Ag TiCte "supported¹' compound.

The minimum bactericidal concentration (MBC) corresponds to the smallest concentration capable to kill 99,9% of bacterial population. The antimicrobial susceptibility testing consisted of a dilution method based on the NCCLS International Standards.

The bactericidal activity of the Ag/TiCte "supported" compounds is mentioned in Table 1 in which the Example 1 was evaluated by means of the study of the minimum bactericidal concentration (MBC) and with it determined the minimum percentage of the Ag NPs mass with average diameter approximately smaller than 15 nm that should be adhered to the T1O2 dispersing agent, and the minimum concentration of the Ag/Ti02 "supported" compound in which the antibacterial composition acts as bactericidal in the dark and, consequently, under Ultraviolet and visible light (UV-vis).

A Luria Bertani type liquid medium was prepared to cultivate bacteria: 1.0% of tryptone, 0.5% of yeast extract and 1.0% of NaCI. The culture medium was placed in test tubes to a volume of 3mL and it was mixed with the Ag/Ti02 compound in quantities from 0.5 - 20.0 mg/mL, respectively. In order to estimate the influence of pure T1O2 on the bacteria growth inhibition, control tests were parallel performed at these same concentrations of that tested for the nanocomposition. Titanium dioxide (T1O2) used as

dispersing agent was also analyzed in the same concentration range to determine the antibacterial capacity it contributes in a dark environment. The mixtures contained in the test tubes were sterilized at 121°C during 15 minutes. Inoculum were prepared in liquid medium of Gram negative bacteria; Salmonella sp; Escherichia coli ATCC 25922 (American Type Culture Collection, Rockville, Md.), as well as of the Gram positive bacteria Staphylococcus aureus ATCC 25923. Bacteria were added to the culture medium mixtures and the different Ag/TiCte compounds, obtained from the formulas presented in Table 1, maintaining a concentration between 10⁷ and 10⁶ CFU/mL respectively. Afterwards, bacteria were incubated in the dark, during the night inside an autoclave, by 10 h at 37°C arid 150 rpm. For each strain was performed the MBC analysis, by means of massive striation in Luria Bertani Agar plates with 100 L of sub- cultivation of each tube. The plates were incubated during 24 h at 37°C to determine the final points of the minimum bactericidal concentration MBC, which is a measure indicating the use of the smallest concentration of the Ag/TiCte "supported" compound for not having presence of bacteria after the incubation. The analysis was made by triplicated for each sample in the different concentrations of 0.5 - 20.0 mg/mL. The result is expressed with a positive sign (+) when there was presence of bacteria growth; and with the sign (-) to indicate the absence of bacterial growth, validating with this a range of compounds and the concentration in which the "supported" Ag/TiCb can be considered as an effective antibacterial composition under the dark and independent of the UV and visible light radiation.

Example 6. Bactericidal activity in the dark of the T1O2 dispersing agent

The MBC microbiological analysis for pure titanium dioxide (Ti02) used as dispersing agent was performed to determine the bactericidal capacity contributed by this dispersing agent in the dark or visible light. As a result it was obtained that within the concentration range of 0.5 - 20.0 mg/mL there was bacteria growth. That corroborates the absorption spectrum of UV-vis light of the Figure in the example 4, where the T1O2 substrate could only have bactericidal activity when exposed to UV radiation.

Table 2. Qualitative evaluation of the growth of bacteria in presence of the T1O2 substrate used as dispersing agent of Ag NPs.

Escherichia Salmonella

Staphylococcus

coli sp

Concentration aureus

Dispersing agent

(mg/mL)

(Gram (Gram

(Gram positive)

negative) negative)

T1O2 0.5 + + +

Ti0₂ 1 + + +

Tio₂ 3 + + +

Ti0₂ 5 + + +

Ti0₂ 8 + + +

^"ΠΟ2 10 + + +

Ti0₂ 15 + + +

Ti0₂ 20 + + +

(+) Presence of bacterial growth; (-) Absence of bacterial growth

Example 7. Ag/TiCte "supported" compounds that did not show bactericidal activity in the dark.

In absence of Ultraviolet radiation and visible light (Uv-vis) at concentrations in the range of 0.5 to 20.0 mg/mL, the Ag Ti02 compounds formed with a content of silver nanoparticles (Ag NPs) lower than 8.5% in weight with regard to the total of the composition were not able to destroy bacteria such as: Salmonella; Escherichia coli and Staphylococcus aureus. This indicates that the silver nanoparticles (Ag NPs) photocatalytically inactive should be present in a quantity enough to perform in sharp cutting way to reach permeability and breaking of the bacteria membrane and achieve their complete elimination.

Table 3. Compositions formulations that do not have bactericidal activity in the

Staphylococcus Escherichia Salmonella

Concentration

Formulation aureus coli sp

(mg/mL) (Gram (Gram

(Gram positive) negative) negative)

1 0.5 - 20.0 + + +

2 0.5 - 20.0 + + +

3 0.5 - 20.0 + + +

4 0.5 - 20.0 + + +

5 0.5 - 20.0 + + +

6 ^•0.5 - 20.0 + + +

7 0.5 - 20.0 + + +

8 0.5 - 20.0 + + +

9 0.5 - 20.0 + + +

10 0.5 - 20.0 + + +

11 0.5 - 20.0 + + +

(+) Presence of growth; (-) Absence of growth

Example 8. Ag Ti02 "supported" compounds with bactericidal activity even in the dark.

In the dark, Ultraviolet and visible light, the Ag/TiC compounds with a minimum Ag NPs percentage of 8.5% in weight with regard to the total of the composition present bactericidal activity, even using a dosage of 10 mg/mL they eradicate bacteria cultures concentrated between 10⁷ to 10⁶ CFU/mL. Without restricting the invention, five Ag Ti02 "supported" compounds are shown in Table 4 as example of bactericidal compositions

capable to act in the dark against Gram negative bacteria such as: Salmonella Escherichia coli and Gram positive bacteria, as for example: Staphylococcus aureus.

Table 4. Examples of the antibacterial composition dosage required in different formulations to originate bactericidal activity in the dark

Staphylococcus Escherichia Salmonella sp

Formulation Concentration aureus cotf

(mg/mL)

(Gram positive) (Gram negative) (Gram negative)

12 4 + + +

5 + + +

6 + + +

7 + + -

8 + +

9 + - -

10 - - -

11 - - -

14 4 + + +

5 + + +

6 + + +

7 + - -

8 + - -

9 + - -

10 - - -

11 - -

11 - - -

15 4 + + +

5 + - +

6 + +

7 +

8 +

9 +

10 -

11 -

16 44 ++ +

5 + +

6 + +

7 +

8 +

9 +

10 -

11 -

17 44 ++ - +

5 + +

6 + +

7 +

8 +

9 +

10 -

11 -

(+) Presence of bacterial growth; (-) Absence of bacterial growth

The five Ag/TiCte "supported" compounds shown in Table 4 are formed with Ag NPs with an average diameter of approximately less than 15nm, just as it is illustrated in Figure 3 for the Example 3. Ag NPs with that diameter and adhered to the T1O2 substrate are photocatalytically inactive as shown in Figure 4, so these require to be present in the antibacterial composition at a concentration in the range of approximately 8.5 to 70% in weight with regard to the total of the composition to generate bactericidal

activity. Therefore, it is demonstrated that the exposition of the Ag NPs mass is decisive to achieve the destruction of bacteria by contact with the antibacterial agent that consists of Ag NPs with average diameter approximately smaller than 15nm.

Table 4 shows how the Gram negative and positive bacteria culture, concentrated between 10⁷ to 10^e CFU/ml, is eradicated with a minimum bactericidal concentration ( BC) of 10 mg per mL of the culture, this using an antibacterial composition that fulfills the minimum content of 8.5% in weight with regard to the total Ag NPs composition with average diameter approximately lower than 15 nm. Such antibacterial composition generates more destruction of Gram negative bacteria, for example the MBC value against Gram negative Salmonella bacteria is of 7 mg/mL and with the Gram negative Escherichia coli is achieved the MBC value with up to 4 mg/mL of an antibacterial composition having a concentration of 18.5% in weight regarding the total of the composition. There is clearly present an increment of the antibacterial activity at the contact with the Gram negative bacteria, this due to an effect of silver ionization from the surface of the Ag NPs. For example, with Escherichia coli it occurs that by increasing from the total of the antibacterial composition the content from 8.5% up to 18.5% in weight the Ag NPs with average diameter approximately smaller than 15 nm, it is required a lower MBC value, reducing the required concentration of the antibacterial composition from 10 mg/mL to 4 mg/mL, as it is shown in Table 4.

Example 9. Substrates that can be used as Ag NPs adhesion and dispersing agents.

The dispersing agent or substrate that can be used as support to the (Ag NPs) antibacterial agent are particles of semiconductors such as Titanium dioxide (T1O2), Zinc oxide (ZnO), Silicon dioxide (S1O2) or even particles of non-semiconductor ceramics such as Zirconium dioxide (ZrCte), Aluminum trioxide (AI2O3), aluminosilicates (S1O2.AI2O3), Tin dioxide (SnC ), Tin oxide (SnO), Antimony oxide (Sb^Os), Beryllium

oxide (BeO), Tellurium oxide (Te02), Indium oxide (ItoCb), Copper oxide (CuO, CU2O), Gallium oxide (GazOs), and their respective hydroxides.

The common characteristic of the support is that these are materials that may have an electro-attractive surface to positive ions (for example, silver ions). The attraction of silver ions and formation of silver nanometric particles on the dispersing agent originates starting from vacancies (0-) formed by the deprotonation of hydroxyl groups (-OH) of the surface of the substrate. Therefore, the formation of silver nanometric particles on the dispersing agent depends on the alkaline condition able to promote a larger number of vacancies (0 ) on the surface of the substrate.

The condition of the hydrogen potential (pH) during the reduction-deposition reaction during the formation of the composition that consists of the dispersing agent supporting Ag NPs with average diameter approximately lower than 15 nm will be characteristic for each type of substrate. For example, Table 5 shows the relation of the pH value required to generate the largest deprotonation of the surface of some metallic oxides and hydroxides mentioned as part of the invention.

Table 5. Condition of pH required with the use of different ceramic substrates for the massive adhesion of Ag NPs.

Substrate type pH

SiOz 10

T1O2 12,

ZrOz 10

CeC-2 11

AI2O3 11

CuO 11

ZnO 12

Example 9. Efficacy of antibacterial composition on Surface of antimicrobial-plastic

The plastic films were fabricated with a material of polyethylene and the antimicrobial composition at a concentration of 100 to 600 ppm, using a melt blender and film extruder (Killion model D.S. Winder).

Antimicrobial composition performance integrated into a matrix that require antimicrobial activity, will be dependent of the de-agglomerating distribution and uniform dispersion.

Results of antimicrobial performances test for it surface types (Japanese Industrial Standard JIS Z 2801) is indicated at the table 6.

Table 6. Percent reduction of incubated e. coli on the plastic surface

Standard JIS Z 2801 specifies the efficacy on bacteria on the surface of antimicrobial products

Procedure:

The inoculum was prepared using Escherichia coli ATCC # 8739. Dilute nutrient broth prepared as described in the test method was used to further dilute the inoculum to a target starting concentration of 2.5- 0 x 10⁵ CFU/mL. The untreated controls were tested in triplicate at Time = 0 and Time = 24 hours to establish organism viability. The treated samples were tested in triplicate at Time = 24 hours. Each sample piece was placed in a sterile Petri dish and then was inoculated with 0.4 mL of the inoculum. The inoculum was then covered the sterile plastic in order to spread the inoculum evenly over the sample surface and hold it in place.

The samples and controls were incubated for 24 hours at 35°C and a relative humidity of at least 90%. At the appropriate time, 10.0 mL of neutralizing broth (SCDLP) was added to the Petri dish. The Petri dish containing the test pieces and the SCDLP was then placed onto a shaker and mixed thoroughly to facilitate the release of the inoculum from the sample surface into the neutralizing broth. Serial dilutions of the neutralizing broth containing the inoculum were plated. All plates were incubated at 35°C for 24- 48 hours. After incubation, bacterial colonies were counted and recorded.

Results can be found in the data tables below. The results pertain only to samples tested.

The number of viable bacteria in the test inoculum was 6.8 x 10⁵ CFU/mL. This is the initial number of bacteria of the starting inoculum.

The value of the antimicrobial activity was calculated according to the formula listed below and recorded as log reduction.

R = ( Ut - Uo) - (A_t - Uo) = Ut - At

Where,

R: antimicrobial activity Uo : average of logarithm numbers of viable bacteria from untreated control at Time= 0

Ut: average of logarithm numbers of viable bacteria from untreated control at Time = 24 hour

At: average of logarithm numbers of viable bacteria from treated sample at Time = 24 hour

Percent reductions are determined by comparing the sample after the contact time to the untreated control sample after the contact time. Percent reduction is translated into log reduction:

90% reduction = 1 log reduction; i.e. 1 ,000,000 reduced to 100,000 is a 1 log reduction

99% reduction= 2 log reduction; i.e. 1 ,000,000 reduced to 10,000 is a 2 log reduction

99.9% reduction= 3 log reduction; i.e. 1 ,000,000 reduced to 1 ,000 is a 3 log reduction

99.99% reduction= 4 log reduction; i.e. 1 ,000,000 reduced to 100 is a 4 log reduction

Claims

1. An antibacterial composition comprising:

a) an antibacterial agent of (Ag NPs) silver nanoparticles of passivated surface; and b) a dispersing agent that consists of a ceramic substrate, where (Ag NPs) silver nanoparticles are bonded to the dispersing agent surface; such Ag NPs have an average diameter of approximately less than 15 nm-.

2. The antibacterial composition according to claim 1 , wherein the Ag NPs antibacterial agent is distributed in homogeneous form on the dispersing agent.

3. The antibacterial composition according to claim 1 , wherein the Ag NPs antibacterial agent comprises a photocatalytically inactive surface.

4. The antibacterial composition according to claim 1 , wherein the dispersing agent supporting the antibacterial agent is a selected semiconductor of Titanium dioxide (T1O2), Zinc oxide (ZnO), Silicon dioxide (S1O2).

5. The antibacterial composition according to claim 1. wherein the dispersing agent is optionally particles of non-semiconductor ceramics such as Zirconium dioxide (Zr02), Aluminum trioxide (AI2O3), aluminosilicates (S1O2.AI2O3), Tin dioxide (SnCte), Tin oxide (SnO), Antimony oxide (Sb^Os), Beryllium oxide (BeO), Copper oxides (CuO, CU2O), Tellurium oxide (ΤβΟ∑), Indium oxide (I^Ch), Gallium oxide (Ga203), and their respective hydroxides.

6. The antibacterial composition according to claim 1 , wherein the bacteria to be eliminated are Gram positive bacteria such as Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Enterococcus,

pyogenes, Enterococcus faecalis, Lactobacillus sp. and Gram negative, such as Escherichia coli, S., Salmonella, Pseudomonas aeruginosa.

7. The antibacterial composition according to claim §1 wherein the bacteria to be eliminated are Gram negative selected of Salmonella sp and Escherichia coli.

8. The antibacterial composition according to claim 6§, wherein the Gram positive bacteria to be eliminated are Staphylococcus aureus.

9. The antibacterial composition according to claim 1 , wherein the Ag NPs have a photocatalytically inactive surface under visible light and wbefeiR-the dispersing agent is not of photocatalytic interest .

10. The antibacterial composition according to claim 1 , wherein the photocatalytically inactive surface has an Ag NPs concentration in the range of 8.5-70% in weight regarding the total composition.

11. The antibacterial composition according to claim 1 , wherein the minimum bactericidal concentration (MBC) is achieved using a dosage of 10 mg/mL of the antibacterial composition constituted from a minimum content of 8.5% in weight of Ag NPs.