JP2009046410A - Antimicrobial composition and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an easily handleable antimicrobial composition with low hazardness to a human body, an animal or a plant. <P>SOLUTION: The antimicrobial composition comprises water, metal nano-particles having antimicrobial actions, nano-particles of a calcium phosphate-based compound and a binder for binding the metal nano-particles to the nano-particles of the calcium phosphate-based compound. The antimicrobial composition comprises Pt nano-particles and/or Ag nano-particles as the metal nano-particles and a water-soluble compound having an amino group and a carboxy group and/or a water-soluble compound having a -HNC=O group as the binder and has the low hazardness because the calcium phosphate-based compound having high biocompatibility is contained. The metal nano-particles are bound through the binder to the calcium phosphate-based compound in the antimicrobial composition and the metal nano-particles contact microbes captured with the calcium phosphate-based compound to exhibit the antimicrobial actions. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to an antibacterial composition and a method for producing the same.

  Antibacterial agents that prevent the growth of microorganisms include synthetic chemicals such as high-concentration alcohol, chlorine, iodine, hypochlorous acid compounds, ozone, hydrogen peroxide, cresol, imidazole compounds (see, for example, Patent Document 1), In addition, natural product extract materials such as eucalyptus oil are known, and they are properly used according to their use and purpose.

  These antibacterial agents are frequently used because of their high antibacterial effects, but they are dangerous if they enter the human body due to accidental ingestion or inhalation of volatile components, and the safe concentration differs from person to person. There are problems such as causing allergic symptoms.

In addition, synthetic chemicals such as imidazole compounds used in the antibacterial composition described in Patent Document 1 have a problem that resistant bacteria are expressed when they are used frequently.
JP 2004-339102 A

  As antibacterial agents other than those described above, for example, lime, photocatalytic titanium oxide, silver ion-containing antibacterial agents, agricultural fungicides, antibiotics administered to livestock, etc., used in the avian influenza outbreak site are known. Of these, lime generates heat and is strongly alkaline due to contact with water, so there is concern about the occurrence of burns and the like due to contact, and it is necessary to pay close attention when using it.

Although photocatalytic titanium oxide can exhibit an excellent antibacterial effect in the presence of ultraviolet rays, the presence of ultraviolet rays is indispensable, and has the disadvantage that it decomposes without distinguishing surrounding organic substances.
Silver ion-containing antibacterial agents have excellent antibacterial properties and high safety, but silver ions are much smaller in size than many cells, so when used for disinfection of skin, etc., they may enter the skin cells. is there.

As agricultural fungicides, chemicals such as Bordeaux liquid, lime sulfur mixture, dithiocarbamate, benzimidazole antibiotics are used, but sprayers use concentration, number of spraying, wearing adequate protective clothing, And attention to the surroundings is necessary.
Antibiotics administered to prevent diseases such as livestock and fish need to consider the development of resistant bacteria and the health of people who eat them.
Therefore, conventional antibacterial agents have high antibacterial effects, but are highly harmful such as toxicity, have the disadvantage of decomposing useful ones and entering cells. It was not easy to use safely and effectively.
This invention is completed based on the above situations, Comprising: It aims at providing the antimicrobial composition with low toxicity with respect to a human body, animals and plants, and being easy to handle.

  That is, the present invention includes water, metal nanoparticles having an antibacterial action, nanoparticles of a calcium phosphate compound, and a binder that binds the metal nanoparticles and the calcium phosphate compound nanoparticles. The particles are Pt nanoparticles and / or Ag nanoparticles, and the binder is a water-soluble compound having an amino group and a carboxyl group and / or a water-soluble compound having a -HNC = O group. It is an antibacterial composition and its manufacturing method.

  In the antibacterial composition of the present invention, metal nanoparticles having antibacterial action and calcium phosphate compound nanoparticles are bound via a binder, and the size of the bound particles is considerably larger than metal ions. , Difficult to enter the cell.

The calcium phosphate compound contained in the antibacterial composition of the present invention is a component having a high affinity with a living body, Pt nanoparticles, Ag nanoparticles, nanoparticles of water-soluble compounds having an amino group and a carboxyl group, and Nanoparticles of water-soluble compounds having —HNC═O groups are components that are less harmful to humans, animals and plants.
Therefore, the antibacterial composition of the present invention hardly enters cells and has low toxicity to human bodies and animals and plants.

In addition, since the calcium phosphate compound nanoparticles contained in the antibacterial composition of the present invention are components that are familiar with biological components, the fungi attached to the calcium phosphate compound nanoparticles cannot be easily detached. , Captured as it is.
And since the metal nanoparticles having an antibacterial action bonded to the calcium phosphate compound via a binding material come into contact with the fungi captured by the calcium phosphate compound nanoparticles, the antibacterial action is exerted reliably. Antibacterial effect is developed early.

  As described above, according to the present invention, it is possible to provide an antibacterial composition that has low toxicity to human bodies and animals and plants and is easy to handle.

  The antibacterial composition of the present invention (hereinafter also simply referred to as “composition”) includes water, metal nanoparticles having antibacterial action, calcium phosphate compound nanoparticles, metal nanoparticles and inorganic compound nanoparticles. And nanoparticles of water-soluble compounds that act as binders that bind the.

  Examples of the metal having an antibacterial action include Pt, Ag, Cu, Zn, Pb, Ni, Cr, bronze and brass, but Ag and Pt are preferable from the viewpoint of high safety and wide use. .

  Despite its antibacterial activity, Ag has long been used in tableware and is safe enough to be used as a food additive on the surface coat of confectionery materials Alazan and fresheners (Nitan). This is particularly preferable because it is inexpensive compared to Pt.

  In the present invention, the metal having the antibacterial action is obtained by pulverizing metal particles into a desired size by a method such as electrolysis or bead mill pulverization, or commercially available metal nanoparticles. Can be used.

  Among the methods of pulverizing the metal into nano-sized particles, the electrolytic method is preferable in that fine particles having an average particle diameter of 10 nm or less can be obtained.

By making the metal having an antibacterial action into nanoparticles, the contact surface area with the fungi can be widened, the antibacterial property can be efficiently exhibited even in a small amount, and it can be uniformly dispersed in water.
In the present specification, the term “fungi” is used as a term meaning microorganisms such as viruses, bacteria, and fungi.

  The average particle diameter of the metal nanoparticles used in the present invention is preferably 3 nm to 50 nm, and more preferably 3 nm to 15 nm. This is because if the average particle diameter of the metal nanoparticles exceeds 50 nm, the stability and dispersibility in water may not be sufficient.

  Along with metal nanoparticles, the calcium phosphate-based compound contained in the composition of the present invention has a property (high biocompatibility) that easily binds to biological proteins and phosphate groups, and has an affinity for humans, animals and plants. Not only is it expensive, it also has the property that fungi are easily attached and do not easily leave.

In the present invention, calcium phosphate compounds include CaHPO ₄ (monetite), CaHPO ₄ .2H ₂ O (calcium monohydrogen phosphate), Ca ₃ (PO ₄ ) ₂ (tricalcium phosphate: TCP), Ca ₄ (PO _{4) 2} O (tetracalcium _{phosphate: TeCP), Ca 10 (PO} 4) 6 (OH) 2 ( _{hydroxyapatite: HAP), Ca 8 H 2} (PO 4) 6 · 5H 2 O ( octacalcium phosphate: OCP), 3Ca ₃ (PO ₄ ) ₂ · Ca (OH) ₂ [Bone Ash (bone ash)], CaHPO ₄ · 2H ₂ O + Ca (OH) ₂ (bone phosphorus) and bone china (firing failure, molding failure, etc.) Slightly soluble calcium phosphates such as (unused) can be used.

Among the calcium phosphate compounds, CaHPO ₄ .2H ₂ O (calcium monohydrogen phosphate), Ca ₃ (PO ₄ ) ₂ (tricalcium phosphate: TCP), Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ (hydroxy) Apatite (HAP) is preferable from the viewpoint of high safety, and HAP is particularly preferable from the viewpoint of high affinity with a living body.

When a calcium phosphate compound other than HAP is dissolved in water at a pH of less than 5, it is converted to HAP, and in the presence of CaCO ₃ (calcium carbonate), at a pH of less than 7, calcium monohydrogen phosphate and CaHPO. _{When 4} is dissolved in water, the conversion to HAP proceeds. In the present invention, the HAP thus obtained may be used.

In the present invention, the calcium phosphate is pulverized by the following method to form nanoparticles.
The above calcium phosphate compounds other than bone china are pulverized into nanoparticles by, for example, a bead mill method (using zirconium balls), and bone china is pulverized until it has an average particle size of about 10 to 100 μm. Further, nanoparticles are formed by a bead mill method.

The average particle size of the calcium phosphate compound is preferably 50 nm to 500 nm, and more preferably 100 nm to 350 nm. This is because when the average particle diameter exceeds 500 nm, it becomes difficult to float and disperse in water.
As with calcium phosphate compounds, it is also possible to use biological glass or titanium metal that is known as a biocompatible material as it is, or use it with apatite coated on its surface. Furthermore, it is not preferable to coat the surface with metal nanoparticles in terms of time and cost.

  Examples of the binder contained in the composition of the present invention for binding metal nanoparticles and calcium phosphate compound nanoparticles include water-soluble compounds having amino groups and carboxyl groups, water-soluble compounds having -HNC = O groups, and the like. Can be used.

  Examples of compounds having an amino group and a carboxyl group include alanine, arginine, cysteine, glutamine, glycine, hydroxyproline, proline, serine, asparagine, histidine, isoleucine, leucine, methionine, phenylalanine, threonine, valine, and lysine (lysine). Examples include water-soluble amino acids.

  Examples of the compound having a —HNC═O group include water-soluble peptides and water-soluble proteins containing the above amino acids, N-polyvinylpyrrolidone polymers, N-dimethylacrylamide polymers, and the like.

The binding material used in the present invention is required to have a function of binding metal nanoparticles and a calcium phosphate compound, a role of stabilizing metal nanoparticles, and dispersibility and solubility in water.
In view of these points, the binding material in the present invention includes water-soluble amino acids such as histidine, cysteine, arginine, lysine, methionine, and serine, water-soluble peptides containing at least one of these amino acids, and water-soluble peptides. The preferred protein is N-polyvinylpyrrolidone (see Test Example 1 for details).

Among the above binder, an amino acid water-soluble, LD ₅₀ by oral administration in rats, more than 10g per body weight 1kg is mostly (only cysteine 3.1 g), particularly preferably because of its high safety.

Next, the manufacturing method of the composition of this invention is demonstrated.
First, metal nanoparticles or commercially available metal nanoparticles pulverized to a predetermined size by the above-described method and a binder are separately dispersed in water to obtain an aqueous dispersion of metal nanoparticles (A) and an aqueous solution of the binder. (C) is prepared, and a calcium phosphate compound is pulverized into nanoparticles (B).
When the thus obtained (A), (B) and (C) are mixed, the composition of the present invention is obtained.

  As described above, in the present invention, since the binder also has a role of stabilizing the metal nanoparticles, the aqueous dispersion (A) of the metal nanoparticles and the aqueous solution (C) of the binder are mixed. It is preferable to add and mix the calcium phosphate compound nanoparticles (B). By mixing in this order, the metal nanoparticles stabilized by the binder are reliably attached to the surfaces of the calcium phosphate compound nanoparticles.

Even if (C) is mixed after mixing (A) and (B), the metal nanoparticles and the calcium phosphate compound nanoparticles are bonded through a binder, but the composition produced by the above method It takes time to reach the same state as.
In addition, it is preferable that the water used in this invention is a pure water. This is because when water containing an anion component (chlorine ion, nitrate ion, sulfate ion) or the like is used, precipitation or color change occurs due to reaction with metal nanoparticles.

The amount of each component in the composition of the present invention (the amount in the composition when actually used) is as follows.
The content of antibacterial metal nanoparticles is selected depending on the type of bacteria to be applied, application (whether or not immediate effect is required, etc.), and the type of binding material. Since it becomes easy to discolor, it is preferable that it is 1 ppm-1000 ppm with respect to the whole composition, and it is more preferable that it is 3-300 ppm.

  The content of the calcium phosphate compound nanoparticles is preferably 5 ppm to 1000 ppm and more preferably 5 ppm to 80 ppm with respect to the entire composition.

  The content of the binder is preferably 1/10 to 10 times the content of the metal nanoparticles. This is because the antibacterial action of the metal nanoparticles cannot be effectively exhibited when the binder is contained in an amount exceeding 10 times the amount of the metal nanoparticles.

In order to prevent pH fluctuation, a phosphate buffer or the like may be added to the composition of the present invention as a component other than the above. Although other antibacterial agents can be added to the composition of the present invention, safety must be taken into consideration.
The composition of the present invention is used, for example, for disinfecting fingers, washing the body of pets such as dogs and cats, used as drinking water for pets and livestock for disease prevention, and for maintaining the freshness of cut flowers. Water for plants, washing water for plants, air-conditioning water for hospitals, etc.

<Test Example 1>
Since the binder used in the present invention is required to have a function of binding the metal nanoparticles and the calcium phosphate compound as well as stabilizing the metal nanoparticles, the following method is used to stabilize the metal nanoparticles. A suitable binder was investigated.
(1) Stability test 1
As an aqueous dispersion of metal nanoparticles, an aqueous dispersion of Ag nanoparticles having a particle diameter of 4 nm and a concentration of 150 ppm [manufactured by Asahi Shokai Co., Ltd., trade name “Sylbarrier”] is used. An aqueous dispersion of Ag nanoparticles with a concentration of 100 ppm was prepared using water (pH 6.5-7.5).
Apart from the above Ag nanoparticle aqueous dispersion, various amino acids shown in Table 1 and pure water were used to prepare an aqueous solution of amino acids containing various amino acids at a concentration of 100 ppm. In this test and the test examples described below, commercially available reagents (L-form) were used as amino acids.

A solution of amino acids and an aqueous dispersion of 100 ppm Ag nanoparticles in a ratio of 1/1 (mass ratio) in a transparent bottle made of polyethylene terephthalate (PET), and then mixed two by two Prepared (final concentrations of Ag nanoparticles and amino acids were 50 ppm each). For comparison, two aqueous dispersions containing only Ag nanoparticles at a concentration of 100 ppm were prepared.
Of the two, one was kept at 40 ° C. for 10 days and the other was kept at 20 ° C. for 30 days without direct sunlight, the presence or absence of discoloration was observed, and the results were judged according to the following criteria and shown in Table 1. Indicated.

◎: No change ○: Some discoloration was observed △: Some discoloration ×: Discoloration

From Table 1, the stability after being allowed to stand at 20 ° C. for 30 days was good for histidine, cysteine, arginine, lysine, methionine, serine, tryptophan, asparagine, glutamine, and phenylalanine.
Of these, histidine, cysteine, arginine, lysine, methionine, and serine had good stability after being left at 40 ° C. for 10 days.

  From the above, it was found that histidine, cysteine, arginine, lysine, methionine, and serine are preferable as the binder used in the present invention.

(2) Stability test 2
The stability test 2 shown below was performed on amino acids whose results of stability test 1 were ◎, ○, and Δ after 30 days of standing at 20 ° C.
Using an aqueous dispersion of Ag nanoparticles having a particle diameter of 4 nm and a concentration of 150 ppm [trade name “Syl Barrier” manufactured by Asahi Shokai Co., Ltd.] and pure water (pH 6.5 to 7.5), 20 ppm An aqueous dispersion of Ag nanoparticles having a concentration of 50 ppm and 100 ppm was prepared.

  An aqueous solution of amino acids containing 100 ppm concentrations of various amino acids whose stability test 1 results after standing at 20 ° C. for 30 days were ◎, ○, and Δ was prepared.

An aqueous solution of amino acid and an aqueous dispersion of Ag nanoparticles were put into a transparent bottle made of PET [1/1 (mass ratio)], and a mixture was prepared for each concentration of Ag nanoparticles. For comparison, an aqueous dispersion containing no amino acid and containing Ag nanoparticles at a concentration of 100 ppm was prepared.
These were left at 20 ° C. for 30 days so that they were not exposed to direct sunlight, the presence or absence of discoloration was observed, and the results were judged according to the following criteria and shown in Table 2.

A: No change.
○: Some discoloration was observed.
Δ: slightly discolored
X: Discolored.

  The concentration of Ag nanoparticles shown in Table 2 is shown as the final concentration after mixing with the aqueous amino acid solution. In the table, Ag nanoparticles having a final concentration of 75 ppm were prepared using the above-described aqueous dispersion of Ag nanoparticles having a concentration of 150 ppm [trade name “Syl Barrier” manufactured by Asahi Shokai Co., Ltd.] Nanoparticles having a final concentration of 150 ppm were prepared using an aqueous dispersion of Ag nanoparticles having a particle diameter of 4 nm and a concentration of 300 ppm [trade name “Syl Barrier” manufactured by Asahi Shokai Co., Ltd.].

  From Table 2, it was found that the higher the silver concentration, the worse the stability.

(3) Stability test 3
N-vinylpyrrolidone polymer was examined as a binder.
As an aqueous dispersion of metal nanoparticles, an aqueous dispersion of Ag nanoparticles having a particle diameter of 4 nm and a concentration of 150 ppm [trade name “Syl Barrier” manufactured by Asahi Shokai Co., Ltd.] was used.
Using this Ag nanoparticle aqueous dispersion and pure water (pH 6.5 to 7.5), an Ag nanoparticle aqueous dispersion having a concentration of 100 ppm was prepared.

  Using an N-vinylpyrrolidone polymer [BASF Corporation, trade name “Luvitec K-30”] 30% aqueous solution and pure water, an aqueous solution of a binder containing the N-vinylpyrrolidone polymer at a concentration of 100 ppm is prepared. did.

  Next, an aqueous solution of the above-mentioned binder and a 100 ppm aqueous dispersion of Ag nanoparticles were mixed in a PET transparent bottle and mixed [1/1 (mass ratio)], and directly irradiated at 40 ° C. for 10 days. When the sample was left out of sunlight and the presence or absence of discoloration was observed, a slight discoloration was observed (corresponding to the criterion Δ shown in the stability tests 1 and 2 above).

<Test Example 2>
Next, the composition of the present invention was prepared and examined for its antibacterial properties.
(1) Preparation of Composition of Example 1 As an aqueous dispersion of metal nanoparticles, an aqueous dispersion of Ag nanoparticles containing 5000 ppm of Ag nanoparticles having a particle diameter of 4 nm [trade name “manufactured by Asahi Shokai Co., Ltd. Sil barrier "] was used.
Histidine was used as a binder, and an aqueous solution of a binder containing histidine at a concentration of 5000 ppm was prepared.

  Hydroxyapatite (manufactured by Ube Materials) is used as the calcium phosphate compound, pulverized to a particle size of 300 nm, and then dispersed in pure water so that the solid content is 10% by mass. A dispersion was prepared.

It is obtained by mixing the Ag aqueous dispersion and the aqueous binder material so that the final concentration is 1000 ppm of Ag nanoparticles, 1000 ppm of histidine, and 10000 ppm of hydroxyapatite, and then adding and mixing the aqueous dispersion of hydroxyapatite. The composition was the composition of Example 1.
In this example and the following examples and comparative examples, pure water was used as the water used for preparing the composition and the aqueous dispersion unless otherwise specified.

(2) Antibacterial test Using a water dispersion containing hydroxyapatite at a concentration of 10,000 ppm as Comparative Example 1 and purified water as Comparative Example 2, antibacterial properties against viruses were examined by the following method.

  The virus used was avian influenza virus [A / whisling swan / schimane / 499/83 (H5N3) isolated from wild swan in Shimane Prefecture in 1983].

First, virus chorioallantoic fluid obtained by propagating the above avian influenza virus using 10-day-old chicken eggs was diluted to about ^106.0 EID ₅₀ /0.1 ml with purified water (hereinafter referred to as virus diluted solution). Was prepared. Note that the _{EID 50} means egg-infectious dose (50% embryonated eggs infectious dose).

Next, the virus dilution was added to each of the composition of Example 1, the aqueous dispersion of Comparative Example 1 and the purified water of Comparative Example 2 (hereinafter also referred to as a test product) so that the total amount was 6.0 ml. 6 ml each was added to prepare a test sample-virus solution.
The test product-virus solution prepared by stirring at 4 ° C. for 1 hour and that stirred for 6 hours were prepared. After stirring, a part of the test product-virus solution was collected and diluted 10-fold, and 0.1 ml was inoculated into the chorioallantoic cavity of 10-day-old chicken eggs.

  After inoculation, the embryonated chicken eggs were cultured at 37 ° C. for 2 days, and then the presence or absence of virus growth in the chorioallantoic cavity was confirmed by hemagglutination (HA) test, and the virus titer in the test sample-virus solution was determined as Reed and Muench. It was calculated by the method. The results are shown below.

  The virus titer of the virus dilution before mixing with the test product was 6.5 (virus inactivation rate 0%). It shows that the inactivation rate of a virus is so high that the numerical value of a virus titer is low. Specifically, when the virus titer is 5.5, the virus inactivation rate is about 90%, and when the virus titer decreases by 1, the virus activity rate decreases by about 1/10.

Example 1 (1 hour stirring) Virus titer 0.5
(Stir for 6 hours) Virus titer -0.5
Comparative Example 1 (1 hour stirring) virus titer 3.3
(Stir for 6 hours) Virus titer 1.8
Comparative Example 2 (1 hour stirring) virus titer 2.5
(6 hours stirring) Virus titer 1.5

  From the above results, when the composition of Example 1 was used, the virus titer was remarkably lowered only by stirring for 1 hour. From this, it was found that the composition of the present invention has a sufficient antibacterial action.

<Test Example 3>
Various compositions with varying concentrations of Ag nanoparticles were prepared and examined for antibacterial properties.
(1) Preparation of compositions of Examples 2 to 9 A composition of Example 2 was prepared in the same manner as in Example 1 except that the final concentration was 1 ppm of Ag nanoparticles, 1 ppm of histidine, and 1 ppm of hydroxyapatite. .

A composition of Example 3 was prepared in the same manner as in Example 1 except that the final concentration was 5 ppm of Ag nanoparticles, 5 ppm of histidine, and 5 ppm of hydroxyapatite.
A composition of Example 4 was prepared in the same manner as in Example 1 except that the final concentration was 10 ppm of Ag nanoparticles, 25 ppm of histidine, and 25 ppm of hydroxyapatite.
A composition of Example 5 was prepared in the same manner as in Example 1 except that the final concentration was 15 ppm of Ag nanoparticles, 5 ppm of histidine, and 5 ppm of hydroxyapatite.
A composition of Example 6 was prepared in the same manner as in Example 1 except that the final concentration was 20 ppm of Ag nanoparticles, 25 ppm of histidine, and 25 ppm of hydroxyapatite.
A composition of Example 7 was prepared in the same manner as in Example 1 except that the final concentration was 25 ppm Ag nanoparticles, 25 ppm histidine, and 25 ppm hydroxyapatite.
A composition of Example 8 was prepared in the same manner as in Example 1 except that the final concentration was 100 ppm of Ag nanoparticles, 100 ppm of histidine, and 100 ppm of hydroxyapatite.
A composition of Example 9 was prepared in the same manner as in Example 1 except that the final concentration was 1000 ppm of Ag nanoparticles, 1000 ppm of histidine, and 1000 ppm of hydroxyapatite.

  A composition of Comparative Example 3 was prepared in the same manner as in Example 1 except that the final concentration was 0 ppm of Ag nanoparticles, 1000 ppm of histidine, and 1000 ppm of hydroxyapatite.

(2) Antibacterial test (silver concentration)
To the total amount of 6.0 ml, 0.6 ml of virus dilution was added to each of the compositions of Examples 2 to 9 and Comparative Example 3 (hereinafter also referred to as test product), and the test product-virus solution was added. Prepared.
The test sample-virus solution using the compositions of Examples 2 to 8 was stirred at 4 ° C for 1 hour, a part of the stirred test sample-virus solution was recovered, diluted 10 times, and 10 days. 0.1 ml each was inoculated into the chorioallantoic cavity of the eggs of the growing eggs.

  Viral titers were measured in the same manner as in the antibacterial test of (2) of Test Example 2 after the cultured chicken eggs after inoculation were cultured at 37 ° C. for 2 days, and the results are shown in Table 3.

(3) Antibacterial test (reaction time)
The antibacterial properties were examined when the stirring time with the virus dilution was changed.
The virus titer was the same as in the antibacterial test (silver concentration) of (2) except that the compositions of Examples 8 and 9 were used and the stirring time of the test article-virus solution was 10 minutes and 30 minutes. The results are shown in Table 3 together with the result of (2).

  From the results shown in Table 3, it was found that if 1 ppm of Ag nanoparticles were included, antibacterial properties were exhibited, and antibacterial properties were developed early as the amount of Ag nanoparticles increased.

<Test Example 4>
Next, the antibacterial property when the composition of the present invention was applied to the human body was examined.
(1) Preparation of composition of Example 10 As an aqueous dispersion of metal nanoparticles, Ag nanoparticles containing 150 ppm of Ag nanoparticles having a particle diameter of 4 nm [trade name “Syl Barrier” manufactured by Asahi Shokai Co., Ltd.] An aqueous dispersion was prepared.
As a binder, methionine was used, and a water solution of the binder containing methionine at a concentration of 100 ppm was prepared.

  Tricalcium phosphate (manufactured by Ube Materials) as a calcium phosphate compound is pulverized with zirconium beads to a mean particle size of 300 nm with a bead mill, and then dispersed in pure water to a concentration of 50 ppm. An aqueous dispersion of tricalcium was prepared.

  Ag water dispersion and binder aqueous solution are mixed, and then tricalcium phosphate aqueous dispersion is added and mixed so that the final concentration is 25 ppm Ag nanoparticles, 25 ppm methionine, and 25 ppm tricalcium phosphate. The composition obtained was used as the composition of Example 10.

(2) Antibacterial test After the composition of Example 10 was put in a spray container and about 5 ml was sprayed on three fingers, 2 minutes passed, 6 minutes passed, and 15 minutes passed, it was present on the fingers. The state of fungi was observed.
Specifically, before the composition of Example 10 was sprayed and the bio-checker TTC which was rubbed with a finger that had passed the predetermined time after spraying was cured at 35 ° C. for 3 days, the state of fungi was observed.

10 ³ colonies were observed for those before spraying, and no colonies were observed for 2 minutes after spraying, 6 minutes after spraying, and 15 minutes after spraying. It was.
From this, it was found that according to the present invention, the antibacterial effect is exhibited in a short time.

<Test Example 5>
Furthermore, the amount of the calcium phosphate compound in the composition of the present invention was examined.
(1) Preparation of compositions of Examples 11 to 13 and Comparative Example 4 As an aqueous dispersion of metal nanoparticles, 150 nm of Ag nanoparticles having a particle diameter of 4 nm [trade name “Syl Barrier” manufactured by Asahi Shokai Co., Ltd.] An aqueous dispersion of Ag nanoparticles contained was prepared.

Methionine was used as a binder, and an aqueous solution of a binder containing methionine at a concentration of 100 ppm was prepared.
Hydroxyapatite (manufactured by Ube Materials) is used as the calcium phosphate compound, and after pulverizing to an average particle size of 100 nm with a bead mill using zirconium beads, it is dispersed in pure water so that the solid content concentration is 10% by mass. To prepare an aqueous dispersion of hydroxyapatite.

  After mixing the Ag aqueous dispersion and the binder dispersion so that the final concentration is 2.5 ppm Ag nanoparticles, 2.5 ppm methionine, and 5 ppm hydroxyapatite, add the aqueous hydroxyapatite dispersion. The composition obtained by mixing was used as the composition of Example 11.

  The obtained composition was used as the composition of Example 12, except that the final concentration was 2.5 ppm of Ag nanoparticles, 2.5 ppm of methionine, and 50 ppm of hydroxyapatite.

  The obtained composition was made the same as that of Example 13, except that the final concentration was 2.5 ppm of Ag nanoparticles, 2.5 ppm of methionine, and 100 ppm of hydroxyapatite.

  The composition obtained in the same manner as in Example 11 except that it did not contain hydroxyapatite was used as the composition of Comparative Example 4.

(2) Antibacterial test An antibacterial test was performed by the following method using human influenza virus A / Aichi / 2/68 (H3N2) and canine parvovirus Cp49 strain.
The compositions of Examples 8 to 10 and Comparative Example 4 were each mixed with the above two viruses, shaken at 37 ° C. for 30 minutes, and then centrifuged to evaluate the number of remaining viruses using the supernatant as a sample.
Specifically, the number of remaining viruses was evaluated by the method of the Institute for Animal Health Safety Science using MDCK cells for human influenza viruses and CRFK cells for canine parvoviruses.
Table 4 shows the test results and the final hydroxyapatite concentration of the compositions of Examples 11 to 13 and Comparative Example 4.

  In the compositions of Examples 11 to 13 containing hydroxyapatite, the number of remaining viruses was remarkably small as compared with the composition of Comparative Example 4 not containing hydroxyapatite.

  This is because the hydroxyapatite nanoparticles have a biocompatibility that they do not easily leave even if the fungi adhere to them, so that the Ag nanoparticles come into contact with the fungi captured by the hydroxyapatite nanoparticles. The antibacterial action could be demonstrated.

  Comparing the number of remaining viruses when the hydroxyapatite concentration is 50 ppm (Example 12) and 100 ppm (Example 13), it is the same for the human influenza virus and for the canine parvovirus. But there was no big difference in the effect.

  From this, it was found that when the hydroxyapatite concentration is 50 ppm or less, the antibacterial property is remarkably improved by increasing the hydroxyapatite concentration.

<Test Example 6>
Furthermore, the amount of the binder in the composition of the present invention was examined.
(1) Preparation of compositions of Examples 14 to 19 As an aqueous dispersion of metal nanoparticles, an aqueous dispersion of Ag nanoparticles containing 150 ppm of Ag nanoparticles having an average particle diameter of 4 nm [manufactured by Asahi Shokai Co., Ltd., The trade name “Syl Barrier”] was used.
As a binder, histidine was used, and an aqueous dispersion of the binder containing histidine at a concentration of 150 ppm was prepared.
Hydroxyapatite (manufactured by Ube Materials) is used as the calcium phosphate compound, and after pulverizing using zirconium beads with a bead mill until the average particle size becomes 100 nm, hydroxyapatite is dispersed in pure water to a predetermined concentration. An aqueous dispersion of apatite was prepared.

  After mixing the Ag aqueous dispersion and the binder aqueous solution so that the final concentration is 5 ppm of Ag nanoparticles, 5 ppm of hydroxyapatite, and 1 ppm of histidine, the aqueous dispersion of hydroxyapatite is added and mixed. The composition obtained was used as the composition of Example 14.

  The composition obtained in Example 15 was made into the composition of Example 15, except that the final concentrations were 50 ppm for Ag nanoparticles, 50 ppm for hydroxyapatite, and 8 ppm for histidine.

  The obtained composition was made the same as that of Example 16, except that the final concentrations were 150 ppm for Ag nanoparticles, 150 ppm for hydroxyapatite, and 25 ppm for histidine.

  The composition obtained in Example 17 was the same as Example 14 except that the final concentration was 5 ppm for Ag nanoparticles, 5 ppm for hydroxyapatite, and 0.5 ppm for histidine.

  The composition obtained in the same manner as in Example 14 except that the final concentration was 50 ppm for Ag nanoparticles, 50 ppm for hydroxyapatite, and 4 ppm for histidine.

  The obtained composition was made the same as that of Example 19 except that the final concentrations were 150 ppm for Ag nanoparticles, 150 ppm for hydroxyapatite, and 15 ppm for histidine.

(2) Antibacterial test An antibacterial test was performed by the following method using Salmonella typhimurum (derived from GP42-Ly guinea pig cervical lymphadenopathy) and Escherichia coli O157 type (Escherichia coli O157: H7 Kitasato University School of Medicine). .

Using Salmonella 10 ⁵ CFU / 50 μl and Escherichia coli O157 type 6.7 × 10 ⁴ CFU / 50 μl, the cells were cultured on an SCD agar medium for 18 hours. ) And suspended in 1 ml of sterile distilled water. 100 μl of this bacterial solution was added to 10 ml of the compositions of Examples 14-19.

The bacterial solution was added dropwise to each of the compositions of Examples 14 to 19 and stirred for several seconds with a vortex, and then immediately inoculated into 50 μl each of three mannitol saline media (shaking and pour 0 minutes).
Separately from the above, the bacterial solution was added dropwise to the compositions of Examples 14 to 19, shaken and shaken for 60 minutes with a shaker, and then inoculated into 50 μl each of three mannitol saline media (shaking and pouring). 60 minutes).
The following shows the results of observing the state of fungi after culturing the medium after inoculating the bacterial solution at 35 ° C. for 3 days.

In all the media inoculated with the shaking / pour time of 60 minutes of the bacterial solution and the composition, no bacterial colonies were observed, and the generation and growth of the bacteria could be suppressed.
In the culture medium inoculated with the composition in which the shaking and pour times of the bacterial solution and the composition were 0 minutes and the Salmonella bacterial solution was dropped, 0 to 5 colonies regardless of the concentration of Ag nanoparticles or histidine Was observed, and its growth could not be suppressed.
Among the culture media inoculated with the composition in which the E. coli solution was dripped, the shaking / pour time of the bacterial solution and the composition was 0 minutes, and the concentration of Ag nanoparticles in the composition was 50 ppm. In the case of 150 ppm, the generation / growth of bacteria could be completely suppressed regardless of the concentration of histidine, but in the case of the Ag nanoparticle concentration of 5 ppm, the generation / growth of bacteria could not be completely suppressed.

  From the results of this test, the concentration of histidine does not affect the antibacterial effect, and if the concentration of Ag nanoparticles is 50 ppm or more, a sufficient antibacterial effect can be obtained even if the shaking and pour times are short. I understood it.

<Test Example 7>
As an aqueous dispersion of metal nanoparticles, an aqueous dispersion of Ag nanoparticles containing 150 ppm of Ag nanoparticles with an average particle size of 4 nm [made by Asahi Shokai Co., Ltd., trade name “Sylbarrier”] is used as a binder. An aqueous dispersion of a binder containing cysteine at a concentration of 150 ppm was prepared using cysteine.
Hydroxyapatite (manufactured by Ube Materials) is used as the calcium phosphate compound, and after pulverizing with a bead mill using zirconium beads until the average particle size becomes 100 nm, it is dispersed in pure water so as to have a predetermined concentration. An aqueous dispersion of apatite was prepared.

  Mix the Ag aqueous dispersion and the aqueous binder solution so that the final concentration is 25 ppm for Ag nanoparticles, 25 ppm for hydroxyapatite, and 25 ppm for cysteine, and then add and mix the aqueous dispersion of hydroxyapatite. The composition obtained was used as the composition of Example 20.

(1) Antibacterial test 1
An antibacterial test was conducted on the fabric coated with the composition of the present invention by the following method.
JIS Z 2801 for a cotton cloth having a size of 50 mm × 50 mm and a cotton cloth not coated with the composition of the present invention after applying the composition of Example 20 at 100 g / m ² and drying. An antibacterial test according to 5.2 was performed. The results are shown in Table 5.
In Table 5, “treatment” indicates that the composition of the present invention was applied, and “no treatment” indicates that the composition of the present invention was not applied.

  From Table 5, it was found that those applied with the composition of the present invention have antibacterial properties against Bacillus subtilis, Pseudomonas aeruginosa, and MRSA (mesitylin-resistant Staphylococcus aureus).

(2) Mold resistance test In the same manner as in (1) Antibacterial test 1 of this test example, the antibacterial test was performed on the cloth coated with the composition of Example 20 by the following method.
A mold resistance test was conducted according to JIS Z 2911-2000 using a cotton cloth having a size of 50 mm × 50 mm after applying the composition of Example 20 at 100 g / m ² .
In this test, Aspergillus niger NBRC 6341, Penicillium funiculosum IAA7013, Paecilomyces variotii IAM5001, Gliocladium vires NBRC 6355, and Chaetumum 47B.
As a result, no fungal growth was observed after 2 weeks and after 4 weeks.
From this, it was found that the composition of the present invention was effective against mold.

(3) Odor Index Test The following test was conducted on an example in which the composition of the present invention was mixed and applied to livestock feed.
A one-year-old hen was allowed to eat a feed mixed with the composition of Example 20 for one week, and then the odor of chicken manure was measured.
The mixing amount of the composition of Example 20 was 3% by mass with respect to the total diet after mixing the composition, and 35 g per day was fed to the hen. The odor of chicken manure was measured using an ODOR CONCENTRATION METER COSMOS XR329III.
As a result, the odor index of chicken manure after ingesting the feed mixed with the composition of the present invention for 1 week was reduced to 1/13 of the odor index when the feed without the composition of the present invention was fed.
From this, it was found that the composition of the present invention has an action of reducing the odor of feces.

<Summary>
According to the above Test Examples 1 to 7, it was found that the antibacterial property can be exhibited if the composition contains Ag nanoparticles at a concentration of 1 ppm or more and 1000 ppm or less.

  Although the antibacterial property improves as the concentration of Ag nanoparticles increases, there is a problem during storage because the stability decreases. Therefore, it is considered that the concentration of Ag nanoparticles in the composition is preferably selected as appropriate according to storage problems, bacterial species to be applied, applications (whether or not immediate effect is required, etc.), and the type of binder. .

<Other embodiments>
The present invention is not limited to the embodiments described with reference to the above description and drawings. For example, the following embodiments are also included in the technical scope of the present invention.
(1) In the above-described examples, the Ag nanoparticles are used as the metal nanoparticles having antibacterial action, but Pt nanoparticles can also be used as the metal nanoparticles having antibacterial action.

  (2) In the above examples, Ag nanoparticles having an average particle diameter of 4 nm were used, but the average particle diameter of the metal nanoparticles having antibacterial action is not limited to 4 nm, and may be 10 nm, 25 nm, 40 nm, etc. Good.

  (3) In the above examples, hydroxyapatite and tricalcium phosphate were used as the calcium phosphate compound, but calcium monohydrogen phosphate, tetracalcium phosphate, bone ash, bone phosphorus, bone china May be used.

  (4) In the above-described examples, compositions using commercially available reagents (L-form) histidine, methionine, and cysteine were exemplified as amino acids, but other amino acids (including synthetic products, D-form, DL-form) and It may contain peptides, proteins, polyvinylpyrrolidone and the like.

  (5) In the above test examples, examples in which the composition of the present invention is applied to avian influenza virus, human influenza virus, canine parvovirus, Salmonella, Escherichia coli O157 type, Bacillus subtilis, Pseudomonas aeruginosa, MRSA, and various molds. As shown, the compositions of the present invention are staphylococci, Shigella, Klebsiella pneumoniae, Vibrio parahaemolyticus, Bacillus cereus, Clostridium botulinum, Clostridium perfringens, Tetanus, Bacillus anthracis, Klebsiella, Legionella, Pseudomonas, Poliovirus, Rotavirus It can also be applied to fungi such as herpes virus.

  (6) In the above test examples, the composition of the present invention was directly used for fungi, applied to human fingers and cloth, or mixed with hen's diet. However, it was applied to fish and plants. It can be used to prevent illness and can be applied to fungi in the air by spraying it into the air.

Claims (6)

Water, metal nanoparticles having antibacterial action, calcium phosphate compound nanoparticles, and a binder that binds the metal nanoparticles and calcium phosphate compound nanoparticles,
The metal nanoparticles are Pt nanoparticles and / or Ag nanoparticles;
The antibacterial composition, wherein the binder is a water-soluble compound having an amino group and a carboxyl group and / or a water-soluble compound having a -HNC = O group. The calcium phosphate compound is composed of CaHPO ₄ .2H ₂ O (calcium monohydrogen phosphate), Ca ₃ (PO ₄ ) ₂ (tricalcium phosphate), Ca ₁₀ (PO ₄ ) ₆ (OH) ₂ (hydroxyapatite). The antibacterial composition according to claim 1, which is one or more of them. The binding material is an amino acid selected from the group consisting of histidine, cysteine, arginine, lysine, methionine, and serine, a peptide containing one or more amino acids, a protein containing one or more amino acids, or a polymer of N-vinylpyrrolidone. The antibacterial composition according to claim 1 or 2, wherein The antibacterial composition according to any one of claims 1 to 3, wherein the metal nanoparticles are contained in an amount of 3 ppm to 600 ppm with respect to the total weight of the antibacterial composition. The antibacterial composition according to any one of claims 1 to 4, wherein an average particle diameter of the metal nanoparticles is 3 nm to 50 nm. It is a manufacturing method of the antibacterial composition according to any one of claims 1 to 5,
A method for producing an antibacterial composition, comprising mixing an aqueous dispersion of the metal nanoparticles and an aqueous dispersion of the binder, and then adding and mixing the nanoparticles of the calcium phosphate compound.