KR100933736B1 - Method for preparing nano-silver particle and detergent composition by using them - Google Patents

Abstract

PURPOSE: An additive for detergent is provided to ensure excellent antibacterial activity, harmful bacteria removal ratio and antibiotic durability, and to increase skin mildness and storage stability while not reducing detergency. CONSTITUTION: A method for preparing nano-silver particle comprises the steps of: (i) mixing trisodium citrate and hydrogen peroxide(H2O2) solution after dispersing silver nitrate(AgNO3) solution; (ii) adding polyvinyl pyrrolidone and sodium borohydride to the mixed solution and stirring them; (iii) removing impurity by filtering the mixed solution; and (iv) evaporating and drying the mixed solution from which impurity is removed.

Description

Method for preparing silver nanoparticles and detergent additives into which silver nanoparticles prepared are introduced {Method for preparing nano-silver particle and detergent composition by using them}

The present invention relates to a method for preparing new silver nanoparticles, and an additive for detergent into which silver nanoparticles prepared by the above method are introduced.

Kitchen detergents are used to remove food debris from utensils and cooking utensils. When washing with bare hands, not only the contamination of utensils but also the moisture and oil of the skin are removed, irritating the skin and making the skin rough. There is a tendency. The main ingredient used in the dishwashing detergent for the cleaning effect of the tableware is a surfactant (Surfactants), but the surfactant is a main ingredient that stimulates the skin as described above.

On the other hand, essential detergents used in kitchen detergents are known to have the following toxicity, that is, environmental hormones are detected in almost all surfactants on the market, the toxicity is high, It is known that it accumulates without decomposition, and consumer distrust of surfactants is spreading, and when these surfactants enter the body, the effects and characteristics on the human body as endocrine disruptors (environmental hormones) are as follows. Is the same as

First of all, it is known that it is not easily decomposed unlike biohormones, while increasing malformations such as atopic dermatitis, asthma, rhinitis, and urethral fever due to a sharp decrease in sperm count, testicular tumors (testicular cancer), neurological dysfunction, and decreased immunity. In addition, they have a negative effect of remaining in the human body and concentrating on the fat and tissue of the human body for many years. Accordingly, the general surfactant market is a natural material with high biodegradability of petroleum-based surfactants such as LAS and AOS. Naturally, there is a trend toward developing a pollution-free, non-toxic environment-friendly product.

On the other hand, dishwashing detergents may use skin protection ingredients to reduce irritation to the skin. The moisturizing and holding action of such skin protection ingredients acts to inhibit dehydration and deoiling of the skin by the active ingredients of the detergent, or the skin It also serves to nourish the body. Commonly used skin protection ingredients are humectants such as glycerin, sorbitol, pyrrolidone, sodium carbonate, protein derivatives, phospholipid derivatives, plant and seaweed extracts.

Patents filed and registered as a technique for adding a skin protective ingredient include U.S. Patent No. 4268406, which specifies a dish detergent composition containing a denatured protein, U.S. Patent No. 3548056.

In addition, although aloe and loofah are used alone, there is a document that describes the effect is excellent, but the skin protection effect was not as great as expected. Some additives have a problem that dishwashing detergents do not remain transparent. In addition, some of the skin ingredients that are excellent in moisturizing skin have a problem of deteriorating cleansing power.

In addition, in recent years, domestic and foreign cleaning agents that come in contact with fruits and vegetables are legalized to use only natural formulations, but now 100% non-toxic detergents have not been released, and existing formulations that satisfy consumer preferences and desires do not have anything special. Situation.

Therefore, the present invention has been made to solve the above problems, and when applied to a detergent composition, of course, it retains the basic ability, such as cleaning power as a detergent composition, even when in contact with the human skin and orally into the human body It is an object of the present invention to provide a detergent additive which is non-toxic and has excellent antibacterial activity and antimicrobial persistence, and a method for preparing silver nanoparticles introduced as a main ingredient in the detergent additive.

As an example for solving the above problems, the method for producing silver nanoparticles of the present invention comprises: 1) a solution of trisodium citrate and hydrogen peroxide (H ₂ O ₂ ) after dispersing a silver nitrate (AgNO ₃ ) solution in water; Mixing step 2) adding polyvinyl pyrrolidone and sodium borohydride to the mixed solution of step 1) and stirring the solution; 3) mixing solution of step 2) Filtering to remove impurities, and 4) evaporating and drying the mixed solution from which impurities have been removed in step 3).

As another example for solving the above problems, the detergent additive of the present invention is manufactured by the above method, characterized in that silver nanoparticles having a diameter in the range of 5 to 100 nm are included in the range of 1 to 100 ppm.

As another example for solving the above problems, the detergent additive of the present invention, the silver nanoparticles having a diameter in the range of 5 to 100 nm and the sugar fatty acid ester (sugar fatty acid ester) produced by the above method is 1: 0.1 to Detergent additive, characterized in that the complex is contained in the range of 1 to 100 ppm bound in a 5 molar ratio.

Hereinafter, the method for producing silver nanoparticles of the present invention will be described in detail for each step.

1) dispersing the silver nitrate (AgNO ₃ ) solution in water and mixing trisodium citrate and hydrogen peroxide (H ₂ O ₂ ) solution.

The silver nitrate of step 1) is reduced by trisodium citrate and hydrogen peroxide, wherein silver nitrate (AgNO ₃ ), trisodium citrate and hydrogen peroxide (H ₂ O ₂ ) are 1: 0.1 to 1: 1. It is suitable for the purpose to keep it in the range from -5M. The silver nitrate solution is preferably used to completely disperse in water in advance, the concentration of the silver nanoparticles obtained depends on the content of the silver nitrate is added, the size of the silver nanoparticles is changed so that the content of the silver nitrate is adjusted accordingly. Mix the silver nitrate solution with trisodium citrate and hydrogen peroxide solution and allow to stir well.

2) adding and stirring polyvinyl pyrrolidone and sodium borohydride to the mixed solution of step 1).

The polyvinylpyrrolidone serves to emulsify the silver particles reduced in step 1), and serves to induce the reduction of the sodium nitrate which is not reduced. The polyvinylpyrrolidone and sodium borohydride may be included in the range of 1 to 0.1: 5 to 5 M with respect to 1 M of silver nitrate (AgNO ₃ ) in terms of preparing particles having a silver nanoparticle size.

The agitation of step 2) is preferably performed at a low temperature in the range of 0 to 25 ° C. in terms of controlling the size, and is preferably made of 50 to 100 rpm.

The agitation of step 2) is performed for 30 minutes to 120 minutes. If the stirring time is short, it is difficult to homogenize, and if the stirring time is long, product deformation tends to occur, so it is preferable to maintain the above range.

3) filtering the mixed solution of step 2) to remove impurities.

The mixed solution mixed and stirred in the step 2) is filtered through a filter membrane to remove impurities, and at the same time to obtain silver nanoparticles of uniform size. In this case, the filtration may be carried out using a filter membrane of several μm in size (0.2 to 0.45 μm).

4) evaporating and drying the mixed solution from which impurities are removed in step 3).

In the step 4), the silver nanoparticles having the impurities removed and the uniform particle size are applied to various evaporation and drying solvents by applying various drying means such as a vacuum oven. As described above, silver nanoparticles having an even diameter in the range of 5 to 100 nm can be obtained. The silver nanoparticles thus obtained can be applied to various industrial fields that require it.

Hereinafter, the additive for detergents of this invention is demonstrated concretely.

Detergent additives of the present invention is characterized in that it comprises a silver nanoparticles prepared by the above-described method, it comprises in the range of 1 to 100 ppm of the total weight.

Detergent additive of the present invention has a characteristic that does not lower the intrinsic function and antimicrobial activity of each component of the conventional detergent composition. Detergent additives of the present invention may comprise a component constituting the usual kitchen and other detergent compositions, it comprises a silver nano-particles described above. Specifically, for example, it may include a surfactant, a metal ion blocking agent, and the like to add these components in a certain ratio and then stirred to achieve a mixed state.

As an example, based on 100 parts by weight of the surfactant used when applied to kitchen detergent composition may include 0.1 to 0.5 parts by weight of the metal ion sequestrant, silver nanoparticles may be used in the range of 1 to 100 ppm. In this case, when the content of silver nanoparticles is less than 1 ppm, the initial antimicrobial properties are shown, but the antimicrobial and antifungal persistence tends to be weakened. When the content of the silver nanoparticles exceeds 100 ppm, the manufacturing cost is increased as well as the composition for detergents. There is a tendency for the storage properties to be lowered because it can precipitate inside.

In particular, the silver nanoparticles prepared according to the present invention exhibits excellent antimicrobial activity against pathogens that are resistant to general antibiotics, and thus may be particularly useful for kitchen detergent compositions. In addition, the silver nanoparticles prepared according to the present invention have a fine and uniform size distribution, so that even when used in powder form, the silver nanoparticles are excellent in storage stability without being precipitated even when stored for a long time.

In addition, the silver nanoparticles prepared according to the present invention are manufactured without using a raw material such as a separate stabilizer, and thus have characteristics that rarely cause problems when applied to kitchen detergents.

The silver nanoparticles used in the detergent additive of the present invention are prepared by the above-described method, and it is preferable to use those having an average particle size of 10 nm or less, and preferably those having an average particle size of 100 nm or less. good.

When introducing the detergent additive containing the silver nanoparticles of the present invention into the kitchen detergent composition, the surfactants that can be used are cationic (±) ionic surfactants, cationic surfactants, anionic surfactants, nonionic surfactants or One kind selected from the group consisting of these mixtures is used, and in the case of the mixture, the mixing ratio can be applied without being particularly limited.

As the amphoteric (±) ionic surfactant, it is preferable to use a betaine-based surfactant, an imidazoline-based surfactant, or a mixture thereof, and as the betaine-based surfactant, cocoamide propyl betain, It is preferable to use lauryl betain or a mixture thereof, and it is preferable to use Cocoamphodiacetate as the imidazoline-based surfactant, and as long as it does not inhibit the present invention, it is known positive. Any of the (±) ionic surfactants may be used.

The anionic surfactant is not particularly limited, but in the group consisting of sodium lauryl ether sulfate, sodium lauryl sulfate, sodium xylene sulfonate or a mixture thereof It is preferable to use one selected.

The nonionic surfactant is not particularly limited, but glycol stearate, amine oxide, coconut diethanol, ethylene oxide adduct, lauryl ether It is preferable to use one selected from the group consisting of cetyl ether, oleyl ether, or mixtures thereof.

The cationic surfactant may be a betaine-based surfactant, an imidazoline-based surfactant, or a mixture thereof. Any of the betaine-based surfactant and the imidazoline-based surfactant may be used.

On the other hand, a large amount of metal ions are contained in general water, rock water or groundwater used in the manufacture of kitchen detergents, and the metal ions may be precipitated or degrade the reactivity by the silver nanoparticles.

Therefore, since the additive for detergent of the present invention includes silver nanoparticles, it is preferable to use a metal ion blocking agent together to prevent a chemical reaction with metal ions contained in water used in the detergent composition for introducing the same.

Preferably, the metal ion blocking agent is added in an amount of 0.1 to 0.5 parts by weight based on 100 parts by weight of the used surfactant. In this case, when the amount of the metal ion blocking agent is mixed to less than 0.1 part by weight, the efficiency of the metal ion blocking agent is mixed. There is a problem in that the silver nanoparticles are discolored or precipitated due to the reaction of other metal ions, and when mixed in excess of 0.5 parts by weight, the antimicrobial and antifungal properties of the silver nanoparticles tend to be lowered.

Meanwhile, as the metal ion blocking agent, sodium phosphate, monosodium phosphate, disodium phosphate, trisodium phosphate, sodium tripoly phosphate, and sodium hexa It is preferable to use a phosphate compound which is one or two or more selected from the group consisting of meta phosphate, sodium hexametaphosphate, tetrasodium pyrophosphate, sodium acid pyroposphate, and the like. In the case of using such a phosphate-based compound, it is more effective in preventing the precipitation of silver (Ag) by preventing color change and giving dispersion, stabilization and thickening effects of silver nanoparticles.

In addition to the surfactants and metal ion blocking agents described above, surfactants and metal ion blocking agents that can be used in the art may be appropriately used according to the choice of those skilled in the art.

The dish detergent composition thus prepared may be prepared as a dish detergent by diluting it in a certain ratio in general water, rock water or ground water.

On the other hand, silver nanoparticles having a diameter in the range of 5 to 100 nm prepared by the above method and sugar fatty acid ester (sugar fatty acid ester) in a composite (hereinafter referred to as nano silver sugar) in a molar ratio of 1: 0.1 to 5 molar ratio It can be used as an additive component for, such a composite may be included in the range of 1 to 100 ppm. When nano silver shoe is used in the state, the antimicrobial effect of sugar fatty acid ester can be increased by five times or more. The sugar is less toxic, fatty acids can be used in the range of 8 to 20 carbon atoms, and generally use a lot of phospholipid (phospholipid).

On the other hand, the detergent additive of the present invention does not inhibit the inherent functions of the components included in the fabric softener, etc., even when introduced into the fabric softener in addition to the kitchen detergent, and excellent antibacterial activity and antimicrobial persistence.

As described above, according to the present invention, fine silver nanoparticles having a uniform particle size distribution can be manufactured by a simple method, and in addition to the antimicrobial and antifungal properties peculiar to silver nanoparticles, pathogenic microorganisms have developed resistance to antimicrobial agents. It is also possible to provide an additive for detergents that exhibits continuous antimicrobial properties.

In addition, kitchen detergents and fabric softeners, such as detergent additives according to the present invention are introduced, not only the antimicrobial and antifungal properties are deteriorated, but also the cleaning power is maintained or raised, the skin irritation is sharply reduced, the skin mildness is excellent Rise, and the storage stability as a product shows the effect improved.

 Hereinafter, the present invention will be described in detail with reference to Examples, but the technical scope of the present invention is not limited by the following Examples.

Example  One. Of silver nanoparticles  Produce

Dissolve 1 mM silver nitrate solution in 10 ml of water, add 10 ml of 30 mM trisodium citrate and 3 ml of hydrogen peroxide (H ₂ O ₂ ) solution, mix, and then add 0.7 mM polyvinylpyrroli. 1 ml of polyvinyl pyrrolidone and 100 mM sodium borohydride were added and stirred at 0 ° C. (100 rpm, 30 minutes).

The impurities were removed by filtration through a 0.2 μm filter membrane, and rotary evaporation was followed by drying in a vacuum oven (45 ° C.) for 4 days to obtain uniform silver nanoparticles in solid form.

Experimental Example  One. Of silver nanoparticles  Check size and shape

After adding sodium borohydride in Example 1, the stirring time was adjusted to 30 minutes, 60 minutes, and 100 minutes to determine the diameter of the silver nanoparticles according to the stirring time.

The shape of the obtained silver nanoparticles was determined by transmission electron microscopy, and the size of the silver nanoparticles was measured by a particle analyzer. In FIG. 1, the stirring time was 60 minutes, and the particle size distribution of the prepared silver nanoparticles was shown. . Figure 2 shows a transmission electron micrograph of the silver nanoparticles prepared under the same conditions. Referring to FIG. 1, silver nanoparticles having a uniform size exhibiting a particle size distribution in a range of 5 to 150 nm may be identified, and in FIG. 2, silver nanoparticles having a uniform size may be identified.

Experimental Example  2. Targeting Escherichia Coli Of silver nanoparticles  Confirm antimicrobial effect

end. Laboratory antimicrobial effect test against Escherichia coli

1) E. coli separation and silver nanoparticles preparation

A) Sampling and preparation of silver nanoparticles

The used strain was used to separate diarrhea from piglet feces, which showed diarrhea and was requested for laboratory diagnosis, and was prepared by the method of Example 1 as an antimicrobial sample, and the particle distribution of 10 nm or less occupied 80%. Silver nanoparticles having the same (used in the same sense as nanosilver or nanosilver-III) [5,000 ppm, Naul Co., Korea] was used (see Fig. 2).

B) medium used

MacConkey agar (Difco) and Blood agar were used to isolate E. coli, and Minca medium (KH ₂ PO ₄ 1.36 g, Na ₂ HPO _{4 ·} 2H ₂ O 10.1) was used to examine pili production. g, MgSO _{4 ·} 7H ₂ O 0.008g, MnCl _{2 ·} 4H ₂ O 0.001g, FeCl · 2H ₂ O 0.000135g, CaCl _{2 ·} 2H ₂ O 0.0004g, Glucose 1g, Casamio acid 1g, Yeast extract 1g, Bacto agar 3 g, 1,000 ml of distilled water, pH 7.5) was used.

To determine the thermophilic enterotoxin, Casamino acid-Yeast extract medium (CYEM: casamino acid 20g, Yeast) was added 1 ml of trace salt solution (MgSO ₄ 5g, MnCl ₂ 0.5g, 0.5% FeCl ₃ solution 10ml, distilled water 90ml). extract 6g, NaCl 2.5g, K ₂ HPO ₄ 8.71g, distilled water 999ml).

 C) E. coli isolation and identification

Fecal samples were planted in MacConkey agar and Blood agar and incubated for 18 to 24 hours, in the case of MacConkey agar plate, 1 to 2 lactose digested colonies were selected, and the blood agar plate showed a smooth white-brown color showing complete hemolysis. (smooth) 1 to 2 colonies were selected for pure separation.

Purely isolated colonies were identified by the biochemical properties shown using an automated microbial identification system (Vitek System, Automicrobic System, AMS).

D) Serological identification

In order to identify the isolates serologically, the OK antiserum was determined by the plate aggregation reaction method using the OK antiserum produced according to the method of Φrskov et al. (1977).

 E) Mother Generation  inspection

The MRHA test (Mannose-resistant hemaglu1tination test) was performed by Jones and Rutter's method (1972). In other words, MRHA test is 50 ppm mannose phosphate buffer solution (NaCl 8g, Na ₂ HPO ₄ 11.5g, KH ₂ PO ₄ 0.2g, KCl 0.2g, D-mannose 5g) , Distilled water 1.000ml, pH 7,2) 0.025ml each was put in a U-shaped microplate (U-form microplate, Dynatech Lab.) And diluted twice. After mixing Alsever solution (glucose 2.05g, sodium citrate 0.8g, NaCl 0.42g, Citric acid 0.055g, distilled water 10ml) and the same amount of blood collected from guinea-pig, 3 parts with phosphate buffer solution (PBS) After washing twice, 0.025ml of 1% guinea pig erythrocytes, made by floating in PBS at 1% (v / v), were mixed dropwise onto a plate with a dropper (Dynatech Lab.), Followed by mixing at 2 to 4 ° C. After standing for 3 hours, the highest dilution factor of antigen in which hemagglutination occurred was determined as MRHA.

bar) Hemolysin  inspection

Hemolysin was proved after hemolysis of the isolated bacteria in blood agar.

four) Heat Toxin ( Heat - labele enterotoxin  : LT  inspection)

In order to prove the thermophilic enterotoxin (LT) of the isolate, the isolate was inoculated into CYEM, centrifuged by shaking culture at 37 ° C. for 18 to 24 hours, and the supernatant was used as an experimental material. Enzyme linked immunosorbent assay (ELISA) was assayed according to the WHO method (1983), and the determination was performed by comparing the mean density of the wells with the average value of the negative control to LT production-positive bacteria when the OD value was 0.2 or more. Judging mainly.

A) Escherichia coli O157 : H7 Preparation of bacteria

The strain used for the analysis of sera and toxin was determined according to the method of Brown et al. (1982) of O157 diarrhea-inducing Escherichia coli isolated from 145 patients with diarrhea who visited a designated hospital. Verotoxin 1 (VT1) Production Escherichia coli and Verotoxin 2 (VT2) Production Escherichia coli VT1: O157: H7 (ACH5), V2: O157: H7 (O59) and V1 + V2: O157: H7 (670), VT2 Cl-0157: H7, VTe 0138, E57 (VTe), VT2 Cl-0157: H7 A total of six species were distributed and used at the Institute of Animal Immunity, Gyeongsang National University.

 E. coli O157 : H7 Serological Identification of Strains

In the serological test of the common pathogen O157: H7 Escherichia coli to be applied, seroaggregation was performed on 6 strains of O-group serotype using Karmail (1985) to determine the coagulation. The biochemical test was carried out in accordance with Edwards and Ewing's method (1986) to confirm that it was additionally O157: H7 Escherichia coli.

2) Culture of Escherichia Coli

E. coli isolates were inoculated in nutrient broth (Difco) and shaken for 18 hours at 37 ° C and 150 rpm. Centrifugation (500 xg, 4min) of this E. coli culture was used to purely separate E. coli, and then diluted with phosphate buffer (0.1M, pH 7.0). If necessary, this centrifugation process was repeated several times to adjust the number of bacteria. Used.

3) Absorbance Measurement of Escherichia Coli

In order to measure the ability of silver nanoparticles to suppress the growth of Escherichia coli, a nutritional broth (Difco) was prepared, the pH was adjusted to 6.0, and the medium solution was dispensed into a 250 ml Erlenmeyer flask, followed by autoclaving. A silver nanoparticle solution diluted by concentration (50ppm, 25ppm, 5ppm, 1ppm) was added to the nutrient broth, and 1 ml of the E. coli culture solution prepared above was dispensed, and then shaken at 37 ° C. at 1 hour intervals from 1 hour. By 12 hours optical density (OD) was measured using an absorbance spectrometer (Spectronic 20, Bauch & Lomb, Germany) (660 nm).

la. Silver nanoparticles  After addition Escherichia coli  Measure

In order to determine the antimicrobial activity of the silver nanoparticles by the concentration of the addition, a nutrient broth was made in a 250 ml flask and inoculated with E. coli and incubated at 37 ° C. These E. coli cultures were adjusted to 2 x 10 ⁷ cfu / ㎖, and then 5 treatments in which silver nanoparticles were added at different concentrations (50ppm, 25ppm, 5ppm, 1ppm) and controls without silver nanoparticles were placed. After adding silver nanoparticles, 10 μl of each treatment was collected at 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, and 12 hours. After mixing well in a cap tube containing 990 μl of phosphate buffer solution, 50 μl of the plate was plated on blood agar medium and cultured for one day to count colonies.

hemp. With general antibiotics Of silver nanoparticles  Antimicrobial Effect Comparison

As a result of testing the antimicrobial activity of silver nanoparticles, it showed excellent antimicrobial activity, but since the final pH range has a weak acid range, the necessity of further checking whether such a pH has an effect on antimicrobial activity has emerged. Experimental zone was set as follows.

One) Experiment  1 (General antimicrobial comparison experiment)

First, in order to determine the antimicrobial activity of the antibiotics OTC and silver nanoparticles by the concentration of the addition, nutrient broth was prepared in a 250 ml flask and inoculated with E. coli O157: K88ac and incubated at 37 ° C. These E. coli cultures were adjusted to 2 x 10 ⁷ cfu / mL, and then, a certain amount of these bacteria were added to physiological saline, and silver nanoparticles and OTC were added to each concentration (50ppm, 25ppm, 5ppm and 1ppm). A control group diluted in dog treatment and physiological saline was set as a comparison experiment.

After 1 hour, 13 hours, and 16 hours after adding OTC and silver nanoparticles, 100 μl of each treatment was collected, mixed well in a cap test tube containing 990 μl of phosphate buffer solution, and 50 μl was smeared on blood agar medium. The colonies were counted by culturing for one day.

2) Experiment  2( pH  Antibacterial Effect According to Condition)

Antimicrobial activity was measured by adjusting the pH of silver nanoparticles to weak acidity and neutrality, and antimicrobial activity was determined by using general antibiotics as a control. To this end, the antimicrobial effects of commercialized antibiotics and silver nanoparticles were compared using the isolated and identified E. coli O157: K88ac species.

At the same time, considering the pH of the silver nanoparticles prepared in the final range of 4.8 to 5.0, it was confirmed whether such an effect on the antimicrobial effect. To this end, OTC (Oxytetracycline-HCl, Korea, Samyang Pharmacy, Lot No. 9503-1) was purchased as a comparative antibiotic and compared with silver nanoparticles.

Experimental conditions were 1 hour, 13 hours, and 16 hours after the composition of the medium in which each of the nanonanoparticles and the OTC were administered in the normal sterile water, and the mixture of 2 x 10 ¹¹ cfu / E. coli culture was mixed. After this period, the antimicrobial effects of silver nanoparticles and OTC were compared, and at the same time, pH 4.8 and pH 6.75 were formulated to examine the difference of antimicrobial effect on the effect of pH of silver nanoparticles.

[result]

1. Test of laboratory antimicrobial effect against Escherichia coli

end. Infectious Escherichia Coli Isolation

The strain used in this experiment was isolated from piglet feces, which had diarrhea due to diarrhea, and serotypes were 088: K-, 0147: K88ac, 09: K103, 015: K-, and 04: K12 for 1 week each, 0157. : K88ac, 015: K-, 09: K103, 020: K101, and unclassified were 26 species, 2 weeks each, 01: K- three weeks, and 0149: K91 seven weeks.

Table 1. Characteristics of Escherichia coli isolated from piglet feces (diarrhea)

Characteristics of Escherichia coli Types serotype F4 F5 F6 LT ST β 0147: K88ac: K89 - + - - - β 0157: K88ac - - + - + β 0157: K88ac + - - - + β 0157: K88ac - - - - + β 0149: K91 + - - - β 0149: K91 - - - - β 0149: K91 - - - - γ 0149: K91 + - - - β 0149: K91 + - - - β 0149: K91 + - - - + β 0149: K91 - - + - + γ 020: K101 + - - - γ 020: K101 - - - - γ 01: K- - - - - β 015: K- - - - - γ 04: K12 - - - - γ 088: K- - - - - β 01: K- - - - - + γ 09: K103: K987 - - - - β 01: K- - - - - + γ nontypable + - - - γ 09: K103: K987 + - - - β 04: K12 - - - - γ nontypable - - - - γ nontypable - - + - γ 015: K- - - + -

Of these serotypes, 0157: K88ac, 01: K-, and 0149: K91 were those producing heat-resistant toxins [Table 1]. In addition, O157: H7 antiserum was used to perform a further confirmation test for recognition of VT1 or VT2 of O157: H7, which is a diarrhea patient-derived Escherichia coli, used in the present experiment. It was determined as VT1 or VT2 of -group serotype.

I. Antimicrobial Activity of Silver Nanoparticles against Escherichia Coli

The antimicrobial activity of E. coli strains used in this experiment was significantly different for 1 to 12 hours after addition of silver nanoparticles between 5 ppm and 50 ppm concentration in addition to 1 ppm silver nanoparticle treatment compared to the control group. ].

Of these, the results of confirming the growth inhibitory effect of the silver nanoparticles in the entire E. coli O157: H7 strain is shown in Figures 3 and 4.

As a result, it was confirmed that the growth of O157: H7 Escherichia coli was generally suppressed when silver nanoparticles were administered at a concentration of 1 ppm to 50 ppm as compared with the control. But. VT2 Cl-Ol57: H- was less than 1 ppm and E. coli strains of VT2 Cl-O157: H7 did not inhibit growth when silver nanoparticles were added at a concentration of 1 ppm or less (Figs. 3B to 3C and P <0.05). . The antimicrobial effect of the O157: H7 strains by serotypes was somewhat different. However, other O157: H7 bacteria showed a marked difference in growth inhibition effect for 1 to 12 hours, showing a very good antibacterial effect [FIG. 3A, P <0.01].

Table 2. Antimicrobial activity after silver nanoparticle treatment by concentration of 26 E. coli isolated from piglet feces (diarrhea) (1 hour after silver nanoparticle treatment, 37 ℃).

Serotypes Antibacterial effects (%) after nano-silver treatment of various concentration control 50 ppm ^* 25 ppm ^* 10 ppm ^* 5 ppm ^* 1 ppm ^** 0147: K88ac: K89 100 1.8 ± 0.7 18.0 ± 6.5 14.7 ± 5.5 24.5 ± 8.9 57.3 ± 13.2 0157: K88ac 100 0.4 ± 0.8 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0157: K88ac 100 0.0 ± 0.0 0.0 ± 0.0 7.0 ± 2.4 9.5 ± 3.4 10.4 ± 5.6 0157: K88ac 100 0.0 ± 0.0 0.0 ± 0.0 4.6 ± 1.2 6.5 ± 3.2 10.0 ± 4.5 0149: K91 100 0.0 ± 0.0 1.5 ± 0.4 3.8 ± 1.5 7.0 ± 4.1 7.9 ± 2.9 0149: K91 100 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.2 ± 0.3 4.2 ± 2.4 0149: K91 100 0.0 ± 0.0 1.2 ± 0.6 0.9 ± 0.8 1.2 ± 0.8 7.1 ± 3.4 0149: K91 100 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.2 ± 0.3 4.7 ± 2.3 0149: K91 100 0.0 ± 0.0 1.2 ± 1.6 0.9 ± 0.2 1.2 ± 0.8 7.1 ± 4.5 0149: K91 100 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0149: K91 100 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 020: K101 100 0.0 ± 0.0 0.0 ± 0.0 26.8 ± 12.0 21.8 ± 13.2 0.4 ± 0.3 020: K101 100 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 01: K- 100 0.0 ± 0.0 0.0 ± 0.0 0.2 ± 0.15 2.0 ± 0.12 2.7 ± 2.3 015: K- 100 0.1 ± 0.2 0.0 ± 0.0 0.1 ± 0.02 2.9 ± 1.3 4.1 ± 1.4 04: K12 100 0.0 ± 0.0 1.5 ± 0.34 4.3 ± 2.4 5.1 ± 1.3 7.6 ± 3.3 088: K- 100 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 01: K- 100 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 09: K103: K987 100 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 18.3 ± 8.7 20.4 ± 12.4 01: K- 100 0.0 ± 0.0 1.7 ± 0.7 1.7 ± 0.3 7.1 ± 3.2 0.0 ± 0.0 nontypable 100 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.6 ± 0.2 09: K103: K987 100 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.6 ± 0.23 0.0 ± 0.0 04: K12 100 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 nontypable 100 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 5.0 ± 6.2 0.0 ± 0.0 nontypable 100 0.0 ± 0.0 0.0 ± 0.0 0.03 ± 0.04 0.0 ± 0.0 0.0 ± 0.0 015: K- 100 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.2 ± 0.3 2.9 ± 3.2  * P <0.01 as compared to control, ** P <0.05 as compared to control

All. Changes in Escherichia Coli Number after Silver Nanoparticles Administration

The antimicrobial activity against E. coli of the experimental group to which 5 ppm silver nanoparticles were added to the control group to which silver nanoparticles were not added is shown in FIG. 4. The colony counts of E. coli strains were significantly decreased compared to the control group within 10 minutes with the addition of 1 ppm to 50 ppm of silver nanoparticles, and the colony counts of the control group increased by 1.21 times on average 10 minutes after the initial administration.

Within 10 minutes after silver nanoparticles treatment, E. coli colonies decreased by 87.7% on average compared to the control (100% standard) when 1ppm silver nanoparticles were treated. E. coli could not be found by up to%. In addition, the colony count of E. coli was significantly reduced to 87% and 84.3%, respectively, when treated with 1 ppm and 5 ppm silver nanoparticles [Table 2].

The antimicrobial effect from 10 minutes to 12 hours after administration of 10 ppm silver nanoparticles was significantly decreased after 10 minutes. In the case of 1ppm to 50ppm silver nanoparticles treatment, all silver nanoparticles at 12 hours E. coli colonies could not be seen in the addition group.

In order to confirm the antimicrobial mechanisms for E. coli and Salmonella, the nanoparticles were treated and confirmed by electron microscopy. As a result, pathogenic bacteria contacted with silver nanoparticles first adhered to the cell wall, and secondly, the cell membrane. It has been investigated that denaturation of cell wall proteins through permeation leads to the killing of bacteria in a short time [FIG. 6].

Also, 1 hour after adding silver nanoparticles, antimicrobial effect of silver nanoparticles was added to whole E. coli, and some of them were aliquoted, plated on nutrient solid medium, and cultured. 57.3 and 20.4% of the E. coli O157: K88ac: K89, O9: K103: K987 E. coli survived less than 10%, and the antimicrobial activity of silver nanoparticles There were some differences depending on the type. However, in the control group, E. coli was continuously grown up to about 6.9 times after 12 hours compared with the initial measurement [Table 2, Figure 5, P <0.01].

Absorption (OD) test results of the antimicrobial inhibition effect of the addition of silver nanoparticles, the silver nanoparticles were confirmed to have a growth inhibitory effect on the bacteria, but whether the results are killing effect or bacteriostatic effect No experiments were conducted to identify colonies and determine killing and bacteriostatic effects.

As a result, the antimicrobial effect of each E. coli O157: H7 in the test group to which 1ppm silver nanoparticles were added to the control group without the silver nanoparticles was as follows. In other words, the number of colonies in E. coli O157: H7 strains was less than 10 minutes when 1 ppm silver nanoparticles were added, compared to the control (100%), VT1: O157: H7 (ACH5), V2: O157: H7 (O59) and V1 + In the case of V2: O157: H7 (670) and VTe 0138, E57 (VTe) decreased from about 20% to 49% [Fig. 4-A, P <0.01].

However, VT2 Cl-0157: H- tended to increase from 102% to 105% [Fig. 4-B], but markedly decreased to 53% after 2 hours after the addition of silver nanoparticles. (P <0.001). Also, in the case of VT2 Cl-0157: H7, E. coli was not killed and bacteriostatic effects even in the silver nanoparticle concentration group of 1 ppm or less [Fig. 4-C]. And, E. coli 0157: H7 in the control group gradually increased in the number of colonies with time after the initial administration [Fig. 4, P <0.01].

Figure 4 (A) is the antimicrobial effect of the concentration of silver nanoparticles added to E. coli VT1 O157: H7 (ACH5). The control group showed 1.8-fold growth after 14 hours compared to the initial administration, and colony did not appear after 14 hours after the treatment of the whole silver nanoparticles, indicating complete death (P <0.01). After 5 minutes of silver nanoparticles were added, the bacteria were reduced by about 38% at 5 minutes and 30% after 30 minutes, and 5.5% after 2 hours. After the last 14 hours, colonies did not appear and completely disappeared (P <0.01).

Figure 4 (B) is the antimicrobial effect of the silver nanoparticles added concentrations for E. coli VT1 O157: H7 (O59). The control group grew 2.72 times after 14 hours compared with the initial administration, and colonies did not appear after 14 hours after the treatment of the whole silver nanoparticles, indicating complete death (P <0.01), and 5 ppm of experiments. After 5 minutes of addition of silver nanoparticles, bacteria were reduced by about 28% compared to the first, and after 30 minutes, 6% and colony disappeared after the last 14 hours. (P <0.01).

Figure 4 (C) is the antimicrobial effect of the concentration of silver nanoparticles added to E. coli VT1 + VT2 O157: H7 (670). The control group grew 5.65 times after 14 hours compared with the initial administration, and colonies did not appear after 14 hours after the treatment of the whole silver nanoparticles, indicating complete death (P <0.01). After 5 minutes of addition of silver nanoparticles, the bacteria decreased by about 69% compared to the first time, and after about 30 minutes, about 48% and 1.56% after 2 hours. After the last 14 hours of decay, colonies did not appear and completely disappeared (P <0.01).

Figure 4 (D) is the antimicrobial effect of the silver nanoparticles added concentrations for E. coli VTe O138, E57 (VTe). The control group showed 6.6-fold growth after 14 hours of initial administration, and no colony was observed after 14 hours of treatment with all the silver nanoparticles, indicating that it was completely killed (P <0.01), and 5 ppm of the experimental group. After 5 minutes of adding silver nanoparticles, the bacteria decreased by about 48% compared to the first time, about 39% after 30 minutes, and 12% after 2 hours, and last 14 hours. The poem was recognized as completely colonized because no colonies appeared (P <0.01).

Figure 4 (E) is the antimicrobial effect of the silver nanoparticles added concentrations for E. coli VTe O138, E57 (VTe). The control group showed 1.15-fold growth after 6 hours of initial administration, and the colony of 5 ppm silver nanoparticles treatment group decreased 1.6% after 6 hours after the entire silver nanoparticles treatment. Sphere was found to have no antimicrobial effect, and the number of microorganisms was decreased continuously when silver nanoparticles of 5 ppm or more were treated (P <0.01).

Figure 4 (F) is the antimicrobial effect of the concentration of silver nanoparticles added to E. coli VT2 Cl-O157: H7. The control group showed 1.02 fold growth after 6 hours of initial administration, and the colony of 5ppm silver nanoparticles treatment group was reduced to about 3% at 6 hours after all silver nanoparticles treatment, but 1ppm silver nano Particle administration was shown to have no antimicrobial effect. When silver nanoparticles of 1 ppm or more were treated, the number of bacteria continued to decrease (P <0.01).

In conclusion, the antimicrobial effects of silver nanoparticles from 10 minutes to 6 hours and 14 hours after E. coli 0157: H. E. coli VT2 Cl-O157: H7 and VT2 Cl-O157: H- The total number of colonies of the four other bacteria was significantly reduced (P <0.01), and the total number of colonies was significantly decreased after 2 hours (P <0.01). Colonies of E. coli O157: H7 were not found (P <0.001).

However, when 10 ppm or more silver nanoparticles were added to E. coli VT2 Cl-O157: H7 and 10 ppm of VT2 Cl-O157: H-, the bacteria were killed after 1 hour and 10 minutes. This result is considered to be the species difference of bacteria. In contrast, the control group continued to proliferate when the total E. coli count was compared with the initial measurement (P <0.01).

la. With general antibiotics Silver nanoparticles  Comparison of Antimicrobial Effects of Formulations

In the present invention, the antimicrobial activity of the detergent additive (nanosilver-III or nanosilver agent) containing the silver nanoparticles, which was the final development agent, was shown to be excellent antimicrobial activity, but the final pH range was weakly acidic, so this pH was There is a necessity to further confirm whether the effect on the antimicrobial activity, the results of this test are as follows [Tables 2 and 3, Figure 5].

First of all, the composition of nano silver-III, a developmental agent, was prepared differently, and the concentration of silver nanoparticles was added in different concentrations. After the addition, the killing result of Escherichia coli was investigated. As a result of the treatment, more than 99% of them were killed within 1 hour, and the mortality rate increased with the passage of time, which was recognized as 100% after the last 16 hours. The expected pH of the manufacturing process did not affect the antimicrobial activity.

In addition, the additives for detergents containing commercially available commercial antibiotic OTC and silver nanoparticles were added to the E. coli solution cultured at the same concentration conditions and at the same time, and the antimicrobial activity over time was examined. The antimicrobial activity was 100% when the concentration of 1ppm or more within the time, while the OTC treatment was shown to show the same antimicrobial activity when the concentration of more than 0.01% (100ppm) after 3 hours.

Considering that it was E. coli isolated from the field, it was expected that it was exposed to antibiotics such as OTC for a long time, and this resulted in the resistance to E. coli.

In addition, these results showed that the silver nanoparticles of the present invention had excellent antimicrobial activity against pathogenic bacteria resistant to general antibiotics, and showed the same result as the antimicrobial activity test for resistant bacteria.

In this experiment, E. coli, the causative agent of diarrhea in Korea, was isolated and the antimicrobial activity of silver nanoparticles was measured under laboratory conditions.

E. coli isolates cultured above a certain level in liquid media showed significant decreases when 1 ppm silver nanoparticles were added as a result of comparing O · D values with control and elapsed time after addition of silver nanoparticles [Table 3, Figure 5 ].

However, the antimicrobial activity of the O · D measurement is insufficient to determine whether or not by the growth inhibition of E. coli or death. Therefore, in order to reconfirm this, E. coli was sufficiently cultured in a liquid medium, and silver nanoparticles were added by concentration (1 ppm to 50 ppm), and then transferred to a solid medium, and the colonies were counted for one day. From now on, the whole E. coli showed an excellent effect (P <0.001) at a low concentration, such as 1 ppm, as well as a sharp decrease in colony counts compared to the control, and for 10 minutes to 12 hours after the administration of silver nanoparticles. The colonies did not appear at all and were judged to have died [Table 3, FIG. 5]. However, it was thought that there was a slight difference in the antibacterial activity of E. coli over time after treatment with silver nanoparticles. The concentration of 50 ppm and 25 ppm showed the fastest reduction effect of E. coli within 10 minutes after silver nanoparticle administration. to be.

Therefore, proper antimicrobial effective concentration test of silver nanoparticles against E. coli should be confirmed.

On the other hand, the report of isolation of Verotoxin (VT) producing Escherichia coli from humans in Korea has not been carried out except for the 1 isolate of E. coli serotype O157 isolated from food poisoning patients in 1994, and caused by verotoxin producing Escherichia coli. There is also little publicity about the importance of becoming a disease (Sandra et al., 1986).

In addition, a clear alternative to prematurely block the contamination of animals that are a host of E. coli O157: H7 and the resulting meat derived from livestock is to use general synthetic antibiotics. In this experiment, E. coli O157: H7, a causative agent of diarrhea and death during infection, was isolated from the domestic and international animals, and the antimicrobial activity of silver nanoparticles was measured under laboratory conditions.

In other words, E. coli O157: H7 isolates cultured above a certain level in liquid medium were compared with silver nanoparticles and the absorbance was compared with the control group.

However, it is necessary to determine whether the effect according to the type of bacteria also shows the same tendency, and in order to reconfirm the effect of E. coli, the silver nanoparticles were added at different concentrations (50ppm, 25ppm, 5ppm and 1ppm). Next, the colonies were directly counted for one day after being transferred to a solid medium, and within 10 minutes after silver nanoparticle administration, the whole E. coli showed an excellent effect (P <0.01) at a low concentration such as 1 ppm compared to the control. Not only the silver nanoparticles were administered, but also 10 minutes to 6 hours and 14 hours after all the silver nanoparticles were added.

However, it was thought that there was a slight difference in the antimicrobial activity of E. coli over time after treatment with silver nanoparticles according to species and concentration. This is the fastest reduction of E. coli at 25 ppm and 50 ppm concentrations within 10 minutes after silver nanoparticle treatment Because it showed an effect [Table 2].

In addition, the total number of colonies of E. coli 0157: H bacteria except for E. coli VT2 Cl-O157: H7, E. coli VTe O138, and E57 (VTe) was significantly reduced (P <0.01). In general, the number of abrupt colonies was significantly decreased after 2 hours, and no colonies of E. coli O157: H7 were found in all silver nanoparticles added groups after 6 hours (P <0.01). However, in the case of E. coli VT2 Cl-O157: H7 and E. coli VTe O138, E57 (VTe), when the silver nanoparticles of 0.01% or more were added, the bacteria were killed within 6 hours. To 5].

Table 3. Antimicrobial Activities of Silver Nanoparticles with Different Concentrations and pH and E. coli O157: k88ac Isolated from Fecal Diarrhea of Piglets Following OTC Treatment

Experimental groups  Nano-silver OTC * pH 4.8 pH 6.75    pH 6.5 1hr 13 hrs 16 hs 3hrs 16 hrs  1hrs 13 hrs control 2,000 # < 2,000 < 2,000 < 2,000 < 2,000 < 2,000 < 2,000 < 50 ppm concentration 0 0 0 0 0 0 0 25 ppm concentration 0 0 0 0 0 131 0 10 ppm concentration 0 0 0 0 0 1448 624 5 ppm concentration 0 0 0 0 0 2,000 < 2,000 <  1 ppm concentration 880 36 8 183 210 2,000 < 2,000 < *: Oxytetracycline HCl (OTC), #: 2 x 10 ⁷ cfu / ml

Experimental Example 3. Investigation of antimicrobial effect and induction of resistance formation from silver nanoparticles

end. Screening of Antimicrobial Effect and Induction of Resistance Formation from Silver Nanoparticles

1) Investigation of antimicrobial effect and induction of resistance formation against drug-resistant and non-resistant bacteria

A) Preparation of E. coli and Silver Nanoparticles

E. coli NCTC 9001 as standard strain and E. coli isolated from piglets requested for laboratory diagnosis due to diarrhea symptoms. The serotype was O157: K88ac. Among them, O157: K88ac produced heat-resistant toxins. The silver nanoparticles prepared in Example 1 were used.

B) Culture of E. coli and drug sensitivity test

Each E. coli strain to be applied to the experiment was inoculated in each nutrient broth (Difco) and shaken for 18 hours at 37 ℃, 150 rpm. Each E. coli culture was centrifuged (500 xg, 4 min) to isolate E. coli, and then diluted with phosphate buffer (0.1M, pH 7.0). It was used for the experiment.

In addition, Escherichia coli O157: K88ac and standard strain E. coli NCTC 9001 isolated from piglet feces were used as a test bacterium for confirming drug retention and antimicrobial test. And, the antimicrobial susceptibility test was tested according to the method of Bauer and Kirby (1966). Antimicrobial susceptible discs are products of BBL, Gentamicin (Gm, 10mcg), Kanamycin (Kanamycin, Km, 30mcg), Neomycin (Neomycin, N, 30mcg), Cephalothin (Cf, 30mcg), Streptomycin Eight antimicrobial agents (Streptomycin, Sm, 10mcg), Tetracycline (Tetracycline, Te, 30mcg), Mycacin (Amikacin, Am, 30mcg), and Chloramphenicol (Cm, 30mcg) were used. Judging by the criteria of the National Committe for Clinical Laboratory Standards (1988).

C) Resistance Formation Effect by Addition of Silver Nanoparticles

In order to determine whether silver nanoparticles were added and resistance generation by generation, after preparing nutritious juice medium in a 250ml flask, each prepared E. coli culture was adjusted to 2 x 10 ⁷ cfu / ㎖ and then inoculated.

In order to confirm whether the test strain is formed by the silver nanoparticles, the experimental group was set to the addition group in which only 5 ppm silver nanoparticles were administered, and the control group without the addition of the silver nanoparticles was set for the entire experimental group, respectively. Incubated at 37 ° C.

In addition, the 5 ppm single silver nanoparticles administration time was 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes and 10 minutes, and 30 minutes thereafter for 1 hour, 2 hours, 3 hours, 10 hours and 18 hours. After this, 10 μl of each treatment was collected, mixed well with a cap tube containing 990 μl of phosphate buffer solution, and 50 μl of this was smeared on blood and MacConkey agar medium, and then cultured for one day. After at least 3 hours elapsed, the surviving hybrids were selected and cultured, and the entire process was repeated up to 10 generations to determine whether silver nanoparticles formed resistance in E. coli.

D) Death after the addition of silver nanoparticles

To determine whether E. coli which survives after a certain time after adding silver nanoparticles is survival due to resistance or difference in concentration of silver nanoparticles, different nutritional broth medium was prepared in a 250 ml flask, and then each E. coli prepared before The culture was adjusted to 2 × 10 ⁷ cfu / ml and then inoculated.

In total, 12 experiments were set up with 5 treatments in which silver nanoparticles were added by concentration (50ppm, 25ppm, 5ppm, and 1ppm) and a control without adding silver nanoparticles, and before adding silver nanoparticles (0 min) 10 μl of each treatment at 30 minutes, 1 hour, 2 hours, 3 hours 4 hours, and 18 hours after the addition, and mixed well in a cap test tube containing 990 μl of phosphate buffer solution. The number of colonies that appeared after a day of culture by agar plated on the agar plate was determined to determine the killing effect, and the colony surviving after a predetermined time (minimum 3 hours) was selected and cultured. When the particles were treated, it was further confirmed whether the viable bacteria survived by resistance or by difference in concentration.

2) Screening of Antimicrobial Effects of Silver Nanoparticles on Resistant Factors

A) Use strain and medium condition

The strain used was E. coli JM83 (hereinafter AMP-JM83) transformed with plasmid pUC19 to be resistant to the same strain as E. coli JM83 strain (hereinafter CNTL-JM83) that is not resistant to ampicillin antibiotics. ) 2 types were used.

LB (50 ppm Yeast extract, Bacto tryptone, 1% NaCl) was used as the liquid medium for E. coli JM83, and 2% Bacto agar was added to LB as the flat medium, and as a medium for selecting antibiotic resistant strains. Ampicillin 50 μg ml ⁻¹ was added and used.

B) transformation by plasmid pUC19

E. coli JM83 to be used as a host was inoculated in LB medium and cultured at 37 ° C. with shaking to incubate at 550 nm until the OD value was 0.45 to 0.5, followed by centrifugation (2,000 × g) to obtain cells. The cells were gently suspended in 15 ml of 0.1 M CaCl ₂ and left on ice for 15 minutes, centrifuged again (2,000 xg) to recover the cells, and then carefully suspended in 3 ml of the solution and stored on ice. Used within the time range.

The transformation was carried out by mixing 10 μl of plasmid and 200 μl of JM 83 competent cell for 30 minutes on ice, then heat shock for 1 minute at 42 ° C. and leaving it on ice for 2 minutes. Then, 1 ml of LB medium was added thereto and left at 37 ° C. for 1 hour, followed by centrifugation (2,000 × g) for 2 minutes at 4 ° C., leaving about 200 μl of solution, and then removing the supernatant. Cells were released from the remaining solution, plated in a plate medium containing 50 μg / ml of ampicillin, and colonies formed by incubating at 37 ° C. for 16 hours were used.

C) Investigation of the sensitivity of CNTL-JM83 and AMP-JM83 to ampicillin

Experimental liquid and solid medium of CNTL-JM83 and AMP-JM83 were prepared by adding 50 μg of ampicillin per ml of medium during the experiment, and then a certain amount of Escherichia coli was plated during the experiment. A susceptibility study to ampicillin of E. coli AMP-JM83 was performed.

D) Plasmid DNA Extraction and Assay

Plasmid DNA extraction of AMP-JM83 artificially introduced ampicillin-resistant plasmid DNA into CNTL-JM83 by treatment with silver nanoparticles was performed by Birnboim et al. (1979), Ish-Horowice et al. (1981), and Sambrook et al. (1989). It implemented by the method of these. Antimicrobial effect and antiplasmidity of plasmid DNA according to antimicrobial experiments treated with silver nanoparticles were confirmed by 0.8% agarose gel electrophoresis (Messing 1983: Norrander et al., 1983; Yonisch-Perron et al., 1985). .

Extraction of the plasmid DNA introduced into AMP-JM83 was carried out by forming a medium (hereinafter referred to as LB (Amp)) in which 50 μg of ampicillin was added per ml of LB medium, followed by selecting colonies grown in LB (Amp) plate medium. Amp) After inoculating 10 ml of medium, the culture was shaken at 37 ° C. for 12-16 hours. When the culture was completed, the cells were transferred to a sterile centrifuge tube and centrifuged at 6,000 rpm for 5 minutes (8,000 xg, 5 min) to recover the cells.

The recovered cells were suspended in 100 µl of Solution I (50 mM glucose, 25 mM Tris-Cl, pH 8.0) and 10 mM EDTA mixture until the cells were completely released, and then solution II (0.2 N NaOH, 1% SDS mixture) was added thereto. After 200 μl was added, the tube was shaken carefully and left to stand on ice for 5 minutes. Add 150 µl of cold prepared solution III (60 ml of 5M potassium acetate, 11.5 ml of glacial acetic acid, 28.5 ml of tertiary deionized water), mix gently, turning the tube 3-4 times, and let it stand on ice for 5 minutes. Centrifuged (35,000 × g, 4 ° C.) for a minute. Subsequently, the supernatant was transferred to a new microfuge tube, and phenol / chloroform was mixed in the same volume as the supernatant, stirred for 5 minutes, and centrifuged again for 10 minutes to remove residual protein. . Only the supernatant containing the double plasmid DNA was taken, transferred to another test tube, mixed well with 95% ethanol, and then treated at low temperature for 5 minutes, followed by centrifugation (35,000 xg, 10 min) to obtain precipitated plasmid DNA. After appropriately drying by vacuum drying, it was dissolved in TE buffer solution (10 mM Tris Acetic acid, 1 mM EDTA pH 8.0) and electrophoresed to examine the profile of DNA.

Electrophoresis was performed by mixing 1 μl of loading buffer and DNA dissolved in TE on a 0.8% agarose gel and loading at 100V for about 20 minutes. When the development is complete, stain with ehtidium bromide solution (5 μl / ml), check the plasmid DNA band with a short wave transillumininator, and use a polaroid UV 55mm camera with a filter. Photographed. In this case, a λ-hind III size marker was used as a standard marker.

Plasmid DNA isolation experiments confirmed abnormality of plasmid DNA against CNTL-JM8 before the first ampicillin resistant plasmid and AMP-JM83 which survived the antimicrobial test after treatment with AMP-JM83 and silver nanoparticles.

hemp) Of silver nanoparticles  Escherichia coli antimicrobial test

Silver nanoparticles prepared in Naul (Korea) by the method of Example 1 were used as silver nanoparticles used in this experiment, and silver nanoparticles content in the stock solution was used by receiving 5,000 ppm of donor, and E. coli CNTL- JM83 and AMP-JM83 strains were inoculated in different nutrient broths (Difco) and shaken for 18 hours at 37 ° C. and 150 rpm. Centrifuge the E. coli culture (500 xg, 4 min) to separate E. coli, and then dilute with phosphate buffer (0.1M, pH 7.0) .If necessary, repeat this centrifugation several times to adjust the bacterial count. Used for.

In order to examine the antimicrobial activity of silver nanoparticles by concentration, nutrient broth was prepared in 250 ml flask and inoculated with E. coli and incubated at 37 ° C. These E. coli cultures were adjusted to 2 x 10 ⁷ cfu / ㎖, and then 5 treatments in which silver nanoparticles were added at different concentrations (50ppm, 25ppm, 5ppm and 1ppm) and controls without silver nanoparticles were placed. After adding silver nanoparticles, 10 μl of each treatment was collected at 1 minute, 2 minutes, 3 minutes, 4 minutes and 5 minutes, 10 minutes, 30 minutes, 1 hour, 3 hours, 5 hours, 12 hours and 24 hours. After mixing well in a cap test tube containing 990 μl of phosphate buffer solution, 50 μl of this was plated on MacConKey solid medium and cultured for one day to count colonies.

[result]

end. Silver nanoparticles  Investigation of antimicrobial effect and induction of resistance formation

One) Drug-resistant bacteria  And immunity In unbearable bacteria  Screening for anti-fungal activity and antimicrobial effect

A) Drug resistance test of E. coli

Table 4 shows the antimicrobial susceptibility test results of E. coli NCTC 9001 and E. coli O157: K88ac isolated from piglets. In other words, E. coli NCTC 9001, which was used as a standard E. coli strain, did not have susceptibility to all antibiotics.E. Coli O157: K88ac isolated from piglets was confirmed to be susceptible to antibiotics. Sensitivity was shown only for two antibiotics, chloroamphenicol (Cm).

I) Of silver nanoparticles  Formation of resistance by continuous treatment by generation Determination of presence

The antimicrobial activity of E. coli in the experimental group to which 5 ppm of silver nanoparticles were added to the control group without the silver nanoparticles was shown in FIGS. 7 to 9.

The colony count of E. coli strains was significantly decreased compared to the control (P <0.001) within 10 minutes when 5 ppm of silver nanoparticles were added up to 10 generations (P <0.001). Increased by an average of 2.0 to 6.6 fold.

In the case of O157: K88ac, which is resistant to E. coli after treatment with silver nanoparticles, E. coli colonies were completely killed in the first generation compared to the control group (100% standard) when treated with silver nanoparticles. The treatment decreased from 0% to 7.4% on average, and no E. coli survived at 18 hours.

In addition, in the case of Escherichia coli NCTC 9001, which is not resistant to general antibiotics, the number of colonies that survived to the first generation and the tenth generation after the administration of the silver nanoparticles was 0.8%. The generation of E. coli colonies did not appear [Fig. 9].

However, colony numbers surviving within 10 hours after 10 minutes were found to survive due to the difference in concentration of silver nanoparticles rather than formation of resistance (P <0.01). In the control group, E. coli bacteria continued to grow up to an average of about 2 to 6.6 times after 18 hours compared with the initial measurement (P <0.01).

C) by concentration Silver nanoparticles  Antibacterial effect

As a result of assaying the antimicrobial effect of E. coli by each unknown concentration with the presence of resistance to the addition of a single concentration of 5 ppm silver nanoparticles as shown in Figures 7 to 9.

The number of colonies of E. coli strains was significantly decreased in 30 minutes after the addition of 1 ppm to 50 ppm silver nanoparticles compared to the control group. Colonies did not appear (P <0.001).

For E. coli O157: K88ac, which has drug resistance, E. coli colonies within 30 minutes after treatment with silver nanoparticles from 1st generation to 10th generation were averaged from 0 to 50% relative to the control (first 100% basis) when treated with silver nanoparticles. E. coli was significantly decreased (P <0.01), which decreased to 6.7%, and averaged from 0% to 12.8% when treated with 0.1% silver nanoparticles and from 0% to 3.58% when treated with 0.01% and 5ppm silver nanoparticles. In addition, the number of colonies of E. coli was significantly decreased from 0 ppm to 37.5%, respectively, when treated with 1 ppm silver nanoparticles (P <0.001).

In the case of non-resistant E. coli NCTC 9001, coliform colonies within 30 minutes after the first to tenth generation of silver nanoparticles were treated with an average of 1.2 to 7.4% relative to the control (first 100%) when treated with 50 ppm silver nanoparticles. E. coli was significantly decreased (P <0.01), with an average of 0.4 to 2.4% when treated with 25 ppm silver nanoparticles and 0 to about 2.6% when treated with 5 ppm and 10 ppm silver nanoparticles. In addition, the number of colonies of E. coli was significantly reduced from 0 to 0.3% compared to the control group even when 1 ppm silver nanoparticles were treated (P <0.001). At 18 hours, no colonies of E. coli were found in all silver nanoparticle addition groups (P <0.001).

Table 4. Susceptibility to Antibacterial Agents of E. coli O157: K88ac and E.coli NCTC 9001

Drugs E. coli strains Antibacterial drug susceptibility Gm Km N Cf Sm Te Am Cm O157: K88ac R R R S R R R S NCTC 9001 S S S S S S S S R: Resistance, S: Sensitivity, Gentamicin (Gm, 10㎍), Kanamycin (Km, 30㎍), Neomycin (N, 30㎍), Cephalothin (Cf, 30㎍), Streptomycin (Sm, 10㎍), Tetracycline ( Te, 30 μg), Amikacin (Am, 30 μg) and Chloramphenicol (Cm, 30 μg)

 2. Screening of Antimicrobial Effects of Silver Nanoparticles on Resistant Factors

end. Antibacterial Test Results of Escherichia Coli

After incubation in phosphate buffer solution (pH 6.0, 37 ℃) of resistant bacteria resistant AMP-JM83 and non-resistant Escherichia coli CNTL-JM83, silver nanoparticles were added up to 1ppm to 50ppm, and culture was collected from 1 minute to 12 hours. The antimicrobial effect of silver nanoparticles was measured by counting colonies that appeared after incubation for 24 hours on MacConKey medium [FIGs. 11 and 12]. As a result, it was confirmed that E. coli CNTL-JM83 and AMP-JM83 both significantly reduced the E. coli between 1 and 30 minutes after administration of silver nanoparticles regardless of the treatment concentration (P <0.001).

The colony count of E. coli continued to decrease after 30 minutes to 12 hours after the addition of 1 ppm to 50 ppm of silver nanoparticles, and no colony of E. coli was observed after 12 hours of silver nanoparticles. (P <0.001), at 24 ppm or less concentration, the total number of bacteria was decreased from 3.5% to 11.3% compared to the control group after 24 hours (P <0.01). In other words, it was recognized that silver nanoparticles showed excellent antimicrobial effect against bacteria having resistance (P <0.01).

I. Results of Susceptibility of AMP-JM83 and CNTL-JM83 to Ampicillin

During the experiment, the experimental liquid and solid medium of AMP-JM83 were incubated with 50 μg of ampicillin per ml of medium, and then E. coli AMP-JM83 plated on solid medium retained ampicillin-resistant plasmid DNA by surviving in LB (Amp) medium. However, CNTL-JM83 Escherichia coli, which was used as a control, was killed in LB (Amp), indicating that no plasmid DNA resistant to ampicillin was formed (FIG. 10).

All. Plasmid DNA Reaction Patterns after Silver Nanoparticles Treatment

Resistant plasmid DNA was isolated by agarose-gel electrophoresis of CNTL-JM83 and AMP-JM83.

That is, in CNTL-JM83 before introducing the plasmid, the band originating from the plasmid did not appear. However, in the case of AMP-JM83 incorporating ampicillin resistant plasmid DNA, the plasmid fraction appeared in the form of a double band in open-circular form and circular form. It was confirmed that the resulting plasmid DNA was introduced.

In addition, the results obtained by screening the surviving hybrids from AMP-JM83 treated with silver nanoparticles and fractionating the resistant plasmid DNA also showed the same results. (P <0.01), the surviving AMP-JM83 was found to be due to the concentration difference of silver nanoparticles and the treatment time, not to be resistant to silver nanoparticles.

The antimicrobial effect of the drug-resistant bacteria is treated when the antimicrobial silver nanoparticles are artificially introduced after the introduction of the drug resistant bacteria and the plasmid DNA of Escherichia coli, and the modification of the plasmid DNA of the surviving resistant E. coli when the silver nanoparticles are treated. I checked the presence or absence.

As the strain, the control group used the first E. coli JM83, and the experiment was performed by setting the strain into which the ampicillin-resistant plasmid DNA was introduced into the same strain as the test strain.

As a result, plasmid DNA fragmentation was confirmed by electrophoresis, whereas the control bacterium CNTL-JM83 showed no plasmid. In the form of a double band, the molecular weight appeared at about 2.0Kb and 3.0Kb, confirming the introduction of plasmid DNA derived from ampicillin.

E. coli AMP-JM83, which survives the treatment of silver nanoparticles, also detected the same pattern of plasmid DNA. As a result, silver nanoparticles showed excellent antibacterial effect against bacteria having drug-resistant plasmid DNA. 12, P <0.01)].

Recently, the majority of Gram-negative bacteria, including E. coli, isolated from clinical materials, are resistant to antimicrobial agents that are inactivated by β-Lactamase, including penicillin, which is mainly associated with the acquisition of plasmid DNA. (Neu 1983: Sabath et al., 1975). It is known that plasmid DNA is involved in resistance transmission, and recently, transposons have been reported to be involved in drug resistance delivery (Cohen et al., 1982: Forbes et al., 1983).

Among these, methicillin, erythromycin, and Rif are chromosomal DNA, and penicillin, gentamycin, and tetracycline are mainly determinants of plasmid DNA. (Carrol et al., 1989: Coleman et al., 1985; Holmberg et al., 1984; Lorian et al., 1986). Lyon et al. (1984) report that various mechanisms for bacterial resistance in antimicrobial agents are known, but plasmid DNA plays an important role as a mediator of drug resistance. They report that resistance to aminoglucoside-based antimicrobials, including gentamicin, is related to deliverable plasmid DNA, but that Bigelowe et al. Reported that it was non-deliverable, and that Witt and Dunnhapt were associated with chromosomal DNA. I've done it.

However, the above results were only general reports on antibiotics, and considering that silver nanoparticles used in the present invention are antimicrobial materials extracted from natural polymer materials, it is assumed that the silver nanoparticles are continuously used. You can not help but think about it.

Therefore, in this experiment, antibacterial activity against E. coli introduced plasmid DNA, which is recognized as a resistance to drug resistance, showed antibacterial activity against bacteria having resistance.

Therefore, in this experiment, E. coli, the causative agent of piglet diarrhea, which is a problem in Korea, is resistant to common antibiotics. The antimicrobial activity of silver nanoparticles was measured under laboratory conditions for E. coli with the plasmid DNA, which plays an important role.

As a result, after culturing in phosphate buffered solution against resistant E. coli CNTL-JM83 and resistant E. coli AMP-JM83, silver nanoparticles were added from 1ppm to 50ppm, and the culture solution was collected from 1 minute to 12 hours. As a result of measuring the antimicrobial effect of silver nanoparticles by counting colonies that appeared after incubation for 24 hours in the medium, E. coli was clearly reduced between 1 and 30 minutes after administration of silver nanoparticles regardless of the concentration of E. coli CNTL-JM83 and AMP-JM83. (P <0.001), resistant plasmid DNA was isolated from CNTL-JM83 and AMP-JM83 by agarose-gel electrophoresis. As a result of the plasmid DNA band in CNTL-JM83 before plasmid DNA was introduced. Did not appear. However, in the case of AMP-JM83 with ampicillin-resistant plasmid DNA, the plasmid DNA fraction appeared in double-band form in open-circular form and circular form, and its molecular weight appeared in about 2.0Kb and about 3.0Kb. It was confirmed that plasmid DNA due to was introduced.

In addition, the results obtained by screening surviving colonies from AMP-JM83 treated with silver nanoparticles and fractionating the resistant plasmid DNA also showed the same results. (P <0.01), the colony of surviving AMP-JM83 was confirmed to be due to the concentration difference and the treatment time of the silver nanoparticles, which is not exhibited resistance by the silver nanoparticles [Figs. 10 to 12].

In addition, E. coli O157: k88ac, which is a field isolate bacterium, is resistant to general antibiotics and has the same killing effect as OTC (Oxytetracycline-HCl) and silver nanoparticles. As a result of adding to E. coli solution incubated at the same time as concentration condition and examining the antibacterial activity over time, silver nanoparticles showed 100% antimicrobial activity at concentration of 1 ppm or more within 1 hour, whereas in OTC treatment, 3 hours In this case, the same antimicrobial activity was observed in the case of treating the concentration of 0.01% (100ppm) or more.

This is expected to be long-term exposure to antibiotics such as OTC according to the E. coli isolated from the field, which was eventually judged to have reduced the killing effect of E. coli as a result of having resistance. In addition, these results showed that silver nanoparticles possessed excellent antimicrobial activity against pathogenic bacteria resistant to general antibiotics, and showed the same results as the antimicrobial activity test for resistant bacteria.

In addition, it was thought that there was a slight difference in the antibacterial activity of E. coli over time after the treatment of silver nanoparticles by concentration, because in this test, the concentration of 0.01% or more showed the fastest reduction effect of E. coli within 10 minutes after silver nanoparticle treatment. Therefore, proper antimicrobial effective concentration test of silver nanoparticles against E. coli should be followed.

Example 2 Preparation of Silver Nanoparticles Additive for Detergent (Nanosilver-III and Nanosilver Sugar)

The silver nanoparticles introduction detergent additives used in Experimental Examples 4 to 7 were introduced with the silver nanoparticles prepared in Example 1, and a surfactant and a metal ion blocking agent for general detergents were applied, and polyvinyl alcohol was used as the surfactant. In addition, polyvinylpyrrolidone was used as the metal ion blocking agent, and the amount used was 0.3% by weight of the metal ion blocking agent based on 100 parts by weight of the surfactant, and the content of the silver nanoparticles was determined by the content applied in the experimental example. Adjusted.

The surfactant, the metal ion sequestrant and the silver nanoparticles were prepared by stirring at 15,000 RPM conditions at room temperature (nanosilver preparation, nanosilver-III).

Nano silver sugar was prepared by adding a sugar fatty acid ester to the mixture of the surfactant, the metal ion blocking agent, and the silver nanoparticles.

Experimental Example 4. Confirmation of basic antimicrobial properties of commercial products and nanosilver-III

1. Materials and Methods

end. material

The materials used in this experiment are as follows.

1) Tong Yang Trading Material: Fabric Softener (Product Name: Popran Iquiss, Popran Greenbouquet, Popran Pinky Rose), Detergent (Product Name: Clear Kitchen)

2) Detergent additives containing silver nanoparticles: Nano silver-I [Ag 5,000 ppm (w / v), Brown type, Naul Corporation], Nano silver-II [Ag 5,000 ppm (w / v), White type, Naul Corporation], Nanosilver-III [including silver nanoparticles prepared by the method according to Example 1, containing 10,000 ppm (w / v) Ag, Naul Corporation], nano silver sugar [method according to Example 1 Containing silver nanoparticles prepared by, containing 100 ppm (w / v) Ag, 16% sugar fatty acid (w / v), Chonnam National University]

3) Antibacterial assay strains: Bacillus sp. CK-1

I. Way

1) Culture of Assay Bacteria and Culture Solution

Standard strains include Bacillus sp. CK-1 was used and prepared Bacillus sp. CK-1 was inoculated in nutrient broth (Difco) and shaken for 18 hours at 37 ℃, 150 rpm. The fungal culture was diluted if necessary to control the number of bacteria and used for this experiment.

2) Antimicrobial Test Condition

Bacillus sp. CK-1 was inoculated and incubated at 37 ° C. These Bacillus sp. CK-1 culture medium was used after adjusting to 2 x 10 ⁷ cfu / ㎖. To determine the presence or absence of antimicrobial properties on commercial products, fabric softeners, dish detergents, and detergent additives containing the silver nanoparticles were introduced into the stock solution (100%, 1x), and 10 times (v / v) in tertiary sterile water, Six treatments diluted with 100-fold (v / v), 250-fold (v / v), 500-fold (v / v) and 1,000-fold (v / v) treatments and control treated with tertiary deionized water were added. [Fig 13].

end) Evenness  Antimicrobial Investigation by Preparation and Concentration Using Medium

In this experiment, we tested the degree of antimicrobial activity of the formulations prepared by the formulation and concentration by contact with the bacteria when the bacteria were spread under the environmental conditions of the solid medium.

To this end, Bacillus sp. After the CK-1 culture solution was smeared once in a solid medium, the diluted diluted formulations were carried out as follows to see the antimicrobial effect upon dropping. Bacillus sp. 50 μl of CK-1 culture was quantitatively collected and spread evenly on the medium, which was used as a material.The treatment for antimicrobial identification was carried out by diluting the preparations, which were prepared by diluting the currently available preparations by concentration, and the control and The comparison was confirmed.

For antimicrobial assay, 20 µl of the diluted solution for each preparation was quantitatively collected and then dipped into a solid broth where bacteria were smeared at regular intervals, followed by incubation at 37 ° C. for 24 hours to grow bacteria formed by the antibacterial effect. The antimicrobial results were confirmed by comparing the size of the low-ring ring with the control group [Table 5].

B) Antimicrobial investigation in case of adding bacteria after diluting by preparation and concentration

In this experiment, Bacillus sp. The antimicrobial activity of CK-1 bacteria over time when contacted with treatment groups prepared by different formulations and concentrations was tested.

That is, after diluting the preparation by the concentration in the broth nutrient liquid medium, Bacillus sp. After adding the same amount (10 μl) of CK-1, the antimicrobial effect was assayed for 3 hours. The assay was also applied by the drop method, and the antimicrobial effect assay was performed in the same manner as above.

[result]

end) Evenness  Antimicrobial Investigation by Preparation and Concentration Using Medium

In the case of smearing the bacteria under solid medium environmental conditions, it was tested to see how the antimicrobial activity of the formulations prepared by the preparation and concentration by contact with the bacteria.

As a result, the antimicrobial activity of the fabric softener, dishwashing detergent, nano silver-I, II and nano silver sugars was found to be over 90% even when diluted up to 500 times, and detergent additives containing silver nanoparticles were introduced. In the case of diluting more than 500 (Ag content, 25ppm) times, the antimicrobial activity was found to be the same [Table 5, Figure 13].

However, when the antimicrobial properties were examined by comparing the silver nanoparticles [nanosilver-I and II] prepared by the same method as in Example 1, the antimicrobial activity was different, which was different depending on the preparation method of the silver nanoparticles. It can be seen that, it was confirmed that the antimicrobial activity of the additive for detergents introduced with silver nanoparticles prepared by the present invention.

 In addition, Bacillus sp. Nano silver sugar [containing 100 ppm of Ag, 16% Sugar fatty acid ester (w / v)] was also used. In the case of dilution of CK-1 by concentration, the results showed that the Ag content of the stock solution was 100ppm, and when diluted 10-fold, the content of Ag (nanosilver-I type) was 10ppm and 500. In the case of diluting the pear, it was found that the antimicrobial effect was more than 90% even at 0.2 ppm, and the addition of Ag to the sugar fatty acid ester enhanced the antimicrobial effect. In other words, when comparing the antimicrobial effect at 90 ppm or more at 5 ppm for Nano Silver-I and the antimicrobial effect at 1 ppm for Nano Silver Sugar, sugar fatty acid has at least 5 times the antimicrobial effect. It was found that the aggregating effect of sugar fatty acid esters has a role of increasing these effects, compared with the result that sugar fatty acid esters do not have an antimicrobial effect against bacteria. 5, FIG. 13].

In conclusion, the sugar fatty acid ester is a natural non-toxic agent and is developed by using a natural biodegradable natural material but having a single sugar fatty acid ester formulation having a function as a surfactant and nanosilver applied thereto. It was confirmed that the development of the phosphorus wellness-type nano silver sugar preparation was possible.

Table 5. Bacillus sp. After addition of commercial products and additives for detergents into which silver nanoparticles were introduced. Effect on the Antimicrobial Activity of CK-1

                 Antimicrobial Effect Antimicrobial Effects of Treatment Agents by Concentration ^* Stock solution 10 times 100 times 250x 500 times 1,000 times Control (tertiary deionized water) +++ NT NT NT NT NT Fabric Softener (Popran Eyes) ^$ +++ +++ +++ +++ +++ - Softener (Popran Pinky Rose) ^$ +++ +++ +++ +++ +++ - Fabric Softener (POFRAN GREENBUKE) ^$ +++ +++ +++ +++ +++ - Kitchen Detergent (Clear Kitchen) ^$ +++ +++ +++ +++ +++ - Nano Silver-I ^& +++ +++ +++ +++ +++ - Nano Silver-II ^% +++ +++ +++ +++ +++ +++ Nano silver sugar ^# +++ +++ +++ ++ ++ -  *: +++: 90% or more killed, ++: 50% or more killed, +: 10% or more,-: No antimicrobial effect, NT: Not measured. $: Textile Softener [Product Name: Popran Iquiss, Popran Greenbouquet, Popran Pinky Rose Co., Ltd. Tong Yang Co., Ltd. [Product Name: Clear Kitchen, Dongyang Co., Ltd.], &: Nano Silver-I [Ag 5,000 ppm content (w / v), White type, Naul Co., Ltd.,%: Nano silver-II [Ag 5,000 ppm (w / v) content, Brown type, Naul Co., Ltd.], #: Nano silver sugar [ Ag 100ppm content (w / v), 16% Sugar fatty acid (w / v), Chonnam National University]

B) Confirmation of antimicrobial activity when bacteria are added after dilution by preparation and concentration

 As a result of testing the degree of antimicrobial activity when contacting the bacteria in the case of direct bacteria in the solid medium in the above experiments, the composition prepared by the formulation and the concentration, the fabric softener, dish detergent, nano silver-I, II And nano silver sugars were diluted up to 500 times, and the antimicrobial activity was found to be greater than 90% [Table 5].

Therefore, in the present experiment, Bacillus sp. When CK-1 bacteria were directly contacted for 3 hours, the antimicrobial activity (killing effect) was further examined [Table 6].

As a result, the control was consistently Bacillus sp. While CK-1 bacteria grow, the antimicrobial effect of the whole fiber softener is greater than 90% when the stock solution is treated. Was found to be absent. In the case of the same fabric softener, in the case of Popran Greenbuque Co., Ltd., Dongyang Co., Ltd., the antimicrobial effect was about 50% even in the undiluted solution. It is presumed that the kind of initial ingredients and their composition ratios have a great difference in antimicrobial activity.

However, in the case of kitchen utensils (CJ, Tong Yang C & T), the dilution ratio is 100 times higher than the antimicrobial activity of 90%. Was considered to be an important factor.

These results, it is expected that it is necessary to additionally determine the detailed antimicrobial effect for each raw material to determine whether there is an effect on the antimicrobial properties and the antimicrobial effect depending on the ingredients of the basic sales agent.

However, as a result of confirming the antimicrobial activity of the detergent additive (nano silver preparation) in which silver nanoparticles were introduced, Bacillus sp. In case of direct contact with CK-1 bacteria, the antimicrobial activity was over 90%. It was investigated to show a sustained antimicrobial effect upon contact with CK-1. In addition, the nanosilver shoe prepared by chelating silver nanoparticles to a sugar fatty acid ester (sugar fatty acid ester) was examined to investigate the antimicrobial activity of the formulation, in this case, the antimicrobial effect was found to be the most excellent.

In view of the fact that silver nanoparticles were 100ppm in the initial raw material, the content of silver nanoparticles was 0.1ppm at 1,000-fold dilution. When the nano silver-I and nano silver-II were treated with 1,000-fold dilution, the amount of nanosilver was 5 ppm, which resulted in an increase of 50-fold antimicrobial effect.

Therefore, nanosilver sugar preparations enhance these antimicrobial effects, as compared with preliminary basic experiments confirming that the original fatty acid esters do not possess antimicrobial effects against bacteria. The antimicrobial effect possessed by the chelated silver nanoparticles was synergized (synergy) and was considered to act as a factor to further double [Table 6].

Table 6. Bacillus sp. After the CK-1 bacteria contacted the microorganisms in different treatment groups and concentrations, the microorganisms were directly contacted and the antimicrobial activity was confirmed over time.

                 Antimicrobial Effect Antimicrobial Effects of Treatment Agents by Concentration ^* Stock solution 10 times 100 times 250x 500 times 1,000 times  Control (tertiary deionized water) +++ NT NT NT NT NT Fabric Softener (Popran Eyes) ^$ +++ + + + + + Softener (Popran Pinky Rose) ^$ +++ + + + + + Fabric Softener (POFRAN GREENBUKE) ^$ ++ + + + + + Kitchen Detergent (Clear Kitchen) ^$ +++ +++ +++ + + + Nano Silver-I (5,000 ppm) ^& +++ ++ ++ +++ +++ +++ Nano Silver-II (5,000 ppm) ^% +++ ++ ++ +++ +++ +++ Nano silver sugar (100ppm) ^# +++ +++ +++ +++ +++ +++  *: +++: 90% or more killed, ++: 50% or more killed, +: 10% or more,-: No antimicrobial effect, NT: Not measured. $: Textile Softener [Product Name: Popran Ikiss, Popran Greenbouquet, Popfran Pinkie Rose, Dongyang C & T], Kitchen Detergent [Product Name: Clear Kitchen, Dongyang C & T], &: Nano Silver-I [Ag 5,000ppm content (w / v), White type, Naul Co., Ltd.],%: Nano silver-II [Ag 5,000ppm (w / v) content, Brown type, Naul Co., Ltd.], #: Nano silver Sugar [Ag 100ppm content (w / v), 16% Sugar fatty acid (w / v), Chonnam National University]

Experimental Example 5. Comparison of antimicrobial activity of synthetic antibiotics, commercially available products and nanosilver-III of the present invention

In this experiment, the basic antimicrobial activity of the commercially available products, commercially available antimicrobial preparations, and commercially antibacterial dog shampoos were tested for the development of well-being detergents using the silver nanoparticle detergent additive (nanosilver-III). After that, when the silver nanoparticles prepared according to the present invention was introduced, it was confirmed how much antimicrobial activity could be amplified.

However, in order to objectively judge the results of antimicrobial activity of the preparation for introducing silver nanoparticles, antimicrobial activity was first tested by applying synthetic antibiotics that are commonly used for medical purposes.

1. Materials and Methods

end. material

1) Commercially available products: Textile softeners (Product name: IKISS, GreenBuke, Popran Pinky Rose), Detergent (Product name: Clear kitchen) [DONGYANG CORPORATION, Korea]

2) Detergent additives containing silver nanoparticles: Nanosilver-I [Ag 5,000ppm (w / v) containing, Brown type, Naul, Korea, manufactured according to a method different from Example 1], Nanosilver- II [Ag 5,000ppm content (w / v), White type, Naul Co., Korea, Manufactured according to a method different from Example 1], Nano silver sugar [Prepared according to the method described in Example 1 containing silver nanoparticles , 100 ppm Ag (w / v), 16% Sugar fatty acid easter (w / v), Chonnam National University], Bioconversion formulation (NTS, Canada), Streptomycin sulfate (Keun Kun Dang, 10% (w / v) solution, Korea) , Anti-Invasive Dog Shampoo (E Company, Korea)]

3) Antibacterial assay strains: Bacillus sp. CK-1, E. coli O157: K88ac

I. Way

1) Culture of Assay Bacteria and Culture Solution

Standard strains include Bacillus sp. CK-1 and E. coli O157: K88ac were used, and the prepared test bacteria were inoculated in nutrient broth (Difco) and shaken for 18 hours at 37 ° C and 150 rpm, and used as experimental materials. Dilution, if necessary, to control the bacteria was used in this experiment

2) Composition and antimicrobial investigation of antimicrobial assay preparation

Bacillus sp. CK-1, E. coli 0157: K88ac was inoculated and incubated at 37 ° C. The culture of these bacteria was adjusted to 1 x 10 ⁷ cfu / ㎖, and each concentration treatment was prepared for each formulation. Concentration composition consisted of six treatments and tertiary sterilized water, which added the above-mentioned materials to stock solution (100%) and tertiary sterilized water, 10 times (v / v), 100 times, 250 times, 500 times and 1,000 times. Treated controls were placed.

The antimicrobial assay was performed as follows. Bacillus sp. 50 μl of CK-1 and E. coli O157: K88ac cultures were added and smeared, and used as a material for the antimicrobial assay. Treatment for antimicrobial confirmation was performed as follows.

In other words, the antimicrobial assay by diluting solution prepared by each preparation and concentration was confirmed by the drip method, which was obtained when 20 μl of each dilution was collected and plated on a juicy agar medium and cultured for one day. The antimicrobial progress was confirmed by comparing the size of the bacterial growth inhibitory ring with the control [Table 7, FIG. 15].

A) Investigation of antimicrobial activity by preparation and concentration using homogenous smear medium

In this experiment, it was confirmed how the antimicrobial activity of the formulations prepared by the formulation and by concentration in contact with the bacteria in the case of spreading the bacteria in the solid medium.

To this end, Bacillus sp. The CK-1 and E. coli O157: K88ac bacteria cultures were once smeared on a solid medium, and then diluted with different concentrations, and the antimicrobial effect was confirmed by the following method.

That is, Bacillus sp. 50 μl of CK-1 and E. coli O157: K88ac cultures were aliquoted and evenly spread on different media, and then used as a material. After diluting each treatment prepared by dilution, compared with the control.

For the antimicrobial assay, the microorganisms formed by the antimicrobial effect were collected by quantitatively collecting 20 μl of the diluted solution for each preparation, and then dripping them at a predetermined interval on a juicy solid medium provoked with the bacteria. The antimicrobial activity was confirmed by comparing the size of the growth inhibitory ring with the control group [Table 7].

B) Antimicrobial effect of bacteria contacted in liquid medium prepared by preparation and concentration

In this experiment, Bacillus sp. The antimicrobial activity of CK-1 and E. coli O157: K88ac in direct contact with liquid medium and after a certain time was tested. That is, the preparation is diluted by concentration in the broth nutrient liquid medium, Bacillus sp. CK-1 and E. coli O157: K88ac were added to each test tube in the same amount (10 μl), and then the antimicrobial effects were tested at 3 and 24 hours. . Juicy solid medium was added dropwise by treatment for 24 hours at 37 ℃ ℃, and the antimicrobial effect was confirmed by assaying the colonies formed at this time [Table 7].

[result]

A) Investigation of antimicrobial activity by preparation and concentration using homogenous smear medium

The antimicrobial properties of various commercially available formulations and the additive for detergents in which the silver nanoparticles of the present invention were introduced were tested for the antimicrobial spectrum of Bacillus and E. coli as the co-bacteria, and the silver nanoparticles prepared according to the present invention. The degree of antimicrobial amplification at the time of introduction of the additive was compared and examined in various angles [FIG. 14].

As a result, first, the bacteria were plated on a solid nutrient medium, and when the formulations prepared according to the formulations and concentrations contacted the bacteria, Bacillus sp. Only the CK-1 bacteria first confirmed how much antimicrobial properties were retained [Table 7, Fig. 15], when the third sterilized water treatment, the bacteria continued to grow, but in the case of fibrous softeners stock solution and stock solution In case of diluting 10 times, the antimicrobial activity was over 90%. However, in the case of dish detergent in the same household goods group, the antimicrobial effect is more than 90% when diluted by 250 times with tertiary sterilized water in the stock solution. As the antibacterial effect of the pear was shown, it was judged as an effect according to the composition difference of the preparation.

Detergent additives containing silver nanoparticles were found to have antimicrobial properties up to 100 (Ag content 50 ppm) in the stock solutions of nanosilver-I and -II. In other words, in case of using only softener, dish detergent and nanosilver-I and II, Bacillus sp. Antimicrobial activity against the CK-1 bacteria showed more than 90% antimicrobial activity in the range from the stock to 250 times. However, the anti-bacterial dog shampoo, which is commercially available by adding a bioconversion preparation made of fatty acid, which is known to have an excellent antifungal effect, and an agent having an anti-worming effect such as pyrethrin, has no antimicrobial effect. This is because the mechanism of insecticide and antifungal mechanism and the antimicrobial activity attempted in the present invention in the control of mold and pests (tooth, mites, fleas, etc.) was investigated.

Therefore, in preparation for the above results, the nano silver sugar preparations, which are expected to be developed, had antibacterial properties even at low concentrations of up to 1,000 times, and had the highest antibacterial effect. Therefore, Bacillus sp. In the case of dilution by concentration for CK-1, the Ag content of the stock solution was 100ppm, and the concentration of Ag (nanosilver-I type) was 10ppm and 500. In the case of diluting the pears, the antimicrobial effect was found to be 90% or more even at 0.2 ppm. When Ag was added to the sugar fatty acid ester, the antimicrobial effect was significantly enhanced.

  That is, the sugar fatty acid esters showed up to 10 times the antimicrobial effect when compared to 90% or more antimicrobial effect at 5ppm for nanosilver-I and -II, 0.1ppm or more for nanosilver sugar. In comparison with the basic experiments in which sugar fatty acid esters do not have an antimicrobial effect against bacteria, the penetrability and diffusion of sugar fatty acids and the function as a chelator of nanosilver preparations have been investigated. It was judged to act as an increasing factor.

However, the necessity of objectively judging the results of antimicrobial efficacy of the proposed formulations to be developed by introducing nanosilver emerged, and the synthetic antibiotics were compared and tested by applying synthetic antibiotics, which are commonly used for medical purposes, as a comparison group. For example, streptomycin sulfate was diluted to 10% (w / v) as a stock solution, and diluted by concentration, and tested for antibacterial activity. The antimicrobial activity was 250% or more in the stock solution and 50% or more in the 500-fold dilution treatment. Although the antimicrobial activity was not observed at the following concentrations, the nanosilver sugar preparation was found to have antimicrobial properties ahead of the synthetic antibiotics. In comparing the antimicrobial effects of different preparations, Bacillus sp. CK-1 bacteria were found to have a sensitivity of 10 to 50 times lower than the E. coli O157: K88ac according to the formulation, and the antibacterial spectrum of each bacterium was different. [Table 7, FIG. 15].

B) Confirmation of the antimicrobial activity of the bacteria contacted in the liquid medium prepared by preparation and concentration by time

In this experiment, Bacillus sp. CK-1 and E. coli O157: K88ac were directly contacted in the liquid medium and tested for antimicrobial activity after a certain time.

That is, after diluting each agent by concentration in the broth nutrient liquid medium, Bacillus sp. After the same amount of CK-1 and E. coli O157: K88ac were added to different test tubes, the antimicrobial effects were tested at 3 and 24 hours. Investigate. By diluting the bacteria-specific formulations by concentration, the optimum time for their antimicrobial effect was examined.

As a result, in case of E. coli O157: K88ac bacteria, after 3 hours, the dish detergent had antimicrobial activity from the stock solution to 250 times, and the fabric softener did not show the antimicrobial activity except the stock solution. However, nanosilver-I, II and nanosilver sugars were found to be antimicrobial within 3 hours, Bacillus sp. In the case of CK-1 bacteria, colonies did not form after 3 hours, and it was judged that they were killed. In other words, Bacillus sp. The antimicrobial activity against CK-1 was significantly higher than that of E. coli O157: K88ac [Fig. 15].

On the basis of the above results, the antimicrobial effect after 24 hours was confirmed by the same method. As a result, the E. coli O157: K88ac bacterium survives at 10 times higher concentrations in the bioconversion agent and the anti-loyal dog shampoo. It was investigated that there was no antimicrobial effect, and dish detergent did not have an antimicrobial effect at a dilution concentration of 250 times or less.

Other formulations showed difference in concentration, but showed antimicrobial activity over a period of 24 hours to give contact time to the bacteria, the antimicrobial activity showed more than 90% [Fig. 16]. Bacillus sp. In the case of CK-1 bacteria, after 24 hours, it was investigated that the antimicrobial activity of more than 90% in the entire experimental group showed similar results to the above experimental results.

Table 7. Antimicrobial Assays for Synthetic Antibiotics, Existing Commercial Products, and Well-Being Nano Silver Materials

Experimental Example  6. Silver nanoparticles  Confirmation of antimicrobial activity according to amount added

1. Materials and Methods

end. material

1) Living Goods Material: Popran Ikiss, Clear Kitchen [Lee Sang Dong Co., Ltd.]

2) Additives for detergents into which silver nanoparticles are introduced: Nano silver-III [containing 5,000 ppm silver nanoparticles prepared by the method according to Example 1, Brown type, Naul, Korea], nano silver shoes [Example 1 Containing 5,000ppm silver nanoparticles prepared by the method according to White Type, Naul, Korea]

3) Antibacterial assay strains: Bacillus sp. CK-1, E. coli O157: K88ac

I. Way

1) Assay bacteria and Culture Solution

Standard strains include Bacillus sp. CK-1 and E. coli O157: K88ac were used as test strains, and the prepared bacteria were inoculated in nutrient broth (Difco) and shaken for 18 hours at 37 ° C. and 150 rpm. The culture solution was diluted if necessary to control the number of bacteria and used for this experiment.

2) Nano silver - III  Antimicrobial Enhancing Effect of Administered Agents

In this experiment, based on the results of antimicrobial investigations of commercially available formulations, the antimicrobial potentiation effect of nanosilver-III at 25ppm and 50ppm concentrations was differently investigated and the antimicrobial activity difference according to time and species on antimicrobial was investigated simultaneously. It was.

end) Nano silver - III  Irradiation of antimicrobial activity by single treatment

After filling the sterilized test tube (2ml) with tertiary sterile water, and nanosilver-III and nanosilver shoe gamanga differently by concentration (25ppm and 50ppm), and then Bacillus sp. Two antibacterial treatments were administered to CK-1 and two treatments to E. coli O157: K88ac. The antimicrobial activity was divided into seven sections of 1 minute, 3 minutes, 5 minutes, 10 minutes, 20 minutes, and 1 hour, 3 hours, starting with the administration of the bacteria to the juicy broth. It was incubated for one day at 37 ° C., followed by E. coli O157: K88ac and Bacillus sp. In addition to the killing time of CK-1 bacteria was confirmed the antimicrobial effect between species.

I) Nano silver product  Confirmation of Antimicrobial Enhancing Effect of Added Commercial Formulations

After diluting the commercial formulation with the stock solution using the stock solution and tertiary sterilized water, the formulation was diluted 10 times (v / v), 100 times, 250 times, 500 times, and 1,000 times, and then the nanosilver-III was 25 ppm and 50 ppm. Respectively, and Bacillus sp. Experimental plots containing 10 μl of CK-1 culture medium or 10 μl of E. coli O157: K88ac at the same time were prepared, and the antimicrobial potentiation effect was examined over time when the final formulation and nanosilver-III were added.

C) by concentration Nano silver - III Of antimicrobial activity increased with time

(One) Nano silver - III  25 ppm  Antimicrobial test at the time of addition

The entire experimental group to determine the antimicrobial activity of each formulation, by concentration, Bacillus sp. Experimental plots were made differently for CK-1 and E. coli O157: K88ac. Final Bacillus sp. The number of bacteria in CK-1 and E. coli O157: K88ac culture solution was adjusted to 1 x 10 ⁹ cfu / ml, and then the concentration of nanosilver-III was added at 25ppm and 50ppm by preparation (fiber softener, dishwashing agent 100 times diluent). One of four formulations was tested for antimicrobial activity.

In other words, in order to assay the antimicrobial effect according to the formulation-specific treatment was used to the final control to a final volume of 1,000μl (v / v) in 2ml Eppendorf tube as a whole. As a control, Bacillus sp. 950 μl of tertiary sterilized water was charged into an Eppendorf test tube and pre-cultured thereto. 50 μl of CK-1 or E. coli O157: K88ac culture was added and used. And, after filling the third sterile water (950 μl) in the Eppendorf test tube and incubated therein Bacillus sp. By adding 50 μl of CK-1 or E. coli O157: K88ac culture and only 25 ppm of nanosilver-III, the antimicrobial activity of the addition of nanosilver-III to the formulation to be marketed and developed is compared. Reviewed.

On the contrary, the treatment group-1 was prepared as follows in order to test the antimicrobial enhancing effect when nanosilver-III was added to the fabric softener.

That is, after diluting the product IKIS (brand name), which is used as a fabric softener, to 100 times (v / v) in tertiary sterile water, it is first filled with 940 μl in an Eppendorf tube tube, and additionally prepared nanosilver- 10 μl of III (5,000 ppm) was stirred and the final Bacillus sp. 50 μl of CK-1 culture medium and E. coli O157: K88ac culture medium were prepared, and 50 μl was administered thereto, and additionally, Bacillus sp. CK-1 treatment and E. coli 0157: K88ac were prepared respectively.

In addition, the treatment group-2, which was formed differently for each bacterium, was subjected to the same conditions as the fabric softener for the antimicrobial effect test when the nano silver-III was added to the dish detergent, but only the dish was prepared as a dishwashing detergent ( Brand name).

The antimicrobial results of each treatment group were confirmed as follows. That is, the antimicrobial assay by the diluting solution prepared by the formulation and nano-silver concentration was confirmed by the time course, and the antimicrobial effect was carried out by the drip method. Sampling times for antimicrobial experiments were subdivided into 8 time periods of 0 minutes, 1 minute, 3 minutes, 5 minutes, 10 minutes, 20 minutes, 1 hour and the last 3 hours.

(2) Nano silver - III  50 ppm  Antimicrobial test at the time of addition

Bacillus sp. The antimicrobial effect of CK-1 and E. coli O157: K88ac when 50ppm of each product and nanosilver-III were treated was as follows.

Final Bacillus sp. The bacterial counts in the CK-1 and E. coli O157: K88ac cultures were adjusted to 1x10 ⁹ cfu / ml, and then added by nanosilver-III (25 ppm, 50 ppm) by formulation (fiber softener, dishwasher 100-fold dilution). Antimicrobial activity was assayed for dog formulations. In other words, in order to assay the antimicrobial effect according to the formulation-specific treatment was used to the final control to a final volume of 1,000μl (v / v) in 2ml Eppendorf tube as a whole.

As a control, Bacillus sp. Was prepared by incubating tertiary sterile water (950 µl) in an Eppendorf tube tube. 50 μl of CK-1 or E. coli O157: K88ac culture was added and used. And, after filling the third sterilized water (950 μl) in the Eppendorf tube tube and pre-cultured Bacillus sp. By adding 50 μl of CK-1 or E. coli O157: K88ac culture, and adding 50 ppm of nanosilver to the experimental group, only 50 ppm of nanosilver-III was added to the final solution. The antimicrobial activity with the case of adding -III was compared and examined.

Treatment group-1 was prepared as follows in order to test the antimicrobial enhancement effect of the addition of nanosilver-III to the fabric softener. In other words, first dilute iKISS (trade name), a product used as a fabric softener, to 100 times (v / v) in tertiary sterile water, and first fill it with 930 μl in an Eppendorf tube tube, and then additionally prepare nanosilver. -III (5,000 ppm) was stirred by administration of 20 µl, and the final Bacillus sp. 50 μl of CK-1 and E. coli O157: K88ac culture medium were added to the test plots, and Bacillus sp. For the antimicrobial test when nanosilver-III was administered to the fabric softener. CK-1 treatment and E. coli 0157: K88ac were prepared respectively. In addition, the treatment group-2, which was formed differently for each bacterium, was subjected to the same conditions as the fabric softener for the antimicrobial effect test when the nano silver-III was added to the dish detergent, but only the dish was prepared as a dishwashing detergent ( Brand name).

[result]

As a result of the introduction of the silver nanoparticles of the present invention, the detergent additive (nanosilver-III) introduced into the commercial formulation, the antimicrobial activity was improved compared to the commercial formulation itself. III ”was confirmed the optimum amount of antimicrobial and the antimicrobial duration according to this can be guaranteed [Table 8 to 11, Figure 17].

As a result of assaying the antimicrobial activity of adding 25 ppm of nanosilver-III to the commercial formulation, E. coli O157: K88ac in the control group without the addition of nanosilver-III continuously increased the bacterial count, In the case of the addition of 25 ppm of -III, E. coli had a mortality rate of 90% or more at 20 minutes and no colony at 30 minutes. However, when 50 ppm of nanosilver-III was added, it showed a killing effect of more than 99% within 3 minutes and 100% within 20 minutes, so that the concentration of nanosilver-III was a major variable on the antimicrobial activity.

As a result of comparing the effect on the antimicrobial activity according to the preparation type and the amount of nano silver-III added with respect to the type of bacteria, it was confirmed that the antimicrobial activity was more than 90% within 1 minute regardless of the added concentration. It was also confirmed to increase further, showing an antimicrobial activity of 99% or more within 1 hour.

Escherichia coli and Bacillus sp. As an antimicrobial effect according to the type of bacteria with CK-1, E. coli Bacillus sp. The antimicrobial activity was slightly weaker depending on the amount of nanosilver-III added compared to CK-1, resulting in 100% killing effect after 30 minutes or more, and when 50 ppm of nanosilver-III was added, the antimicrobial activity was 100% within 20 minutes. A killing effect was shown.

Table 8. Confirmation of antimicrobial potentiation effect after nanosilver formulation containing silver nanoparticles (25ppm) to Bacillus sp

         Antimicrobial Effect Treatment by Time Antimicrobial Effect by Addition of Silver Nanoparticles (25ppm) 0 min 1 minute 3 minutes 5 minutes 10 minutes 20 minutes 1 hours 3 hours Control (tertiary sterilization water + Bacillus sp. CK-1 bacteria) - - - - - - - - Treatment Tool 1 (Fiber Softener + Nano Silver-III + Bacillus sp. ND +++ +++ +++ +++ +++ +++ +++ Treatment group 2 (kitchen detergent + nano silver-III + Bacillus sp. CK-1 bacteria) ND ++ +++ +++ +++ +++ +++ +++ *: +++: 90% or more killed, ++: 50% or more killed, +: 10% or more,-: No antimicrobial effect, ND: Not tested.

Table 9. Confirmation of antimicrobial potentiation effect after nanosilver preparation containing silver nanoparticles (50ppm) in Bacillus sp

            Antimicrobial Effect Treatment by Time Antimicrobial Effect by Addition of Silver Nanoparticles (50ppm) 0 min 1 minute 3 minutes 5 minutes 10 minutes 20 minutes 1 hours 3 hours Control (tertiary sterilization water + Bacillus sp. CK-1 bacteria) - - - - - - - - Treatment Tool 1 (Fiber Softener + Nano Silver-III + Bacillus sp. ND +++ +++ +++ +++ +++ +++ +++ Treatment 2 (Kitchen detergent + Nano silver-III + Bacillus sp . CK-1 bacteria) ND ++ +++ +++ +++ +++ +++ +++ +++: 90% or more killed, ++: 50% or more killed, +: 10% or more,-: no antimicrobial effect, ND: not tested.

Table 10. Confirmation of antimicrobial potentiation effect over time after treatment with E. coli nanosilver formulation containing silver nanoparticles (25ppm)

            Antimicrobial Effect Treatment by Time Antimicrobial Effect by Addition of Silver Nanoparticles (25ppm) 0 min 1 minute 3 minutes 5 minutes 10 minutes 20 minutes 1 hours 3 hours Control (tertiary sterilization water + E. coli O157: K88ac bacteria) - - - - - - - - Treatment Zone 1 (Fiber Softener + Nano Silver-III + E. coli O157: K88ac) ND +++ +++ +++ +++ +++ +++ +++ Treatment Group 2 (Kitchen detergent + Nano silver-III + E. coli O157: K88ac bacteria) ND ++ ++ +++ +++ +++ +++ +++ +++: 90% or more killed, ++: 50% or more killed, +: 10% or more,-: no antimicrobial effect, ND: not tested.

Table 11. Confirmation of antimicrobial potentiation effect over time after treatment with E. coli nano-silver preparations containing silver nanoparticles (50ppm)

            Antimicrobial Effect Treatment by Time Antimicrobial Effect by Addition of Silver Nanoparticles (50ppm) 0 min 1 minute 3 minutes 5 minutes 10 minutes 20 minutes 1 hours 3 hours Control (tertiary sterilization water + E. coli O157: K88ac bacteria) - - - - - - - - Treatment Zone 1 (Fiber Softener + Nano Silver-III + E. coli O157: K88ac) ND +++ +++ +++ +++ +++ +++ +++ Treatment Group 2 (Kitchen Detergent + Nano Silver-III + E. coli O157: K88ac) ND +++ +++ +++ +++ +++ +++ +++ +++: 90% or more killed, ++: 50% or more killed, +: 10% or more,-: no antimicrobial effect, ND: not tested.

Experimental Example 7. Antimicrobial effect assay against laundry contaminants of products incorporating Nanosilver-III

This experiment is an optimal method for obtaining antimicrobial effects when applied to real life while testing whether silver nanoparticles prepared according to the present invention is capable of killing microorganisms contaminated with a detergent additive (nanosilver-III), such as towels and laundry. It was conducted to establish the amount of use and application time.

1. Materials and Methods

end. material

1) Commercially available formulations: Popran Ikiss (Fiber Softener), Clear Kitchen (Kitchen Detergent) [Long Dong Dong Co., Ltd.]

2) Detergent additives containing silver nanoparticles: Nanosilver-III (Containing silver nanoparticles, Ag 5000ppm, Brown type, Naul, Korea), Nanosilver Sugar (Example 1 Containing silver nanoparticles, Ag 100ppm, 16% sugar fatty acid ester (w / v), Naul, Korea)

3) Antibacterial assay strains: Bacillus sp. CK-1, E. coli O157: K88ac

4) Contaminated laundry material: commercial bath scrubber towel, commercial towel

  I. Way

  1) Culture of Assay Bacteria and Culture Solution

Standard strains include Bacillus sp. CK-1 and E. coli O157: K88ac were used as test strains, and the prepared bacteria were inoculated in nutrient broth (Difco) and shaken for 18 hours at 37 ° C. and 150 rpm. The bacterial culture was diluted if necessary to control the bacterial count.

2) Antimicrobial Black

A) Confirmation of the killing effect of commercial scrubber contaminants for bath

(1) Investigation of Antimicrobial Activity by Concentration of Nanosilver Agents against Escherichia Coli and Bacillus

Towels used for long-term bathing at home were purchased and the conditions for testing the killing effect of contaminants present with long-term use were set as follows.

As medium conditions, 200 ml of nutrient broth in each sterilized 500 ml Erlenmeyer flask was dispensed, and five were prepared. The experimental plot was prepared as follows. First, the antimicrobial activity of the nanosilver preparations treated with 25ppm and 50ppm, respectively, was investigated first to predict the killing effect on contaminants present in a commercial bath towel.

As a composition of Experiment 1-1 (control) for comparison with the treatment after adding only the culture bacteria to the broth nutrient medium, 200 ml of the Erlenmeyer flask divided into the flask was set to the condition of adding 1 ml of E. coli culture, Experiment 2 (Control) was the same condition as Experiment-1, but Bacillus sp. Only 1 ml of CK-1 was administered. And, after culturing for 24 hours at 37 ℃ was confirmed the survival number of the bacteria according to the formulation treatment.

Experiment-3 for comparing the antimicrobial effects of the control and the nanosilver preparations at different concentrations in each medium was 25 ppm of the nanosilver preparation in the flask in which the culture medium was dispensed, and Experiment 4 was 50 ppm of the nanosilver preparation. After the addition, 1 ml of E. coli O157: K88ac bacteria were added thereto. In the case of experimental zone-5 and experimental zone-6, the same treatment as that of experimental zone-3 and 4 was performed, but Bacillus sp. 1 ml each of CK-1 bacteria was changed and other treatment conditions were performed in the same manner.

The killing rate of bacteria according to each experimental group was experimental group-1, experimental group-2, and nanosilver preparations, which were added when the bacteria were added to the normal medium, and the experimental group-3 to the experimental group separately added for each concentration and bacteria. Compared with 6.

In other words, the killing rate of bacteria and the number of bacteria according to the growth were determined as time elapsed after the addition of the nanosilver agent after the composition of each experimental group (before administration, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours). , 6 hours, 8 hours and 18 hours) uniformly aliquot 1ml each, and then dilute it in a step and smear the same amount in a juicy solid nutrient medium and incubate at 37 ° C for 24 hours to survive. Was calculated and compared with the control.

(2) Confirmation of killing effect on commercial scrubber contaminants for bath

In the above test results, when E. coli and Bacillus bacteria were administered to the nutrient broth, it did not affect the growth of the bacteria, and the results showed that the antimicrobial effect was excellent when the nanosilver agent was administered by concentration. The treatment of the product containing the nano-silver agent against unknown contaminants present in the study was judged to have no problem in determining the killing effect on the contaminant. Was carried out.

Towels for the contaminant killing effect were purchased for a long-term (more than 1 year) bath in the general household, these were cut into the same size (3cm x 1cm x 1cm) in a sterile state and prepared as test pieces.

As a medium condition for confirming the antimicrobial effect, nine aliquots of nutrient broth in each sterilized 500 ml Erlenmeyer flask were prepared, and the experimental conditions for this were as follows.

Experimental sphere-7 (control) was prepared under the condition that the prepared test pieces were sufficiently washed in tertiary sterile water for 10 minutes and then administered to the growth measuring medium.

Experimental zone-8 was diluted 100-fold with commercially available fiber softener, Popran Iquis, to 25 ppm of nanosilver agent, and in the case of experimental group-9, 50 ppm of nanosilver agent was diluted in the same diluted dilution of Popran Iquis. The added solution was made up. In the case of Experiment-10 and Experiment-11, after diluting 100 times of the clear kitchen among commercial detergents in a sterile flask, 25ppm (Experimental-10) and 50ppm (Silver-11) ) Was added and the experimental scrubber sections were stagnated for 10 minutes to confirm the killing effect of contaminants present in the experimental scrubber sections.

As a control, less than the contaminated section scrubber was sufficiently washed in tertiary deionized water, and then immersed in a medium composed of 25 ppm (test-12) and 50 ppm (test-13) nanosilver preparation, followed by washing in tertiary sterile water. One experiment was set.

The entire experimental group (Experimental Group 7 to Experimental Section 13) was diluted 100-fold dilutions of commercially available formulations and nanosilver formulations with respect to contaminated scrubber fragments, but the amount of nanosilver formulations was adjusted to 25 ppm and 50 ppm in each solution. After immersing the test scrubber section for 10 minutes, it was rinsed thoroughly and washed thoroughly in tertiary deionized water for 10 minutes to complete the process of completely removing the remaining agent in the experimental section, and then administered to the growth measuring medium. Was carried out. For antimicrobial assays over time, 1,000 μl of sterile test tubes were stored and stored in the freezer. First, 50 μl of the microorganisms were precisely taken and placed on a juicy solid nutrient medium and smeared, and then colonies formed upon incubation at 37 ° C. for 24 hours. The number was confirmed and antimicrobial results were obtained compared to the control.

In addition, the antimicrobial assay by diluting solution prepared by each formulation and concentration was also confirmed by the drip method. Each diluted solution was collected by 20 μl and plated on agar broth at regular intervals and cultured for one day. The antimicrobial activity was further confirmed by comparing the size of the bacterial growth inhibitory ring in one case with the control.

(3) long term In a commercial towel  Confirmation of antimicrobial effect against contaminants present

In the results of Experiment 6, the administration of E. coli and Bacillus bacteria to the nutrient broth did not affect the growth of the bacteria. In this experiment, it was judged that there was no problem in judging the killing effect of contaminants by introducing a nano silver agent against unknown contaminants present in the present study. We confirmed the killing effect of the developmental formulation on.

Towels for testing the killing effect of contaminants were purchased for a long-term (more than 1 year) bath in a general household. As a control towel, sterilization was completed and fresh towels sold in department stores were used. And, these were cut into the same size (3cm x 1cm x 1cm) in a sterile state to prepare a test piece. The test zone according to the entire experiment was carried out with a total of 13 treatment zones. In other words, the experimental group-A is a control group that confirms the growth of E. coli by administering 1 ml of E. coli to the nutrient broth (LB medium), the experimental group-B is a control group administered 1 ml of Bacillus to the LB medium, and the experimental group-C is a common towel section 3 After washing for 10 minutes in sterile water was used as a culture treatment for confirming the viable bacteria of the remaining contaminated bacteria administered in LB medium.

Experimental-D was immersed in a towel towel 100-fold solution for 10 minutes, and washed in tertiary sterile water for 10 minutes, and then administered to LB medium. Treated with 10 minutes of Franc Iris for 10 minutes and thoroughly washed with tertiary sterile water for 10 minutes and then administered to LB medium to determine whether the remaining contaminated bacteria cultured, Experimental-F is 100% of Popran Iris After immersing in a solution to which 50 ppm of the nanosilver preparation was added for 10 minutes, the solution was washed with tertiary sterilized water for 10 minutes and administered to LB medium to measure remaining contaminants. The control towel section was immersed in a 100-fold solution of Popran Iris for 10 minutes and then washed with tertiary sterile water for 10 minutes and administered to LB medium. Submerged in 100 times solution for 10 minutes, washed in tertiary sterilized water for 10 minutes and administered to LB medium. After treatment with 3 sterile water and then administered to the LB medium, the treatment group cultured the remaining contaminants, experimental group-J was immersed for 10 minutes after forming a solution containing 50 ppm of nanosilver preparation in a 100-fold solution of commercial towels After treatment with 3 sterile water, the treatment group incubated with contaminants remaining in LB medium, and Experimental-K, the control towel section was immersed for 10 minutes after forming a solution containing 50 ppm of nanosilver agent in 100-fold solution of clear kitchen. , After washing with tertiary sterile water was used as a treatment incubation of contaminating bacteria remaining in the LB medium.

For the antimicrobial assay against contaminants present in the commercial towels for each formulation, immediately after the treatment of the formulation, 0 hours, 1 hour to 8 hours elapsed, and 16 hours later, a total of 9 Samples were collected in sterile Eppendorf test tubes with time after time intervals of dog intervals, and stored at a low temperature (4 ° C), and collected hourly in a juicy solid medium at the time of sample collection. 20 µl each sample was carefully collected from the sample of Test A and Test M, which were instilled at regular intervals on a juicy agar medium and cultured for one day. And antimicrobial activity were compared and examined.

(4) Nano silver  Addition by concentration Development  Confirmation of time to kill cultured contaminants

In this experiment, the contaminants isolated from commercial scrubbers were treated to check the killing effect for each concentration and time when the nanosilver preparations were treated differently by concentration. Experimental conditions for this were as follows.

In the case of the control group, the culture group was set as a treatment group in which 1 ml of each was added to the sterilized water, and the nano silver agent treatment group was 25 ppm of the nano silver agent to the cultured contaminant solution. Treated with 30ppm, 35ppm, 40ppm, 45ppm and 50ppm was added, and the treatment using anti-competent dog shampoo was diluted 100 times with the third sterilized water, and then the nano-silver agent Antibacterial assay was carried out with the treatment tool containing 25 ppm and the treatment tool added to 50 ppm.

In addition, 1 ml of each contaminant culture medium was added to the different composition at the same time in such a state that the composition was completed, and the antimicrobial results were determined by setting it from 1 hour to 16 hours.

Antimicrobial activity was determined by dripping method and measuring the colony count according to survival over time. In other words, immediately after the addition of the bacteria to the treatment composition prepared for each concentration as 0 hours, sterilization by a total of nine intervals from 1 hour to 8 hours elapsed in one hour unit, and 16 hours after the end Samples were accurately collected in 1 ml each in an Eppendorf test tube and stored at low temperature (4 ° C).

At the time point at which the sampling of the hourly sample is completed, 50 μl of the sample collected every hour in the juicy solid medium is dropped into the petri dish, and the bacterial colony count generated by incubating at 37 ° C. for 24 hours is measured. By comparing with the control group, the effect of the treatment of the nanosilver preparation preparation preparation on the antimicrobial activity was confirmed.

In addition, the antimicrobial assay was confirmed in parallel with the drip method, and after the preparation, 20 μl of samples collected by time was precisely collected, and the size of the bacterium growth inhibitory ring that was inoculated at a predetermined interval on the juicy agar medium and incubated for one day was compared with the control. The antimicrobial progress was confirmed in parallel.

[result]

This experiment was carried out in Experimental Example 1 to Experimental Example 6, the household goods, raw materials constituting them, and additives for detergents incorporating the silver nanoparticles prepared according to the present invention, and “well-being” having strong antimicrobial properties. As the development of “type household goods” is completed, the formulations that have introduced the above results are manufactured and used to test them for the killing of microorganisms contaminated with towels, laundry, etc., which are the most basic to the use of household goods, and applied to real life. When the use time for obtaining the antimicrobial effect was confirmed.

end. Escherichia coli Bacillus Against fungi Nano silver  Antimicrobial investigation by concentration

Prior to experiments for the purchase of towels used for long-term bathing in homes and testing for the killing effect of contaminants present with long-term use, the effects of nanosilver preparations against E. coli and Bacillus on the concentration of antimicrobial agents The effect was investigated. To this end, 25 ppm and 50 ppm of nanosilver preparations were prepared in nutrient broth, and then cultured therein to E. coli O157: K88ac and Bacillus sp. The addition of CK-1 bacteria and incubation was confirmed by comparing the antimicrobial effect by time and control compared to the control [Table 12].

As a result, for the control, E. coli 0157: K88ac and Bacillus sp. Regardless of CK-1 bacteria, bacteria showed a tendency to continuously grow, but in the case of treatment of nanosilver preparations by concentration, the germ of survival was not detected at 0 hours immediately after the treatment of nanosilver preparations. It was investigated to kill, and also colonies did not appear even after a long time was shown to continue to show antimicrobial activity [Table 12 and 13, Figure 22 and 23].

I. For commercial scrubber contaminants Development  Confirmation of death effect

Towels used for long-term bathing at home were purchased, and experiments were carried out to test the killing effect of contaminants present with long-term use.

To this end, purchase towels used for bathing, and use the nano towels, commercial fabric softeners (popran iris), and commercial kitchen detergents (clear kitchen) for the killing effect of contaminants that exist as long-term bath towels are used. For this purpose, the nano-silver preparation was prepared to contain 25 ppm and 50 ppm, and then immersed for 10 minutes, and then sufficiently washed with tertiary deionized water to cultivate the bacteria that survived the preparation after treatment in a nutrient broth. The number of bacterial colonies that appeared was confirmed by the antimicrobial effect compared to the control (test group-7).

As a result, in the case of experimental group-7, which was only washed in tertiary sterile water, the contaminating bacteria showed the result of the continuous growth of the bacteria, but in the other experimental groups (Experimental Group 8 to Experimental 13). The contaminants were killed during the 10 minutes of immersion of the preparation, and no further growth of the bacteria occurred even after a long time. These results suggest that the developmental agents will be effective in killing remaining contaminants when they are in contact with contaminants for at least 1 minute and up to 10 minutes when used in the field (home) [Tables 12 and 13, Fig. 18]. ]).

All. Long term In a commercial towel  For contaminants present Development  Confirm antimicrobial effect

In the results of the experiment, the administration of E. coli and Bacillus bacteria to nutrient broth did not affect the growth of bacteria, and the antimicrobial effect of the administration of nanosilver preparations by concentration and the presence of commercial bath towels. In this experiment, development of contaminants remaining in commercial towels that have been used for a long period of time in general households has been considered. The killing effect of the formulation was confirmed [Table 14].

As a result, the results of the antimicrobial investigation in the case of using the drip method showed that E. coli O157: K88ac and Bacillus sp. In the case of washing the contaminated germs in the test zones A and B administered only CK-1 and the commercial towel only in the third sterile water, the number of bacteria continued to increase as time passed. In the case of A and B, the bacterial count showed the maximum growth after 6 hours in the case of commercial towels from the initial administration.

However, in the case of Experiment D and Experiment M, the contaminants present before treatment of the preparation showed 10 cfu of bacteria per 50 μl of the commercial towel and the control towel, but the preparation was treated for 10 minutes. In 1 hour, no bacteria were detected, and the same result was also observed after 16 hours of elapsed time, and the antimicrobial effect was found to be high [Table 14, Fig. 19].

These results showed the same tendency as the fabric softener and the dish detergent before or after the introduction of the nanosilver preparation, presumably due to the first low levels of contaminants present in the control towel and the control towel. The antimicrobial activity was judged to be excellent antimicrobial activity, which is considered to be the difference in antimicrobial activity due to the high and low levels of contaminants.

la. Confirmation of concentration and time of nanosilver agent concentration and development agent treatment on contaminant killing for cultured contaminants

In this experiment, we examined the killing effect by concentration and time when the nanosilver preparations were treated differently by concentration in contaminants isolated from commercial scrubber.

As a result, as a result of the antimicrobial investigation by applying the drip method, the number of bacteria was not decreased even in the control group, but the control group and the nano-silver agent treatment group were treated with nano silver preparations at 25 ppm to 50 ppm. Compared with the time, it was found that the antimicrobial activity was 99% or more after 1 hour after contact with the nanosilver agent-added preparation and the contaminating bacteria [Table 16, FIG. 22].

However, this drip method has a problem in quantitative evaluation of antimicrobial properties, and detailed antimicrobial properties were examined by comparing the number of bacterial colonies that survived the treatment. Although the number of bacterial colonies was shown, the number of bacteria showed a tendency to increase continuously over time [Fig. 22].

However, when the nanosilver preparations were treated by concentration, the first approximately 800 contaminant colonies were about 10 viable colonies after 1 hour after contact with the preparations containing the nanosilver preparations, and after 3 hours. Most contaminants were killed and no colony appeared after 8 hours, and the preparations with nanosilver preparations showed excellent antimicrobial activity regardless of the concentration when the nanosilver preparations were treated in the range of 25ppm to 50ppm. Antimicrobial activity was found to be consistent.

Table 12. Killing Effect of Developmental Agents on Commercial Scrubber Contaminants for Bath

Table 13. Killing Effect of Developmental Agents on Commercial Scrubber Contaminants for Bath

Table 14. Antimicrobial Effects of Developmental Agents over Time on Contaminants in Commercial Towels (Drip Method Application Experiments)

Table 15. Confirmation of concentration and time of nanosilver concentration and development agent treatment on the killing of contaminated bacteria for cultured contaminated bacteria (Drip method application experiment)

                   Antimicrobial Treatment Antimicrobial effect by time after treatment by concentration of nanosilver agent ^* 0 hours 1 hours 3 hours 8 hours 16 hours Control (third distilled water) - - - - - Ag25ppm - +++ +++ +++ +++ Ag30ppm - +++ +++ +++ +++ Ag35ppm - +++ +++ +++ +++ Ag40ppm - +++ +++ +++ +++ Ag45ppm - +++ +++ +++ +++ Ag50ppm - +++ +++ +++ +++  *: +++: 90% killed, ++: 50% killed, +: 10% or more,-: No antimicrobial effect, 0 hours: Number of bacteria before nanosilver administration, 1 to 16 hours: After administration of nanosilver Elapsed time

Table 16. Concentration and time test of nanosilver concentration and development agent treatment on the killing of contaminated bacteria for cultured contaminated bacteria (colonial count according to viable bacteria)

                    Antimicrobial Treatment Antimicrobial effect by time after treatment by concentration of nanosilver agent ^* 0 hours 1 hours 3 hours 8 hours 16 hours  Control (tertiary deionized water treatment) 840 10,000 << 10,000 << 10,000 << 10,000 <<  Ag25ppm 847 5 3 5 0  Ag30ppm 810 6 0 0 0  Ag35ppm 821 2 0 0 0  Ag40ppm 835 2 0 0 0  Ag45ppm 887 16 2 0 0  Ag50ppm 856 One One 0 0 10,000 <<: Expression value when excess colony exists, 0 hours: Number of bacteria before administration of nanosilver, 1 to 16 hours: Elapsed time after administration of nanosilver preparation

Experimental Example 8. Antimicrobial test of well-developed well-being nano silver sugar product

1. Materials and Methods

end. material

1) Nano silver preparation: Nano silver sugar-I (containing silver nanoparticles prepared according to the method of Example 1, Ag 25ppm, flavored brown type, containing 16% Sugar fatty acid easter (w / v), Chonnam National University), Nano silver sugar-II (containing silver nanoparticles prepared according to the method of Example 1, containing 25 ppm Ag, fragrance free, Brown type, containing 16% sugar fatty acid (w / v), Chonnam National University), nano silver-IV (AgNO ₃ 10,000 ppm, Naul Corporation)

2) Comparative treatment agent: EG agent (ethylen glycol, pure chemistry, Japan)

3) Antibacterial assay strain: Bacillus sp. CK-1

I. Way

1) Culture of Assay Bacteria and Culture Solution

Standard strains include Bacillus sp. CK-1 was used and prepared Bacillus sp. CK-1 was inoculated in nutrient broth (Difco) and shaken for 18 hours at 37 ℃, 150 rpm. When necessary, the bacterial culture was diluted and used for the experiment by controlling the number of bacteria.

2) Antimicrobial Black

Bacillus sp. The culture solution inoculated with CK-1 and incubated at 37 ° C. was prepared by adjusting to 1 × 10 ⁷ cfu / ml using tertiary sterile water.

In addition, the composition of the treatment to test the effect on the antimicrobial effect when the prepared culture medium was added as follows.

As a control, tertiary deionized water was used to fill a sterile test tube. Based on this, the final "well-being preparation" nanosilver suga-I, nano silver suga-II, nano silver-IV treatment and nano silver As a comparison of the sugar fatty acid ester contained in the sugar preparation, EG preparation (ethylen glycol) was prepared by diluting as follows. In other words, by using the third sterile water for each formulation, it was added to the six treatment and dilute to 10 times (v / v), 100 times, 250 times, 500 times and 1,000 times and the control treated with normal water.

The antimicrobial assay using the prepared solution and the prepared preparation was carried out in the following order. First, Bacillus sp. 50 μl of CK-1 culture medium is evenly spread, and 20 μl of each diluted solution prepared according to the formulation and concentration is collected and Bacillus sp. Antimicrobial results were confirmed by comparing the size of the bacterial growth-lowering rings formed by incubating the CK-1 bacteria with broth nutrient solid medium at regular intervals and incubating at 37 ° C. for one day.

[result]

As a result, the drop of tertiary deionized water as a control did not show antimicrobial activity, and E.G used in place of the sugar fatty acid ester in the nanosilver sugar preparation was also found to have no antimicrobial activity.

In addition, in this study, nanosilver-induced nanosilver-IV was prepared by preparing nanosilver-IV containing 10,000 ppm, and the antimicrobial activity thereof was also investigated. The nanosilver preparation showed antimicrobial activity up to 1,000 times (10 ppm). As a result, the effect of antimicrobial activity was found to be an important variable [Table 17, Figure 23].

Developed as a final “well-being formulation” but adjusted the content of nanosilver to 25ppm and added flavor to it (nanosilver sugar-I) and compared to anti-fragrance-free nano silver sugar-II As a result, it was found that the antimicrobial activity was maintained up to 10 times when the nano silver sugar-II was treated, but the antimicrobial activity was decreased by the dilution factor. It was confirmed that the careful selection is required because the antimicrobial properties of the scent selectivity affect the production of “well-being preparations” using nanosilver because it does not appear [FIG. 23].

Eventually, antimicrobial activity was affected by the presence or absence of fragrance. In the undiluted solution containing 25 ppm of nanosilver, the antimicrobial activity showed more than 90%, but when diluted 10 times, only 50% or more antimicrobial activity appeared. The effects of dilution of nanosilver and sugar fatty acid esters and additives were sensitive to the increase and decrease of effects.

Table 17. Antimicrobial test of final well-being nano silver sugar products

             Antimicrobial effect Antimicrobial effect upon specific, concentrations ^# treatment agent ^* Stock solution 10 times 100 times 250x 500 times 1,000 times   Control (tertiary sterilization water treatment) - NT NT NT NT NT   EG - - - - - -   Nano Silver-IV +++ +++ +++ +++ +++ +++   Nano silver sugar-I +++ ++ - - - -   Nano Silver Sugar-II +++ ++ - - - - *: +++: 90% or more killed, ++: 50% or more killed, +: 10% or more,-: No antibacterial effect, #: Dilution ratio of the preparation to tertiary sterilized water (v / v), NT: Not Testing, Nano Silver Sugars-I (Ag 25ppm, Brown Type with Flavor, 16% Sugar Fatty Acid (w / v), Chonnam National University), Nano Silver Sugars-II (Contains 25 ppm Ag, Unscented Brown Type, 16% Contains sugar fatty acid (w / v), Chonnam National University), Nanosilver-III (containing 10,000 ppm of AgNo ₃ , Naul), EG (ethylen glycol, Pure Chemical, Japan)

Figure 1 shows the diameter distribution of the silver nanoparticles prepared by Example 1.

Figure 2 is a SEM photograph of the silver nanoparticles prepared by Example 1.

Figure 3 is a photograph showing the pathological antimicrobial activity of silver nanoparticles (5 ppm) against E. coli O157: H7 over time [A: O157: H7ACH5 (VT1), B: O157: H759 (VT2) , C: O157: H7670 (VT1 + VT2), D: VTe O138, E57 (VTe). E: VT2 Cl- O157: H-, F: VT2 Cl-O157: H7). 1: control, 2: 30 minutes after treatment, 3: 1 hour after treatment, 4: 8 hours after treatment].

Figure 4 is a graph showing the antimicrobial activity against E. coli (E. coli O157: H7) after 5 minutes to 6 hours after the silver nanoparticles treatment ((A): E. coli VT1 O157: H7 (ACHT5), (B) : E. coli VT2 O157: H7 (O59), (C): E coli VT1 + VT2 O157: H7 (670), (D) E. coli VTe O138, E57 (VTe), (E): E. coli VT2Cl -O157: H-, (F): E. coli VT2 Cl-O157: H7,-●-: control,-■-: 50ppm nano-silver,-▲-: 25ppm nano-silver,-▼-: 10ppm nano-silver,-◆-: 5ppm nano-silver, -◎-: 1 ppm nano-silver].

Figure 5 is a graph showing the antimicrobial activity after the treatment of various concentrations of silver nanoparticles for 26 kinds of E. coli isolated from piglets feces (diarrhea) [37 hours after silver nanoparticles treatment].

FIG. 6 shows pathological micrographs showing antimicrobial activity after silver nanoparticle treatment according to concentrations (1 ppm to 50 ppm) for various pathogens (Escherichia coli, S. gallinarum, Bacillus cereus and Staphylococcus aureus) isolated from pig and chicken feces. [A: photograph of SEM, B: photpgraph of TEM].

7 is a graph showing the antimicrobial activity of the silver nanoparticles according to the continuous treatment for each generation of E. coli NCTC 9001 [(A) 1st generation, (B) 5th generation, (C) 10th generation,-●-: control ,-■-: 50 ppm silver nanoparticles,-▲-: 25 ppm silver nanoparticles,-▼-: 10 ppm silver nanoparticles,-◆-: 5 ppm silver nanoparticles,-◎-: 1 ppm silver nanoparticles].

8 is a graph showing the antimicrobial activity of the silver nanoparticles according to the continuous treatment for each generation of E. coli O157: K88ac [(A) 1st generation, (B) 5th generation, (C) 10th generation,-●-: Control,-■-: 50ppm silver nanoparticles,-▲-: 25ppm silver nanoparticles,-▼-: 10ppm silver nanoparticles,-◆-: 5ppm silver nanoparticles,-◎-: 1ppm silver nanoparticles].

9 is a graph showing the antimicrobial activity of the silver nanoparticles according to the continuous treatment of E. coli ((A) E. coli O157: K88ac, (B) E. coli O157: K88ac) generation by generation [-●-: 1 generation,-■ -: 5th generation,-▲-: 10th generation].

Fig. 10 shows a medium in which the ampicillin concentration of E. coli (E. coli Jm83) into which drug-resistant plasmid DNA is introduced and (B) Escherichia coli without drug-resistant plasmid DNA is introduced is 50ug / ml. It is a photograph showing the survival rate.

11 is a graph showing the antimicrobial activity of E. coli JM83 after 1 hour and 24 hours after silver nanoparticle treatment [(A): CNTL-JM 87 (without tolerant plasmid DNA) and (B): AMP-JM87 (with tolerant plasmid DNA),-●-: control,-■-: 50ppm silver nanoparticles,-▲-: 25ppm silver nanoparticles,-▼-: 10ppm silver nanoparticles,-◆-: 5ppm silver nanoparticles].

12 is a graph showing the antimicrobial activity of E. coli JM83 after 1 minute and 12 hours after 5 ppm silver nanoparticles treatment [A: CNTL-JM87 (without tolerant plasmid DNA), B: AMP-JM87 ( with tolerant plasmid DNA),-●-: control,-■-: 5ppm silver nanoparticles].

Figure 13 is a photograph showing the type of the formulation applied in Experimental Example 4 [1: fiber softener (product name: Popran Iquix, Dongyang Co., Ltd.), 2: kitchen detergent (product name: clear kitchen, Dongyang Co., Ltd.), 3: Fabric softener (Product name: Popran Greenbouquet, Tong Yang Co., Ltd.), 4: Fiber softener (Product name: Popran Pinky Rose, Tongyang Co., Ltd.), 5: Nano silver-I (Ag 5,000 ppm, Brown type, Naul Corporation) , 6. Nano silver sugar (containing 100 ppm silver nanoparticles Ag prepared according to Example 1, 16% sugar fatty acid (w / v)), 7: nano silver-II (containing 5,000 ppm Ag, White Type, Naul Co., Ltd.) )].

14 is a photograph showing the kind of the preparation used in Experimental Example 5 [1: fiber softener (product name; Ikiss), 2: kitchen detergents (product name: clear kitchen), 3: fiber softener (product name; Greenbuque), 4: fiber softener (product name: Popran Pinkyrose), 5: nano silver-I (Ag content 5,000 ppm, Brown type, Naul Corporation), 6. nano silver sugar (silver nanoparticles Ag prepared in accordance with Example 1 100 ppm Containing: 16% sugar fatty acid (w / v), Chonnam National University), 7: nanosilver-II (Ag content 5,000ppm, white type), 8: bioconversion agent (NTS, Canada), 9: streptomycin 10% (Chong Kun Dang, Korea), 10: Anti-loyal dog shampoo (E company, Korea)].

15 is a photograph showing the results of confirming the antimicrobial activity according to the concentration of the synthetic antibiotics and the existing product and well-being nano silver material as a result of Experimental Example 5 Popran Pinky Rose (4), Dongyang Co., Ltd., Kitchen Detergent [Product Name: Clear Kitchen (2), Dongyang Co., Ltd.], Nano Silver-I [5, Ag contains 5,000 ppm, w / v, White type, Naul Co., Nano Silver-II [7, containing Ag 5,000 ppm (w / v), Brown type, Naul Co., Ltd., nano silver sugar [6, prepared according to the method of Example 1 Silver nanoparticles, 100 ppm Ag, 16% sugar fatty acid (w / v)], Bioconversion preparation (8, 100%, v / v, Chonnam National University), Streptomycin sulfate (9, 100%, w / w, Chong Kun Dang ), Anti-loyal dog shampoo (10, 100%, v / v, E, Korea)], x10 to x1,000: dilution ratio of the preparation to tertiary deionized water, A: Bacillus sp. CK-1, B: E. coli O157: K88ac]

16 shows E. coli 0157: K88ac (A) and Bacillus sp. It is a photograph showing the results of confirming the antimicrobial effect according to the fungal species upon direct contact with CK-1 (B) bacteria for 24 hours [1: Textile softener (product name: IKIS, Tong Yang Co., Ltd.), 2: dish detergent (product name: clear kitchen) , Tong Yang Co., Ltd., 3: Fiber softener (Product name: Greenbuke, Dongyang Co., Ltd.), 4: Fiber softener (Product name: Popran Pinky Rose, Tong Yang Co., Ltd.), 5: Nano silver-I (containing 5,000 ppm of Ag, Brown type, Naul Co., Ltd., 6: Nano silver sugar (including silver nano particles prepared by the method according to Example 1, 100 mg of Ag, 16% sugar fatty acid (w / v), 7: Nano silver-II (Ag 5,000) ppm-containing, White Type, Naul Co., Ltd., 8: Bioconversion preparation (100%, v / v, Chonnam National University), 9: streptomycin sulfate (100%, w / w, Chong Kun Dang), 10: anti-loyal dog shampoo (10, 100%, v / v, E company, Korea)], x10 to x1,000: dilution ratio of the preparation to tertiary deionized water].

17 is a photograph showing the results of confirming the antimicrobial enhancement effect according to the addition amount to E. coli O157: K88ac bacteria after the addition of silver nanoparticles addition amount (25ppm and 50ppm) to the commercial formulation [A: control, B : After 1 minute of contact with the agent, C: After 3 minutes, D: After 5 minutes, E: After 10 minutes, F: After 20 minutes, G: After 1 hour, H: After 3 hours, 1: tertiary sterilization water + nanosilver-III 25 ppm + E. coli O157: K88ac bacteria, 2: softener + nanosilver-II 50 ppm + E. coli O157: K88ac bacteria, 3: dishwashing detergent + nano silver-III 25 ppm + E. coli O157: K88ac bacteria, 5: tertiary sterilization water + nanosilver-III 50 ppm + E. coli O157: K88ac bacteria, 6: softener + nanosilver-III 50 ppm + E. coli O157: K88ac bacteria, 7: dishwashing detergent + nano silver-III 50 ppm + E. coli O157: K88ac bacteria].

Figure 18 is designed to confirm the killing effect of the development agent against the scrubber contaminants among household goods performed in Experimental Example 7.

Figure 19 is designed to confirm the killing effect of the development agent against the commercial towel contaminants of the utility carried out by Experimental Example 7.

20 is a photograph showing the results of the investigation of the antimicrobial activity of the concentration of nanosilver-III against Bacillus and E. coli bacteria carried out by Experimental Example 7 [1: 1ml E.coli O157: K88ac culture solution in LB medium added , 2: Bacillus sp. In LB medium. 1 ml of CK-1 culture solution added, 3: 25 ppm nanosilver-III in LB medium + 1 ml of E. coli O157: K88ac solution, 4: 50 ppm nanosilver-III in LB medium + E. coli O157: K88ac solution 1 ml addition, 5: 25 ppm nanosilver-III in LB medium + Bacillus sp. 1 ml of CK-1 bacteria solution, 6:50 ppm of nanosilver-III in LB medium + 1 ml of Bacillus sp. CK-1 bacteria solution].

21 is a photograph showing the results of the antimicrobial investigation by concentration and time-lapse of nano silver-III against E. coli and Bacillus bacteria [1: E. coli O157: K88ac in LB medium 1 ml of culture was added, 2: Bacillus sp. 1 ml of CK-1 culture solution added, 3: 25 ppm nanosilver-III in LB medium + 1 ml of E. coli O157: K88ac solution, 4: 50 ppm nanosilver-III in LB medium + E. coli O157: K88ac solution 1 ml addition, 5: 25 ppm nanosilver-III in LB medium + Bacillus sp. Add 1 ml of CK-1 bacteria solution, add 50 ppm of nanosilver-III in 6: LB medium + Bacillus sp. 1 ml of CK-1 bacteria solution), 1-6 (hours): preparation method (elapsed time)].

22 is a photograph showing the results of confirming the concentration and time of the nano-silver concentration and development agent treatment on the killing of contaminants for the contaminated bacteria cultured by Experimental Example 7 (colonial count investigation according to viable bacteria) [ (1: Ag25ppm administration, 2: Ag 30ppm administration, 3: Ag 50ppm administration, 4: anti-competent dog shampoo: after diluting the anti-worm dog shampoo solution 100 times with tertiary sterilized water, nanosilver-III 25 ppm to 50 ppm), 0 hours: bacterial count before nanosilver-III administration, 1-16 hours: elapsed time after administration of nanosilver-III)].

23 is a photograph showing the results of confirming the antimicrobial activity of the nano-silver sugar product performed by Experimental Example 8 [A: EG preparation (ethylen glycol, pure chemistry, Japan), B: nanosilver-IV (in Example 1 Silver nanoparticles prepared by the same method, AgNo3 10,000ppm, Naul Co., Ltd., C: Nano silver sugar-I (Ag 25ppm, Brown type, 16% sugar fatty acid (w / v) containing, Chonnamdae), D: Nano silver sugar preparation-II (containing 25 ppm of Ag, fragrance-free Brown type, containing 16% Sugar fatty acid (w / v), Chonnam National University), x10 ~ x1,000: dilution ratio of the preparation to tertiary sterilized water, 37 ℃, 24 hours incubation, test strain: Bacillus sp. CK-1.].

Claims (8)

1) dispersing the silver nitrate (AgNO ₃ ) solution in water and then mixing trisodium citrate and hydrogen peroxide (H ₂ O ₂ ) solution, 2) adding and stirring polyvinylpyrrolidone and sodium borohydride to the mixed solution of step 1), 3) filtering the mixed solution of step 2) to remove impurities, and 4) evaporating and drying the mixed solution from which impurities are removed in step 3) Method for producing silver nanoparticles comprising a. The method according to claim 1, Silver nitrate (AgNO ₃ ), trisodium citrate and hydrogen peroxide (H ₂ O ₂ ) of the step 1) is a method of producing silver nanoparticles, characterized in that the range of 1: 0.1 to 1: 0.5 to 5M. The method according to claim 1, Polyvinyl pyrrolidone and sodium borohydride of step 2) is characterized in that it is included in the range of 0.1 to 1: 0.5 to 5 M with respect to 1 M of silver nitrate (AgNO ₃ ). Method for producing silver nanoparticles. The method according to claim 1, Agitation of the step 2) is a method for producing silver nanoparticles, characterized in that carried out in the range 0 to 25 ℃. The method according to claim 1, Agitation of the step 2) is a method for producing silver nanoparticles, characterized in that performed for 30 to 120 minutes. The method according to claim 1, Filtration of step 3) is a method for producing silver nanoparticles, characterized in that carried out using a 0.2 to 0.45 ㎛ filter membrane. The additive for detergents prepared by the method of any one of claims 1 to 6, wherein the silver nanoparticles having a diameter in the range of 5 to 100 nm are included in the range of 1 to 100 ppm. Claims 1 to 6 of the nanoparticles having a diameter in the range of 5 to 100 nm prepared by the method of any one selected from sugar fatty acid (sugar fatty acid) in a 1: 1 to 100 ppm range of the combined complex Detergent additives, characterized in that included.

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