KR20110061311A - Antibiotic composition using low molecular weight silver-alginate and manufacturing method thereof and biotic pesticide using the same - Google Patents

Abstract

PURPOSE: An antibacterial composition using low molecular silver-alginate and a biological agricultural formulation using the same are provided to enhance antibacterial activity and to replace an organometallic antibacterial agent. CONSTITUTION: An antibacterial composition contains alginate and eugenol. The alginate is a silver alginate with silver ions. A method for preparing the antibacterial composition comprises: a decomposing alginate into low molecules; a step of reacting silver nitrate with low molecular alginate to silver alginate; and a step of mixing the silver alginate with eugenol. A biological agricultural formulation contains the antibacterial composition as an active ingredient.

Description

Antibiotic composition using low molecular weight silver-alginate and preparation method thereof, and biopesticide preparation using the same {Antibiotic composition using low molecular weight silver-alginate and manufacturing method etc. and biotic pesticide using the same}

The present invention relates to an antimicrobial composition using a low molecular weight silver-alginate and a method for preparing the same, and a biopesticide preparation using the same, wherein the antimicrobial composition and low-molecular weight of alginate of a polymer and silver and eugenol are added to improve antimicrobial properties and the preparation thereof. A method and a biopesticide preparation using the same.

Chemical pesticides, which have been used for decades, are resistant to pests and can only be controlled by using more pesticides or more lethal pesticides over time. Disturbance, water pollution and pesticide residues from soils and agricultural products are becoming a major problem. In addition, it causes serious social problems that act as fatal hazards to the human body that consumes crops directly.

As a result, there is an urgent need for low-toxic, non-toxic, pollution-free natural pesticides or biological pesticides to realize eco-friendly agriculture. However, most natural pesticides and natural microorganisms have a weak control effect or extremely limited effects. In addition, due to its high price, it is not effective and economically unfavorable by farmers, so it has not succeeded as a substitute for chemical pesticides to realize eco-friendly agriculture.

Alginate, on the other hand, is a polysaccharide contained in the cell wall or cytoplasm of brown algae and is composed of α-L-glulonate [G] and β-D-manruronate [M]. (heteroblock), a homopolymer block of the GG block or a heteropolymer block such as the MG block (heteropolymer block) is a polysaccharide consisting of alternating α-1,4 or β-1,4 bonds.

The physical and chemical properties of alginate are determined by the M / G ratio and block composition. The ratio of M / G varies depending on the type of algae, but in general, the ratio of M / G is high when the alginate content is high. G-blocks are formed in a rigid chain as opposed to M-blocks and crosslinked by gel formation by divalent cations. It is reported that the alginate has an excellent ability to remove heavy metals with a lower M / G ratio. It is also known that metal salts containing carboxyl groups may be used as antibacterial agents.

Lee et al. (J. Kor. Fish. Soc. 33 (1), 32, 2000) is a method of separating and extracting seaweed kelp with dilute Na ₂ CO ₃ . The effect of the viscosity of the Na-alginate solution on the metal ion binding capacity was investigated. The higher the viscosity of the alginate was, the higher the metal ion binding capacity was. In particular, it has been reported that alginate (Ag-alginate, Cu-alginate, etc.) has an antimicrobial effect on Escherichia coli and staphylococci above a certain concentration of Ag and Cu at pH 7.

A technique for biopesticides using alginic acid is disclosed in Korean Laid-Open Patent No. 2002-0014190.

However, the biopesticides use plant extracts as a main raw material, but the composition is very complicated and there is a problem in that the antibacterial activity is low since the alginate is simply used as it is.

The present invention has been made to solve the above problems, low molecular weight of the polymer alginic acid by mixing the eugenol to the alginate having silver ions, antimicrobial composition excellent in antimicrobial activity and preparation method thereof, and bio-pesticide formulations using the same The purpose is to provide.

The antimicrobial composition of the present invention for achieving the above object is characterized in that it contains an alginate and eugenol and has antimicrobial activity.

The alginate is characterized in that the silver alginate having silver ions.

The alginate is a low molecular weight alginate having a molecular weight of 3500 to 15000 Da.

And the production method of the antimicrobial composition of the present invention for achieving the above object comprises the decomposition step of decomposing alginate to low molecular weight; A silver substitution step of producing silver-alginate by reacting silver nitrate with the low molecular weight alginate obtained in the decomposition step; And a reaction step of reacting the silver-alginate mixture by mixing eugenol.

The decomposition step is characterized in that the alginate is low molecular weight of 3500 to 15000 Da by adding ultrasonic peroxide to the alginate and sonication.

In the reaction step, the silver alginate and ethanol were mixed and dissolved in a volume ratio of 1: 10, and the eugenol was added at 0.1 to 10.0 w / v%, and then irradiated with ultrasonic waves of 20 kHz to 40 kHz at 30 ° C. to 80 ° C. It is characterized by reacting for 30 minutes.

And the biological pesticide formulation of the present invention for achieving the above object is characterized in that it contains the antimicrobial composition as an active ingredient.

According to the present invention as described above it is possible to provide an antimicrobial composition having a high antimicrobial activity and a method for preparing the same, and a biopesticide formulation using the same by the composition of the alginate mixed with eugenol excellent antimicrobial activity.

In addition, the present invention can prepare an antimicrobial composition having further improved antimicrobial properties by substituting the metal of the low molecular weight alginate with silver ions.

The antimicrobial composition has an excellent antimicrobial property has a useful replacement of the organometallic antimicrobial agent that caused a safety problem for the human body.

Hereinafter, the antimicrobial composition of the present invention according to a preferred embodiment of the present invention, a preparation method thereof, and a biological pesticide preparation using the same will be described in detail.

 In the present invention, the term "antimicrobial" means not only to inhibit the growth of pathogenic microorganisms such as bacteria (fungi), fungi, etc., but also to sterilize by inhibiting their survival, further limiting the growth of bacteria It includes the meaning of bacteriophages without an increase in the number of the above.

The antimicrobial composition of the present invention contains alginate and eugenol.

The alginate is a polysaccharide contained in the cell wall or cytoplasm of brown algae, and is a linear, unbranched chemical polymer containing α-L-glulonate and β-D-manruronate residues. Alginates extracted from brown algae are mostly high molecular weight polymers (about 4 million Da or more). The polymer alginate has a high viscosity, and there are many difficulties in use because of poor solvent properties.

Therefore, it is preferable to use a low molecular alginate obtained by decomposing the high molecular alginate to make it low molecular. In order to improve the yield of the silver alginate which will be described later and dissolved in water, it is particularly preferable that the molecular weight is 3500-15000 Da as the low molecular weight alginate.

Alginates useful for low molecular weight in the present invention also include monovalent salts of alginates, such as sodium and potassium alginates, silver alginates, as well as esterified forms of alginates, such as propylene glycol alginates. All such esterified forms are included within the definition of alginate salts used in the present invention. Further examples of alginates that may be used in the present invention are magnesium alginate and 3-ethanol amine alginate. Such alginate salts may be used alone or two or more together. And the best alginates in the present invention are low molecular weighted silver alginates.

Eugenol is a colorless or pale brown transparent liquid, separated from natural essential oils such as clove oil, nutmeg and cinnamon. Eugenol used in the present invention is well mixed with low molecular weight silver alginate and shows high synergy effect in antibacterial action.

The antimicrobial composition of the present invention is prepared by mixing eugenol with silver alginate dissolved in ethanol. Such antimicrobial compositions are provided in the form of polymers in which the carboxyl groups of alginates and the hydroxy of eugenol are fully or partially crosslinked.

The antimicrobial compositions of the present invention can be used to inhibit biological pathogens and inhibit or kill the growth of pathogenic bacteria. The antimicrobial composition according to the present invention exhibits strong antimicrobial activity against a wide variety of fungi such as bacteria, yeasts and molds. Examples of bacteria include anthrax ( Bacillus) anthracis ), Escherichia coli coli ), subtilis bacteria ( Bacillus subtilis ), Staphylococcus aureus ), Pseudomonas aeruginosa ), Klebsiella aerogenes ), Proteus mirabilis ), and an example of yeast is Candia solani ), Saccharomyces cerevisiae ), Zygosaccharomyces rouxii , and the like. Examples of fungi are Aspergillus niger ), Penicillium chrysogenum ).

The antimicrobial composition of the present invention may further comprise one or more pharmacologically acceptable carriers, excipients and diluents in addition to silver alginate and eugenol. As used herein, `` pharmacologically acceptable '' refers to a substance that is physiologically acceptable and does not normally cause an allergic or similar reaction when administered to humans.

The antimicrobial composition of the present invention can be formulated using methods known in the art as aqueous solutions, powders, granules, tablets, pills, dragees, capsules, solutions, gels, syrups, slurries and suspensions and the like. Furthermore, the compositions according to the invention can be formulated in various forms.

In addition, the antimicrobial composition of the present invention can be used as a biopesticide formulation. Biopesticide preparations can be used in soil spraying, foliar fertilization, etc. with proper dilution in water. The biopesticide preparation is preferably used as a bacteriostatic agent for crops in which antibacterial and bacteriostatic properties are required, particularly for anthrax in peppers.

In addition, the antimicrobial composition of the present invention is a necessity that requires antibacterial, in particular, plastic products which are prohibited to use organometallic antimicrobial agents, preferably antibacterial mat, antibacterial toothbrush, antibacterial filter, antibacterial deodorant and antibacterial dressing (gauze, bandages, etc. Fibers) and the like.

Hereinafter, the manufacturing method of the antimicrobial composition of this invention is demonstrated. The method for producing an antimicrobial composition of the present invention comprises a decomposition step of largely decomposing alginate to lower molecular weight, a silver substitution step of reacting silver nitrate with low molecular alginate obtained in the decomposition step to form silver alginate, and the silver-alginate salt. A reaction step of reacting by mixing the eugenol.

1. Disassembly

The polymer alginate used as a starting material in the present invention may be used by using a commercially available product or by using a known method from brown algae.

In order to lower the molecular weight of the polymer alginate, an appropriate amount of chloroform solution may be added to the ethanol solution of the alginate, and the molecular weight may be separated in the order of high molecular weight. In order to obtain a low molecular weight alginate having a molecular weight of 3500 Da and 15000 Da, the addition amount of chloroform is changed from 5 parts by weight to 35 parts by weight to obtain a low molecular weight alginate. In addition, by adding hydrogen peroxide to the alginate and sonication it is possible to lower the alginate to a molecular weight of 3500 to 15000 Da. In addition to the above-described method, the alginate may be enzymatically decomposed using cellulase, a glycolysis enzyme, to obtain a low molecular weight alginate, or may be obtained using an acid or an alkali. In addition, of course, the alginate of 15000 Da can be purchased and used in the molecular weight 3500Da marketed.

2. Silver substitution

Next, the low molecular weight silver alginate is obtained by substituting the metal ions (Na, K, Ca, etc.) of the low molecular weight alginate with silver. To obtain low molecular weight silver alginate, silver nitrate (AgNO ₃ ) was dissolved in primary distilled water, and then dropping the low molecular weight silver alginate in an aqueous solution of silver nitrate using a dropping funnel, followed by reaction at room temperature for about 1 hour. The precipitate is washed three times with primary distilled water and ethanol and dried under reduced pressure.

At this time, the content of silver in the low molecular weight silver alginate can be adjusted by changing the concentration of silver nitrate. In order to increase the antimicrobial activity in the silver substitution step, copper nitrate and titanium dioxide may be selectively added together with silver nitrate to react with alginate.

3. Reaction step

Next, the obtained low molecular weight silver alginate is mixed with eugenol to carry out a reaction step.

In the reaction step, silver alginate and ethanol were mixed and dissolved in a volume ratio of 1: 10, eugenol was added at 0.1 to 10.0 w / v%, and then irradiated with ultrasonic waves of 20 kHz to 40 kHz at 30 ° C. to 80 ° C. for 30 minutes. It is preferable to react. The concentration of eugenol was adjusted to a range of 0.1-10.0 w / v%, but is not limited to this concentration.

In general, the ultrasonic wave is a high frequency region having a frequency of about 1 to 10 MHz, which is used for medical devices or nondestructive examination, and a low frequency region having a relatively low frequency of 15 to 100 kHz, which is used for welding of a washer, plastic, or metal. There is an ultrasound. In the present invention, the ultrasonic wave having a vibration range of 20kHz to 40kHz, which is a low frequency region, is sonicated in order to effectively induce the binding of two molecules of alginate and eugenol.

In the ultrasonic treatment, the temperature is performed at 30 ° C. to 80 ° C., preferably at 40 ° C. to 50 ° C. If the temperature is less than 30 ℃, the molecular motion of the alginate molecule is limited, so that the condensation reaction by the ultrasonic wave does not occur smoothly, and if it exceeds 80 ℃, the alginate is easily pyrolyzed.

By sonication, the OH group of eugenol and the carboxyl group of low molecular weight silver alginate cause partial condensation. Molecular weight rapidly increases until the reaction time 30 minutes, the rise of molecular weight almost disappears from 30 minutes or more.

Hereinafter, the antimicrobial composition of the present invention will be described through the following examples. However, the following examples are intended to illustrate the present invention in detail, and the scope of the present invention is not limited to the following examples.

(Example)

5000 ml of water and 500 ml of hydrogen peroxide were added to 100 g of sodium alginate (molecular weight 150,000 Da) of Junsei Chemical, and sonicated at 50 kHz for 30 minutes at 50 ° C. to prepare a low molecular weight alginate having a molecular weight of 10,000 Da.

43.5 g of the prepared low molecular weight alginate was uniformly dissolved in 100 ml of primary distilled water, and 2.5 g of silver nitrate (AgNO ₃ ) was dissolved in 35 ml of primary distilled water using a dropping funnel, followed by 1 hour at room temperature. After the reaction, the precipitated silver alginic acid salt was recovered. The recovered silver alginate was washed three times with primary distilled water and ethanol and dried under reduced pressure. Next, the low molecular weight silver alginate and ethanol were mixed in a volume ratio of 1:10, and the silver alginate was dissolved in ethanol. Eugenol was added at 5.0 w / v% and sonicated at 30 kHz for 30 minutes at a reaction temperature of 50 ° C. An antimicrobial composition of the invention was prepared.

Experimental Example 1 Determination of Silver Content of Low-Molecular Silver Alginate

In order to measure the silver content of the low molecular weight silver alginate obtained in the above example, inductively coupled plasma mass spectrometry (ICP-MS) was performed.

As a result, the low molecular weight silver alginate prepared in the Example obtained by preparing the low molecular weight alginate and then substituting it with silver was 28% silver, and the alginate -COO ^- Na ⁺ was replaced by -COO ^- Ag ⁺ about 78%. It was confirmed.

Experimental Example 2: Viscosity Change of Antimicrobial Composition According to Reaction Time

In order to establish the control conditions of the optimum antimicrobial composition according to the ultrasonic irradiation, the viscosity change of the antimicrobial composition with the ultrasonic irradiation time was investigated.

As a result, it was confirmed that the intrinsic viscosity value increased as the reaction time increased, because the dimerization occurred due to partial condensation of the carboxyl group of the eugenol OH and the low molecular weight silver alginate salt. However, such condensation reactions are considered to be partial, not overall, and the unreacted low molecular weight silver alginate and eugenol appear to be present in the composition in a mixed state. Viscosity of the composition is rapidly increased to 30 minutes reaction time, from 30 minutes or more it was confirmed that the increase in molecular weight almost disappeared.

Experimental Example 3: Antimicrobial Test of Antimicrobial Composition

An antimicrobial test was conducted to evaluate the antimicrobial properties of the antimicrobial compositions prepared in the above examples. Antimicrobial test method which is an important pathogenic bacteria as bacteria disclose the control piece and the test piece with KS KO693 method (Clinical Escherichia coli Inoculated and cultured, shaken in a certain amount of liquid to extract the cultured bacteria, and the number of bacteria present in the liquid was measured to calculate the reduction rate in the antimicrobial test piece.

% Reduction = ((B-A) / B) x 100,

(A: the number of bacteria regenerated from the test specimen cultured through a constant contact time after inoculation, B: the number of bacteria regenerated from a control specimen cultured through a constant contact time after inoculation)

First, autoclave sterilization of the nutrient medium (LB medium) and the tube (15 ml), respectively, and then pyrimethanil (Pyrimethanil SC), silver nano, low molecular weight silver alginate, and 0.2 g of the antimicrobial composition of the Example 4 ml of nutrient medium (LB medium) was dispensed, and the culture solution was incubated for 24 h, inoculated in a sterile tube 20 µl, and then cultured at 37 ° C. for 24 h. Dilutions were inoculated by inoculating 500-fold dilutions (2.6 × 10 ⁵ CFU / ml) of the optimally cultured bacteria. In addition, the number of bacteria before the test (growth colony) and the number of bacteria after the test (growth colony) were respectively measured, and the antimicrobial and bacteriostatic properties were evaluated according to the following criteria, and the results are shown in Table 1. (): The number of bacteria after the test is less than 1 / 10,000 of the number of bacteria before the test ○: The number of bacteria after the test is 1 / 10,000 or more to less than 1 / 1,000 of the number of bacteria before the test Δ: The number of bacteria after the test is 1 / 1,000 or more and less than 1/100 x: The number of bacteria after a test is 1/100 or more of the number of bacteria before a test.)

division Whether bacteria grow Antimicrobial activity Bacteriostatic Pyrimethanyl Yes ○ ○ Nano silver Yes △ △ Low Molecular Silver Alginate No ○ ◎ Example No ◎ ◎

As a result, as shown in Table 1, the antimicrobial properties of pyrimethanyl, a conventional fungicide, was better than nano silver. And it was confirmed that the antimicrobial activity of low molecular weight silver alginate compared to pyrimethanyl. This is believed to be the result of the combination of silver with alginate to increase the antibacterial activity. And the antimicrobial composition of the example showed the best antibacterial bacteriostatic properties. This is due to the combination of the antibacterial activity of silver alginate and that of eugenol with antimicrobial activity greatly increased.

On the other hand, anthrax ( Bacillus) according to the concentration of the antimicrobial composition of the present invention The antimicrobial activity against anthracis ) was tested and the results are shown in FIGS. 1 to 4. The bacteriological test was carried out using a conventional flat plate smear method. After strain cultured in an antimicrobial composition of Example 50, 100, 1000ppm concentration in petri dishes at 35 ℃ for 24 hours and then the number of individual strains were measured.

Referring to Figures 1 to 4, it can be seen that a significant decrease in anthrax in Figures 2 to 4 with the addition of the antimicrobial composition of the embodiment compared to Figure 1 without adding the antimicrobial composition as a control. In FIG. 2, the reduction rate was 80%, FIG. 3 was 60%, and FIG. 4 was 99.9%.

As described above, the antimicrobial composition of the present invention is expected to have an excellent antimicrobial activity and be usefully used as an antimicrobial agent.

The terminology or words used in this specification and claims are not to be construed as being limited to the common or dictionary meanings, and the inventors properly define the concept of terms in order to best explain their invention in the best way. It should be interpreted as meaning and concept corresponding to the technical idea of the present invention based on the principle that it can.

Therefore, the configurations shown in the embodiments of the present specification are only exemplary embodiments of the present invention and do not represent all of the technical ideas of the present invention, and various equivalents may be substituted for them at the time of the present application. It should be understood that there may be variations.

The antimicrobial composition of the present invention can be used as a biopesticide formulation. The biopesticide preparation is preferably used as a bacteriostatic agent for crops in which antibacterial and bacteriostatic properties are required, particularly for anthrax in peppers.

In addition, the antimicrobial composition of the present invention is a necessity that requires antibacterial, in particular, plastic products which are prohibited to use organometallic antimicrobial agents, preferably antibacterial mat, antibacterial toothbrush, antibacterial filter, antibacterial deodorant and antibacterial dressing (gauze, bandages, etc. Fibers) and the like.

1 is a photograph showing the results of anthrax culture without adding the antimicrobial composition,

Figure 2 is a photograph showing the anthrax culture result of adding 100 ppm of the antimicrobial composition,

Figure 3 is a photograph showing the anthrax culture result added 500 ppm antimicrobial composition,

Figure 4 is a photograph showing the anthrax culture result of adding 1000 ppm antimicrobial composition.

Claims (7)

An antimicrobial composition comprising alginate and eugenol and having antimicrobial activity. The antimicrobial composition according to claim 1, wherein the alginate is silver alginate having silver ions. The antimicrobial composition of claim 1, wherein the alginate is a low molecular weight alginate having a molecular weight of 3500 to 15000 Da. A decomposition step of decomposing alginate to lower molecular weight; A silver substitution step of producing silver alginate by reacting silver nitrate with the low molecular weight alginate obtained in the decomposition step; Reaction step of reacting by mixing the eugenol to the silver-alginate; method of producing an antimicrobial composition comprising a. The method of claim 4, wherein the decomposing step comprises adding hydrogen peroxide to the alginate and sonicating the alginate to lower the molecular weight to a molecular weight of 3500 to 15000 Da. The method of claim 4, wherein the reaction is performed by dissolving the silver alginate and ethanol in a volume ratio of 1: 10, and then adding the eugenol at 0.1 to 10.0 w / v%, and then at 20 to 20 kHz at 30 ° C to 80 ° C. Method for producing an antimicrobial composition, characterized in that for 30 minutes by irradiating 40kHz ultrasonic waves. A biopesticide preparation, comprising the antimicrobial composition of any one of claims 1 to 3 as an active ingredient.

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