KR101386228B1 - Extracts from Angelica gigas using methanol and ethyl acetate for inhibition of biofilm formation of microbes - Google Patents

Abstract

The present invention relates to the use of the solvent-fractionated fraction using ethyl acetate in the methanol extract of Angelica gigas as an effective ingredient to inhibit the biofilm formation of microorganisms. Angelica used in the present invention provides a method for inhibiting microbial biofilm formation, which is safe for various uses including food as a safe medicinal plant.

Description

Extracts from Angelica gigas using methanol and ethyl acetate for inhibition of biofilm formation of microbes}

The present invention relates to the use of ethyl acetate (ethyl acetate) solvent fraction in the methanol extract of Angelica gigas as a method for inhibiting the biofilm formation of microorganisms.

Microorganisms are found almost everywhere in nature, and they are an indispensable entity that plays an important role in the circulation of substances in the ecosystem, and it causes a great deal of economic loss due to the presence of unwanted microorganisms in undesirable places and times. The most serious problem with microorganisms is that they cause disease in human body, crops and livestock, threatening not only economic loss but also life.

Various methods have been developed to control the contamination and growth of unwanted microorganisms, and as a powerful method for controlling microorganisms, antibiotics are used which selectively kill or inhibit the growth of microorganisms. However, since the abuse of antibiotics causes the appearance of antibiotic-resistant bacteria, it can not be effectively used in a situation where the use of antibiotics is desperately desired. Therefore, a microorganism growth regulator which utilizes a new action mechanism different from existing antibiotics is desperately needed It is true. The best way to prevent unwanted microbial contamination is to regulate microbial growth in the early stages of contamination and growth.

When the microorganism grows in the liquid, it can easily remove the contamination source by removing the contaminated liquid. Generally, however, the microorganism does not grow uniformly in the medium as in the laboratory, attaches to the surface of the container to perform community life, (Referred to as a " biofilm "). The biofilm imparts a protective action against physical stresses such as shear force and heat to the microorganism and inhibits the transfer of compounds that kill microorganisms such as pH, antibiotics, hydrogen peroxide, and hyphochlorite, Protects microorganisms. The microorganisms growing in the biofilm are resistant to various stresses than the general microorganisms because their physiology changes, making it difficult to remove the microorganisms forming the biofilm and provide the main cause of microbial contamination. Therefore, inhibiting the formation of biofilm can easily eliminate the contamination of unwanted microorganisms. In addition, if biofilm formation is suppressed to remove most of the contaminated microorganisms easily, it may maximize its efficiency in further sterilization or sterilization. In addition, if the formation of biofilm is suppressed only without affecting the growth of microorganisms, the selectivity for resistant strains is not selected, and thus the appearance of resistant strains is expected to be blocked to a great extent.

Therefore, if biofilm formation of microorganisms is inhibited, the contamination by microorganisms can be minimized, and economic damage can be reduced.

The present invention has been made in view of the above necessity, the problem to be solved in the present invention provides a method for inhibiting the biofilm formation of microorganisms using a solvent fraction using ethyl acetate in the methanol extract of Angelica gigas. I would like to.

In order to solve the above problems, it provides a microbial biofilm formation inhibitor containing the fraction obtained by extracting the Angelica gigas with methanol and then fractionating the solvent using ethyl acetate as an active ingredient.

In the above method can be extracted using ethyl acetate directly from Angelica

In this method it can be extracted or fractionated using a solvent similar to ethyl acetate.

A functional food having an effect of inhibiting microbial biofilm formation containing the extract prepared by the above method as an active ingredient.

There is provided a hygiene cleaner having an effect of inhibiting microbial biofilm formation containing an extract prepared by the above method as an active ingredient.

A preservative for the microbial biofilm formation inhibitory effect containing the extract prepared by the above method as an active ingredient is provided.

Angelica has been ingested for many years as a herbal medicine and is safe for the human body. In the present invention, by presenting a new functional microorganism, which inhibits the formation of biofilms, it is possible to create new economic benefits by using various products. .

1 shows that the formation of biofilms was hardly observed as an effect of inhibiting the formation of biofilms of food poisoning bacteria Yersinia enterocolitica when the Angelica extract was treated with 4.25 g / L.
Figure 2 is Xanthomonas auria which is a causative strain of rice leaf blight when treated with 4.25 g / L Angelica extract ( Xanthomonas oryzae , the formation of a biofilm was not observed at all.
Fig. 3 is a graph showing the results of a food poisoning strain Yersinia Enterocolitica ) shows the reduction of biofilm formation according to the concentration when the Angelica extract was treated at low concentration.
Figure 4 shows the decrease in the formation of biofilm according to the concentration of the Angelica extract at low concentrations in the formation of a biofilm of Xanthomonas oryzae , the causative strain of rice leaf blight of rice.

Hereinafter, the present invention will be described in detail.

The present invention is extracted by soaking for 12 hours at room temperature with 1,500 ml of methanol per 100g, extracted, concentrated, dried and dissolved in 250 ml of water. Thereafter, 750 ml of ethyl acetate is mixed, mixed well, and left to stand for 3 hours to separate a solvent to obtain a fraction of an ethyl acetate layer. Concentrate the obtained fractions, dry them, and dissolve them in water.

Ethyl acetate can be used to extract the active substance directly from Angelica gigas without extraction of methanol.

As the extraction solvent used in the above method, it is preferable to use a nonpolar solvent. More preferably, ethyl acetate is used.

It is a microbial biofilm formation inhibitor using as an active ingredient the fraction obtained from the ethyl acetate layer of Angelica gigas prepared by the above method.

[Example]

1. Preparation of Angelica Extract

After extracting by soaking 1,500 ml of methanol per 100g at room temperature for 12 hours, it is concentrated, dried and dissolved in 250 ml of water. Thereafter, 750 ml of ethyl acetate is mixed, mixed well, and left to stand for 3 hours to separate a solvent to obtain a fraction of an ethyl acetate layer. Concentrate the obtained fractions, dry them, and dissolve them in water.

2. The composition of the medium used in this example is as follows.

YGC  Badges (100 ml )

Glucose 5 g

Yeast extracts 0.5 g

CaCO ₃ 1.25 g

Agar 1.5 g

Water make up 100 ml

210 medium (100 ml )

Sucrose 0.5 g

Casein hydrolysates 0.8 g

Yeast extracts 0.4 g

K ₂ HPO ₄ 0.3 g

MgSO ₄ 7H ₂ O 30 mg

Water make up 100 ml

XOM2  badge( pH  6.5)

L-methionine 670 [mu] M

Na-glutamate H ₂ O 10 mM

KH ₂ PO ₄ 14.7 mM

MnSO ₄ 40 uM

Fe (III) -EDTA 240 uM

MgCl ₂ 5 mM

D-xylose 0.18%

TYE  Badges (100 ml )

Tryptone 1 g

Yeast extracts 0.5 g

(Agar) 1.5 g

Water make up to 100 ml

M63  Badges (100 ml )

5x M63 medium 80 ml

20% Glucose 1 ml

20% MgSO ₄ 0.1 ml

Water make up to 100 ml

5x M63  (100 ml )

KH ₂ PO ₄ 1.5 g

K ₂ HPO ₄ 3.5 g

_{(NH 4) 2 SO 4 1} g

FeSO ₄ (1 mg / ml) 0.25 ml

3. Biofilm Formation Assay

In the present invention, in order to measure the formation of a biofilm, microorganisms were inoculated in an amount of 100 ul per well using a microplate of 96 wells of polyvinyl chloride as much as an absorbance of 0.05 at a wavelength of 600 nm After culturing the microorganism in order to quantify the amount of the biofilm, 100 μl of a 1% crystal violet solution was exposed to the microplate on which the supernatant was removed for 15 minutes Thereafter, the excess crystal violet was removed with water, and the crystal violet which was not removed by the biofilm was eluted with 100 μl of 95% ethanol and the absorbance was measured with a microplate reader at a wavelength of 595 nm.

4. Yersinia Bio-film formation of enterocolitica

Freeze-dried Yersinia Enterocolitica is streaked on a TYE plate and incubated at 26 ° C for 2 days. After inoculating 5 ml of TYE medium, incubate in a test tube at 26 ° C for 2 days at 250 rpm. The cell concentration of the culture medium is measured by Abs600. Place 100 μl of M63 medium in a 96-well microplate. Using the measured cell concentration value, inoculate the sample so that Abs600 = 0.05. After incubation at 26 ° C for 6 hours or 24 hours, the amount of biofilm is measured using crystal violet.

5. Xanthomonas Biofilm formation of oryzae

The lyophilized Xanthomonas The oryzae are streaked on a YGC plate and incubated at 28 ° C for 2 days. After inoculating in 5 ml of 210 medium, incubate for 2 days at 250 rpm in a test tube at a temperature of 28 ° C. The cell concentration of the culture medium is measured by Abs600. Place 100 μl of XOM2 medium in a 96-well polyvinyl chloride (PVC) microplate. Using the measured cell concentration value, inoculate the sample so that Abs600 = 0.05. After incubation at 28 ° C for 6 hours or 24 hours, the amount of biofilm is measured using crystal violet.

6. Experiment to Inhibit Formation of Microbial Biofilm Using Angelica Extracts Presented in the Present Invention

Yersinia by adding previously prepared Angelica extract The results of the changes in the biofilm formation degree of enterocolitica and Xanthomonas oryzae are shown in FIG. 1 to FIG.

Claims (3)

A composition for inhibiting biofilm formation of Yersinia enterocolitica or Xanthomonas oryzae containing the extract of Angelica gigas with methanol, followed by fractions extracted with ethyl acetate as an active ingredient.
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