KR101717533B1 - Composition comprising essential oil for inhibiting Biofilm and Methods therefor - Google Patents

Abstract

The present invention relates to a composition for inhibiting biofilm and a method for inhibiting biofilm formation, and more particularly to a composition for inhibiting biofilm comprising at least one substance selected from the group consisting of pepper oil, cananga oil and myrrh oil, To a method of inhibiting film formation. The composition for inhibiting biofilm may further comprise cis-nerolide and may be used to control the expression of a gene associated with a biofilm or a toxic factor, to inhibit hemolytic activity of Staphylococcus aureus, to form biofilm of Staphylococcus aureus And is effective for prevention and treatment of diseases caused by Staphylococcus aureus. Therefore, it can be usefully used in various fields such as medicines, health foods, and cosmetics.

Description

[0001] The present invention relates to a composition for inhibiting biofilm comprising essential oil as an active ingredient,

The present invention relates to a composition for suppressing biofilm of a pathogenic microorganism and a method for suppressing biofilm that does not induce drug resistance of a pathogenic microorganism and a method for suppressing the biofilm.

In some parts of the bacterium that are infected, there is a mucus, a colony of bacteria surrounded by a polymer matrix. The mucous bacterial complex formed by the bacteria is called a biofilm, a bacterium or an biofilm. Biofilm is a complex of bacterial colonies, which is a solid biological surface, surrounded by an outer membrane, a polymer substrate composed of polysaccharides and polypeptides, which is a non-biological surface. Communicate, and defend against the outside world. Biofilm makes it possible for bacteria to survive under various environmental stresses including antibiotics.

These biofilms are often found in relation to bacterial infectious diseases in addition to the natural environment. It may be formed in a person's organs, in the form of plaque in the teeth, or in industrial equipment or medical implants. Because of this, biofilms have been of interest to scholars studying periodontal disease, pneumonia associated with cystic fibrosis, and earache in the middle ear. In the 2002 report, the US National Institutes of Health estimated that up to 80% of bacterial populations are spreading pathogens through biofilm formation.

Antibiotics, which have shown efficacy against planktonic bacteria, tend to lose efficacy when bacteria form biofilms. When a bacterium forms a biofilm, the outer membrane of the biofilm can not penetrate the antibody, so that the host immune system can be disabled and the resistance of the bacteria to the antibiotic can be increased to about 1,000 times. The cause of the increase in resistance to antibiotics due to the biofilm formation has not yet been clarified. The first is "ecological change of microorganisms". When the biofilm is formed, the binding force between the bacteria is strengthened, and as a result, bacterial colonization does not spread well and bacterial growth is also lowered. This weakens the dependence on exchange with the environment, slows down metabolism, and results in a lower susceptibility to antibiotics.

The second is the physical properties of the "outer membrane composed of mucopolysaccharides". The mucopolysaccharides that form the outer membrane have electrical properties and tend to bind with antibiotics, thus interfering with the spread of antibiotics by binding with antibiotics. In other words, antibiotics are not transmitted to individual bacteria and it is difficult to exert their effects.

The third is the "production of inhibitors", which is an estimate related to the general antibiotic resistance acquisition mechanism. The most commonly known inhibitors of antibiotic efficacy inhibitors are beta-lactamases produced by pseudomonas. When a biofilm is formed, bacteria that are not resistant to the biofilm tend to acquire resistant gene related genes through horizontal gene transfer from surrounding resistant bacteria and become resistant bacteria. That is, when the biofilm is formed in the infected area, it can be regarded as an antibiotic resistant state.

Because of these reasons, the formation of biofilm makes antibiotics, which are widely used for the treatment of infectious diseases, difficult to treat, resulting in weakening of the therapeutic effect of antibiotics and chronic bacterial infection. In this case, as described above, the susceptibility of bacteria to antibiotics is low, so it is hardly effective using antibiotics. To overcome them, simply over-prescribing antibiotics increases the antibiotic resistance of bacteria. In other words, bacterial infection with biofilm means that treatment with antibiotics alone is no longer effective treatment. In particular, the infection caused by the bacteria forming the biofilm becomes more serious due to the multi-drug resistance bacteria which are resistant to various antibiotics.

Therefore, in order to solve the above problem, it is necessary to develop a therapeutic agent capable of destroying the outer membrane existing in a biofilm or a biofilm.

1. Korean Patent Publication No. 10-20100043579.

Accordingly, it is an object of the present invention to provide a composition for inhibiting biofilm.

It is another object of the present invention to provide a method for inhibiting biofilm formation.

In order to accomplish the above object, the present invention provides a composition for inhibiting biofilm comprising, as an active ingredient, at least one essential oil selected from the group consisting of pepper oil, cananga oil and myrrh oil.

The composition may further comprise cis-nerolidol.

The composition for inhibiting biofilm may be provided as a pharmaceutical composition or a health food.

According to another aspect of the present invention, there is provided a method for inhibiting biofilm formation comprising the steps of treating at least one substance selected from the group consisting of pepper oil, cananga oil and myrrh oil to a surface of an organism or an organism .

The material may further comprise cis-nerolidol.

The composition for inhibiting biofilm comprising at least one substance selected from the group consisting of pepper oil, pepper oil, cananga oil and myrrh oil according to the present invention may further comprise cis-nerolide, Film can be inhibited and the drug resistance of Staphylococcus aureus is not induced, so that it can be useful for the prevention or treatment of various diseases. Therefore, it can be usefully used in various fields such as medicines, health foods, cosmetics and the like.

FIG. 1 is a schematic view of an embodiment of the present invention, wherein the composition is selected from the group consisting of bay, black pepper, cade, cananga, cedarwood, frankincense, lovage root, myrrh, It was confirmed that the effect of inhibiting the formation of biofilm of Staphylococcus aureus in origanum, sandalwood, thyme red and vetiver haiti oil,
2 is a result of confirming the inhibitory effect of pepper oil, cananga oil and myrrh oil on biofilm formation,
FIG. 3 shows the results of confirming the inhibitory effect of trans-nerolidol and cis-nerolidol on the formation of biofilm of Staphylococcus aureus.
FIG. 4 shows the results of confirming the effects of three essential oils and cis-nerolite in human blood hemolysis by Staphylococcus aureus,
Fig. 5 shows the results of examining the effect of the three essential oils and cis-neroli stone on the growth of Staphylococcus aureus.
Figure 6 shows the results of performing CFU analysis and comparing it with optical density data to confirm cell viability in the presence of essential oil,
FIG. 7 shows the results of confirming the toxicity of Staphylococcus aureus of pepper oil and the effect of regulating the expression of genes related to biofilm,
Fig. 8 shows the results of confirming the antitoxic effect of the three essential oils and cis-nerolide in the nymphs of the beautiful nymphs.

Hereinafter, the present invention will be described in detail.

Conventionally used antibiotics and biocide treatment are harmful to the human body and frequently cause a multipledrug resistance due to microorganisms. Accordingly, the inventors of the present invention have attempted to develop a therapeutic strategy capable of inhibiting pathogenic microorganisms without inducing multidrug resistance, and there has been proposed a method of treating a pathogenic microorganism by using an oil selected from the group consisting of essential oils such as pepper oil, Of the total amount of the pharmaceutical composition of the present invention is inhibited to form biofilm upon treatment with Staphylococcus aureus, thereby completing the present invention.

The Staphylococcus aureus is a gram-positive bacterium and is a pathogenic microorganism causing pyogenic, abscess formation, various purulent infections, and sepsis. The average resistance rate to the antibiotic methicillin tested in Korea is 73%, which is the highest level in the world. This means that 73% of Staphylococcus aureus does not die even with the use of antibiotics. In addition, Staphylococcus aureus has many species that can form a biofilm, and when a biofilm is formed, it is impossible to penetrate the drug, and thus it shows a state of chronic infection. Because of this, the formation of biofilms by Staphylococcus aureus results in chronic infections. The treatment of diseases caused by Staphylococcus aureus with biofilms is particularly difficult compared to the treatment of diseases caused by biofilms formed by other bacteria. Because of the difficulty of drug delivery due to biofilm even when drugs are used for the treatment of diseases, antibiotic - based treatment is not effective due to the characteristics of Staphylococcus aureus, Therefore, biofilm treatment by Staphylococcus aureus is not a method dependent on conventional antibiotics, but a new approach is needed.

Accordingly, the present invention provides a composition for inhibiting biofilm comprising at least one essential oil selected from the group consisting of pepper oil, cananga oil and myrrh oil.

The composition may further comprise nerolide, and the nerolide may be trans-nerolidol or cis-nerolidol, more preferably a cis-nerolide.

The biofilm is a biofilm produced by Staphylococcus aureus.

The composition may comprise 0.4 to 20% (v / v) of one or more substances selected from the group consisting of pepper oil, cananga oil and myrrh oil, for a total of 100% (v / v).

In addition, the composition may comprise from 0.15 to 1.26% (v / v) trans-neroli stone or cis-nerolite, respectively, for a total of 100% (v / v).

According to an embodiment of the present invention, 0.01% (v / v) of cis-nerolidol, farnesol and valencene in the components of the pepper oil, cananga oil and myrrh oil, , The inhibition of the biofilm formation of Staphylococcus aureus can be inhibited, and cis-nerolide has a greater inhibitory effect than trans-nerolidol.

In addition, according to another embodiment of the present invention, the pepper oil, cananga oil, myrrh oil and cis-nerolite can inhibit hemolytic activity of Staphylococcus aureus.

In addition, according to another embodiment of the present invention, the pepper oil can control the expression of a gene associated with a biofilm or toxic factor of Staphylococcus aureus.

The composition for inhibiting biofilm comprising at least one substance selected from the group consisting of pepper oil, cananga oil and myrrh oil according to the present invention as an active ingredient can inhibit the biofilm formed by Staphylococcus aureus on the surface.

The composition may further comprise nerolide, and the nerolide may be trans-nerolidol or cis-nerolidol, more preferably a cis-nerolide.

The surface may include the surface of an organism or an organism in which the microorganism is capable of forming a biofilm, and may preferably be a tissue, an organ, or a medical article derived from an organism.

For example, various artificial implants applied to biological tissues such as various medical apparatuses, medical equipment, medical facilities / facilities, as well as various living tissues / organs including epithelial cells, bones, teeth, It can be used for implants.

The at least one essential oil selected from the group consisting of pepper oil, cananga oil and myrrh oil can be used in an amount effective to prevent or inhibit biofilm formation. The effective amount can be determined according to the conditions including the kind of the pathogenic microorganisms forming the biofilm, the kind and the area of the surface to be treated, the degree of reduction of the desired biofilm formation, the treatment time point and the like, Based on your knowledge of film formation, you will be able to select the appropriate concentration.

The biofilm inhibitor of the present invention can be prepared in various forms in liquid form, spray form or solid form, for specific purposes in the home, industrial, medical, and environmental fields.

Accordingly, the present invention can provide a composition for inhibiting biofilm comprising, as an active ingredient, at least one essential oil selected from the group consisting of pepper oil, cananga oil and myrrh oil, and neroli stone.

The nerolideol may be trans-nerolidol or cis-nerolidol, more preferably cis-nerolide.

In addition, the biofilm inhibiting composition may be provided as a pharmaceutical composition.

The pharmaceutical composition may be used for the prevention or treatment of all inflammatory diseases caused by Staphylococcus aureus, preferably toxic shock syndrome (TSST), sepsis, pneumonia, inflammatory bowel disease or severe skin May be used for infection, but is not limited thereto.

The pharmaceutical composition may be prepared by methods known in the art (e.g., Remington's Pharmaceutical Science (current edition; Mack Publishing Company, Easton PA), itself or in combination with a pharmaceutically acceptable carrier, excipient, diluent, Granules, tablets, capsules, injections, and the like. They may also be formulated for oral use (tablets, suspensions, granules, emulsions, capsules, syrups, etc.), parenteral formulations (sterile injectable aqueous or oleaginous suspensions) ≪ / RTI >

In addition, the pepper oil, cananga oil, myrrh oil, and cis-nolyol stone constituting the pharmaceutical composition according to the present invention are components derived from natural products and have already been prescribed for other fields and medical uses, and thus are safe.

In addition, the biofilm inhibiting composition may be provided as a health food.

The health food of the present invention may contain a sweetening agent, a flavoring agent, a physiologically active ingredient, a mineral, and the like in addition to the above-mentioned materials, and may contain preservatives, emulsifiers, acidifiers, thickeners and the like as needed.

The health food may be manufactured in the form of various foods, beverages, gums, tea, vitamin complex, health functional foods and the like. For example, it can be manufactured in the form of gum or tea to inhibit the formation of biofilm in the oral cavity to prevent corrosion of teeth, prevention of tartar formation, prevention and improvement of periodontal disease, and improvement of mouthfeel.

The present invention also provides a method for inhibiting biofilm formation comprising treating at least one substance selected from the group consisting of pepper oil, cananga oil and myrrh oil to the surface of an organism or an organism.

The substance may further comprise nerolide, and the nerolite may be trans-nerolidol or cis-nerolidol, more preferably cis-nerolide.

The inhibition method may be carried out by contacting at least one member selected from the group consisting of pepper oil, cananga oil, myrrh oil and cis-neriol styrene with Staphylococcus aureus, or contacting tissues derived from an organism in which Staphylococcus aureus can proliferate or proliferate , The surface of an organ or a medical article to prevent or suppress the formation of the biofilm in advance.

The surface may comprise a surface of a material used in a home, industry, environment, medical field as well as a surface derived from a living body. This means that the surface of an organism or an organism in which Staphylococcus aureus can form a biofilm can do.

For example, it may be the surface of an organism-derived tissue or organ such as a skin or a tooth, and may be used for various medical facilities, equipment, instruments, temporary or permanent artificial implants such as lenses, prosthetic valves, pacemakers, The surface of a medical article such as a catheter, but the scope of the present invention is not limited thereto.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

< Example  1> Experimental method

1. Strain, essential oil and chemical preparation

Staphylococcus aureus ATCC 6538 was cultured in LB medium at 37 ° C.

All essential oils were purchased from Berje, Jin Aromatics (Anyang, Gyeonggi Province, Korea) or Sigma-Aldrich (St. Louis, USA).

After adding a small amount of essential oil (<0.05%) to the LB medium, the solution was thoroughly mixed for 1 minute using a vortex mixer.

For the measurement of cell growth, the optical density was measured at 600 nm using a spectrophotometer (UV / VIS Spectrophotometer, Opizen, Korea) and the cell survival of the cells was analyzed by a colony-forming unit (CFU) Respectively.

2. crystal - Violet biofilm analysis

Fixed biofilm formation assays were performed on 96-well polystyrene plates (SPL Life Sciences, Korea).

When cells showed an initial turbidity of 0.05 at 600 nm, they were inoculated into a total volume of 300 μl of LB medium and cultured at 37 ° C. for 24 hours in the absence or presence of essential oil.

To quantify total biofilm formation, the biofilm was stained with crystal violet, dissolved in 95% ethanol and then measured at 570 nm (OD570).

3. Confocal  Laser microscope

Staphylococcus aureus was cultured in 96-well plates and stained with carboxyfluorescein diacetate succinimidyl ester (Invitrogen, Molecular Probes, Inc, Eugene, USA).

The stained Staphylococcus aureus biofilm was visualized through a confocal laser microscope (Nikon Eclipse Ti, Tokyo) Ar laser (excitation wavelength: 488 nm, emission wavelength: 500 to 550 nm) and 20x objective lens.

Color confocal images were removed using NIS-Elements C version 3.2.

4. Scanning Electron Microscope, SEM ) Based Membrane  analysis

A nylon filter (0.5 x 0.5 mm) was placed in a 96 well plate with 300 μL of cells showing an initial turbidity of 0.05 at 600 nm.

Cells and nylon filters were incubated together to form biofilm cells at 37 ° C for 24 hours without shaking.

The cells were then fixed with glutaraldehyde (final concentration 2.5%) and formaldehyde (formaldehyde, final concentration 2%) and cultured overnight at 4 ° C.

Cells were fixed with 1% osmium and irradiated using a S-4100 scanning electron microscope (Hitachi, Tokyo, Japan) at 15 kV and then magnified at a magnification ranging from 2000x to 10000x .

5. Hemolysis analysis

The dissolution efficiency of human red blood cells was measured using cultures of Staphylococcus aureus grown in the presence of essential oils or compounds.

Staphylococcus aureus was diluted 1: 100 in LB medium and cultured for 16 hours with or without essential oil or compound, with shaking at 250 rpm.

Human red blood cells were centrifuged at 890xg for 2 minutes and washed 3 times with PBS buffer.

Cell cultures (including cells and culture supernatant) (200 μL) were added to human red blood cells diluted in PBS buffer (330 μL red blood cells per 10 mL PBS buffer).

To confirm hemolytic activity, red blood cells and Staphylococcus aureus mixture were incubated at 37 ° C for 1 hour at a rate of 250 rpm.

The supernatant was collected by centrifugation at a rate of 16,600 x g for 10 minutes and the optical density of the supernatant was measured at 543 nm.

6. Isolation and quantification of RNA Real-time RT- PCR

To investigate gene expression, Staphylococcus aureus was shake-cultured for 8 hours at a rate of 250 rpm in the presence or absence of 0.01% black pepper.

Before the samples were taken, RNase inhibitor (RNase inhibitor, RNAlater, Ambion, TX, USA) was added to the cells and the mixture was immediately cooled with dry ice and 95% ethanol for 30 seconds to prevent RNA degradation. And centrifuged at a speed of 3 minutes.

Cell pellets were then immediately frozen in dry ice and stored at -80 ° C.

Total RNA was isolated using a Qiagen RNeasy mini kit (Valencia, CA, USA), and all the DNA was removed and 30 units of DNase1 were treated for 15 minutes in the purified RNA.

qRT-PCR was performed using a quorum-sensing gene ( agrA ), a Zinc metalloproteinase aureolysin ( aur ), a serine protease ( clp ), an α-hemolysin gene, hla ), intercellular adhesin gene ( icaA ) and icaD, transglycosylase gene ( isaA3 ), staphylococcal accessory regulator A ( sarA ), nucleic acid hydrolysis was used to probe a gene (genes nuclease) and the nuc1 nuc2, enterotoxin B (enterotoxin B, seb) and protease Sigma factor (polymerase sigma factor gene, sigB) transcription of a gene.

The primer pairs used in qRT-PCR were as in SEQ ID NOS: 1 to 26, and the 16S rRAN housekeeping gene was used as an internal standard.

Real-time qRT-PCR was performed using a step-on real-time PCR system (StepOneTMReal-Time PCR system, Applied Biosystems, Foster City, CA) and SYBR Green master mix (Applied Biosystems, Foster City, USA).

7. Pretty Little nematode  Life analysis

Ten microliters of overnight staphylococcal culture was applied to NGM plates in the presence or absence of three essential oils and cis-nerolyte.

L4 / immature adult fer-15; fem-1 (L4 / young adult fer-15; fem-1) were placed in lawns and infected and cultured at 25 ° C. Respectively. At this time, nematodes crawling toward the wall from the culture dish were excluded from the analysis.

Survival of the nematodes was analyzed by the Kaplan-Meier method and the significance of survival difference was measured using a log-rank test (STATA6; STATA, College station, TX).

< Example  2> Effect of 83 essential oils on biofilm formation

To identify the new anti-biofilm compounds, 83 essential oils were tested in 96-well plates and only 0.01% (v / v) of each essential oil was used to minimize the antimicrobial effect.

The growth of Staphylococcus aureus and the effect of 83 essential oils on biofilms are shown in Table 1 below.

Overall, 83 essential oils controlled the biofilm formation of Staphylococcus aureus and showed different efficiencies.

In particular, as shown in Fig. 1, twelve essential oils showed strong biofilm formation inhibitory effect.

It has been found that it can be used in many different ways, including bay, black pepper, cade, cananga, cedarwood, frankincense, lovage root, myrrh, origanum ), Sandalwood, thyme red and vetiver haiti oil inhibited the biofilm formation of Staphylococcus aureus by more than 75%.

In particular, nine out of 83 essential oils showed antibacterial activity.

In comparison to the untreated control group, 0.01% (v / v) of Bay, Cade, Cedarwood, frankincense, ruby root, Oriental sandalwood, timely red, And decreased cell growth.

The results showed that pepper oil, cananga oil and myrrh oil inhibited the biofilm formation of Staphylococcus aureus without reducing the growth of Staphylococcus aureus.

< Example  3> pepper oil, Kananga  oil And Myrrh  Confirmation of inhibitory effect of oil on biofilm formation

Since Staphylococcus aureus forms a biofilm on the bottom and sides of a 96-well plate, the effect of inhibiting biofilm formation of pepper oil, cananga oil and myrrh oil was confirmed using a confocal laser microscope.

As a result, it was confirmed that all three essential oils inhibited the biofilm formation of Staphylococcus aureus in a concentration-dependent manner (Fig. 2A).

In addition, microscopic observation confirmed that all of the essential oils of 0.01% inhibited the formation of the bottom of the biofilm (FIG. 2B).

In addition, the adhesion of cells on the nylon filter surface was observed using SEM.

As a result, it was observed that some of the biofilm cells adhered to the nylon surface in the presence of any one of the three essential oils (Fig. 2C).

< Example  4> trans- Neroli Stone (trans- nerolidol ) And Cis - Of cis-nerolidol  Confirming the inhibitory effect of Staphylococcus aureus on biofilm formation

Since pepper and myrrh are known to contain more than 100 compounds, the effect of inhibiting the biofilm formation of 24 commercially available compounds among them is investigated and the results are shown in Fig.

As a result, it was confirmed that the concentration of 0.01% (v / v) of farnesol, cis-nerolite and valencene markedly inhibited the formation of biofilm of Staphylococcus aureus.

In addition, pepper oil contains 0.4% trans-nerolidol, cananga oil contains 1.26% trans-nerolite and 0.15% cis-nerolide, and myrrh oil contains 0.6% cis- As shown in Figs. 3B and 3C, cis-neroliol has a greater inhibitory effect than trans-neroli stone.

Specifically, 0.01% (v / v) cis-nerolite reduced the biofilm formation of Staphylococcus aureus by more than 80%, while trans-nerolite had an inhibitory effect of 45%.

Confocal microscopy also showed that 0.01% (v / v) of cis-neroliol significantly inhibited the formation of the bottom of the biofilm (FIG. 3D).

< Example  5> pepper oil, Kananga  oil, Myrrh  Oil and cis-Neroli stone of Anti-hemolytic  Check activity

Staphylococcus aureus secretes α-toxin, which causes hemolysis and contributes to biofilm formation. Therefore, we investigated the effects of three essential oils and cis - nerolites on human blood hemolysis by Staphylococcus aureus.

As a result, all three essential oils and cis-nerolites inhibited hemolysis in a dose-dependent manner (Figs. 4A to 4D).

In particular, 0.0005% (5 ppm) of cis-nerolide reduced hemolysis by 95% or more (FIG. 4D).

In addition, analysis of hemolysis inhibition effect of 24 compounds of 5ppm showed that hematopoietic effect of parnesol, cis-nerolite and trans-neroli stone was inhibited by Staphylococcus aureus, The stone was most effective at inhibiting (Fig. 4E).

< Example  6> Three essential oils and Cis - Neroli Stone  Identification of the effect on the growth of Staphylococcus aureus

The effects of the three essential oils and cis-neroli stone on the growth of Staphylococcus aureus were investigated. As a result, the three essential oils and cis-nerolite showed a 0.05% inhibition effect on the growth of Staphylococcus aureus at a maximum concentration of 0.01% 5).

To confirm cell viability in the presence of essential oil, CFU analysis was performed and compared with optical density data (Figure 6).

From the above results, it was confirmed that the hemolysis and biofilm formation inhibitory effect of the three essential oils and cis-neroli stone on Staphylococcus aureus were not due to their antibacterial activity.

< Example  7> Toxicity of Staphylococcus aureus in pepper oil and its effect on the regulation of expression of biofilm-related genes

The effect of antioxidant activity and antimicrobial activity of pepper oil, which inhibited biofilm formation most significantly among the three oils, was examined.

To do this, we used real-time qRT-PCR to investigate the differences in expression of biofilm and toxic factor related genes and important regulatory genes in Staphylococcus aureus.

The control genes hla, nuc1, nuc2 and sarA were significantly reduced by pepper oil, and the expression of hla was suppressed 10 times (FIG. 7).

< Example  8> Beautiful From a nematode  Three essential oils and Cis - Neroli Stone  Identification of anti-toxic effects

Since Staphylococcus aureus can kill pretty nematodes, it was used to confirm the toxicity of the three essential oils and cis-neroli stone (Fig. 8).

Similar to the biofilm formation and hemolytic inhibitory activity (Figs. 2 to 4) identified in the above examples, the three essential oils and cis-nerolites prolong survival of the nymphs in the presence of Staphylococcus aureus.

However, the 0.01% concentration of cis-nerolideol shortened the survival of the rather small nematode. This means that high concentrations of cis-nerolideol may be toxic to nematodes (Fig. 8B).

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that such detail is solved by the person skilled in the art without departing from the scope of the invention. will be. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> Research Cooperation Foundation of Yeungnam University <120> Composition containing essential oil for inhibiting Biofilm and          Methods therefor <130> ADP-2015-0193 <160> 26 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 16S rRNA forward primer <400> 1 tgtttgacga tgtttgagca 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 16S rRNA reverse primer <400> 2 ccttcctcca gttcagatgc 20 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> agrA forward primer <400> 3 tgataatcct tatgaggtgc tt 22 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> agrA reverse primer <400> 4 cactgtgact cgtaacgaaa a 21 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> aur forward primer <400> 5 accgtgtgtt aattcgtgtg cta 23 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> aur reverse primer <400> 6 atggtcgcac attcacaagt tt 22 <210> 7 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> clp forward primer <400> 7 caggtaccat cacttcatc 19 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> clp reverse primer <400> 8 ggttcacaaa ttgatgacaa cg 22 <210> 9 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> hla forward primer <400> 9 cggcacattt gcaccaataa ggc 23 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> hla reverse primer <400> 10 ggtttagcct ggccttcagc 20 <210> 11 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> icaA forward primer <400> 11 ctggcgcagt caatactatt tcgggtgtct 30 <210> 12 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> icaA reverse primer <400> 12 gacctcccaa tgtttctgga accaacatcc 30 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> icaD forward primer <400> 13 atggtcaagc ccagacagag 20 <210> 14 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> icaD reverse primer <400> 14 agtattttca atgtttaaag caa 23 <210> 15 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> isaA3 forward primer <400> 15 gctcaaatca tggctcaacg t 21 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> isaA3 reverse primer <400> 16 ttgattcacg agcgatgatt g 21 <210> 17 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> nuc1 forward primer <400> 17 cacctgaaac aaagcatcct aa 22 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> nuc1 reverse primer <400> 18 tatacgctaa gccacgtcca t 21 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> nuc2 forward primer <400> 19 atggacgtgg cttagcgtat 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> nuc2 reverse primer <400> 20 tgacctgaat cagcgttgtc 20 <210> 21 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> sarA forward primer <400> 21 caaacaacca caagttgtta aagc 24 <210> 22 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> sarA reverse primer <400> 22 tgtttgcttc agtgattcgt tt 22 <210> 23 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> seb forward primer <400> 23 tgttcgggta tttgaagatg g 21 <210> 24 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> seb reverse primer <400> 24 cgtttcataa ggcgagttgt t 21 <210> 25 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> sigB forward primer <400> 25 aagtgattcg taaggacgtc t 21 <210> 26 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> sigB reverse primer <400> 26 tcgataacta taaccaaagc ct 22

Claims (12)

A composition for inhibiting biofilm, comprising cis-nerolidol, wherein said cis-nerolidol inhibits biofilm formation by Staphylococcus aureus. The method according to claim 1,
Wherein the composition further comprises at least one essential oil selected from the group consisting of pepper oil, caraway oil and myrrh oil. delete delete 3. The method of claim 2,
Characterized in that the composition comprises 0.4 to 20% (v / v) of at least one essential oil selected from the group consisting of pepper oil, cananga oil and myrrh oil, for a total of 100% (v / v) / RTI &gt; The method according to claim 1,
Wherein the composition comprises 0.15 to 1.26% (v / v) cis-nerolide, based on 100% (v / v) of the total. delete delete delete Treating cis-nerolidol on the surface of an organism or an organism other than a human, wherein the cis-nerolidol is a biofilm by Staphylococcus aureus, And inhibiting the formation of the biofilm. 11. The method of claim 10,
Wherein the step of treating the surface of an organism or an organism other than human comprises further treating at least one essential oil selected from the group consisting of pepper oil, caraway oil and myrrh oil.










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