KR101625891B1 - Composition for inhibiting quorum sensing or anti-bacterial composition comprising Nymphaea tetragona georgi extract - Google Patents

Abstract

The present invention relates to a composition for inhibiting the recognition of a quorum water, a composition for antibiosis or a composition for feed addition, which comprises a water extract as an active ingredient. The water extract according to the present invention has no toxicity and has a quorum sensing inhibitory activity which interferes with the communication between microorganisms, thereby effectively preventing the propagation of microorganisms and formation of a biofilm, and when used in combination with existing antimicrobial agents, And can be effectively used as a composition for inhibiting the recognition of a quorum, an antimicrobial composition or a composition for adding a feed.

Description

[0001] The present invention relates to a composition for inhibiting the recognition of a quorum of water or an antimicrobial composition comprising a water extract as an active ingredient.

The present invention relates to a composition for inhibiting the recognition of a quorum water, a composition for antibiosis or a composition for feed addition, which comprises a water extract as an active ingredient.

When bacteria reach a certain level of cell density, they use the chemical language they produce to transmit signals to and from each other, thereby regulating the expression of specific genes. The mechanism by which gene expression depends on the density of individuals is called quorum sensing (QS). The quorum sensing mechanism is a mechanism in which each bacterial individual accumulates low-molecular signaling substances such as autoinducer or pheromone into the cell to induce active proliferation and to induce gene expression by filling a certain quorum Refers to a series of biological phenomena. Bacteria are formed by the formation of biofilm, virulence, bioluminescence, antibiotic production or conjugation of tumor-derived plasmids (Ti plasmid) through the quorum sensing mechanism And the like.

Biofilm is a typical function of bacteria through communication. Biofilm refers to a smooth mucous layer on the surface formed by extracellular polymeric materials and attached microorganisms secreted during the metabolism of microorganisms. When microorganisms attach to various inanimate objects and organisms' surfaces to form a biofilm and form populations / communities, there are various environmental conditions such as depletion of nutrients, changes in acidity, changes in osmotic pressure, changes in temperature, oxidative shock, It is known that the resistance to stress is significantly increased. It also causes many ailments while staying in human organs. Therefore, development of an antibacterial agent for inhibiting the formation of biofilm as described above is required.

On the other hand, the water lily (Nymphaea tetragona georgi) is a plant belonging to the family Ranunculaceae and it is cultivated in the whole country for ornamental use in our country. Seeds and rootstocks contain a lot of starch and contain alkaloids called nupalidins and are used as gastrointestinal drugs. In addition, the water extract has excellent anti-stress or antidepressant effect and is known to be used as food, food additive or medicine (Patent No. 10-0876440). It is known that it has antioxidative activity, and cosmetic composition (Patent Publication No. 10-2011-0085371, 10-2012-0057197).

As described above, various pharmacological effects of the water extract are known, but it is not known at all that the water extract has a quorum sensing inhibitory activity, and there is no research on this.

Accordingly, the present inventors have sought to find a new substance having a quorum-suppressing activity that can prevent the propagation of bacteria by interfering with the communication of germs as well as killing the bacteria. As a result, the water extract has a superior quercetin recognition inhibitory activity The present invention has been completed.

It is an object of the present invention to provide a composition for inhibiting the recognition of a quorum water, a composition for antimicrobial use or a composition for feed addition comprising a water extract as an active ingredient.

In order to achieve the above object, the present invention provides a composition for suppressing the recognition of a quorum water, a composition for antibiosis or a composition for adding a feed comprising an extract of a water lily as an active ingredient.

The water extract according to the present invention has no toxicity and has a quorum sensing inhibitory activity which interferes with the communication between microorganisms, thereby effectively preventing the propagation of microorganisms and formation of a biofilm, and when used in combination with existing antimicrobial agents, And can be effectively used as a composition for inhibiting the recognition of a quorum, an antimicrobial composition or a composition for adding a feed.

1 is a schematic view showing the process of obtaining a water extract according to the present invention.
FIG. 2 is a graph showing the quercetin recognition inhibitory activity of the water extract of Chromobacterium bisreflex (A: water extract extract 2.5 mg / ml, B: water extract 1.25 mg / ml, C: water extract 0.625 mg / D: water extract extract 0.3125 mg / ml, F: furanone 10 μg / disc, M: methanol).
FIG. 3 is a graph showing the quercetin perception inhibitory activity of Pseudomonas aeruginosa extract of water extract (A: water extract extract 5 mg / ml, B: water extract 2.5 mg / ml, C: water extract 1.25 mg / Water extract: 0.625 mg / ml, E: water extract: 0.3125 mg / ml, F: water extract: not added).
FIG. 4 shows the effect of water extract on the biofilm formation of Pseudomonas aeruginosa (A: no water extract, B: water extract 5 mg / ml, C: water extract 10 mg / 20 mg / ml).
Fig. 5 is a graph showing antimicrobial activity against Pseudomonas aeruginosa of a water extract.
FIG. 6 is a graph showing antimicrobial activity against Chromobacterium bilecy when water extracts and conventional antimicrobial agents (amikacin, tyrosine, tetracycline, amoxicillin, magobactycoxin or gentamicin) are used in combination.
FIG. 7 is a graph showing the salmonellosis inhibitory effect of the feed composition containing the water extract. FIG.

Hereinafter, the present invention will be described in more detail.

The present invention provides a composition for suppressing the recognition of a quorum water, a composition for antimicrobial use or a composition for feed addition, which comprises a water extract as an active ingredient.

The water extract which is an active ingredient in the composition of the present invention can be obtained by the following method.

First, the water lily is washed with water to remove impurities, sedimented and dried. The water lily can be cultivated or marketed without limitation. After the dried water lily is pulverized, an appropriate amount of solvent is added so that it is completely immersed. The extraction method can be impregnated or warmed at room temperature. The extraction solvent is not limited thereto, and at least one solvent selected from water, alcohols having 1 to 4 carbon atoms, and a mixed solvent thereof may be used. In one embodiment of the present invention, the dried water lily powder is shaken at a weight ratio of powder 1: ether 5 to 15, and then the ether layer as an upper layer liquid is removed. The remaining lower layer ether is evaporated in a vacuum drier, The dried water lily powder is successively extracted with 100% ethanol, 40 to 60% ethanol and hot water, and then the independently obtained extracts are mixed at a weight ratio of 1: 2: 1 to 2: 1 to 2. The extract is concentrated, lyophilized, dissolved in an organic solvent, and the supernatant is filtered and purified to obtain a final water extract.

The water extract according to the present invention has no toxicity and has a quorum sensing inhibiting activity which interferes with the communication between microorganisms and can effectively block the propagation of microorganisms and the formation of biofilm. When used in combination with existing antimicrobial agents It has an excellent antimicrobial synergistic effect.

Therefore, the water extract according to the present invention can be widely used for a variety of life tools and medical instruments that need to prevent the infection of bacteria or prevent the formation of biofilm, and can be used as a therapeutic agent for diseases caused by antibacterial agents and strains, A composition for addition, and the like.

Examples of the strain exhibiting a quorum-sensing inhibitory activity or antimicrobial activity include gram-negative bacteria and gram-positive bacteria. Examples of the bacteria include C. violaceum, Pseudomonas aeruginosa, Pseudomonas aeruginosa, E. aeruginosa, E. coli, P. hemolytica, S. aureus, S. cholerasuis, S. typhimurium, S. typhimurium, ), And the like.

The compositions of the present invention may further comprise suitable carriers, excipients or diluents conventionally used. Examples of the carrier, excipient or diluent which can be contained in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose , Methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

The composition of the present invention can be used as a preservative, antimicrobial agent, antifungal agent, immunizing agent including sodium benzonate, allantoin, sodium propionate, citral, betaglucan, Enhancing agents, and the like.

The composition of the present invention may be administered orally or parenterally in a conventional manner. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, do. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, lactose, Gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, syrups and the like. Various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included in addition to water and liquid paraffin, which are simple diluents commonly used. have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the non-aqueous solvent and suspension include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

The content of the active ingredient according to the present invention in the preparation can be appropriately selected depending on the degree of absorption, inactivation rate, excretion rate, age and condition of the active ingredient in the body, and is not limited thereto. For example, when the composition of the present invention is formulated into a feed, the content of the water extract may be added in an amount of 0.1% to 20%, preferably 0.1% to 10% (weight%) based on the total weight of the feed, But is not limited thereto.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples.

Example 1. Preparation of water extract

The water lily (Nymphaea tetragona GEORGI) collected from the Gyeongju lotus in Gyeongbuk province was used. First, the water lily roots were washed cleanly, then dried in a size of about 1 cm and dried (less than 10% moisture). The dried water lily roots were pulverized, and then the powder was shaken at a weight ratio of 1: ether 10, and the mixture was allowed to stand to remove the ether layer as an upper layer liquid. The remaining lower ether was evaporated with a vacuum drier, and the dried water lily powder was sequentially extracted with 100% (v / v) ethanol (weight ratio of ethanol: water liquor = 20: 1), 50% ethanol and hot water . Each of the independently obtained extracts was mixed at a weight ratio of 1: 1: 1 through the above procedure, and then concentrated and lyophilized to obtain a powdered water-extract. The powdery state water extract was dissolved in methanol, and the supernatant was filtered and purified by RP-Sepak column to obtain a final water extract.

The above-mentioned manufacturing process is shown in FIG. 1 as a schematic diagram.

EXPERIMENTAL EXAMPLE 1. Verification of quorum-recognition inhibitory activity of water extracts

In order to examine the quan- tity recognition inhibitory activity of the water extract obtained in Example 1, it was confirmed that quercetin peroxidase (C. violaceum (ATCC12472)) and Pseudomonas aeruginosa Pseudomonas aeurginosa (PAO1)).

The quercetin recognition inhibition activity using the chromobacterium viremia was confirmed by the following experiment. First, LB (Luria-Britani) agar medium was inoculated with 5x10 ⁶ cfu / ml of chromobacterium bimetallicum, and then poured into petri dishes (90 x 15 mm) to prepare LB agar plates. Water extracts of various concentrations (2.5-0.3125 mg / ml) were dropped on a sterilized paper disk (Ø = 8 mm), dried, and placed on the LB agar plate and cultured at 30 ° C for 24 hours. After that, pigment inhibition rings at various concentrations were measured (digital vernier calipers, Mitotoyo, Japan). Furanone was used as a positive control and methanol was used as a negative control. The results are shown in Fig.

As shown in FIG. 2, it was confirmed that the water extract of the present invention inhibited the quorum recognition of chromobacterium-biasedis in a concentration-dependent manner.

In addition, quercetin recognition inhibition activity using Pseudomonas aeruginosa was confirmed by the following experiment. Various concentrations (2.5-0.3125 mg / ml) of water extracts were added to the LB agar medium containing 0.5% glucose, which was then plated. A single colony of Pseudomonas aeruginosa was inoculated at the center of the plate and cultured at 37 ° C for 48 hours. As control, LB agar plates without water extract were used. The results are shown in Fig.

As shown in Fig. 3, it was confirmed that the water extract of the present invention had a superior quorum recognition performance inhibiting the migration of Pseudomonas aeruginosa.

Experimental Example 2. Effect of Water Extract on the Inhibition of Quorum Identification of Chromobacterium bisrefil

As shown in Experimental Example 1, in order to reexamine the effect of the water extract on quorum recognition perception of chromobacterium viremia, violacein pigment inhibitory activity was measured. First, 5 × 10 ⁶ cfu / ml of chromobacterium bimetallicum was inoculated into LB medium containing water extracts of various concentrations (2.5-0.625 mg / ml) and cultured for 24 hours at 30 ° C. with shaking. Furanone was used as a positive control, and LB medium containing methanol as a negative control was used. Violacein pigments were extracted and quantified in each medium according to the method described in elsewhere (Choo et al. 2006). The results are shown in Table 1.

Sample Concentration (mg / ml) Violacien (Mean ± SD) Water extract 2.5 0.087 ± 0.000 Water extract 1.25 0.125 ± 0.002 Water extract 0.625 0.142 ± 0.004 Furanone 0.01 0.081 0.011 Negative control group - 0.500 + 0.011

As shown in Table 1, the water extract of the present invention inhibited the production of violacein pigment in a concentration-dependent manner, and thus it was confirmed that the water extract extract inhibited the quorum recognition of chromobacterium-biasedis.

Experimental Example 3: Effect of water extract on the inhibition of quorum recognition by Pseudomonas aeruginosa

As shown in Experimental Example 1, the activity of inhibiting Pyocyanin production and the activity of LasA protease were measured in order to reexamine the effect of the water extract on the quorum recognition perception of Pseudomonas aeruginosa. Pyocyanin (1-hydroxy-5-methyl-phenazine) is one of the compounds produced by Pseudomonas aeruginosa and is a characteristic blue-green oxidation-reduction active substance. It expresses genes regulated by quorum recognition As a final signaling factor.

First, 3 ml of chloroform was added to Pseudomonas aeruginosa grown in an LB medium containing 5 ml of various concentrations (2.5-0.625 mg / ml) of water extracts to measure phycocyanin production inhibitory activity. Followed by vortexing for a few seconds. The aqueous layer was discarded and the chloroform layer was mixed with 1 ml of 0.2N HCI. The mixture was vortexed for 30 seconds and centrifuged at 8000 x g for 10 minutes. The absorbance of the upper layer of the pink layer was measured at a wavelength of 520 nm using a microplate reader (Molecular device, Sunnyvale, Calif., USA). Furanone was used as a positive control, and LB medium was used as a negative control.

In order to measure the activity of LasA protease, Staphylococcus aureus was cultured overnight in LB medium, and 10 ml of the strain was boiled for 10 minutes and centrifuged at 10,000 × g for 10 minutes. The pellet was suspended in 0.02 M Tris buffer (pH = 8.5). 900 μl of Staphylococcus aureus suspension was added to 100 μl of the culture supernatant of Pseudomonas aeruginosa and absorbance was measured at a wavelength of 600 nm at an interval of 1 hour using a microplate reader. Furanone was used as a positive control, and LB medium was used as a negative control. The results are shown in Table 2.

Sample (mg / ml) Pyocyanin
(μg / mL) LasA activity Water extract (2.5) 1.37 ± 0.01 0.0005 0.001 Water extract (1.25) 1.49 ± 0.15 0.0151 0.001 Water extract (0.625) 2.43 + - 0.48 0.0376 + 0.021 Furanone (0.01) 1.40 ± 0.4 - Negative control group 2.51 + 0.04 0.0373 0.003

As shown in Table 2, the water extract of the present invention inhibited the production of phyisianine of Pseudomonas aeruginosa in a concentration-dependent manner and inhibited the activity of LasA which decomposes Staphylococcus aureus. Thus, the water extract extract of Pseudomonas aeruginosa Of the quasi-

Experimental Example 4. Effect of Water Extract on Biofilm Survival

Biofilm viability experiments were carried out by modifying the method of elsewhere (Karen and Iain 2008) in order to verify the quercetin recognition inhibitory activity of the water extract obtained in Example 1 above. More specifically, 7.5 ml of LB medium containing 0.5% glucose was placed in a 50 ml tube, and sterilized slides were added thereto to form a biofilm of Pseudomonas aeruginosa, which was then washed with 1x PBS. The slides were transferred to fresh 50 ml tubes containing LB medium containing water extracts of various concentrations (5-20 mg / ml) and cultured at 30 ° C for 24 hours. After incubation, the biofilm was identified as BacLight live / dead stain. The results are shown in Fig.

As shown in FIG. 4, when the water extract of the present invention was added, it was confirmed that the green area, which means living cells, was smaller than the control group.

Experimental Example 5. Antimicrobial Activity of Water Extract

5-1. Measurement of MIC (minimal inhibitory concentration) and MBC (minimal bactericidal concentration)

The MIC and MBC of S. aureus, Pseudomonas aeruginosa, and Chromobacterium bimetallicum obtained in Example 1 were measured according to the guidelines of Clinical and Laboratory Standards Association (CLSI, 2007). More specifically, when the extract was inoculated into the bacterial suspension and cultured for 24 hours, the lowest concentration without any noticeable growth was determined as MIC. When the concentration after MIC was applied to TSA and cultured for 24 hours, 99.9 % Of colonies were determined as MBC. Marbofloxacin was used as a positive control. The results are shown in Table 3.

Strain training Magoblockocaine MIC (mg / ml) MBC (mg / ml) MIC (μg / ml) MBC (μg / ml) S. aureus 5 20 0.5 2 P. aeurginosa 10 20 2 8 C. violaceum 5 10 0.25 8

As shown in Table 3, it was confirmed that the water extract of the present invention had antimicrobial activity against Staphylococcus aureus, Pseudomonas aeruginosa and Chromobacterium bresenius.

5-2. Time-Kill assay

Pseudomonas aeruginosa (5x10 ⁵ cfu / ml) was inoculated into MHB (Mueller Hinton Broth) medium containing water extracts of various concentrations (20-0.624 mg / ml) and cultured at 37 ° C for 24 hours. To measure the number of bacteria at 1, 2, 4, 8, 12 and 24 hours later, 0.1 ml of each sample was taken and diluted 10-fold. Twenty microliters of even (2, 4, 6, 8 th) diluted samples were placed on TSA plates and incubated at 37 ° C for 24 h. The results are shown in Fig.

As shown in FIG. 5, the water extract of the present invention inhibited the growth of Pseudomonas aeruginosa from a concentration of 5 mg / ml, and inhibited bacterial activity at a concentration of 20 mg / ml.

Experimental Example 6. Antimicrobial activity test using water extract and conventional antimicrobial agent

6-1. Diffusion method

In order to verify the antimicrobial activity of the water extract obtained in Example 1 and the existing antimicrobial agent, antimicrobial activity was confirmed by the diffusion method using chromobacterium bimetallicus. 60 μl of each sample (water extract and existing antimicrobial agent (amikacin, tyrosine, tetracycline, amoxicillin, magofoxacin or gentamycin) was adsorbed on each paper disk and the test medium was incubated overnight at 37 ° C. The results are shown in Fig.

As shown in FIG. 6, the water extract of the present invention was found to have an adduct or synergistic action with existing antimicrobial agents (amikacin, tyrosine, tetracycline, amoxicillin, marbophloxacin or gentamicin).

6-2. Checkerboard method

Based on the above results, the following experiment was conducted by using a checkerboard method to confirm the combined effect of water extract and marbloxacin. Target strains include E. coli (ATCC 25922), P. hemolytica ( ATCC 55518), S. aureus ( ATCC 29213), S. cholerasuis 7001), was used Salmonella typhimurium (S. typhimurium). The target strains were subcultured for 24 hours in agar medium, adjusted to 0.5 McFarland ( ⁵ × 10 ⁵ CFU / ml), inoculated into 96-well plates containing ENR and TMP, cultured at 37 ° C. for 24 hours And the growth of each strain was confirmed. The interaction between water extracts and marbophlogasin was evaluated by fractional inhibitory concentration index. In general, antibiotics are considered to be synergistic when FIC≤0.5, additive effect / indefinite if 0.5 <FIC <2, and antagonism if FIC≥2. The equation for obtaining FIC is as follows.

Strain type
Magoblockocaine KVCP-1 extract Co-administration FIC index
(Based on moppel width god)
Interaction
(μg / ml) (μg / ml) (μg / ml) E. coli
(ATCC 25922)
0.063 500 0.031 / 500 1.5
(0.5) Synergism (synergistic action of marbrophloxacin by water extract) P. hemolytica
(ATCC 55518)
0.125 500 0.063 / 500 1.25
(0.25) Synergism (synergistic action of marbrophloxacin by water extract) S. aureus
(ATCC 29213)
0.25 500 0.125 / 500 1.5
(0.5) Synergism (synergistic action of marbrophloxacin by water extract) S. cholerasuis
(ATCC 7001)
0.125 500 0.031 / 500 1.25
(0.25) Synergism (synergistic action of marbrophloxacin by water extract) S. typhimurium
(field isolated.)
0.063 500 0.031 / 500 1.5
(0.5) Synergism (synergistic action of marbrophloxacin by water extract)

As shown in Table 4, it was confirmed that the water extract and marbophloxacin of the present invention showed an adverse effect on each strain. Thus, it was confirmed that the water extract can be used as an antimicrobial enhancer to enhance the antimicrobial activity of existing antimicrobial agents Respectively.

Experimental Example 7. Safety Test of Water Extract

To confirm the stability of the water extract obtained in Example 1, an acute toxicity test was conducted. The test group was divided into control (control, 0.85% NaCl), water extract extract low dose (500 mg / kg), water extract extract medium (1,000 mg / kg) and water extract extract high dose (2,000 mg / kg) Specific pathogen free (SPF) rats of the Sprague-Dawley strain were fasted overnight, and each sample was orally administered to the rats using a single dose, followed by observation for 14 days. The results are shown in Table 5.

gender density
(mg / kg) Date (days) after administration Final mortality (%) LD ₅₀ One 2 3 4 5 6 7 8 9 10 11 12 13 14 cock 2,000 0 ^a) 0 0 0 0 0 0 0 0 0 0 0 0 0 0 > 2,000 mg / kg 1,000 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 500 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Control 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0  female 2,000 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 > 2,000 mg / kg 1,000 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 500 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Control 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ^a) Number of dead animals

As shown in Table 5, when the water extract of the present invention was administered at a dose of 2,000 mg / kg, the mortality rate was 0% in both cancer and animal models, and no clinical symptoms related to toxic symptoms were observed Respectively.

Experimental example 8. GC-MS analysis of water extract

GC / MS (Gas Chromatography Coupled Mass Spectroscopy) analysis was performed at Kyungpook National University joint lab habit to identify the major components contained in the water extract obtained in Example 1. [ The results are shown in Table 6.

Experimental Example 9. Inhibition of Salmonella Inhibition by Feed Additives Containing Water Extracts

In order to confirm the inhibitory effect of feed additive including water extracts on Salmonella, the following experiment was carried out using a weedy pig in a nursery pig farm in Chilgok - myeon, In the negative control (n = 10), only 3% of the water extracts were added to the feed. All the experimental groups were inoculated with salmonella and the group with no water extract (n = 10), group with 0.1% water extract (n = 10), group with 1% water extract (n = 10) = 10). The fecal samples were collected at 1, 2, and 3 weeks after the inoculation of Salmonella to investigate the growth of Salmonella. The results are shown in Fig.

As shown in Fig. 7, the growth of Salmonella was inhibited from the 2nd week of the group supplemented with 3% water extract, and the growth of Salmonella was inhibited from the 3rd week after the addition of the 0.1% water extract.

Claims (8)

Water roots powder: Ether was shaken at a weight ratio of 1: 5 to 15, and the mixture was allowed to stand to remove the ether layer as an upper layer. The resulting powder was dissolved in ethanol; 40 to 60% (v / v) aqueous ethanol solution; C. violaceum containing as an active ingredient a water extract obtained by extracting each of the extracts with water and water and then mixing the extracts in a weight ratio of 1: 2: 1 to 2: 1: 2, (Pseudomonas aeruginosa), E. coli, P. hemolytica, S. aureus, S. cholerasuis, and Salmonella typhi And S. typhimurium. &Lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt;
delete The antimicrobial composition according to claim 1, wherein the mixing weight ratio is 1: 1: 1.
delete delete delete delete A feed additive composition having an antibacterial activity comprising a water extract as an active ingredient,
The water lily extract was prepared by shaking water lily root powder: ether in a weight ratio of 1: 5 to 15, and removing the ether layer as an upper layer liquid. The resulting powder was dissolved in ethanol; 40 to 60% (v / v) aqueous ethanol solution; Wherein the extracts are mixed with each other at a weight ratio of 1: 2: 1 to 2: 1: 2.

Images