KR101880650B1 - Microorganism capable of degrading ethanol and acetaldehyde, composition and kit comprising the same - Google Patents

Abstract

Provided are a microorganism having ability to degrade ethanol and acetaldehyde, and a composition and a kit comprising the same. The microorganism, and the composition and the kit comprising the same, according to certain aspects, may be used to remove one or more of ethanol and acetaldehyde in a sample.

Description

Microorganism capable of degrading ethanol and acetaldehyde, composition and kit comprising the same}

It relates to a microorganism having the ability to decompose ethanol and acetaldehyde, and a composition and kit comprising the same.

Lactobacillus is a genus of Gram-positive, facultative anaerobic or microaerophilic, rod, non-sporogenic bacteria. Lactobacillus is a major part of the lactic acid bacter (LAB) group.

Alcohol consumed by humans is easily absorbed by the diffusion action in the stomach and upper part of the small intestine. Alcohol metabolism occurs mainly in the liver tissue, where alcohol is oxidized to acetaldehyde by alcohol dehydrogenase (ADH), and acetaldehyde is converted to acetic acid by aldehyde dehydrogenase (ALDH). .

Acetaldehyde, a metabolite produced by alcohol oxidation, is a highly reactive toxic substance. Acetaldehyde covalently binds to various proteins in the liver to alter liver function and structure. Through binding with tubulin, the polymerization of microtubules is reduced, causing damage to protein secretion, causing hepatocytes to swell. The formation of acetaldehyde adduct impairs the activity of some enzymes. Directly or through binding to GSH, acetaldehyde favors lipid peroxidation. Acetaldehyde alters various mitochondrial functions, especially after chronic ethanol consumption, which sensitizes the mitochondria to the toxic effects of acetaldehyde. In cultured myofibroblasts, acetaldehyde stimulates collagen production. It has been reported to be the cause of liver fibrosis in chronic alcohol intakes. In addition, chronic alcohol intake can cause fatty liver, alcoholic hepatitis, and cirrhosis. The acetaldehyde-protein adduct stimulates the production of antibodies against acetaldehyde epitopes. This immune response contributes to the exacerbation or perpetuation of alcohol-induced liver damage. In addition, acetaldehyde causes hangover.

However, even according to the prior art described above, there is a need for bacteria of the genus Lactobacillus having alcohol and acetaldehyde decomposition activity.

Unexamined Patent Publication No. 10-2013-0092182

One aspect is Lactobacillus brevis ( Lactobacillus brevis ) LMT1-73 (accession number KCTC-13412BP) and Lactobacillus fermentum (Lactobacillus fermentum) LMT2-75 (accession number KCTC-13413BP) selected from the group consisting of ethanol and acetaldehyde resolution. Provides the microbes that have it.

Another aspect provides a composition comprising the microorganism or its lysate.

A kit for use in removing one or more of ethanol and acetaldehyde from a sample containing the microorganism and a diluent or a carrier is provided.

One aspect is Lactobacillus brevis ( Lactobacillus brevis ) LMT1-73 (accession number KCTC-13412BP) and Lactobacillus fermentum (Lactobacillus fermentum) LMT2-75 (accession number KCTC-13413BP) selected from the group consisting of ethanol and acetaldehyde resolution. Provides the microbes that have it.

The microorganisms are excellent in ethanol resistance, ethanol and/or acetaldehyde resolution, as well as acid resistance, bile acid resistance, and intestinal fixation. The microorganism was isolated from kimchi.

Another aspect provides a composition comprising the microorganism or its lysate.

The composition may contain a food-acceptable diluent or carrier. The diluent may be water, a medium, or a buffer such as PBS. The carrier may be a conventional excipient, a disintegrant, a binder, a lubricant, a thickener, or a filler. The diluent or carrier is lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, poly It may be vinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil. In addition, the composition may include a lubricant such as magnesium stearate and talc.

The composition may be for use in removing one or more of ethanol and acetaldehyde from the sample. The term “removal” refers to reducing the concentration of one or more of ethanol and acetaldehyde in a sample, and includes completely removing it. The sample may be a bodily fluid. The sample may be intestinal fluid or blood. The intestinal fluid may be gastric juice, duodenal fluid, small intestine fluid, or large intestine fluid.

The composition may be an oral dosage form. The composition may be a granule, powder, liquid, tablet, capsule, or dry syrup. The composition may be, for example, a culture obtained by culturing the microorganism in a medium, or a dried product thereof.

The composition may be food. The food may be a dairy product, a food for preventing alcoholic liver disease or relieving a hangover, or a food additive. The dairy product may be fermented milk, butter, cheese, or powdered milk. The food may be a health functional food. The health functional food may be a health functional food for preventing alcoholic liver disease or relieving a hangover. The food may also be beverages, confectionery, diet bars, chocolate, pizza, ramen, other noodles, gums, and ice cream.

The food may include ingredients that are commonly added during food production, and include, for example, proteins, carbohydrates, fats, nutrients, flavoring agents, and flavoring agents.

Carbohydrates used in food production include monosaccharides such as glucose and fructose; Dissaccharides such as maltose, sucrose, oligosaccharides, and the like; And polysaccharides, for example, common sugars such as dextrin, cyclodextrin, and the like, and sugar alcohols such as xylitol, sorbitol, and erythritol. In addition, natural flavoring agents and synthetic flavoring agents such as saccharin and aspartame may be used as flavoring agents. The natural flavoring agent may be a stevia extract such as taumatin, rebaudioside A, and glysirhizin.

Health functional foods are meant to bring specific health effects when ingested.

In the composition, the microorganism may be in the range of 0.01 to 50% by weight, or 0.1 to 20% by weight based on the weight of the composition. ^{In addition, the composition may include cells of 10 5} to 1x10 ⁹ CFU/g, or 1x10 ⁵ to 1x10 ⁸ CFU/g based on the weight of the composition.

Another aspect is the decomposition of ethanol and acetaldehyde selected from the group consisting of Lactobacillus brevis LMT1-73 (accession number KCTC-13412BP) and Lactobacillus fermentum) LMT2-75 (accession number KCTC-13413BP). It provides a method for removing at least one of ethanol and acetaldehyde from a subject, comprising administering to the subject having a microorganism. The method may be to prevent or treat a disease associated with the accumulation of ethanol and/or acetaldehyde in the body. The disease may be for preventing alcoholic liver disease or a hangover. The subject may be a mammal. The mammal may be a human or a non-human mammal.

Another aspect provides a kit for use in removing one or more of ethanol and acetaldehyde from a sample comprising the microorganism and a diluent or carrier. The kit may be provided separately from the microorganism and a diluent or carrier.

The microorganism according to an aspect, a composition and a kit including the same, may be used to remove one or more of ethanol and acetaldehyde from a sample.

1 is a diagram showing the extent to which the selected strain adheres to intestinal epithelial cells.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1: Isolation and identification of microorganisms with alcohol and acetaldehyde decomposition ability

In this example, microorganisms having excellent alcohol and acetaldehyde decomposition ability were isolated and identified from kimchi. As a result, this microorganism has excellent decomposition ability of alcohol and acetaldehyde in blood, excellent acid resistance, bile resistance, and intestinal fixation, and thus excellent stability in the intestine.

1. Isolation and identification of strains

(1) Isolation of strain

20 g of kimchi prepared directly at home stored at 4°C was aseptically taken, diluted in 180 ml of 0.85% NaCl solution, and homogenized for 5 minutes with a stomacher. A kimchi sample was prepared by gradually diluting the homogenized kimchi in a tube containing 9 ml of a sterilized 0.85% NaCl solution. Kimchi samples were plated on agar plate medium (MRS medium (Difco, USA)) and cultured at 37° C. for 2 to 3 days, and the colonies that appeared were separated by morphology and color, and purified again. The isolated colonies were cultured in MRS liquid medium of pH 6.8 at 37° C. for 24 hours to select 230 colonies whose pH decreased to 4.5 or less. The selected strain was tested for alcohol resistance, alcohol and acetaldehyde degradation, and finally two strains with excellent alcohol and acetaldehyde degradation and intestinal stability, namely, Lactobacillus brevis LMT1-73 (accession number KCTC-13412BP ), and Lactobacillus fermentum LMT2-75 (accession number KCTC-13413BP) were selected.

(2) In vitro alcohol tolerance confirmation

(2.1) Alcohol tolerance test

The isolated 230 strains were cultured in a medium containing ethanol at different concentrations to confirm resistance to alcohol.

Specifically, ethanol was added to the MRS liquid medium to be 5%, 10%, 15%, and 20%, and the final 10 ml was sterilized with a membrane filter and used as a medium. Each of the 230 strains activated in the MRS liquid medium ^{was added in an amount of 10 7} CFU/ml, followed by static culture at 37°C. The degree of growth of the strain was confirmed by measuring the absorbance at _{OD 600 nm.} Measurements were taken before cultivation and 4 hours after cultivation. Table 1 shows the ethanol resistance of the two selected strains.

number Strain Alcohol concentration (v/v %) 5 10 15 20 One LMT1-73 +++ +++ ++ ++ 2 LMT2-75 +++ +++ +++ +++

In Table 1, +, ++, and +++ indicate that 80% or more, 90% or more, and 100% cells were grown when compared to the control group cultured by adding ethanol and the same amount of D.W, respectively.

As shown in Table 1, Lactobacillus fermentum LMT2-75 and Lactobacillus brevis LMT1-73 showed high growth even in MRS liquid medium containing 20% alcohol.

(2.2) Alcohol tolerance comparison

Alcohol resistance of the two strains identified as having high alcohol resistance in (2.1) was compared with other strains of the same species and alcohol resistance. As comparative strains, type strains Lactobacillus brevis KCTC3498 ^T and Lactobacillus fermentum KCTC3112 ^T were used, and these can be purchased commercially from KCTC.

The alcohol tolerance experiment was performed in the same manner as described in (2.1), except that a comparative strain as shown in Table 2 was used. Table 2 shows the results of the alcohol tolerance test between strains.

number Bell Strain Alcohol concentration (v/v %) 5 10 15 20 One L.brevis LMT1-73 +++ +++ ++ ++ 2 KCTC3498 ^T ++ + + ++ 3 L.fermentum LMT2-75 +++ +++ +++ +++ 4 KCTC3112 ^T ++ ++ + +

In Table 2, +, ++, and +++ are as shown in Table 1, and-indicates that the cells do not grow. As shown in Table 2, the two selected strains had excellent alcohol resistance compared to the comparative strains of the same species.

(3) Confirmation of in vitro alcohol and acetaldehyde resolution

(3.1) Confirmation of ethanol resolution

The two isolated strains LMT1-73 and LMT2-75 were cultured in MRS liquid medium at 37° C. for 18 hours to obtain a culture with a cell concentration of 10 ⁹ CFU/3 ml, and 15 containing 3 ml of this culture. ml test tubes were prepared. After ethanol was added to the tube to a final concentration of 10 (v/v)%, the tube lid was closed to prevent air from entering and allowed to stand at 37° C. for 4 hours.

Next, the culture was centrifuged at 3000 rpm to remove cells, the supernatant was taken, and the alcohol concentration in the supernatant was measured. Specifically, 100 ul of the supernatant and 6 mM NAD+ were mixed in 1.0M Tris/HCl (pH8.8) buffer to make a final 3 ml, and then allowed to stand at room temperature for 5 minutes. After measuring the absorbance at OD _{340 nm (A 1} ) by adding alcohol dehydrogenase (ADH) (Sigma) to measure the absorbance at 340 nm (A ₂ ) to confirm the consumption of NADH, and this Converted to consumption. Ethanol consumption was calculated according to the following calculation formula. The results are shown in Table 3.

Ethanol consumption (%) = △ A = ( A 2 -A 1) sample - (A ₂ -A ₁₎ Blanc

Ethanol residual amount (g/L) = (0.7256/6.3)*△A

Ethanol consumption (%) = (Ethanol residual amount sample / ethanol residual amount control)*100

number Strain Ethanol consumption (%) One LMT1-73 97.9 2 LMT2-75 98.6

As shown in Table 3, the two selected strains directly degraded about 98% ethanol. This indicates that these strains can be used to break down alcohol in the intestine or in the body. This indicates that the strain can relieve hangovers or protect organs such as the liver from alcohol.

(3.2) Confirmation of acetaldehyde resolution

The two isolated strains LMT1-73 and LMT2-75 were cultured in MRS liquid medium at 37° C. for 18 hours to obtain a culture with a cell concentration of 10 ⁹ CFU/2.7 ml, and 2.7 ml of this culture was contained. ml test tubes were prepared. After acetaldehyde was added to the tube to a final concentration of 0.01 M, the tube lid was closed so that air did not enter and allowed to stand at 37° C. for 4 hours.

After the reaction was completed, the culture was filtered through a 0.2 um membrane filter, and an acetaldehyde detection kit (r-biopharm) was used for the filtrate sample from which cells were removed according to the manufacturer's instructions. The amount of residual acetaldehyde was measured. Acetaldehyde consumption was calculated according to the following calculation formula. The results are shown in Table 4.

Acetaldehyde consumption _{(%) = (A 2 -A} 1) sample - (A ₂ -A ₁₎ Blanc

Acetaldehyde residual amount (g/L) = (0.7158/6.3)*△A

Acetaldehyde consumption (%) = (Acetaldehyde residual amount sample / Acetaldehyde residual amount control)*100

number Strain Acetaldehyde consumption (%) One LMT1-73 98.2 2 LMT2-75 95.0

As shown in Table 4, the two strains selected consumed 98.2% and 95.0% of acetaldehyde, respectively. This indicates that these strains can reduce the harmful symptoms that occur to the body after alcohol consumption by reducing the concentration of acetaldehyde, which is known to be the main cause of hangovers produced by alcohol oxidation.

(4) Identification of in vivo alcohol and acetaldehyde resolution

The selected strain was orally administered with alcohol to rats, and the effect on blood alcohol and acetaldehyde concentrations was confirmed. Rats were used as male SD (Sprague Dawley)-based rats (Orient Bio Co., Ltd., Seongnam, Korea) of 5 to 6 weeks of age (140 g to 160 g in weight).

The rats were freely ingested with solid feed and tap water, and after pre-breeding for 1 week, the rats were divided into a normal group, a control group, and an experimental group (3 animals per group). The normal group was administered with PBS to rats, the control group was administered with 40% EtOH, and experimental groups 1 and 2 were each (Lactobacillus brevis LMT1-73 1x10 ⁸ CFU/rat/day + 40% EtOH) and (Lactobacillus Fermentum LMT2-75) 1x10 ⁸ CFU/rat/day + 40% EtOH) was administered.

After preliminary rearing and fasting for 18 hours, the experimental groups were ^{administered 1×10 8} CFU/day of live bacteria per rat after being turbid in a buffer solution (Phosphate Buffered Saline:PBS, biosesang).

After 30 minutes, 1.5 ml of ethanol at a concentration of 40 (v/v)% was orally administered to the control group and the experimental group, and 1.5 ml of blood was collected by cardiac bleeding after 5 hours. The collected blood was centrifuged at room temperature and 13,000 rpm for 10 minutes to separate plasma. The concentration of ethanol was measured in plasma using an ethanol measurement kit (Roche, USA). The results are shown in Table 5.

Control Experimental group 1 Experimental group 2 Alcohol residual amount (%) 100 24 79.3

As shown in Table 5, it was confirmed that the amount of alcohol detected in plasma was low in the two experimental groups compared to the control group.

In addition, the concentration of acetaldehyde in plasma was measured using an acetaldehyde measurement kit (Roche, USA), which is an ethanol decomposition product. The results are shown in Table 6.

Control Experimental group 1 Experimental group 2 Acetaldehyde residual amount (%) 100 4.4 47.6

As shown in Table 6, acetaldehyde was detected low in plasma compared to the control group in both experimental groups. Therefore, it is shown that the strain can reduce the harmful symptoms that occur to the body after alcohol consumption by reducing the concentration of alcohol and acetaldehyde in the experimental animal model.

(5) Genetic analysis of the selected strains

(5.1) 16S rDNA analysis

PCR was performed using the primer sets of SEQ ID NO: 3 and SEQ ID NO: 4 and the genomes of the two isolated strains LMT1-73 and LMT2-75 as a template to obtain a 16S rDNA amplification product. The nucleotide sequence of the amplification product was confirmed through sequencing. As a result, the 16S rDNAs of LMT1-73 and LMT2-75 have nucleotide sequences of SEQ ID NOs: 1 and 2, respectively.

In addition, the nucleotide sequence of the 16S rDNA was analyzed using NCBI blast (https://www.ncbi.nlm.nih.gov/). As a result, the 16S rDNAs of LMT1-73 and LMT2-75 had 99.9% and 100.0% sequence identity with Lactobacillus brevis species and Lactobacillus fermentum species, respectively. In addition, as a result of phylogenetic tree analysis, LMT1-73 was the same as Lactobacillus brevis species, and LMT2-75 was the same as Lactobacillus fermentum species. As a result, the LMT1-73 and LMT2-75 strains were identified as new strains belonging to the new Lactobacillus brevis species and Lactobacillus fermentum species. These two strains were named Brevis ( Lactobacillus brevis ) LMT1-73 and Lactobacillus fermentum (Lactobacillus fermentum) LMT2-75, respectively. On December 5, 2017, it was deposited under the deposit numbers KCTC 13412BP and KCTC 13413BP.

(5.2) Identification of enzyme genes involved in alcohol and acetaldehyde degradation

LMT1-73 and LMT2-75 were cultured in MRS liquid medium at 37° C. for 18 hours, and the cells were recovered. Genomic DNA was obtained from the cells using a genomic DNA kit. Using the primer set shown in Table 7 as a primer and performing PCR using the genomic DNA as a template, ADH, ALDH and bifunctional acetaldehyde-CoA/alcohol dehydrogenase (bifunctional acetaldehyde-CoA/alcohol dehydrogenase: ADHE) The presence of the gene was confirmed.

As a result, ADH, ALDH and ADHE gene amplification products were obtained for LMT1-73 and LMT2-75. Sequencing was performed on these products, and the obtained sequences were compared with other sequences using NCBI blast. As a result, the ADH, ALDH and ADHE genes of LMT1-73 were identical to WP_011668736, WP_011668306, and WP_024855276, respectively, ADH, ALDH and ADHE genes were consistent with NZ_CP019030, CP002033.1 and NC_010610.1, respectively.

number Strain Target gene Primer (SEQ ID NO:) One LMT1-73 L. brevis ADH 5 6 2 L. brevis ALDH 7 8 3 L. brevis ADHE 9 10 4 LMT2-75 L. fermentum ADH 11 12 5 L. fermentum ALDH 13 14 6 L. fermentum ADHE 15 16

(6) Investigation of morphological and physiological characteristics of the selected strains

(6.1) Morphological characteristics analysis

The two selected species LMT1-73 and LMT2-75 were plated on MRS agar plate medium, cultured at 37°C, and the morphology of colonies formed was observed. Table 8 shows the morphological characteristics of LMT1-73 and LMT2-75.

LMT1-73 LMT2-75 shape circle circle size 2 mm 1 mm color cream color cream color Opacity opacity opacity bump Convex Convex surface lubricity lubricity Aerobic growth + + Anaerobic growth + +

(6.2) Sugar fermentation properties of the selected strains

Sugar fermentation properties were investigated using the API 50 CHL kit (Biomerieux, France) according to the supplier's experimental guidelines. Table 9 shows the sugar fermentation properties of LMT1-73 and LMT2-75.

LMT1-73 LMT2-75 Glycerol - - Erythritol - - D-Arabinose - - L-arabinose + + D-ribose + + D-Xylose + - L-Xylose - - D-Adonitol - - Metyro-β D-xylopyranoside - - D-galactose + + D-glucose + + D-Fructose + + D-mannose - + L-Sorbos - - L-Rhamnos - - Dulcitol - - Inositol - - Mannitol - - D-sorbitol - - Methyl αD-mannopyranoside - - Methyl αD-glucopyranoside + - N-acetylglucosamine + - Amigdalin - - Arbutin - - Esculin - - Salicin - - D-cellobiose - + D-maltose + + D-lactose - - D-Mellibiose + - D-Sacarose - - D-Trehalose - - Inulin - - D-Melegitos - - D-Raffinose - - ARMIDON - - Glycogen - - Xylitol - - Gentibios - - D-Turanos - - D-lyxose - - D-Tagatos - - D-fucos - - L-fucos - - D-arabitol - - L-arabitol - - Potassium glunate + - Potassium 2-ketoglunate - - Potassium 5-ketoglunate + -

(7) Intestinal stability of the selected strain

(7.1) Acid resistance Research

In order for the selected strain to exert its efficacy as a probiotic in the intestine, it must pass through a low pH stomach after ingestion.

The two selected strains were inoculated in a sterilized MRS liquid medium and cultured at 37° C. for 16 hours. Then, the selected strain was inoculated in an MRS liquid medium sterilized by adjusting the pH to 2.5 with HCl in an amount of 1%, and cultured at 37° C. for 2 hours. Samples immediately after strain inoculation and after 2 hours incubation were collected, diluted in MRS liquid medium, plated on MRS plate medium, incubated at 37° C. for 24 hours, and then the number of colonies on the plate medium was counted to measure the number of cells. As a control, the number of cells was counted in the same manner in the MRS (pH 6.8) liquid medium in which the pH was not adjusted. As comparative strains, type strains Lactobacillus brevis KCTC3498 ^T and Lactobacillus fermentum KCTC3112 ^T were used, and these can be purchased commercially from KCTC. Table 10 shows the acid resistance measurement results.

Cells (CFU/ml) LMT1-73 LMT2-75 KCTC3498 ^T KCTC3112 ^T MRS (pH 6.8) 6.7x10 ⁹ 8.6x10 ⁹ 5.5x10 ⁹ 3.9x10 ⁹ MRS (pH 2.5) 9.0x10 ⁸ 3.6x10 ⁹ 5.8x10 ⁸ 9.1x10 ⁶

As shown in Table 10, the selected strains were superior to the comparative strains in resistance to acidity at pH 2.5. Specifically, the selected LMT1-73 and LMT2-75 survived 13.4% and 41.2%, whereas the comparative strain Lactobacillus brevis KCTC3498 ^T And Lactobacillus fermentum KCTC3112 ^T survived only 10.6% and 0.2%, respectively. The characteristics of these selected strains indicate that the proper number of cells is maintained at a pH lower than pH 3, which is close to the physiological pH of the stomach, so that the number of viable cells can be stably maintained even at a low pH due to secretion of gastric acid, and the intestinal reach rate will be very high when ingested. .

(7.2) Bile resistance Research

The two selected strains were cultured in a medium containing bile acids, and the effect of bile acids on the growth of the two strains was confirmed.

Specifically, the selected strain was inoculated in a sterilized MRS liquid medium and cultured at 37°C for 24 hours, and considering that the concentration of bile salts in the intestine was around 0.1%, 0.3% bile salts (Sigma, USA) The strain was inoculated in an amount of 1% in the MRS liquid medium containing this, and incubated at 37° C. for 2 hours, respectively. Samples immediately after strain inoculation and after 2 hours incubation were collected, diluted in MRS liquid medium, plated on MRS plate medium, cultured at 37° C. for 24 hours, and then the number of colonies on the plate medium was counted to measure the number of strain cells. As a control, culture was performed in the same manner in MRS liquid medium not containing 0.3% bile hydrochloric acid, and the number of strain cells was counted. As comparative strains, type strains Lactobacillus brevis KCTC3498 ^T and Lactobacillus fermentum KCTC3112 ^T were used, which are commercially available from KCTC. Table 11 shows the results of measuring the bile salts.

Cells (CFU/ml) LMT1-73 LMT2-75 KCTC3498 ^T KCTC3112 ^T MRS 6.7x10 ⁹ 8.6x10 ⁹ 5.5x10 ⁹ 3.9x10 ⁹ MRS (0.3% bile salt) 8.7x10 ⁸ 1.3x10 ⁸ 6.1x10 ⁸ 3.9x10 ⁷

As shown in Table 11, the selected strain maintained an appropriate number of cells even at 0.3% higher than 0.1% similar to the actual concentration in the intestine. Specifically, the selected strains LMT1-73 and LMT2-75 survived 12.9% and 1.5%, respectively, whereas the comparative strains Lactobacillus brevis KCTC3498 ^T and Lactobacillus fermentum KCTC3112 ^T survived 11.7% and 1.0%, respectively. Therefore, this indicates that the selected LMT1-73 and LMT2-75 can survive sufficiently in the intestine of humans or animals, and the intestinal reach rate will be very high.

* (7.3) Investigation of fixation in the intestine

The selected strain was co-cultured with Caco-2, an intestinal epithelial cell, and the cells of the selected strain bound to the epithelial cells were counted, and the degree to which the selected strain adhered to the intestine was measured. Caco-2 cells were human epithelial colorectal adenocarcinoma cells, and were purchased from the Korea Cell Line Bank (KCLB 30037.1).

Specifically, Caco-2 cells were added to a well of a culture 96-well plate by making ^{7x10 4} _{cells/100 µl using a cell culture medium, and cultured in 5% CO 2} and 37°C conditions to form a cell monolayer. . The culture plates and media used were DMEM (Dulbecco's modified Eagle's medium) (Gibco, USA) containing 96-well cell culture plates (Corning, USA) and 10% fetal bovine serum (FBS) (Gibco, USA). ) It was a medium.

Next, LMT1-73 and LMT2-75 cultured in MRS liquid medium were washed with Phosphate Buffered Saline (PBS), and then suspended in DMEM medium to which antibiotics were not added, and the Caco-2 cell monolayer The strain was added so that the amount of the strain was 1x10 ⁷ _{CFU, and incubated for 2 hours under 5% CO 2} and 37°C conditions. To remove the cells that did not adhere to Caco-2 cells, wash 5 times with PBS, remove the adhered cells with 100 µl of 0.1% Triton x-100, and spread them on MRS solid medium, and then at 37°C for 24 hours. After culturing during, the number of colonies on the plate medium was counted to investigate the intestinal fixation of the selected strains.

1 is a diagram showing the extent to which the selected strain adheres to intestinal epithelial cells. As shown in FIG. 1, the selected LMT1-73 and LMT2-75 were attached by 73.7% and 72.9%, respectively, whereas the comparative strains Lactobacillus brevis KCTC3498 ^T and Lactobacillus fermentum KCTC3112 ^T were 68.0%, and 58.3%, respectively. Is attached.

Therefore, the selected strains Lactobacillus brevis LMT1-73 and Lactobacillus fermentum LMT2-75 showed superior fixation power to Caco-2 cells, which are intestinal epithelial cells, than the comparative strains.

<110> Medytox Inc. <120> Microorganism capable of degrading ethanol and acetaldehyde, composition and kit comprising the same <130> PN121795KR <160> 16 <170> KopatentIn 2.0 <210> 1 <211> 1369 <212> DNA <213> Lactobacillus brevis LMT1-73 <400> 1 ttgcactgat ttcaacaatg aagcgagtgg cgaactggtg agtaacacgt gggaaatctg 60 cccagaagca ggggataaca cttggaaaca ggtgctaata ccgtataaca acaaaatccg 120 catggatttt gtttgaaagg tggcttcggc tatcacttct ggatgatccc gcggcgtatt 180 agttagttgg tgaggtaaag gcccaccaag acgatgatac gtagccaacc tgagagggta 240 atcggccaca ttgggactga gacacggccc aaactcctac gggaggcagc agtagggaat 300 cttccacaat ggacgaaagt ctgatggagc aatgccgcgt gagtgaagaa gggtttcggc 360 tcgtaaaact ctgttgttaa agaagaacac ctttgagagt aactgttcaa gggttgacgg 420 tatttaacca gaaagccacg gctaactacg tgccagcagc cgcggtaata cgtaggtggc 480 aagcgttgtc cggatttatt gggcgtaaag cgagcgcagg cggtttttta agtctgatgt 540 gaaagccttc ggcttaaccg gagaagtgca tcggaaactg ggagacttga gtgcagaaga 600 ggacagtgga actccatgtg tagcggtgga atgcgtagat atatggaaga acaccagtgg 660 cgaaggcggc tgtctagtct gtaactgacg ctgaggctcg aaagcatggg tagcgaacag 720 gattagatac cctggtagtc catgccgtaa acgatgagtg ctaagtgttg gagggtttcc 780 gcccttcagt gctgcagcta acgcattaag cactccgcct ggggagtacg accgcaaggt 840 tgaaactcaa aggaattgac gggggcccgc acaagcggtg gagcatgtgg tttaattcga 900 agctacgcga agaaccttac caggtcttga catcttctgc caatcttaga gataagacgt 960 tcccttcggg gacagaatga caggtggtgc atggttgtcg tcagctcgtg tcgtgagatg 1020 ttgggttaag tcccgcaacg agcgcaaccc ttattatcag ttgccagcat tcagttgggc 1080 actctggtga gactgccggt gacaaaccgg aggaaggtgg ggatgacgtc aaatcatcat 1140 gccccttatg acctgggcta cacacgtgct acaatggacg gtacaacgag tcgcgaagtc 1200 gtgaggctaa gctaatctct taaagccgtt ctcagttcgg attgtaggct gcaactcgcc 1260 tacatgaagt tggaatcgct agtaatcgcg gatcagcatg ccgcggtgaa tacgttcccg 1320 ggccttgtac acaccgcccg tcacaccatg agagtttgta acacccaaa 1369 <210> 2 <211> 1272 <212> DNA <213> Lactobacillus fermentum LMT2-75 <400> 2 aacagatgct aataccgcat aacagcgttg ttcgcatgaa caacgcttaa aagatggctt 60 ctcgctatca cttctggatg gacctgcggt gcattagctt gttggtgggg taacggccta 120 ccaaggcgat gatgcatagc cgagttgaga gactgatcgg ccacaatggg actgagacac 180 ggcccatact cctacgggag gcagcagtag ggaatcttcc acaatgggcg caagcctgat 240 ggagcaacac cgcgtgagtg aagaagggtt tcggctcgta aagctctgtt gttaaagaag 300 aacacgtatg agagtaactg ttcatacgtt gacggtattt aaccagaaag tcacggctaa 360 ctacgtgcca gcagccgcgg taatacgtag gtggcaagcg ttatccggat ttattgggcg 420 taaagagagt gcaggcggtt ttctaagtct gatgtgaaag ccttcggctt aaccggagaa 480 gtgcatcgga aactggataa cttgagtgca gaagagggta gtggaactcc atgtgtagcg 540 gtggaatgcg tagatatatg gaagaacacc agtggcgaag gcggctacct ggtctgcaac 600 tgacgctgag actcgaaagc atgggtagcg aacaggatta gataccctgg tagtccatgc 660 cgtaaacgat gagtgctagg tgttggaggg tttccgccct tcagtgccgg agctaacgca 720 ttaagcactc cgcctgggga gtacgaccgc aaggttgaaa ctcaaaggaa ttgacggggg 780 cccgcacaag cggtggagca tgtggtttaa ttcgaagcta cgcgaagaac cttaccaggt 840 cttgacatct tgcgccaacc ctagagatag ggcgtttcct tcgggaacgc aatgacaggt 900 ggtgcatggt cgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc 960 aacccttgtt actagttgcc agcattaagt tgggcactct agtgagactg ccggtgacaa 1020 accggaggaa ggtggggacg acgtcagatc atcatgcccc ttatgacctg ggctacacac 1080 gtgctacaat ggacggtaca acgagtcgcg aactcgcgag ggcaagcaaa tctcttaaaa 1140 ccgttctcag ttcggactgc aggctgcaac tcgcctgcac gaagtcggaa tcgctagtaa 1200 tcgcggatca gcatgccgcg gtgaatacgt tcccgggcct tgtacacacc gcccgtcaca 1260 ccatgagagt tt 1272 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 agagtttgat cmtggctcag 20 <210> 4 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 ggttaccttg ttacgactt 19 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 cttatgcagg gatttgtggg actg 24 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 cttgttccag actaacttcg tggc 24 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 ggtgctaaga acattacccg ttgg 24 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 ccaagtccgt aatgagaacc cttc 24 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 gatcaatacc ccatctgcta tcgg 24 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 gtgggtactt cacgtgagtc ttag 24 <210> 11 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 gaaaatctgt gttcccactc gctg 24 <210> 12 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 ggtggtcctg gtaatttaag ctgg 24 <210> 13 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 gggcaagtat gtacttcctc caag 24 <210> 14 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 cttaattcac gaccgtaacc ggag 24 <210> 15 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 ctactctccg gtgaaaagct gag 23 <210> 16 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 cttgatccct ggcatatcct tgag 24

Claims (10)

Lactobacillus brevis LMT 1-73 (Accession No. KCTC-13412BP), a microorganism having the ability to degrade ethanol and acetaldehyde. A composition comprising the microorganism and diluent or carrier according to claim 1, for the prevention of alcoholic liver disease or for the removal of hangover. The composition of claim 2, for use in removing one or more of ethanol and acetaldehyde in a sample. 4. The composition of claim 3, wherein the sample is a intestinal fluid, including blood, duodenum, small intestine, or intestinal fluid, or blood. The composition according to claim 2, which is a food. The composition according to claim 5, wherein the food is a dairy product or a food additive. 7. The composition of claim 6, wherein the dairy product is fermented milk, butter, cheese, or powdered milk. The composition of claim 2, wherein the composition is in the form of a liquid, a powder, a granule, a tablet or a capsule. A kit for use in removing one or more of ethanol and acetaldehyde in a sample comprising the microorganism and diluent or carrier of claim 1. delete

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