MICROORGANISM HAVING ABILITY TO DEGRADE ETHANOL AND ACETALDEHYDE, AND COMPOSITION AND KIT EACH INCLUDING THE SAME

Abstract

Provided are a microorganism having an ability to degrade ethanol and acetaldehyde, and a composition and a kit each including the microorganism.

Claims

1. A microorganism having ethanol and acetaldehyde degradation ability, the microorganism being selected from the group consisting of Lactobacillus brevis LMT1-73 (Accession No. KCTC-13412BP) and Lactobacillus fermentum LMT2-75 (Accession No. KCTC-13413BP).

2. A composition comprising one or more of the microorganism of claim 1 and a diluent or carrier.

3. The composition of claim 2, wherein the composition is suitable for use in removing one or more of ethanol and acetaldehyde from a sample.

4. The composition of claim 3, wherein the sample is blood or is an intestinal fluid including gastric juice, duodenal juice, small intestinal fluid, or large intestinal fluid.

5. The composition of claim 2, wherein the composition is a food.

6. The composition of claim 5, wherein the food is a dairy product, a food for preventing alcoholic liver diseases or relieving hangovers, or a food additive.

7. The composition of claim 6, wherein the dairy product is fermented milk, butter, cheese, or milk powder.

8. The composition of claim 2, wherein the composition is in the form of a liquid, a powder, granules, a tablet, or a capsule.

9. A kit comprising the microorganism of claim 1 and a diluent or carrier, wherein the kit is used to remove one or more of ethanol and acetaldehyde from a sample.

10. A method for removing one or more of ethanol and acetaldehyde from a sample of a subject in need thereof, comprising administering the composition of claim 2 to the subject.

11. The method of claim 10, wherein the sample is blood or is an intestinal fluid including gastric juice, duodenal juice, small intestinal fluid, or large intestinal fluid.

12. The method of claim 10, wherein the composition is a food.

13. The method of claim 12, wherein the food is a dairy product, a food for preventing alcoholic liver diseases or relieving hangovers, or a food additive.

14. The method of claim 13, wherein the dairy product is fermented milk, butter, cheese, or milk powder.

15. The method of claim 10, wherein the composition is in the form of a liquid, a powder, granules, a tablet, or a capsule.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0026] These and/or other aspects will become apparent and more readily appreciated from the following description of the embodiments, taken in conjunction with the accompanying drawings in which:

[0027] FIG. 1 shows mucoadhesion of selected strains to intestinal epithelial cells.

BEST MODE FOR CARRYING OUT THE INVENTION

[0028] Hereinafter, the present disclosure will be described in more detail with reference to Examples. However, these Examples are for illustrative purposes only, and the scope of the present disclosure is not intended to be limited by these Examples.

EXAMPLE 1: ISOLATION AND IDENTIFICATION OF MICROORGANISM HAVING ABILITY TO DEGRADE ALCOHOL AND ACETALDEHYDE

[0029] In this example, microorganisms having an excellent ability to degrade alcohol and acetaldehyde were isolated from kimchi, and identified. As a result, it was found that the microorganisms had excellent ability to degrade alcohol and acetaldehyde in blood, acid resistance, bile acid resistance, and intestinal mucoadhesion to show excellent stability in the intestine.

1. Isolation and Identification of Strain

(1) Isolation of Strain

[0030] 20 g of kimchi directly prepared at home and stored at 4 C. was taken aseptically, and diluted in 180 ml of 0.85% NaCl solution, and homogenized using a stomacher for 5 minutes. The homogenized kimchi was serially diluted in a tube containing 9 ml of sterile 0.85% NaCl solution to prepare kimchi samples. Each of the kimchi samples were spread on MRS (Difco, USA) agar plate and incubated at 37 C. for 2 days to 3 days. The appeared colonies were classified according to their morphology and color, and purely isolated. Each of the isolated colonies was cultured in MRS liquid medium at pH 6.8 and 37 C. for 24 hours, and 230 kinds of colonies, of which culture medium had pH of 4.5 or less, were selected. The selected strains were tested for alcohol resistance, and alcohol and acetaldehyde degradation ability. Finally, two strains of Lactobacillus brevis LMT1-73 (Accession No. KCTC-13412BP) and Lactobacillus fermentum LMT2-75 (Accession No. KCTC-13413BP) having excellent alcohol and acetaldehyde degradation ability and intestinal stability were selected.

(2) In Vitro Test of Alcohol Resistance

(2.1) Test of Alcohol Resistance

[0031] 230 kinds of the isolated strains were cultured in media containing different concentrations of ethanol to examine alcohol resistance.

[0032] In detail, ethanol was added to MRS liquid medium at a concentration of 5%, 10%, 15%, or 20%, and final 10 ml of the media was used after being sterilized using a membrane filter. Each of 230 kinds of the activated strains was added to the MRS liquid medium at a density of 10.sup.7 CFU/ml, followed by stationary culture at 37 C. Growth of the strains was examined by measuring absorbance at 0D.sub.600 nm. The measurement was performed before culture and at 4 hours after culture. Table 1 shows ethanol resistance of the selected two strains.

TABLE-US-00001 TABLE 1 Alcohol concentration (v/v %) No. Strain 5 10 15 20 1 LMT1-73 +++ +++ ++ ++ 2 LMT2-75 +++ +++ +++ +++ In Table 1, +, ++, and +++ represent 80% or more, 90% or more, and 100% cell growth, as compared with control groups which were cultured by adding ethanol and D.W in equal amounts, respectively.

[0033] As shown in Table 1, Lactobacillus fermentum LMT2-75 and Lactobacillus brevis LMT1-73 showed high growth even in MRS liquid media containing 20% alcohol.

(2.2) Comparison of Alcohol Resistance

[0034] Alcohol resistance of the two strains which were confirmed to have high alcohol resistance in (2.1) was compared with that of other species of strains. As comparative strains, Lactobacillus brevis KCTC3498T and Lactobacillus fermentum KCTC3112T which are type strains were used, and they are commercially available from KCTC.

[0035] The alcohol resistance test was performed in the same manner as in (2.1), except that comparative strains as shown in the following Table 2 were used. Table 2 shows results of comparing the alcohol resistance between strains.

TABLE-US-00002 TABLE 2 Alcohol concentration (v/v %) No. Species Strain 5 10 15 20 1 L. brevis LMT1-73 +++ +++ ++ ++ 2 KCTC3498.sup.T ++ + + ++ 3 L. fermentum LMT2-75 +++ +++ +++ +++ 4 KCTC3112.sup.T ++ ++ + + In Table 2, +, ++, and +++ are the same as in Table 1, and represents no cell growth. As shown in Table 2, the two selected strains showed excellent alcohol resistance, as compared with the comparative strains of the same species.

(3) In Vitro Test of Alcohol and Acetaldehyde Degradation Ability

(3.1) Test of Ethanol Degradation Ability

[0036] Each of the two isolated strains, LMT1-73 and LMT2-75, was cultured in MRS liquid medium at 37 C. for 18 hours to obtain a culture at a cell density of 10.sup.9 CFU/3 ml. A 15 ml test tube containing 3 ml of the culture was prepared. Ethanol was added to the tube at a final concentration of 10 (v/v)%, and the tube was air-tightened with a cap, and left at 37 C. for 4 hours.

[0037] Next, the culture was centrifuged at 3000 rpm to remove cells, and a supernatant was taken. An alcohol concentration in the supernatant was measured. In detail, 100 l of the supernatant and 6 mM NAD+ were mixed in 1.0 M Tris/HCl (pH 8.8) buffer at a final volume of 3 ml, and left at room temperature for 5 minutes. Absorbance at OD.sub.340 nm was measured (A.sub.1), and alcohol dehydrogenase (ADH) (Sigma) was added and then absorbance at OD.sub.340 nm was measured (A.sub.2). The final concentration of ethanol was obtained according to the following Equation (ethanol detection kit, Cat. No. 10 176 290 035, r-biopharm/Roche). The results are shown in Table 3.

[0038] Difference value of the absorbance, A=(A.sub.2A.sub.1)sample-(A.sub.2-A.sub.1) blank

[0039] Final concentration of ethanol (g/L)=(0.7256/6.3)*A

Ethanol consumption (%)=(Ethanol retention in sample/Ethanol retention in control)*100

TABLE-US-00003 TABLE 3 Ethanol consumption No. Strain (%) 1 LMT1-73 97.9 2 LMT2-75 98.6

[0040] As shown in Table 3, the two selected strains directly degraded about 98% of ethanol, indicating that these strains may be used in degrading alcohol in the intestine or body, and may relieve hangovers or may protect organs such as liver from alcohol.

(3.2) Test of Acetaldehyde Degradation Ability

[0041] Each of the two isolated strains, LMT1-73 and LMT2-75, was cultured in MRS liquid medium at 37 C. for 18 hours to obtain a culture at a cell density of 10.sup.9 CFU/2.7 ml. A 15 ml test tube containing 2.7 ml of the culture was prepared. Acetaldehyde was added to the tube at a final concentration of 0.01 M, and the tube was air-tightened with a cap, and left at 37 C. for 4 hours.

[0042] After completion of the reaction, the culture was filtered using a 0.2 m membrane filter, and a cell-removed filtrate sample was used to measure acetaldehyde retention in the sample using an acetaldehyde detection kit (r-biopharm) in accordance with the manufacturer's instructions. The final concentration of acetaldehyde was obtained according to the following Equation (acetaldehyde detection kit, Cat. No. 10 668 613 035, r-biopharm/Roche). The results are shown in Table 4.

[0043] Difference value of the absorbance, A=(A.sub.2-A.sub.1)sample-(A.sub.2-A.sub.1)blank

[0044] Final concentration of acetaldehyde (g/L)=(0.7158/6.3)*A

Acetaldehyde consumption (%)=(Acetaldehyde retention in sample/Acetaldehyde retention in control)*100

TABLE-US-00004 TABLE 4 Acetaldehyde consumption No. Strain (%) 1 LMT1-73 98.2 2 LMT2-75 95.0

[0045] As shown in Table 4, the two selected strains consumed 98.2% and 95.0% of acetaldehyde, respectively, indicating that these strains may decrease the concentration of acetaldehyde which is produced by alcohol oxidation and known as a major cause of hangovers, thereby reducing harmful symptoms that may occur in the body after alcohol ingestion.

(4) In Vivo Test of Alcohol and Acetaldehyde Degradation Ability

[0046] Rats were orally administered with alcohol and the selected strain to examine its effect on blood concentrations of alcohol and acetaldehyde. The rats were 5 to 6-week old male SD(Sprague Dawley) rats (weighing 140 g to 160 g) (Orient Bio, Inc., Seongnam, Korea).

[0047] The rats were preliminary raised with free access to solid feed and tap water for 1 week, and divided into a normal group, a control group, and an experimental group (three rats per group). The normal group indicates rats which were administered with PBS, the control group indicates rats which were administered with 40% EtOH, and Experimental group 1 and Experimental group 2 indicate rats which were administered with (Lactobacillus brevis LMT1-73 110.sup.8 CFU/rat/day+40% EtOH) and (Lactobacillus fermentum LMT2-75 110.sup.8 CFU/rat/day+40% EtOH), respectively.

[0048] After being preliminary raised and fasted for 18 hours, the experimental groups were administered once with the live strain of 110.sup.8CFU/day per rat suspended in phosphate buffered saline (PBS, biosesang).

[0049] 30 minutes later, the control group and the experimental groups were orally administered with 1.5 ml of 40 (v/v)% ethanol, and 5 hours later, 1.5 ml of blood was collected by cardiac blood collection. The collected blood was centrifuged at room temperature and 13,000 rpm for 10 minutes to separate blood plasma. Ethanol concentrations in the blood plasma were measured using an ethanol assay kit (Roche, USA). The results are shown in Table 5.

TABLE-US-00005 TABLE 5 Control Experimental Experimental group group 1 group 2 Alcohol retention (%) 100 24 79.3

[0050] As shown in Table 5, the amounts of alcohol detected in the blood plasma of the two experimental groups were lower than that of the control group.

[0051] Further, acetaldehyde concentrations in the blood plasma were measured using an assay kit of acetaldehyde which is an ethanol degradation product (Roche, USA). The results are shown in Table 6.

TABLE-US-00006 TABLE 6 Control Experimental Experimental group group 1 group 2 Acetaldehyde retention (%) 100 4.4 47.6

[0052] As shown in Table 6, the amounts of acetaldehyde detected in the blood plasma of the two experimental groups were lower than that of the control group. Accordingly, it was confirmed that the strains may reduce alcohol and acetaldehyde concentrations in experimental animal models, thereby reducing harmful symptoms that may occur in the body after alcohol ingestion.

(5) Genetic Analysis of Selected Strains

(5.1) 16S rDNA Analysis

[0053] PCR was performed using a primer set of SEQ ID NO: 3 and SEQ ID NO: 4, and genomes of the two isolated strains, LMT1-73 and LMT2-75 as a template to obtain 16S rDNA amplification products, respectively. Nucleotide sequences of the amplification products were confirmed by sequencing. As a result, 16S rDNAs of LMT1-73 and LMT2-75 have nucleotide sequences of SEQ ID NOS: 1 and 2, respectively.

[0054] Further, the nucleotide sequences of 16S rDNAs were analyzed using NCBI blast (http://www.ncbi.nlm.nih.gov/). As a result, 16S rDNAs of LMT1-73 and LMT2-75 had sequence homology of 99.9% and 100.0% to the species Lactobacillus brevis and Lactobacillus fermentum, respectively. Results of phylogenetic analysis also showed that LMT1-73 corresponds to the species Lactobacillus brevis, and LMT2-75 corresponds to the species Lactobacillus fermentum. Consequently, LMT1-73 and LMT2-75 strains were found to be novel strains belonging to the species of Lactobacillus brevis and Lactobacillus fermentum. These two strains were designated as Lactobacillus brevis LMT1-73 and Lactobacillus fermentum LMT2-75, respectively and deposited at the Korean Collection for Type Cultures (KCTC) of the Korea Research Institute of Bioscience and Biotechnology under accession number KCTC 13412BP and KCTC 13413BP on Dec. 5, 2017, respectively.

(5.2) Identification of Genes of Enzymes Associated with Alcohol and Acetaldehyde Degradation

[0055] LMT1-73 and LMT2-75 were cultured in MRS liquid medium at 37 C. for 18 hours, respectively to recover cells. Genomic DNAs were obtained from the cells using a genomic DNA kit. PCR was performed using a primer set shown in Table 7 as primers and the genomic DNA as a template to confirm the presence of ADH, ALDH, and bifunctional acetaldehyde-CoA/alcohol dehydrogenase (ADHE) genes.

[0056] As a result, amplification products of ADH, ALDH, and ADHE genes were obtained from LMT1-73 and LMT2-75. These products were subjected to sequencing, and the obtained sequences were compared with other sequences using NCBI blast. As a result, ADH, ALDH, and ADHE genes of LMT1-73 corresponded to WP_011668736, WP_011668306, and WP_024855276, and ADH, ALDH, and ADHE genes of LMT2-75 corresponded to NZ_CP019030, CP002033.1 and NC_010610.1, respectively.

TABLE-US-00007 TABLE 7 Primer No. Strain Target gene (SEQ ID NO.) 1 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) Morphological and Physiological Characterization of Selected Strains

(6.1) Morphological Characterization

[0057] The two selected LMT1-73 and LMT2-75 strains were spread on an MRS agar plate, respectively and cultured at 37 C. Morphologies of the colonies were examined. Table 8 shows morphological characteristics of LMT1-73 and LMT2-75.

TABLE-US-00008 TABLE 8 LMT1-73 LMT2-75 Shape Circular Circular Size 2 mm 1 mm Color Cream color Cream color Opacity Opaque Opaque Elevation Convex Convex Surface Smooth Smooth Aerobic growth + + Anaerobic growth + +

(6.2) Sugar Fermentation Characteristics of Selected Strains

[0058] Sugar fermentation characteristics were examined using an API 50 CHL kit (Biomerieux, France) in accordance with the supplier's experimental guidelines. Table 9 shows sugar fermentation characteristics of LMT1-73 and LMT2-75.

TABLE-US-00009 TABLE 9 LMT1-73 LMT2-75 Glycerol Erythritol D-arabinose L-arabinose + + D-ribose + + D-xylose + L-xylose D-adonitol Methyl--D-xylopyranoside D-galactose + + D-glucose + + D-fructose + + D-mannose + L-sorbose L-rhamnose Dulcitol Inositol Mannitol D-sorbitol Methyl D-mannopyranoside Methyl D-glucosamine + N-Acetyl Glucosamine + Amygdaline Arbutin Esculin Salicin D-Cellobiose + D-maltose + + D-lactose D-melibiose + D-saccharose D-trehalose Inulin D-melezitose D-raffinose Amidon Glycogen Xylitol Gentiobiose D-turanose D-lyxose D-tagatose D-fucose L-fucose D-arabitol L-arabitol Potassium gluconate + Potassium 2-ketogluconate Potassium 5-ketogluconate +

(7) Intestinal Stability of Selected Strains

(7.1) Examination of Acid Resistance

[0059] The selected strains are required to pass through the stomach of low pH after ingestion to exert their probiotic efficacy in the intestine.

[0060] Each of the two selected strains was seeded in a sterile MRS liquid medium, and then cultured at 37 C. for 16 hours. Next, the selected strain was seeded at an amount of 1% in sterile MRS liquid medium of which pH was adjusted to pH 2.5 with HCl, and cultured at 37 C. for 2 hours. Immediately and 2 hours after seeding, the sample was recovered and diluted with a MRS liquid medium, and spread on an MRS agar plate, and cultured at 37 C. for 24 hours. Then, the number of colonies on the agar plate was counted to measure the number of cells. As a control group, the experiment was performed in the same manner, except that MRS (pH 6.8) liquid medium was used without pH adjustment, and the number of cells was counted. As comparative strains, Lactobacillus brevis KCTC3498T and Lactobacillus fermentum KCTC3112T which are type strains were used, and these strains are commercially available from KCTC. Table 10 shows results of measuring acid resistance.

TABLE-US-00010 TABLE 10 Cell (CFU/ml) LMT1-73 LMT2-75 KCTC3498.sup.T KCTC3112.sup.T MRS (pH 6.8) 6.7 10.sup.9 8.6 10.sup.9 5.5 10.sup.9 3.9 10.sup.9 MRS (pH 2.5) 9.0 10.sup.8 3.6 10.sup.9 5.8 10.sup.8 9.1 10.sup.6

[0061] As shown in Table 10, the selected strains had excellent resistance against the acid of pH 2.5, as compared with the comparative strains. Specifically, 13.4% and 41.2% of the selected LMT1-73 and LMT2-75 were found to survive, respectively. In contrast, only 10.6% and 0.2% of Lactobacillus brevis KCTC3498T and Lactobacillus fermentum KCTC3112T which are comparative strains were found to survive, respectively. The selected strains showed characteristics of maintaining the proper number of cells at pH of lower than 3 close to the physiological pH of the stomach, indicating that the number of live cells may be stably maintained even at the low pH due to gastric juice and the cells may maintain a very high survival rate until they reach the intestine.

(7.2) Examination of Bile Acid Resistance

[0062] Each of the two selected strains was cultured in a medium containing bile acid to examine effects of bile acid on growth of the two strains.

[0063] In detail, each of the selected strains was seeded in a sterile MRS liquid medium, and then cultured at 37 C. for 24 hours. Each of the strains was seeded at an amount of 1% in MRS liquid medium containing 0.3% bile salts (Sigma, USA), considering that the concentration of bile salt in the intestine is about 0.1%, and cultured at 37 C. for 2 hours. Immediately and 2 hours after seeding, the sample was recovered and diluted with a MRS liquid medium, and spread on an MRS agar plate, and cultured at 37 C. for 24 hours. Then, the number of colonies on the agar plate was counted to measure the number of cells. As a control group, the experiment was performed in the same manner, except that MRS liquid medium without 0.3% bile salts was used, and the number of cells was counted. As comparative strains, Lactobacillus brevis KCTC3498 .sup.T and Lactobacillus fermentum KCTC3112T which are type strains were used, and these strains are commercially available from KCTC. Table 11 shows results of measuring bile acid resistance.

TABLE-US-00011 TABLE 11 Cell (CFU/ml) LMT1-73 LMT2-75 KCTC3498.sup.T KCTC3112.sup.T MRS 6.7 10.sup.9 8.6 10.sup.9 5.5 10.sup.9 3.9 10.sup.9 MRS (0.3% bile 8.7 10.sup.8 1.3 10.sup.8 6.1 10.sup.8 3.9 10.sup.7 salt)

[0064] As shown in Table 11, the selected strains maintained the proper number of cells even at 0.3% bile salts higher than 0.1% which is similar to the actual concentration in the intestine. Specifically, 12.9% and 1.5% of the selected LMT1-73 and LMT2-75 strains were found to survive, respectively. In contrast, only 11.7% and 1.0% of Lactobacillus brevis KCTC3498T and Lactobacillus fermentum KCTC3112T which are comparative strains were found to survive, respectively. Accordingly, the selected LMT1-73 and LMT2-75 strains may survive well in the intestine of a human or an animal, and may maintain a very high survival rate until they reach the intestine.

(7.3) Examination of Intestinal Mucoadhesion

[0065] Each of the selected strains was co-cultured with Caco-2 intestinal epithelial cells, and the cell number of the selected strain that adhered to the epithelial cells was counted to examine mucoadhesion of the selected strain. Caco-2 cell which is a human epithelial colorectal adenocarcinoma cell was purchased from the Korean cell line bank (KCLB 30037.1).

[0066] In detail, Caco-2 cells were diluted at a density of 710.sup.4 cells/100 l using a cell culture medium, and added to each well of a 96-well culture plate, and cultured under conditions of 5% CO.sub.2 and 37 C. to allow formation of a cell single layer. The used culture plate and medium were a 96-well cell culture plate (Corning, USA) and DMEM (Dulbecco's modified Eagle's medium) (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA).

[0067] Next, LMT1-73 and LMT2-75 each cultured in MRS liquid medium were washed with phosphate buffered saline (PBS), and suspended in a DMEM medium without antibiotics, and added at a density of 110.sup.7 CFU to the Caco-2 cell single layer, and cultured under conditions of 5% CO.sub.2 and 37 C. for 2 hours. To remove cells that did not adhere to Caco-2 cells, the cells were washed with PBS five times, and the adhered cells were detached using 100 l of 0.1% Triton x-100, spread on an MRS solid medium, and cultured at 37 C. for 24 hours. The number of colonies on the agar plate was counted to examine mucoadhesion of the selected strains.

[0068] FIG. 1 shows mucoadhesion of the selected strains to intestinal epithelial cells. As shown in FIGS. 1, 73.7% and 72.9% of the selected LMT1-73 and LMT2-75 strains were found to adhere, respectively. In contrast, 68.0%, and 58.3% of Lactobacillus brevis KCTC3498T and Lactobacillus fermentum KCTC3112T which are comparative strains were found to adhere, respectively.

[0069] Accordingly, the selected Lactobacillus brevis LMT1-73 and Lactobacillus fermentum LMT2-75 strains showed excellent mucoadhesion to Caco-2 cells which are intestinal epithelial cells, as compared with the comparative strains.

[0070] It should be understood that embodiments described herein should be considered in a descriptive sense only and not for purposes of limitation. Descriptions of features or aspects within each embodiment should typically be considered as available for other similar features or aspects in other embodiments.

[0071] While one or more embodiments have been described with reference to the figures, it will be understood by those of ordinary skill in the art that various changes in form and details may be made therein without departing from the spirit and scope of the disclosure as defined by the following claims.