KR102479732B1 - Limosilactobacillus reuteri MG5458 strain and composition for preventing, improving or treating alcoholic fatty liver comprising the same - Google Patents

Abstract

The present invention relates to a novel Limosilactobacillus reuteri MG5458 strain and a composition comprising the same for preventing, alleviating, or treating alcoholic fatty liver. The Limosilactobacillus reuteri MG5458 strain of the present invention is separated from food, thereby being safe, exhibits properties suitable for probiotics, has excellent lipid peroxidation inhibitory activity, alanine aminotransferase (ALT) inhibitory activity, or aspartate aminotransferase (AST) inhibitory activity, and thus can be widely used in various fields for the prevention, alleviation, and treatment of alcoholic fatty liver.

Description

Limosilactobacillus reuteri MG5458 strain and a composition for preventing, improving or treating alcoholic fatty liver comprising the same

The present invention relates to a novel Rimosyllactobacillus reuteri MG5458 strain and a composition for preventing, improving or treating alcoholic fatty liver comprising the same.

Alcoholic liver disease (ALD) is caused by chronic drinking and includes cirrhosis, fibrosis, hepatitis, and liver cancer. In particular, according to the World Health Organization (WHO), at least 3 million people die from alcoholic fatty liver disease (AFLD), a common liver disease in many countries. The 3-month short-term mortality of patients with severe alcoholic steatohepatitis is very high at 40-50%. Since there are few treatments available for AFLD, the discovery of new therapies useful for AFLD is needed.

Alcohol is metabolized primarily through oxidation, catalyzed by alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH). In this metabolism, cytochrome P450 2E1 (CYP2E1), which is co-activated with ADH, induces oxidative stress, leading to an imbalance between reactive oxygen species (ROS) production and elimination. However, ROS levels are reduced by the expression of antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX). Alcohol intake also inhibits peroxisome prolifera-tor-activated receptor α (PPARα) to delay fatty acid oxidation and activates SREBP1C (sterol regulatory element-binding transcription factor 1C), which can induce fatty liver, to increase adipogenesis. Therefore, a functional food that exhibits antioxidant activity and regulates lipid metabolism in hepatocytes can be a therapeutic agent for preventing AFLD.

Lactic acid bacteria (LAB), the most commonly used probiotics, are live microorganisms that provide health benefits by improving the balance of the host's gut microbiota. Recently, LABs, especially the genera Lactobacillus and Bifidobacterium , have been proven therapeutic based on scientific studies showing a variety of health benefits, including prevention of diarrhea, anti-allergic effects and immune system regulation.

Korea Patent Registration No. 10-2390927 Korea Patent Registration No. 10-2049700 Korea Patent Registration No. 10-1853603 Korea Patent Registration No. 10-1955276

Accordingly, the inventors of the present invention have completed the present invention by discovering a new strain of the genus Rimosyllactobacillus and confirming its preventive, ameliorative and therapeutic effects on alcoholic fatty liver.

Therefore, an object of the present invention, Rimosilactobacillus reuteri ( Limosilactobacillus reuteri ) MG5458 strain (Accession Number: KCTC14694BP); To provide; and a composition for preventing, improving or treating alcoholic fatty liver comprising the same.

In order to achieve the above object, the present invention provides a Limosilactobacillus reuteri MG5458 strain (Accession Number: KCTC14694BP).

In addition, the present invention provides a pharmaceutical composition for preventing or treating alcoholic fatty liver comprising the strain, its culture medium or a cell-free extract thereof.

In addition, the present invention provides a food composition for preventing or improving alcoholic fatty liver comprising the strain, its culture medium or a cell-free extract thereof.

In addition, the present invention provides a health functional food composition for preventing or improving alcoholic fatty liver comprising the strain, its culture medium or a cell-free extract thereof.

The Rimosyllactobacillus reuteri MG5458 strain according to the present invention is not only safe when isolated from food, but also exhibits properties suitable for probiotics, and has excellent lipid peroxidation inhibitory activity, alanine aminotransferase (ALT) inhibitory activity, or aspartate aminotransfer It has enzyme (aspartate aminotransferase, AST) inhibitory ability, and can be widely used in various fields for the prevention, improvement, and treatment of alcoholic fatty liver.

1 is a diagram showing the results of measuring the activity of aldehyde dehydrogenase (ALDH) according to LAB (lactic acid bacteria) treatment.
Figure 2 is a view showing the results of observing the morphology of HepG2 cells according to ethanol and MG5458 treatment.
Figure 3a is a diagram showing the results of measuring the total glutathione content according to ethanol and MG5458 treatment.
Figure 3b is a diagram showing the results of measuring lipid peroxidation according to ethanol and MG5458 treatment.
Figure 4a is a diagram showing the results of measuring the level of alanine aminotransferase according to ethanol and MG5458 treatment.
Figure 4b is a diagram showing the results of measuring the level of aspartate aminotransferase according to ethanol and MG5458 treatment.
Figure 5a is a diagram showing the results of analyzing the mRNA expression of alcoholic fatty liver-related factors according to ethanol and MG5458 treatment.
Figure 5b is a diagram showing the results of analyzing the mRNA expression of adipogenesis-related factors according to ethanol and MG5458 treatment.
Figure 5c is a diagram showing the results of analyzing the mRNA expression of lipid oxidation factors according to ethanol and MG5458 treatment.

Hereinafter, the present invention will be described in detail.

According to an aspect of the present invention, the present invention provides Limosilactobacillus reuteri MG5458 strain (Accession Number: KCTC14694BP).

In the present invention, the genus Limosyllactobacillus is a thermophilic heterofermentative lactic acid bacteria produced in 2020 by division in Lactobacillus. Most strains of the genus Rimosyllactobacillus exhibit the characteristic of producing exopolysaccharide from sucrose. There are currently 31 species or subspecies of the genus Rimosyllactobacillus, most of which have been isolated from the human or animal intestinal tract. Among the various strains, Rimosyllactobacillus reuteri is used as a model organism to evaluate the host adaptation of lactobacilli to the human and animal gut.

The Rimosyllactobacillus reuteri MG5458 is isolated from food and is characterized as a safe strain when ingested into the body. In addition, the Limosyllactobacillus reuteri MG5458 was patented at the Center for Biological Resources on September 1, 2021, and was given accession number KCTC14694BP.

In an embodiment of the present invention, the strain preferably includes the nucleotide sequence of SEQ ID NO: 1. The nucleotide sequence of SEQ ID NO: 1 is a 16S rRNA gene, and is a site used when identifying a strain.

In an embodiment of the present invention, the strain may have lipid peroxidation inhibitory activity, alanine aminotransferase (ALT) inhibitory activity or aspartate aminotransferase (AST) inhibitory activity.

In an embodiment of the present invention, as a result of calculating the lipid peroxidation inhibitory ability of the strain using a malondialdehyde (MDA) calibration curve, it was experimentally confirmed that the MDA level was significantly low. This means that the strain significantly inhibits lipid peroxidation.

In an embodiment of the present invention, the strain may have acid resistance or basic resistance.

In this Example, after creating a gastrointestinal environment using simulated gastric juice and intestinal juice, the cell viability of the strain was evaluated. As a result, it was confirmed that the strain had a high survival rate in the pH range of 3 to 8. In addition, it was confirmed that the strain had an adhesive ability of about 96% in the gastrointestinal tract environment. This means that the strain has excellent acid resistance and basic resistance.

The strain of the present invention may have sensitivity or resistance to various antibiotics, and may be appropriately prescribed or ingested according to its inherent characteristics.

In an embodiment of the present invention, the strain may have sensitivity to one or more antibiotics selected from the group consisting of ampicillin, gentamicin, kanamycin, streptomycin, tetracycline, chloramphenicol, erythromycin, and clindamycin.

According to another aspect of the present invention, the present invention provides a composition for preventing, improving or treating alcoholic fatty liver comprising a Limosyllactobacillus reuteri MG5458 strain, a culture thereof, or a cell-free extract thereof. The composition includes a pharmaceutical composition, food or health functional food composition.

In the present invention, the culture medium means that the strain is inoculated and cultured in a liquid medium, and the cell-free extract means the supernatant obtained by removing the strain from the liquid culture medium, which can be used interchangeably with the culture filtrate.

In the present invention, the term, prevention, may refer to any action that suppresses or delays the onset of alcoholic fatty liver by administering the composition for preventing or treating alcoholic fatty liver according to the present invention to a subject.

In the present invention, the term, treatment, may refer to any action that improves or benefits the symptoms of alcoholic fatty liver by administering the composition according to the present invention to a subject suspected of having alcoholic fatty liver disease.

In the present invention, the term improvement may refer to any activity that at least reduces a parameter related to a condition to be treated, for example, the severity of a symptom.

In the present invention, the term, subject, may refer to all animals, including humans, who have or are likely to develop alcoholic fatty liver disease.

The present invention provides a pharmaceutical composition for preventing or treating alcoholic fatty liver comprising the strain, its culture medium, or a cell-free extract thereof.

Since the strain of the present invention exhibits excellent lipid peroxidation inhibitory activity, alanine aminotransferase inhibitory activity, or aspartate aminotransferase inhibitory activity, alcoholic fatty liver can be effectively prevented or treated.

In addition, since the strain of the present invention has selective resistance and sensitivity to antibiotics, it can be selected and co-administered together when taking antibiotics with resistance, and can be effectively removed from the body through taking antibiotics with sensitivity, so it can be safely taken. this is possible

The pharmaceutical composition according to the present invention may include a pharmaceutically effective amount of the strain, its culture medium, or a cell-free extract thereof alone, or may include one or more pharmaceutically acceptable carriers, excipients or diluents.

The above pharmaceutically effective amount refers to an amount sufficient to exert an effect of preventing, improving or treating alcoholic fatty liver. The term "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not usually cause allergic reactions such as gastrointestinal disorders and dizziness or similar reactions when administered to humans.

In addition, the pharmaceutical composition according to the present invention is formulated according to conventional methods into oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories and sterile injection solutions. can Suitable formulations known in the art are preferably those disclosed in the literature (Remington's Pharmaceutical Science, recently, Mack Publishing Company, Easton PA). Carriers, excipients and diluents that may be included include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate, propylhydroxy benzoate, talc, magnesium stearate, mineral oil, and the like. When formulating the pharmaceutical composition, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations contain at least one excipient such as starch, calcium carbonate, sucrose, and lactose in the composition. , gelatin, etc. are mixed and prepared. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. there is. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogeratin and the like may be used.

The pharmaceutical composition of the present invention is the amount of active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human thought by a researcher, veterinarian, physician or other clinician, that is, the symptom of the disease or disorder being treated. It can be administered in a therapeutically effective amount that is an amount that induces remission. It is apparent to those skilled in the art that the therapeutically effective dosage and frequency of administration of the pharmaceutical composition of the present invention will vary depending on the desired effect. Therefore, the optimal dose to be administered can be easily determined by those skilled in the art, and depends on the type of disease, the severity of the disease, the content of the active ingredient and other ingredients contained in the composition, the type of formulation, and the age, weight, and general health of the patient. Condition, sex and diet, administration time, administration route and secretion rate of the composition, treatment period, can be adjusted according to various factors including drugs used simultaneously. For desirable effects, the pharmaceutical composition of the present invention may be administered in an amount of 1 to 10,000 mg/kg/day, preferably 1 to 200 mg/kg/day, and may be administered once a day or several times. It can also be administered in divided doses.

The pharmaceutical composition of the present invention can be administered to a subject by various routes. All modes of administration are contemplated, eg oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine intrathecal or intracerebrovascular injection.

In addition, when the composition of the present invention is a food or health functional food composition, the type of food is not particularly limited, and may include all foods in a conventional sense. Non-limiting examples of foods to which the substance can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea , drinks, alcoholic beverages, and vitamin complexes. When using the composition as a food additive, the composition may be added as it is or used together with other foods or food ingredients, and may be appropriately used according to conventional methods.

In the present specification, food means a natural product or processed product containing one or more nutrients, preferably means that it can be directly eaten through a certain degree of processing process, and is a conventional meaning, and the food The composition is intended to include all foods, food additives, health functional foods and beverages.

Foods to which the composition of the present invention can be added include, for example, various foods, beverages, gum, candy, tea, vitamin complexes, and functional foods. In addition, in the present invention, the food includes special nutritious food (eg, formula milk, infant food, baby food, etc.), processed meat product, fish meat product, tofu, jelly, noodles (eg, ramen, noodles, etc.), health supplement food, seasoning food ( For example, soy sauce, soybean paste, red pepper paste, mixed soybean paste, etc.), sauces, confectionery (eg, snacks), dairy products (eg, fermented milk, cheese, etc.), other processed foods, kimchi, pickled foods (various types of kimchi, pickles, etc.), beverages (eg, fruit and vegetable beverages, soy milk, fermented beverages, ice cream, etc.), natural seasonings (eg, ramen soup, etc.), vitamin complexes, alcoholic beverages, alcoholic beverages, and other health supplements, but are not limited thereto. The food, beverage or food additive may be prepared by a conventional manufacturing method.

In the present invention, the term, health functional food refers to a food manufactured and processed by using a specific ingredient as a raw material or by extracting, concentrating, refining, mixing, etc. a specific ingredient contained in a food raw material for the purpose of health supplement. It refers to food designed and processed to sufficiently exert biological control functions such as biological defense, regulation of biological rhythm, prevention and recovery of disease, etc. say that

When the strain according to the present invention, its culture medium, or its cell-free extract is used as a health functional food, it can be added as it is or used together with other foods or food ingredients, which can be selected and used appropriately as needed. . In addition, the strain to be added, its culture medium, or its cell-free extract can be suitably determined depending on the purpose of use.

Since the strain according to the present invention, its culture medium, or its cell-free extract ensures biosafety and shows an effect of increasing activity in proportion to the concentration, it is obvious that it can be used in an appropriate amount without being limited to a specific range.

In addition, there is no particular limitation on the type of health functional food in which the strain according to the present invention can be used. For example, ramen, other noodles, beverages, tea, drinks, alcoholic beverages, various soups, meat, sausages, bread, chocolate, candy, confectionery, pizza, chewing gum, dairy products including ice cream, or vitamin complexes.

In particular, the strain according to the present invention, its culture medium, or its cell-free extract exhibits viability against gastric acid or bile acid in the digestive tract, lipid peroxidation inhibitory activity, alanine aminotransferase inhibitory activity or aspartate aminotransferase inhibitory activity, It can be added to various foods for the purpose of improving and preventing alcoholic fatty liver to show the function, and according to the selection of a person skilled in the art, it is mixed with other appropriate auxiliary ingredients and known additives that can be commonly contained in health functional foods can be used The known additives include other microorganisms that can be used together with the strain according to the present invention.

Redundant content is omitted in consideration of the complexity of the present specification, and terms not otherwise defined in the present specification have meanings commonly used in the technical field to which the present invention belongs.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1. Isolation of bacterial strains and preparation of cell-free extracts

All lactic acid bacteria (LAB) strains used in this study were collected from MEDIOGEN (Jecheon, Korea). The LABs used in this study, Lactobacilus brevis MG5250 and Limosilactobacillus reuteri MG5458, were derived from food, and Lacticaseibacillus rhamnosus MG4502, Lactiplantibacillus plantarum MG4229, Limosilactobacillus fermentum MG4244, and Limosilactobacillus reuteri MG4224 were derived from humans. In addition, L. fermentum MG590, a probiotic that relieves alcoholic fatty liver, was used as a positive control. All strains were cultured in MRS medium (de Man, Rogosa and Sharpe, Difco, Detroit, MN, USA) for 18 h at 37 °C under anaerobic conditions.

Among the LABs, MG5458 is Limosilactobacillus reuteri MG5458, and includes a 16S rRNA gene represented by the nucleotide sequence of SEQ ID NO: 1. The Limosilactobacillus reuteri MG5458 was patented at the Center for Biological Resources on September 1, 2021, and was given accession number KCTC14694BP.

Cell-free extracts of each strain were prepared as follows. After culturing each strain, it was collected by centrifugation (4000 rpm, 4 °C for 20 minutes). The collected pellets were lyophilized and resuspended in 10 mg/mL phosphate-buffered saline (PBS). The suspension was homogenized for 50 seconds using a sonicator (KFS-150N; Korea Process Technology Ltd., Seoul, Korea) and placed on ice for 1 minute (repeated 3 times). The suspension was then centrifuged (4000 rpm) at 4 °C for 10 min. The supernatant was filter sterilized using a 0.22 μm polytetrafluoroethylene membrane filter (ADVANTEC, Tokyo, Japan) and kept at -80 °C until use.

Example 2. ALDH (aldehyde dehydrogenase) activity

ALDH activity was measured by the following method. 10 μL of cell-free extract, 700 μL of distilled water, 375 μL of 1 M Tris-HCl buffer (pH 8.8, Sigma-Aldrich, St.Louis, MO, SUSA) and 150 μL of 25 mM NAD ⁺ (Sigma-Aldrich) was reacted for 10 minutes to prepare a mixed sample. ALDH (5 U/mL, Sigma-Aldrich) was added to the mixed samples. Optical density in kinetic mode (10 min intervals for 90 min) was determined at 340 nm using a microplate reader (EPOCH2, Biotek, Winooski, VT, USA). The protein content of cell-free extracts was determined using the Bradford assay (Bio-Rad, Hercules, CA, USA). ALDH activity was calculated as the molar extinction coefficient of NADH (6.22 mM ⁻¹ cm ⁻¹ ) and expressed as units/mg protein/min. As a control of this experiment, PBS buffer solution was added instead of the cell-free extract. The results of measuring ALDH activity are shown in FIG. 1 .

As shown in Figure 1, MG5250, MG4502, MG4229, MG4224 and MG5458 increased the ALDH activity compared to the control group. In particular, it was confirmed that MG5458 had significantly higher ALDH activity than other strains.

Accordingly, LAB strain MG5458 was used in the experiments described below.

Example 3. Cell culture

HepG2 cells (88065, KCLB, Seoul, Korea) were cultured in minimum essential medium (MEM) containing 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (PS; Gibco). ; Gibco, MT, USA). HT-29 cells (30038, KCLB) were cultured in DMEM (Gibco) with 10% FBS and 1% PS at 37 °C in a 5% CO ₂ incubator. Cells were subcultured when 70%-80% confluent.

Example 4. Cell viability assay

Cell viability was analyzed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. HepG2 cells were seeded in 96-well plates at 4 x 10 ⁵ cells/mL. After overnight growth, cells were treated with cell-free extract for 1 hour, followed by treatment with or without ethanol for 24 hours. MTT solution (0.2 mg/mL) was added and cells were further incubated for 2-4 hours. After incubation, formazan crystals in each well were dissolved in DMSO. Absorbance at 550 nm was measured using a microplate reader. Experimental results were repeated three times and expressed as mean ± standard deviation (SD), and statistical processing was performed by Student's t-test using IBM SPSS statistics version 21.0 software (Inc., IBM, USA) *** ; Significance was tested at the level of p<0.005. The results of confirming the cytotoxicity and protective effect are shown in Table 1.

Lactic acid bacteria (μg/mL) Cell viability (%) Control + 3% Ethanol Untreated 100.00 ± 5.68 55.40 ± 2.07 Limosilactobacillus reuteri
MG5458 50 91.26 ± 1.18 82.60 ± 1.31 ^*** 100 91.14 ± 6.32 89.59 ± 0.96 ^***

As shown in Table 1, LAB strain MG5458 showed no cytotoxicity in HepG2 cells.

In addition, the survival rate of HepG2 cells induced by ethanol was reduced by about 55% compared to the control group not treated with ethanol. Nevertheless, it was confirmed that the LAB strain MG5458 treatment group increased the viability of HepG2 cells treated with ethanol (ie, cytoprotective effect).

Therefore, HepG2 cell damage was induced by 3% ethanol treatment in subsequent experiments.

Using a microscope, the morphology of HepG2 cells with or without ethanol treatment was observed. Cell observation results are shown in FIG. 2 .

As shown in FIG. 2, it was confirmed that the ethanol-treated group (EtOH (3%)) was modified in cell shape and decreased in cell number compared to the untreated group (control). However, it was confirmed that the LAB strain MG5458 treatment group (EtOH (3%) + MG5458) maintained the original cell shape and number even after treatment with ethanol.

The above result means that the LAB strain MG5458 not only has no cytotoxicity to hepatocytes, but also protects cells from cell damage caused by ethanol treatment.

Example 5. Measurement of lipid peroxidation and glutathione (GSH) content

Lipid peroxidation and glutathione content measurements were performed according to the manufacturer's instructions (Cayman Chemical, Ann Arbor, MI, USA) and normalized to protein content using Bradford analysis.

플레이트에서 5 × 10⁶ 세포를 무세포 추출물의 존재 또는 부재하에 1시간 동안 배양하였다. 배양된 세포를 24시간 동안 에탄올(3%)로 자극하였다. 배양 후 지질 과산화는 540 nm에서 마이크로플레이트 판독기를 사용하여 측정하고 말론디알데히드(malondialdehyde, MDA) 보정 곡선을 사용하여 계산하였다.Specifically, for the evaluation of lipid peroxidation, 100

5×10 ⁶ cells in the plate were cultured for 1 hour with or without cell-free extract. Cultured cells were stimulated with ethanol (3%) for 24 hours. After incubation, lipid peroxidation was measured using a microplate reader at 540 nm and calculated using a malondialdehyde (MDA) calibration curve.

To measure the GSH content, cells (4 x 10 ⁵ cells/mL) were cultured in a 6-well plate for 1 hour regardless of the presence of cell-free extracts. After incubation, they were stimulated with ethanol (3%) for the next 24 hours. After stimulation with ethanol, absorbance was measured at 405 nm using a microplate to determine the total glutathione content. The experimental results were expressed as mean±standard error (SD), and statistical significance between each group was determined by one-way ANOVA and Duncan's multiple range test at the p<0.05 level. It was verified using SPSS statistics version 21.0 software (Inc., IBM, USA).

Total GSH and lipid peroxidation measurement results are shown in Figures 3a and b, respectively.

As shown in Fig. 3a, the GSH content of ethanol-treated cells decreased 0.56 times compared to the control group. However, it was confirmed that the LAB strain MG5458 treatment group increased the GSH content to the level of the ethanol untreated control group.

As shown in Figure 3b, the MDA level of the ethanol-treated cells increased compared to the control group. However, it was confirmed that the MDA level was significantly reduced in the LAB strain MG5458 treatment group.

Example 6. Measurement of liver damage

Liver damage was measured by alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels using commercially available assay kits (Cayman Chemical) and by protein content using the Bradford assay. normalized. Specifically, cells (5 x 10 ⁵ cells/mL) in a 6-well plate were cultured for 1 hour in the presence or absence of a cell-free extract. Then, cells were stimulated with 3% ethanol for 24 hours. After stimulation with ethanol, ALT and AST levels in cell lysates were measured using a microplate reader at 340 nm. The experimental results were expressed as mean±standard error (SD), and statistical significance between each group was determined by one-way ANOVA and Duncan's multiple range test at the p<0.05 level. It was verified using SPSS statistics version 21.0 software (Inc., IBM, USA). The results of evaluating ALT and AST levels are shown in Figures 4a and b, respectively.

As shown in Figure 4a, the ALT level of ethanol-treated cells increased 1.58 times compared to the control group. However, it was confirmed that the ALT level of the LAB strain MG5458 treatment group decreased to that of the ethanol untreated control group.

As shown in Figure 4b, the AST level of ethanol-treated cells increased compared to the control group. However, it was confirmed that the AST level of the LAB strain MG5458 treatment group (100 μg/ml) was reduced to that of the ethanol untreated control group.

Example 7. mRNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)

mRNA from HepG2 cells was isolated using 0.5 mL of NuceloZOL (MA-CHEREY-NAGEL GmbH & Co. KG, Dueren, Germany) according to the manufacturer's instructions. cDNA was synthesized from 1 μg of total RNA using reverse transcriptase premix (Intron, Seongnam-Si, Korea).

The mRNA expression of alcoholic fatty liver-related factors, lipogenesis-related factors, and lipid oxidation factors was analyzed by qRT-PCR. Specifically, qRT-PCR was performed using the CFX Connect Real-Time PCR Detection System (Bio-Rad), and target gene expression was assessed using the iQ™ SYBR® Green Supermix (Bio-Rad). Relative expression of target genes was normalized to GAPDH expression and analyzed by the 2 ^-ΔΔCT method. The experimental results were expressed as mean±standard error (SD), and statistical significance between each group was determined by one-way ANOVA and Duncan's multiple range test at the p<0.05 level. It was verified using SPSS statistics version 21.0 software (Inc., IBM, USA). The mRNA expression analysis results of alcoholic fatty liver-related factors, lipogenesis-related factors, and lipid oxidation factors are shown in FIGS. 5a to c.

As shown in Figure 5a, the expression level of CYP2E1 under oxidative stress in ethanol-treated HepG2 cells was significantly increased by 1.77 times compared to ethanol-untreated cells (control), and the expression levels of SOD, CAT and GPX were 0.67-fold and 0.81-fold, respectively. times, 0.76 times significantly changed. However, LAB strain MG5458-treated group reversed the expression levels of mRNA CYP2E1, SOD, CAT, and GPX. In addition, mRNA expression related to lipid metabolism was investigated to confirm the effect of LAB on ethanol-induced HepG2 cells.

As shown in Figure 5b, the expression levels of SREBP1C and fatty acid synthase (FAS), which are adipogenesis-related factors, increased 5.54-fold and 7.18-fold compared to the control group after ethanol treatment in HepG2 cells. However, it was confirmed that the LAB strain MG5458 treatment group significantly reduced the mRNA expression of SREBP1C and FAS.

As shown in Figure 5c, HepG2 cells treated with ethanol showed lipid oxidation factors such as PPAR, acyl-CoA oxidase (ACO), and carnitine palmitoyltransferase-1 (CPT-1), and the expression level of each factor was higher than that of ethanol-untreated cells. It was confirmed that it decreased by 0.21, 0.67, and 0.44 times. However, it was confirmed that the LAB strain MG5458 treatment group significantly reduced the mRNA expression of PPAR, ACO, and CPT-1.

The above results show that ethanol-induced HepG2 cells activate CYP2E1 to stimulate antioxidant enzymes (SOD, CAT and GPX) and fat metabolism-related factors (SREBP1C, FAS, PPAR, ACO and CPT-1), and LAB This means that strain MG5458 is effective in restoring ALFD through regulation of antioxidant enzyme expression and lipid metabolism pathway.

Example 8. Probiotic properties

8-1. Antibiotic susceptibility and hemolysis assay

Antibiotic susceptibility was measured using antibiotic strips according to the manufacturer's instructions (bioMerieux, Marcy-l'Etoile, France). Antibiotic resistance was confirmed according to EFSA (European Food Safety Authority) guidelines. The antibiotic susceptibility analysis results are shown in Table 2.

Antimicrobiotics ¹ L. reuteri MG5458 Ampicillin S(0.75) Gentamicin S(2) Kanamycin S(6) Streptomycin S(24) Tetracycline S(2) Chloramphenicol S(3) Erythromycin S(0.25) Clindamycin S(0.016) 1. Susceptible (S) and resistant (R) strains according to the microbiology cut-off values from the EFSA guideline. The minimum inhibitory concentrations are indicated in parentheses (μg/mL).

As shown in Table 2, it was confirmed that the LAB strain MG5458 has sensitivity to ampicillin, gentamicin, kanamycin, streptomycin, tetracycline, chloramphenicol, erythromycin and clindamycin.

Hemolysis assay was performed using trypsin agar (BD Bioscience, NJ, USA) plates (MBCell, Seoul, Korea) containing 5% (w/v) sheep blood. Areas were observed as green colonies (α-hemolysis), clear areas (β-hemolysis) and no color change (γ-hemolysis).

As a result of hemolytic analysis, it was confirmed that the LAB strain MG5458 was γ-hemolytic (ie, no hemolytic activity was observed).

8-2. Gastrointestinal Tract (GIT) stability and adhesion

The survival rate of the simulated GIT was evaluated according to the Maragkoudakis method with minor modifications. Specifically, LABs were cultured for 18 hours and washed twice with PBS (pH 7.4) after centrifugation (4000 xg for 5 minutes at 4 °C). Collected LAB was resuspended at 10 ⁸ CFU/mL in simulated gastric fluid containing 3 g/L pepsin (adjusted to pH 3 and 4 with 1 N HCl) for 2 hours. LAB was then resuspended in simulated intestinal fluid containing pH-adjusted 1 g/L pancreatin (adjusted to pH 7 and 8 with 1 N NaOH) and incubated at 37 °C for 4 h. LAB was measured by counting viable cells using MRS agar plates.

The adhesion of LAB was evaluated using HT-29 cells. After seeding HT-29 cells (1 x 10 ⁵ cells/mL) in a 12-well plate, they were cultured for 24 hours at 37°C and 5% CO ₂ conditions. LAB was cultured in MRS medium at 37°C for 24 hours. HT-29 cells were resuspended in DMEM without FBS and PS at 1 x 10 ⁸ CFU/mL and administered to the cells. After 2 hours, the cells were washed twice with PBS and then detached. The number of viable LABs was determined by plate counting on MRS agar and calculated as log CFU/mL.

The results of confirming the stability and adhesion of the LAB strain MG5458 under simulated gastrointestinal conditions are shown in Table 3.

Experiment L. reuteri MG5458 Stimulated gastric fluid
(Log CFU/mL) Initial 7.57 ± 0.04 pH 3 7.58 ± 0.07 pH 4 7.58 ± 0.06 pH 7 7.51 ± 0.00 pH 8 7.44 ± 0.06 Adhesion ability
(Log CFU/mL) Initial 7.23 ± 0.04 Adherent 6.96 ± 0.03 Cell adhesion (%) 70.76 ± 0.865

As shown in Table 3, it was confirmed that all strains had a cell viability of 98% or more in the stimulated gastrointestinal environment. In addition, it was confirmed that all strains had about 96% adhesive ability. The above result means that the LAB strain MG5458 is stable in the gastrointestinal tract environment and has excellent adhesive ability. In addition, it can be seen that the LAB strain MG5458 has excellent pH stability, that is, acid resistance and basic resistance.

In the above, specific parts of the present invention have been described in detail, and for those skilled in the art, it is clear that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Biological Resource Center KCTC14694BP 20210901

<110> MEDIOGEN Co.,Ltd. <120> Limosilactobacillus reuteri MG5458 strain and composition for preventing, improving or treating alcoholic fatty liver including the same <130> 1-50 <160> 1 <170> KoPatentIn 3.0 <210> 1 <211> 1441 <212> DNA <213> artificial sequence <220> <223> 16S rRNA <400> 1 tgcagtcgta cgcactggcc caactgattg atggtgcttg cacctgattg acgatggatc 60 accagtgagt ggcggacggg tgagtaacac gtaggtaacc tgccccggag cgggggataa 120 catttggaaa cagatgctaa taccgcataa caacaaaagc cgcatggctt ttgtttgaaa 180 gatggctttg gctatcactc tgggatggac ctgcggtgca ttagctagtt ggtaaggtaa 240 cggcttacca aggcgatgat gcatagccga gttgagagac tgatcggcca caatggaact 300 gagacacggt ccatactcct acgggaggca gcagtaggga atcttccaca atgggcgcaa 360 gcctgatgga gcaacaccgc gtgagtgaag aagggtttcg gctcgtaaag ctctgttgtt 420 ggagaagaac gtgcgtgaga gtaactgttc acgcagtgac ggtatccaac cagaaagtca 480 cggctaacta cgtgccagca gccgcggtaa tacgtaggtg gcaagcgtta tccggattta 540 ttgggcgtaa agcgagcgca ggcggttgct taggtctgat gtgaaagcct tcggcttaac 600 cgaagaagtg catcggaaac cgggcgactt gagtgcagaa gaggacagtg gaactccatg 660 tgtagcggtg gaatgcgtag atatatggaa gaacaccagt ggcgaaggcg gctgtctggt 720 ctgcaactga cgctgaggct cgaaagcatg ggtagcgaac aggattagat accctggtag 780 tccatgccgt aaacgatgag tgctaggtgt tggagggttt ccgcccttca gtgccggagc 840 taacgcatta agcactccgc ctggggagta cgaccgcaag gttgaaactc aaaggaattg 900 acgggggccc gcacaagcgg tggagcatgt ggtttaattc gaagctacgc gaagaacctt 960 accaggtctt gacatcttgc gctaacctta gagataaggc gttcccttcg gggacgcaat 1020 gacaggtggt gcatggtcgt cgtcagctcg tgtcgtgaga tgttgggtta agtcccgcaa 1080 cgagcgcaac ccttgttact agttgccagc attgagttgg gcactctagt gagactgccg 1140 gtgacaaacc ggaggaaggt ggggacgacg tcagatcatc atgcccctta tgacctgggc 1200 tacacacgtg ctacaatgga cggtacaacg agtcgcaaac tcgcgagagt aagctaatct 1260 cttaaagccg ttctcagttc ggactgtagg ctgcaactcg cctacacgaa gtcggaatcg 1320 ctagtaatcg cggatcagca tgccgcggtg aatacgttcc cgggccttgt acacaccgcc 1380 cgtcacacca tgggagtttg taacgcccaa agtcggtggc ctaaccttta tggagggagc 1440 c 1441

Claims (8)

Limosilactobacillus reuteri MG5458 strain (accession number: KCTC14694BP). The strain according to claim 1, wherein the strain comprises the nucleotide sequence of SEQ ID NO: 1. The strain according to claim 1, wherein the strain has lipid peroxidation inhibitory ability, alanine aminotransferase (ALT) inhibitory ability or aspartate aminotransferase (AST) inhibitory ability. The strain according to claim 1, wherein the strain has acid resistance. The strain according to claim 1, wherein the strain is sensitive to one or more antibiotics selected from the group consisting of ampicillin, gentamicin, kanamycin, streptomycin, tetracycline, chloramphenicol, erythromycin and clindamycin. Claims 1 to 5 of any one of the strains, their culture or containing a cell-free extract thereof, preventing or treating alcoholic fatty liver pharmaceutical composition. Claims 1 to 5 of any one of the strain, its culture, or containing a cell-free extract thereof, preventing or improving alcoholic fatty liver food composition. Claims 1 to 5 of any one of the strains, their culture medium, or their cell-free extracts, preventing or improving alcoholic fatty liver health functional food composition.

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