TWI480045B - Prevention and/or alleviation of alcoholic liver disease with a mixture of four lactic acid bacteria strains - Google Patents

Description

Use a mixture of 4 strains of lactic acid bacteria to prevent and/or alleviate alcoholic liver disease

The present invention relates to the use of a mixture comprising Lactobacillus acidophilus LASW, Enterococcus faecium TM39, Lactobacillus fermentum LF33, and Lactobacillus plantarum BCRC 10069 for prevention. And/or alleviation of alcoholic liver disease (ALD), wherein the Lactobacillus acidophilus LASW, Enterococcus faecalis TM39, and Lactobacillus fermentum LF33 are deposited in the food industry development research under the registration numbers BCRC 910276, 910248, and 910554, respectively. The Center for the Conservation and Research of Biological Resources (BCRC of FIRDI).

Alcoholic liver disease is a liver injury caused by alcohol overconsumption, which can be divided into the following three stages according to pathophysiology: (1) fatty liver (fatty Liver) [also known as hepatic steatosis]: Hepatocytes cannot effectively metabolize triglyceride (TG) due to excessive fatty acid synthesis or transport from the liver to the liver. In turn, a large amount of fat [including triglycerides, fatty acids, and cholesterol esters] accumulate in the liver and cause liver lesions; (2) alcoholic hepatitis (AH): chronic alcohol Consumption changes gut permeability and increases absorption of endotoxins released by bacteria present in the intestine, making the liver Dirty macrophages [ie, Kupffer cells] release large amounts of proinflammatory cytokines [such as tumor necrosis factor-α (TNF-α)] and freedom. Free radicals, thereby forming oxidative stress, which leads to inflammation of the liver cells and apoptosis (apoptosis); and (3) chronic hepatitis with hepatic fibrosis or sclerosis ( Cirrhosis): When hepatocytes continue to produce an inflammatory response, hepatic stellate cells (HSCs) are activated and begin to proliferate and transform into myofibroblasts, while secreting Accumulation of a large number of extracellular matrices [including type I and III collagen and fibronectin], which leads to liver fibrosis, and excessive liver fibrosis leads to liver Scarring and necrosis, and finally cirrhosis.

At present, most of the clinical is by interrupting alcohol consumption (also known as abstinence) and with nutrition therapy [including intake of vitamins and dietary minerals). Diet] to relieve liver damage or to avoid liver damage. In addition, drugs commonly used to treat alcoholic liver disease can be classified into the following three categories: (1) corticosteroids: it can be used to treat severe alcoholic liver disease; (2) colchicine: It has anti-inflammatory and anti-fibrotic effects and is therefore useful for inhibiting liver fibrosis; and (3) anti-cytokine drug: for example, due to welfare (infliximab) and Pentoxifylline, which can be used to inhibit TNF-α production, thereby reducing liver inflammation caused by TNF-α.

However, the above-mentioned treatment methods or drugs still have problems in clinical application and are prone to side effects. Therefore, researchers in the field are working to develop drugs that can effectively treat alcoholic liver disease without causing unwanted side effects.

Lactic acid bacteria (LAB) are a group of Gram-positive bacteria that ferment sugars and use lactic acid as the main metabolite. They are ubiquitous in dairy products, pickles, and intestinal mucosa of humans or animals. Lactobacillus has been widely accepted for its morphological and physiological characteristics including: (1) globular (cocci) or rod (rod); (2) lack of cytochrome and catalase; (3) no formation Endogenous spores; and (4) not motility.

Lactic acid bacteria are generally recognized as safe (GRAS) and are well-known and widely used probiotics. Lactic acid bacteria have been found to have the effects of inhibiting the growth of gastrointestinal pathogens, alleviating lactose intolerance, immunoregulation, anti-cancer, and lowering blood pressure. Can be used as a probiotic lactic acid bacteria used in many species, such as Lactobacillus (Lactobacillus), Lactococcus (Lactococcus), Pediococcus (Pediococcus), Streptococcus (Streptococcus) and Enterococcus (Enterococcus) and so on.

Studies have shown that certain lactic acid bacteria strains have the effect of relieving alcoholic liver disease. For example, in AKIrina et al . (2008), Alcohol , 42: 675-682, AKIrina et al . found a disease with alcohol-induced liver injury compared to a healthy control group. Suffering from a modified bowel flora, short-term oral supplementation with Bifidobacterium bifidum and Lactobacillus plantarum 8PA3 reconstitutable enteric phase, and in alcohol-induced liver The improvement in injury is more pronounced than standard treatment (ie, alcohol withdrawal and vitamin B ₁ and B ₆ supplementation).

In S. Segawa et al . (2008), International Journal of Food Microbiology , 128: 371-377, S. Segawa et al . found that heat-killed Lactobacillus brevis SBC8803 can be used by Inhibition of TNF-α in the liver and overexpression of sterol regulatory element-binding proteins (SREBPs) to improve alcohol-induced liver damage and fat accumulation [ie fatty liver ( Fatty liver)] is thus expected to be used to alleviate alcoholic liver disease.

In CB Forsyth et al . (2009), Alcohol , 43: 163-172, CB Forsyth et al found that gastric tube feeding of Lactobacillus rhamnosus GG in a rat model of alcoholic liver disease (gavage) ) can significantly reduce the severity of alcoholic steatohepatitis (ASH), alcohol-induced gut hyperpermeability and alcohol-induced tissue (including intestinal and liver) and systemic oxidative stress ( Systemic oxidative stress). Therefore, Lactobacillus rhamnosus GG is expected to be useful for ameliorating and/or preventing alcoholic liver disease.

In addition, a master's thesis by Liang Xinwen from the Bioindustry Research Institute of Hongguang University of Science and Technology [name: "The immunomodulation activity of the multiple lactic acid bacteria combination and its application in the multispecises combination of lactic acid bacteria" )]], Liang Xinwen from different sources of lactic acid bacteria strains [including the Biosource Collection and Research Center (BCRC) purchased from the Food Industry Research and Development Institute (FIRDI) in Taiwan) And the strains isolated from the laboratory] can stimulate macrophages to produce a large amount of interferon-γ (IFN-γ), TNF-α and nitric oxide (NO) and have good A bacteriostatic effect and a potential strain of intestinal sorption capacity. Next, these potential strains were subjected to different combinations to prepare a mixture containing different lactic acid bacteria strains, which were then fed to mice for 10 weeks, and then infected with Salmonella typhimurium to investigate the mice. The effectiveness of these mixtures in boosting the immunity of these mice. The experimental results show that a mixture containing Lactobacillus acidophilus LASW, Enterococcus faecium TM39, Lactobacillus fermentum LF33, and Lactobacillus plantarum BCRC 10069 has an increased peritoneum. The phagocytic ability of macrophages and the inhibition of the spleen and liver of mice infected with Salmonella typhimurium.

Upon investigation, the applicant unexpectedly discovered that the mixture containing Lactobacillus acidophilus LASW, Enterococcus faecalis TM39, Lactobacillus fermentum LF33 and Lactobacillus plantarum BCRC 10069 can be alcoholic in addition to the immunomodulatory effect. Liver-lesioned mice produce beneficial prophylactic and/or ameliorating effects. Therefore, the mixture containing 4 strains of lactic acid bacteria is expected to be available for prevention and/or Or relieve alcoholic liver disease.

Summary of invention

Thus, in a first aspect, the present invention provides a pharmaceutical composition for preventing and/or alleviating alcoholic liver disease comprising Lactobacillus acidophilus LASW, Enterococcus faecalis TM39, Lactobacillus fermentum LF33, and Lactobacillus plantarum BCRC 10069, wherein the Lactobacillus acidophilus LASW, Enterococcus faecalis TM39, and Lactobacillus fermentum LF33 are deposited with the Bioresource Conservation and Research Center of the Food Industry Development Research Institute under the registration numbers BCRC 910276, 910248, and 910554, respectively.

In a second aspect, the present invention provides a food product for preventing and/or alleviating alcoholic liver disease comprising Lactobacillus acidophilus LASW, Enterococcus faecalis TM39, Lactobacillus fermentum LF33, and Lactobacillus plantarum BCRC 10069.

In a third aspect, the present invention provides a method for alleviating an individual having or suspected of having an alcoholic liver disease, comprising administering to the individual a solution comprising Lactobacillus acidophilus LASW, Enterococcus faecalis TM39, fermentation A mixture of Lactobacillus LF33 and Lactobacillus plantarum BCRC 10069.

The above and other objects, features and advantages of the present invention will become apparent from

Detailed description of the invention

It is to be understood that if any of the previous publications is quoted here, the prior publication does not constitute an acknowledgement that in Taiwan or any other country, the pre-existing publication forms a common general knowledge in the art. Part of it.

For the purposes of this specification, it will be clearly understood that the term "comprising" means "including but not limited to" and the term "comprises" has a corresponding meaning.

All technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention pertains, unless otherwise defined. A person skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which can be used to practice the invention. Of course, the invention is in no way limited by the methods and materials described.

In developing drugs that can be used to prevent and/or alleviate alcoholic liver disease, the Applicant found that: one strain containing four strains of lactic acid bacteria (including Lactobacillus acidophilus LASW, Enterococcus faecalis TM39, Lactobacillus fermentum LF33, and Lactobacillus plantarum BCRC) The mixture of 10069) has industrial application potential in this respect. Thus, the present invention discloses a mixture comprising Lactobacillus acidophilus LASW, Enterococcus faecalis TM39, Lactobacillus fermentum LF33, and Lactobacillus plantarum BCRC 10069 for use in the preparation of a medicament for preventing and/or alleviating alcoholic liver disease. use.

Lactobacillus acidophilus LASW, Enterococcus faecalis TM39 and Lactobacillus fermentum LF33 have been registered on September 14, 2004, April 14, 2004, and June 26, 2012, under the registration numbers BCRC 910276, BCRC 910248, and BCRC 910554 is deposited at the Biosource Collection and Research Center (BCRC) of the Food Industry Research and Development Institute (FIRDI) (300 Food Road, Hsinchu, Taiwan).

In particular, the applicant confirmed by an in vivo animal test that when a mixture containing the above four strains of lactic acid bacteria according to the present invention is administered to a mouse having alcoholic liver disease, many of them Pathological symptoms (including liver cell damage, liver dysfunction, and accumulation of fat in the liver, etc.) were significantly improved. Accordingly, the present invention provides a pharmaceutical composition for preventing and/or alleviating alcoholic liver disease comprising Lactobacillus acidophilus LASW, Enterococcus faecalis TM39, Lactobacillus fermentum LF33, and Lactobacillus plantarum BCRC 10069.

As used herein, "alleviating" or "alleviation" means treating, reducing, ameliorating, relieving, or controlling a disease (disease) Or one or more clinical signs of a disorder, and lowering, stopping, or reversing the severity of a condition or symptom being treated (symptom) Progress of progress.

According to the present invention, the alcoholic liver disease comprises a condition selected from the group consisting of fatty liver, alcoholic hepatitis, liver fibrosis, and cirrhosis.

According to the present invention, the pharmaceutical composition has a bacterial concentration ranging from 10 ⁷ to 10 ⁹ CFU/mL. In a preferred embodiment of the invention, the pharmaceutical composition has a bacterial concentration ranging from 10 ⁸ to 10 ⁹ CFU/mL.

According to the present invention, in the pharmaceutical composition, the Lactobacillus acidophilus LASW, Enterococcus faecalis TM39, Lactobacillus fermentum LF33, and Lactobacillus plantarum BCRC The relative ratio of 10069 is 1:1:1:1 (v/v/v/v).

In accordance with the present invention, the pharmaceutical composition can be made into a dosage form suitable for oral administration using techniques well known to those skilled in the art, including, but not limited to, solutions. Suspension, emulsion, powder, tablet, pill, syrup, lozenge, troche, elixir , chewing gum, capsule, slurry and the like.

The pharmaceutical composition according to the present invention may further comprise a pharmaceutically acceptable carrier which is widely used in pharmaceutical manufacturing techniques. For example, the pharmaceutically acceptable carrier can comprise one or more agents selected from the group consisting of: solvents, buffers, emulsifiers, suspending agents (suspending) Agent), decomposer, disintegrating agent, dispersing agent, binding agent, excipient, stabilizing agent, chelating agent ), preservative, wetting agent, lubricant, diluent, absorption delaying agent, liposome, sweetening agent , flavoring agents, coloring agents, and the like. The selection and quantity of these reagents falls within the professional literacy and routine skills of those skilled in the art.

The present invention also provides a method for alleviating an individual having or suspected of having an alcoholic liver disease, comprising administering to the individual a A mixture of Lactobacillus acidophilus LASW, Enterococcus faecalis TM39, Lactobacillus fermentum LF33, and Lactobacillus plantarum BCRC 10069.

According to the present invention, the dosage and the number of administrations vary depending on the severity of the disease to be ameliorated, the route of administration, and the weight, age, physical condition and response of the individual to be improved. The choice of dosage and frequency of administration falls within the professionalism and routine skills of those who are familiar with the technology.

Further, the present invention also contemplates that the mixture comprising the above four strains of lactic acid bacteria can be used to prepare a health food or over-the-counter medicine for preventing and/or alleviating alcoholic liver disease.

Accordingly, the present invention provides a food product for preventing and/or alleviating alcoholic liver disease comprising Lactobacillus acidophilus LASW, Enterococcus faecalis TM39, Lactobacillus fermentum LF33, and Lactobacillus plantarum BCRC 10069.

According to the present invention, the four strains of lactic acid bacteria can be used as food additives, added by raw materials during preparation of raw materials, or added during food production, and any edible property. The materials are formulated together into food products for human and non-human animals.

According to the invention, the type of food product includes, but is not limited to, fluid milk products such as milk, concentrated milk, fermented milk, such as yogurt. , sour milk, frozen yogurt, lactic acid bacteria-fermented beverages; milk powder; ice cream; cream cheeses; Dry cheeses; soybean milk; fermented soybean milk; vegetable-fruit juices; fruit juices; sports drinks; confectionery; jelly (jellys ); candies; health foods; animal feeds; and dietary supplements.

Furthermore, the food product according to the present invention can be manufactured in the form of an instant instant food containing lyophilized or spray-dried (spray-) which can be eaten directly. Dried bacterial powder. For the preparation of food products, see, for example, US 6,872,565 B2, US 7,244,424 B2, US 7,270,994 B2, US 7,172,777 B2 and US 6,872,411 B1.

Detailed description of the preferred embodiment

The invention is further described in the following examples, but it should be understood that these examples are for illustrative purposes only and are not to be construed as limiting.

Example General experimental materials: 1. Prepare a mixture of four lactic acid bacteria strains:

The Lactobacillus acidophilus LASW, Enterococcus faecium TM39, and Lactobacillus fermentum LF33 used in the following experiments were respectively on September 14, 2004, in April 2004. On the 14th and June 26th, 2012, the Bioresource Conservation and Research Center (Biosource) of the Food Industry Research and Development Institute (FIRDI) was deposited in Taiwan under the registration numbers BCRC 910276, BCRC 910248 and BCRC 910554. Collection and Research Center, BCRC) (300 Food Road, Hsinchu City, Taiwan), and Lactobacillus plantarum BCRC 10069 is a biological resource conservation and research center purchased from the Food Industry Development Institute of Taiwan.

First, Lactobacillus acidophilus LASW, Enterococcus faecalis TM39, Lactobacillus fermentum LF33, and Lactobacillus plantarum BCRC 10069 were inoculated separately into MRS Broth (Difco, USA), and then in a constant temperature incubator (37). The culture was carried out in °C for 24 hours. Thereafter, each lactic acid bacteria culture was centrifuged at 10,000 g for 10 minutes at 4 ° C, respectively, and then the supernatant was removed, and the pellets were washed with phosphate buffered saline (PBS). Two times, and then suspended in an appropriate amount of PBS and adjusted to a concentration of 10 ⁹ CFU/mL (counted by plate count medium). Next, the obtained bacterial liquid of each Lactobacillus acidophilus LASW, Enterococcus faecalis TM39, Lactobacillus fermentum LF33, and Lactobacillus plantarum BCRC 10069 is 1:1:1:1 (v/v/v/v) The ratios were mixed evenly to give a mixture having a bacterial concentration of 10 ⁹ CFU/mL for subsequent experiments.

2. Preparation of feed:

The preparation of normal diet and ethanol-containing diet is referred to CSLieber and LM Decarli (1982), Alcoholism-Clinical and Experimental Research ., 6: 523-531 and CSLieber and LM Decari (1989), respectively. , Alcohol and Alcoholism ., Method 24: 197-211, and partially modified. Formulations for normal feeds and feeds containing alcohol are shown in Table 1 below.

3. Experimental animals:

Male C57BL/6N mice used in the examples below (C57BL/6N mice) (7 weeks old, weighing about 18-20 g) was purchased from BioLasco Taiwan Co., Ltd. All experimental animals were housed in an independent air-conditioned animal room with light and dark for 12 hours, room temperature maintained at 22 ± 2 ° C and relative humidity maintained at 55 ± 5%, and the water was in the second item above. The prepared normal feed is sufficiently supplied. The treatment of experimental animals and all experimental procedures are in accordance with the provisions of the Animal Protection Act of Taiwan and are based on the guidelines of the Animal Care Committee of the Council of Agriculture (Taiwan). get on.

General experimental method: A. Statistical analysis:

In the following examples, the experimental data is expressed as "mean ± standard error of the mean (SEM)". All data were analyzed by analysis of variance (ANOVA) followed by Duncan's multiple range test to assess differences between groups. If the statistical analysis obtained is p < 0.05, it represents statistical significance.

Example 1. Evaluation of the ameliorating effect of a mixture of four strains of lactic acid bacteria of the present invention on mice with alcoholic liver disease

In this experiment, the applicant used alcohol-containing feed to induce alcoholic liver disease in mice, and by measuring the Tianmen in the serum of these mice The activity of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) was used to examine the liver function of mice, and the triglyceride contained in the liver of mice was analyzed. TG), and histological examination of the liver was performed to evaluate the alleviation effect of the mixture of the four strains of the lactic acid bacteria of the present invention on alcoholic liver disease.

experimental method: A. Treatment of animals:

Male C57BL/6N mice were randomly divided into one normal control group, one pathological control group, one lactic acid bacteria control group, and one experimental group (n=8 per group). From the first 3 days after the start of the experiment, the normal feed used to feed the pathological control group and the experimental group mice was changed day by day into a different one according to the second item "Preparation of feed" of the above "Experimental Materials". Alcohol feeds at concentrations (16.75 mL/L, 33.5 mL/L, and 50.25 mL/L, respectively) were used to allow the mice to gradually adapt to alcohol-containing feeds. As for the normal control group and the lactic acid bacteria control group, the normal feed was continuously fed.

On the first day from the start of the experiment, the pathological control group and the experimental group of mice were fed daily to prepare an alcohol-containing feed according to the second item "Preparation of feed" of the above "Experimental Materials" (alcohol concentration was 67 mL/L), in order to induce alcoholic liver disease in these mice. As for the normal control group and the lactic acid bacteria control group, the mice were fed with normal feed.

In addition to feeding the feed, each group of mice was further administered orally with a gastric tube needle (0.9 x L 50 mm gavage needle, Popper & Sons) once a day and continued until the 28th after the start of the experiment. At the end of the day, mice in the normal control group and the pathological control group were orally administered with 200 μL of ddH ₂ O, while the lactic acid bacteria control group and the experimental group of mice were orally administered with 200 μL based on the above "experimental materials". A mixture containing four strains of lactic acid bacteria prepared in the first item "Preparation of a mixture containing four strains of lactic acid bacteria".

On the 14th day and the 28th day after the start of the experiment, the blood was collected from the orbital position of the mouse in the second hour after oral administration in each group of mice, and then the obtained blood was allowed to stand. The mixture was allowed to coagulate at room temperature for 2 hours, followed by centrifugation at 2,000 rpm for 10 minutes at 4 ° C, and the resulting serum sample was taken for analysis of the following item B.

In addition, on the 28th day after the start of the experiment, the mice of each group were sacrificed by isoflurane after the completion of the blood collection in the eye socket, and then the liver was taken out to carry out the analysis of the following items C and D.

B. Analysis of the activity of aspartate transaminase and alanine transaminase in serum:

The activity analysis of aspartate transaminase and alanine transaminase in the serum of each group of mice was carried out by the Dahua Medical Laboratory Center. The experimental data obtained were analyzed in accordance with the method described in "A. Statistical Analysis" of the "General Experimental Method" above.

C. Analysis of the concentration of triglyceride:

The concentration analysis of the triglyceride was carried out using the Randox Triglycerides Assay Kit (TR 210, Randox Laboratories Ltd., UK) and according to the manufacturer's instructions. Briefly, 0.05 g of each liver of each group of mice obtained in item A above was immersed in a 0.25% sucrose solution (containing 1 mM EDTA) at a ratio of 1:9 (w/v). Then, it was ground at 4 ° C for 5 minutes by a homogenizer, and the formed liver homogenate was carried out using an organic solvent containing chloroform and methanol [chloroform:methanol=2:1 (v/v)]. Extraction, followed by evaporation to remove the organic solvent, thereby obtaining a dried product. Then, the obtained dried product was dissolved in distilled water containing 5% fatty acid-free bovine serum albumin, followed by centrifugation at 10,000 g for 30 minutes at 4 °C. Thereafter, 10 μL of each of the formed supernatants was added, and then 1 mL of RI reagent (Reagent RI) was added and uniformly mixed, and then the resulting mixture was subjected to a reaction at 37 ° C for 5 minutes. Finally, the mixture was measured for absorbance (OD ₅₀₀ ) at a wavelength of 500 nm using a spectrophotometer (Ultrospec 2100 pro, GE healthcare).

Absorbance (OD ₅₀₀₎ connected respectively in accordance with a predetermined standard triglyceride having a known concentration itself relative to its OD ₅₀₀ value is converted into triglyceride concentration (mg / group measured each Dl), the triglyceride concentration of the normal control group was then taken as 100%, and the percentages of the triglyceride concentrations of the remaining groups relative to the normal control group were respectively calculated. The calculated experimental data was analyzed in accordance with the method described in "A. Statistical Analysis" of the "General Experimental Method" above.

D. Histopathological examination:

The liver samples of each group of mice obtained in the above item A were each taken to have a volume of 1 cm ³ of tissue samples, and fixed at 10% foreground at room temperature for one day. Then, the fixed liver tissue samples were sequentially subjected to dehydration at different concentrations (including 30%, 50%, 70%, 95%, and 99.5%) of ethanol. Thereafter, the dehydrated liver tissue sample is subjected to clearing with xylene, followed by embedding with paraffin, and then subjected to slicing, thereby obtaining a thickness of 5 μm. Tissue sections.

Thereafter, the resulting tissue sections are stained by using hematoxylin-eosin and according to techniques well known and used by those skilled in the art, and the stained tissue sections are made by using a handstand. A fluorescent photomicrography system (TE2000-S) (Nikon, Japan) was observed and photographed at a magnification of 200 X.

Result : A. Analysis of the activity of aspartate transaminase and alanine transaminase in serum:

Figure 1 and Figure 2 show the activity of aspartate transaminase and alanine transaminase measured in the serum of each group of mice on the 14th day and the 28th day after the start of the experiment, respectively. .

As can be seen from Fig. 1 and Fig. 2, there was no significant difference in the activity of aspartate transaminase and alanine transaminase in the serum of the lactic acid bacteria control group compared with the mice of the normal control group. The activity of aspartate transaminase and alanine transaminase in the serum of the pathological control group was significantly increased, indicating that the alcohol-containing feed induced alcoholic liver disease in mice and caused liver cell damage. And liver dysfunction. Compared with the mice in the pathological control group, the activities of aspartate transaminase and alanine transaminase in the serum of the experimental group were significantly reduced and showed statistical significance ( p < 0.05). ). The results of this experiment show that a mixture containing four strains of lactic acid bacteria according to the present invention can effectively improve liver cell damage and liver dysfunction caused by alcoholic liver disease.

B. Analysis of the concentration of triglyceride:

Figure 3 shows the percentage of triglyceride concentration measured in the liver of each group of mice at the 28th day after the start of the experiment. As can be seen from Fig. 3, there was no significant difference in the concentration of triglyceride in the liver of the lactic acid bacteria control group compared with the mouse of the normal control group, and the concentration of the triglyceride in the liver of the pathological control group. It is significantly increased, which means that alcohol-containing feed causes a large amount of triglyceride to accumulate in the liver of mice and cause alcoholic liver disease. In contrast to the mice in the pathological control group, the triglyceride concentration in the liver of the experimental group was significantly reduced and exhibited statistical significance ( p < 0.05). The results of this experiment show that the mixture containing four strains of lactic acid bacteria according to the present invention can effectively reduce the accumulation of triglyceride in the liver of mice, thereby achieving the effect of improving alcoholic liver disease.

C. Histopathological examination:

Fig. 4 shows the results of observation of tissues obtained from the liver of each group of mice by hematoxylin-eosin staining on the 28th day after the start of the experiment. As can be seen from Figure 4, compared with the normal control group, lactic acid bacteria There was no significant difference in liver tissue sections between the control mice, and there was a large amount of fat accumulation in the liver tissue sections of the pathological control mice, indicating that the alcohol-containing feed caused a large amount of fat accumulation in the liver of mice. And cause alcoholic liver disease. Compared with the pathological control group, the accumulation of fat in the liver tissue sections of the experimental group was significantly reduced. The results of this experiment show that the mixture containing four strains of lactic acid bacteria according to the present invention has a phenomenon of improving liver fat accumulation caused by alcoholic liver disease. Therefore, the mixture containing four strains of lactic acid bacteria is expected to be useful for preventing and/or alleviating alcoholic liver disease.

All of the patents and documents cited in this specification are hereby incorporated by reference in their entirety. In the event of a conflict, the detailed description of the case (including definitions) will prevail.

While the invention has been described with respect to the specific embodiments of the invention, it will be understood that many modifications and changes can be made without departing from the scope and spirit of the invention. It is therefore intended that the invention be limited only by the scope of the appended claims.

Figure 1 shows the activity of aspartate transaminase measured in the serum of each group of mice on the 14th day and the 28th day after the start of the experiment, where "*" means: when normal compared to control group, p <0.05; and "#" represents: when compared to the pathological control group, p <0.05; Figure 2 shows the first 14 days after the experiment began, and 28 days, in groups of small The activity of alanine transaminase measured in the serum of the mouse, wherein "*" means: when compared with the normal control group, p <0.05; and "#" means: when compared with the pathological control group, p <0.05; Figure 3 shows the percentage of triglyceride concentration measured in the liver of each group of mice at the 28th day after the start of the experiment, where "*" indicates: when compared with the normal control group, p <0.05; and "#" means: when compared with the pathological control group, p <0.05; and Figure 4 is a tissue section staining map showing that from the 28th day after the start of the experiment, from each group The tissue obtained in the liver of the mouse was observed by hematoxylin-eosin staining. the result of.

Claims (12)

A pharmaceutical composition for preventing and/or alleviating alcoholic liver disease, comprising Lactobacillus acidophilus LASW, Enterococcus faecalis TM39, Lactobacillus fermentum LF33, and Lactobacillus plantarum BCRC 10069, wherein the Lactobacillus acidophilus LASW, Enterococcus faecalis TM39 and Lactobacillus fermentum LF33 were deposited with the Bioresource Conservation and Research Center of the Food Industry Development Research Institute under the registration numbers BCRC 910276, 910248 and 910554, respectively. The pharmaceutical composition according to claim 1, wherein the alcoholic liver disease comprises a condition selected from the group consisting of fatty liver, alcoholic hepatitis, liver fibrosis, and cirrhosis. The pharmaceutical composition of claim 1, further comprising a pharmaceutically acceptable carrier. The pharmaceutical composition of claim 3, wherein the pharmaceutically acceptable carrier comprises one or more reagents selected from the group consisting of solvents, buffers, emulsifiers, suspending agents, Decomposers, disintegrants, dispersants, binders, excipients, stabilizers, chelating agents, preservatives, wetting agents, lubricants, diluents, absorption delaying agents, liposomes, sweeteners, flavoring agents, and Staining reagent. For example, the pharmaceutical composition of claim 1 is in a dosage form for oral administration. The pharmaceutical composition according to claim 5, wherein the dosage form is selected from the group consisting of a solution, a suspension, an emulsion, a powder, a tablet, a pill, a syrup, a lozenge, a tablet, and a mash. Agents, chewing gums, thick pastes and capsules. a mixture comprising Lactobacillus acidophilus LASW, Enterococcus faecalis TM39, Lactobacillus fermentum LF33, and Lactobacillus plantarum BCRC 10069 for use in the preparation of a medicament for preventing and/or alleviating alcoholic liver disease, wherein the Lactobacillus acidophilus LASW, Enterococcus faecalis TM39, and Lactobacillus fermentum LF33 are deposited in the Bioresource Conservation and Research Center of the Food Industry Development Research Institute under the registration numbers BCRC 910276, 910248, and 910554, respectively. The use of claim 7, wherein the alcoholic liver disease comprises a condition selected from the group consisting of fatty liver, alcoholic hepatitis, liver fibrosis, and cirrhosis. The use of the seventh aspect of the patent application, wherein the pharmaceutical product is in a dosage form for oral administration. The use according to claim 9 wherein the dosage form is selected from the group consisting of a solution, a suspension, an emulsion, a powder, a tablet, a pill, a syrup, a lozenge, a tablet, an elixir, Chewing gum, thick paste and capsules. A food product for preventing and/or alleviating alcoholic liver disease, comprising Lactobacillus acidophilus LASW, Enterococcus faecalis TM39, Lactobacillus fermentum LF33, and Lactobacillus plantarum BCRC 10069, wherein the Lactobacillus acidophilus LASW, feces Enterococcus genus TM39 and Lactobacillus fermentum LF33 are deposited with the Bioresource Conservation and Research Center of the Food Industry Development Research Institute under the registration numbers BCRC 910276, 910248 and 910554, respectively. The food product of claim 11, wherein the alcoholic liver disease comprises a condition selected from the group consisting of: fatty liver, Alcoholic hepatitis, liver fibrosis, and cirrhosis of the liver.