Monascus red fungus, compositions for the prevention or treatment of non-alcoholic fatty liver disease, and uses of the same Monascus red fungus

Abstract

To provide a Monascus pilosus strain, a composition for preventing or treating nonalcoholic fatty liver disease, and uses of the Monascus pilosus strain.SOLUTION: A main purpose of the present invention is to provide a Monascus pilosus and a composition for preventing or treating nonalcoholic fatty liver disease, and the composition comprises the Monascus pilosus and / or its fermented product, or comprises a functional ingredient including its fermented product. Another objective of the present invention is to provide a use of the Monascus pilosus in the manufacture of a composition for the prevention or treatment of nonalcoholic fatty liver disease. The composition of the present invention not only has the efficacy of reducing the accumulation of total cholesterol and triglycerides in nonalcoholic fatty liver disease caused by obesity but also has the efficacy of reducing serum liver function markers Aspartate aminotransferase and Alanine aminotransferase, therefore the composition of the present invention has the potential for preventing or treating nonalcoholic fatty liver disease and related metabolic diseases (for example, hyperlipidemia).SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to the technical field of preventing or treating non-alcoholic fatty liver disease, and particularly to the technical field of a composition for preventing or treating non-alcoholic fatty liver disease, which contains Monascus, its Monascus fermented product or a functional component such as the Monascus fermented product.

Background Art

[0002] Non-alcoholic fatty liver disease (NAFLD) is one of the most common liver diseases in Western countries, and the prevalence rate in general adults accounts for about 20% - 30%. In obese patients and diabetic patients, the prevalence rate has been shown to reach as high as 70% - 90%. According to the research of domestic research institutions, the prevalence rate of non-alcoholic fatty liver disease is shown to be about 11.5%. In recent years, insulin resistance has been known as an important cause of non-alcoholic fatty liver disease. Therefore, patients with non-alcoholic fatty liver disease have a clear association with metabolic syndrome. Evidence increasingly shows that non-alcoholic fatty liver disease is associated with an increased risk of developing cardiovascular disease, and this risk increases with the increase in the severity of non-alcoholic fatty liver disease.

[0003] Non-alcoholic fatty liver disease (NAF) refers to fatty liver disease in which excessive fat accumulates in liver cells, and is not caused by alcohol. Generally speaking, it is normal for the liver to contain some fat, however, if 5% to 10% or more of the liver's weight is fat, it is called fatty liver disease. NAF often occurs in overweight, obese, or people with diabetes, high cholesterol, or high triglycerides. Rapid dieting and an unbalanced diet can also lead to NAF. The harm that NAF poses to the body cannot be ignored. Over time, fatty liver can lead to cirrhosis, and in severe cases, liver cancer or liver failure. NAF can directly lead to decompensated cirrhosis, hepatocellular carcinoma, and recurrence of transplanted livers, as well as influencing the progression of other chronic liver diseases and contributing to the development of type 2 diabetes and atherosclerosis. Non-alcoholic fatty liver disease can cause liver damage similar to that of alcoholism, and in the most serious cases, it can progress to cirrhosis or liver failure.

[0004] Gut microbiota refers to the vast number of bacteria that inhabit the human digestive tract. Its influence on various physiological and pathological phenomena in the human body is quite clear and important. The interaction between fatty liver and gut microbiota is closely related, and this relationship is called the gut-hepatic axis. The composition and metabolites of gut microbiota affect hepatic lipid metabolism, inflammatory responses, and the degree of fibrosis. Several studies have found that patients with non-alcoholic fatty liver disease (NAL) exhibit reduced gut microbiota diversity, making them more susceptible to dysbiosis. Furthermore, abnormal gut microbiota produce harmful substances such as phenylacetic acid, which enter the liver via the hepatic portal vein, promoting the formation and progression of fatty liver. Therefore, regulating the gut microbiota ecosystem, for example by increasing the intake of beneficial bacteria, can be helpful in improving fatty liver. [Overview of the project] [Problems that the invention aims to solve]

[0005] Therefore, in order to prevent or treat non-alcoholic fatty liver disease caused by overweight and obesity, the inventors of this application have diligently researched and invented, resulting in the research, development, and completion of the present invention's Aspergillus oryzae, composition for the prevention or treatment of non-alcoholic fatty liver disease, and uses of the Aspergillus oryzae. In daily life, obese and hyperlipidemia patients can prevent or treat non-alcoholic fatty liver disease using the composition of the present invention, which is easily available and can be taken without adverse effects on the body.

[0006] The present invention primarily aims to provide Monascus pilosus and a composition for the prevention or treatment of non-alcoholic fatty liver disease, wherein the composition contains Monascus pilosus and / or its fermented product, or contains monascinol obtained by purifying such fermented product, and the composition for the prevention or treatment of non-alcoholic fatty liver disease can be provided for oral administration. [Means for solving the problem]

[0007] To achieve the above objectives, the present invention proposes a novel Monascus pilosus, which has been deposited with the National Industrial, Food and Marine Bacteria Collection Ltd (NCIMB Ltd) in the United Kingdom under accession number NCIMB 44103.

[0008] Furthermore, the present invention provides a composition for the prevention or treatment of non-alcoholic fatty liver disease, which comprises an effective amount of a red yeast rice ferment, the red yeast rice ferment, which can be produced by fermenting a substrate using Monascus pilosus, and the Monascus pilosus is deposited with the National Industrial, Food and Marine Bacteria Collection Ltd (NCIMB Ltd) in the United Kingdom under accession number NCIMB 44103.

[0009] The non-alcoholic fatty liver disease (NAL) that can be prevented or treated by the compositions of the present invention described above includes NAL caused by at least one condition selected from the group consisting of obesity, hypertension, hyperlipidemia, and diabetes. Furthermore, such compositions can reduce the serum liver function markers AST and ALT, and can suppress the accumulation of fat in liver tissue. In addition, such compositions can reduce the content of total cholesterol (TC) and triglycerides (TG) in the liver, and can improve the accumulation of lipid droplets and fat infiltration in the liver caused by NAL. Moreover, such compositions have the function of improving the richness of beneficial intestinal bacteria and the function of altering the composition of intestinal bacteria.

[0010] In addition, the substrate in the composition of the present invention described above is a mixture of rice, yam, or related carbohydrates.

[0011] In addition, the effective amount in the composition of the present invention described above is such that an adult ingests at least 0.5 grams of the red yeast rice ferment daily, which contains 1.5 milligrams of monascinol, of which the red yeast rice ferment contains at least one functional component, and the functional component comprises at least one selected from the group consisting of monascinol, ankaflavin, and monascin.

[0012] The compositions of the present invention described above may be food compositions, pharmaceutical compositions, feed compositions, nutritional supplement compositions, dietary supplement compositions, or food additive compositions.

[0013] Furthermore, the present invention also provides a composition for the prevention or treatment of non-alcoholic fatty liver disease, which contains an effective amount of a functional component, the functional component being extracted from a red yeast rice ferment, the red yeast rice ferment can be produced by fermenting a substrate using Monascus pilosus, and the Monascus pilosus is deposited with the National Industrial, Food and Marine Bacteria Collection Ltd (NCIMB Ltd) in the United Kingdom under accession number NCIMB 44103.

[0014] The non-alcoholic fatty liver disease (NAL) that can be prevented or treated by the compositions of the present invention described above includes NAL caused by at least one condition selected from the group consisting of obesity, hypertension, hyperlipidemia, and diabetes. Furthermore, such compositions can reduce the serum liver function markers AST and ALT, and can suppress the accumulation of fat in liver tissue. In addition, such compositions can reduce the content of total cholesterol (TC) and triglycerides (TG) in the liver, and can improve the accumulation of lipid droplets and fat infiltration in the liver caused by NAL. Moreover, such compositions have the function of improving the richness of beneficial intestinal bacteria and the function of altering the composition of intestinal bacteria.

[0015] In addition, the functional component in the composition of the present invention described above comprises at least one selected from the group consisting of monascinol, ankaflavin, and monascin. The substrate in the composition of the present invention described above is a mixture of rice, yam, or related carbohydrates, and the effective amount is such that an adult ingests at least 1.5 milligrams to 12 milligrams of the functional component daily when used alone, or at least 6 milligrams of the functional component daily.

[0016] Furthermore, the present invention provides a use for Monascus pilosus for preparing compositions for the prevention or treatment of non-alcoholic fatty liver disease, wherein the Monascus pilosus is deposited with the National Industrial, Food and Marine Bacteria Collection Ltd (NCIMB Ltd) in the United Kingdom under accession number NCIMB 44103.

[0017] As used in this text, the term "obesity" is defined as a condition characterized by the accumulation and deposition of abnormal or excessive amounts of fat, which poses a risk to health. Irregular eating habits, excessive food intake, lack of exercise, endocrine disorders, genetic factors, psychological factors, and medications can all contribute to obesity. Furthermore, obesity increases the risk of developing diseases such as arteriosclerosis, cardiovascular disease (stroke and ischemic cardiovascular disease), hypertension, diabetes, hyperlipidemia, and fatty liver.

[0018] As used in this text, "hyperlipidemia" refers to a condition in which there is an excess of lipids or fats in the body, such as cholesterol and triglycerides. The term "hypertension" refers to a condition in which an individual's blood pressure (the pressure exerted by blood on the blood vessel walls) is measured higher than normal, and studies have shown that triglycerides are positively associated with blood pressure (including systolic and diastolic blood pressure).

[0019] The term "hepatic steatosis" used in this text refers to a common condition caused by the excessive accumulation and deposition of fat in the liver. Even a small amount of fat in a healthy liver, when it accounts for 5% to 10% of the liver's weight, can be problematic and lead to various diseases, such as angina pectoris, myocardial infarction, stroke, arteriosclerosis, pancreatitis, or other similar conditions. Furthermore, fatty liver is classified into alcoholic fatty liver, which is caused by alcohol consumption, and non-alcoholic fatty liver disease (NAFLD), which is not caused by alcohol.

[0020] As used herein, the term "non-alcoholic fatty liver disease" refers to fatty liver caused by excessive accumulation of fat in the liver without alcohol as the cause, and obesity, hypertension, hyperlipidemia (e.g., excessive cholesterol or triglycerides), and diabetes, etc., which are also mentioned in the literature, may all cause factors for fat accumulation in the liver.

Brief Description of the Drawings

[0021] [Figure 1] It is a diagram showing the process of identifying and discriminating the strain of Monascus purpureus of the present invention. [Figure 2] It is a phylogenetic tree showing the phylogenetic relationship after alignment of the β-tubulin (β-tubulin) sequence of Monascus purpureus of the present invention. [Figure 3] It is a phylogenetic tree showing the phylogenetic relationship after alignment of the ITS sequence of Monascus purpureus of the present invention. [Figure 4] It is the result showing the species-specific PCR analysis of Monascus purpureus of the present invention. [Figure 5] It is the result showing the PCR analysis of the pksCT gene of Monascus purpureus of the present invention. [Figure 6] It is a photograph showing the ascocarp morphology of Monascus purpureus of the present invention. [Figure 7] It is a liver pathological tissue section image showing the effects of the red yeast rice of the present invention and its Monascinol on high-fat diet mice for a long period. [Figure 8] It is a diagram showing the relative abundance of the intestinal flora listed in the top 10 rankings through high-throughput sequencing regarding the effects of the red yeast rice of the present invention and its Monascinol on the intestinal flora of high-fat diet mice for a long period. [Figure 9] It is a diagram showing the comparison of the richness of the flora between each test group (L008, H008, and Msol groups) and the HFD group. [Figure 10] It is a diagram showing the partial least squares discriminant analysis (PLS-DA) of the β-diversity of the intestinal flora of each test group (ND, HFD, L008, H008, and Msol groups).

Best Mode for Carrying Out the Invention

[0022] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Hereinafter, the details of the present invention will be explicitly described using examples, but these examples are merely illustrative and not restrictive, and the present invention is not limited to these examples. Unless otherwise stated, the materials used in the present invention are preferably all commercially available ones that are easily obtainable, and the available routes shown below are merely examples.

[0023] This invention provides a novel Monascus pilosus, which has been deposited with the National Industrial, Food and Marine Bacteria Collection Ltd (NCIMB Ltd) in the United Kingdom under accession number NCIMB 44103. In addition, this invention provides a composition for the prevention or treatment of non-alcoholic fatty liver disease, which contains an effective amount of a fermented Monascus pilosus, and which is obtained by fermenting the Monascus pilosus deposited with the National Industrial, Food and Marine Bacteria Collection Ltd (NCIMB Ltd) in the United Kingdom under accession number NCIMB 44103. Furthermore, the present invention also provides a composition for the prevention or treatment of non-alcoholic fatty liver disease, which contains an effective amount of a functional component, the functional component being extracted from a red yeast rice ferment, the red yeast rice ferment, which can be produced by fermenting a substrate using Monascus pilosus, and the Monascus pilosus being deposited with the National Industrial, Food and Marine Bacteria Collection Ltd (NCIMB Ltd) in the United Kingdom under accession number NCIMB 44103. In addition, the functional component is monascinol, and the substrate is a mixture of rice, yam, or related carbohydrates. Moreover, experiments conducted using high-fat diet-induced non-alcoholic fatty liver mice confirmed that the Monascus pilosus and the composition prepared as described above have efficacy in preventing or treating non-alcoholic fatty liver disease.

[0024] The aforementioned strains of Monascus purpurea of ​​the present invention also include progeny or mutant strains of its subculture, but still possess the same species characteristics, genomics, or uses (used to prevent or treat non-alcoholic fatty liver disease) as those of the present invention.

[0025] The compositions described herein may include, but are not limited to, applications applicable to the present invention such as foods, beverages, health foods, animal drinking water additives, animal feed additives, veterinary pharmaceutical compositions and human pharmaceutical compositions, food additives, and beverage additives.

[0026] The terms "prevention" and "treatment" mean that, compared to compositions that do not use the Monascus red yeast rice, its fermented product, or an extract of the fermented product of the present invention, the compositions of the present invention can effectively prevent the development of non-alcoholic fatty liver disease or effectively treat non-alcoholic fatty liver disease.

[0027] The term "effective dose" refers to the effective amount of a functional ingredient that can effectively prevent or treat non-alcoholic fatty liver disease, and is also called the "therapeutic effective dose" or "improvement effective dose." Furthermore, the term "pharmaceutically acceptable" means that the substance or composition must be compatible with the other components of the compound and be harmless to the patient.

[0028] The compositions of the present invention refer to those which, using techniques familiar to those familiar with this art, can be prepared in combination with a pharmaceutically acceptable vehicle to form a dosage form applicable to the compositions of the present invention, the above-mentioned Monascus red yeast rice, its fermented product, or an extract of the fermented product, and such dosage forms include, but are not limited to, solutions, emulsions, suspensions, powders, tablets, pills, orally disintegrating tablets, lozenges, troches, capsules, and other dosage forms similar to or applicable to the present invention.

[0029] The above composition may also contain, as needed, one or more types of solubilizers, buffers, preservatives, colorants, fragrances, flavorings, excipients, etc., that are commonly used in the pharmaceutical field.

[0030] In another preferred embodiment, the compositions provided by the present invention may be further added to edible materials and prepared as food products or health maintenance products, the edible materials of which include water, fluid milk products, milk, concentrated milk, fermented milk such as yogurt, frozen yogurt, sour milk and lactic fermenting beverages, milk powder, ice cream, cream cheese, dry cheese, soybean milk, fermented soybean milk, fruit and vegetable juice, juice, sports drink, confectionery, jelly, baby food, health food, animal feed This includes, but is not limited to, feed, herbal medicine, and dietary supplements.

[0031] Furthermore, the present invention also provides a method for preventing or treating non-alcoholic fatty liver disease, which involves providing an effective amount of the aforementioned composition to a person with excess liver fat and is used for preventing or treating non-alcoholic fatty liver disease.

[0032] In addition, the present invention also provides a method or use for preparing compositions for the prevention or treatment of non-alcoholic fatty liver disease using the aforementioned Monascus red yeast rice, Monascus red yeast rice ferment, or extracts of the Monascus red yeast rice ferment.

[0033] The administration route of the composition for the prevention or treatment of non-alcoholic fatty liver disease provided by the present invention is not particularly limited, as it can be appropriately adjusted as required. Preferred administration routes include oral administration in a suitable dosage form.

[0034] <Example 1: Novel identification and identification of Monascus pilosus species according to the present invention> 1. Basis for and process of identifying bacterial species. Traditional classification and identification of Monascus purpurea are primarily based on colony morphology, with colony size and color being the main criteria (Hawksworth and Pitt, 1983). However, some Monascus purpurea strains are not easily classified based on colony appearance (e.g., Monascus pilosus and Monascus ruber), so molecular typing methods are sometimes employed to differentiate Monascus purpurea species. Currently, the primary method for molecular typing Monascus purpurea is the alignment of β-tubulin and ITS sequences (Park et al., 2004). However, this invention improves the accuracy of species identification by simultaneously employing polymerase chain reaction (PCR) and pksCT gene PCR, developed in the laboratory by the inventors, in addition to the alignment of β-tubulin and ITS sequences, thereby further enhancing the accuracy of species identification (see Figure 1).

[0035] 2. Strain culture and deoxyribonucleic acid (DNA) extraction Potato dextrose broth (PDB) medium was purchased from Difco (Becton-Dickinson Diagnostic System, Sparks, MD, USA). For DNA extraction, the test strain was cultured in PDB medium, incubated at 28°C for 7 days, then the cells were collected, ground in liquid nitrogen, and then extracted and dried in a vacuum oven. 0.1 grams (g) of the dried cell powder was then weighed and placed in a 2 ml (mL) microcentrifuge tube, and DNA extraction was performed using the QIAamp DNA Mini Kit (QIAGEN NV, purchased from Velno, The Netherlands).

[0036] 3. Alignment analysis of ITS sequences and β-tubulin sequences, and species specificity PCR analysis.

[0037] (1) PCR amplification of the ITS sequence The total volume of the PCR reaction is 25 μL, containing 1x PCR buffer, 0.05 mM of four types of deoxynucleoside triphosphates (dNTPs), 5 U of ExSel high fidelity DNA polymerase (purchased from Bertec Enterprise Co., Ltd., Taipei), 0.2 μM of primer ITS1 (SEQ ID NO: 1) and primer ITS4 (SEQ ID NO: 2), and 0.16 μg of template DNA. The PCR reaction conditions are as follows: First, denaturation is performed at 95°C for 5 minutes. Then, 35 cycles are performed, each consisting of 30 seconds at 95°C, 30 seconds at 62°C, and 1 minute at 70°C. Finally, 10 minutes at 70°C is performed, followed by storage at 4°C. After confirmation via electrophoresis, the product is sent to Genomics Bioscience Technology Co., Ltd. (Taipei, Taiwan) for DNA sequencing.

[0038] (2) PCR amplification of β-tubulin sequence The total volume of the PCR reaction is 25 μL, containing 1x PCR buffer, 0.05 mM dNTPs, 5 U of ExSel high fidelity DNA polymerase, 0.12 μM β-tubulin forward primer (primer β-tubulin F, SEQ ID NO: 3) and β-tubulin reverse primer (primer β-tubulin R, SEQ ID NO: 4) (Park et al., 2004), and template DNA (0.16 μg). The PCR reaction conditions are as follows: first, denaturation at 95°C for 5 minutes. Subsequently, 35 cycles are performed, each consisting of 30 seconds at 95°C, 2 minutes at 55°C, and 2 minutes at 70°C. Finally, 10 minutes at 70°C is performed, followed by storage at 4°C. After confirmation by electrophoresis, the product is sent to Genomics Bioscience Technology Co., Ltd. for DNA sequencing.

[0039] (3) Species specificity of the red yeast fungus Monascus purpureus (M. purpureus) by PCR The species-specific PCR for M. purpureus is designed based on the residual fragment of the unique monacolin K biosynthesis gene mokH within the M. purpureus genome, and can effectively differentiate whether or not a particular Aspergillus species belongs to M. purpureus. The total volume of the PCR reaction is 25 μL, containing 1x PCR buffer, 0.05 mM dNTPs, 5 U of DNA polymerase (SupeTherm GOLD DNA polymerase), 0.12 μM MPuS1 primer (primer MPuS1, SEQ ID NO: 5) and MPuS2 primer (primer MPuS2, SEQ ID NO: 6), and template DNA (0.16 μg). The PCR reaction conditions are as follows: first, denaturation at 95°C for 10 minutes. Subsequently, 35 cycles are performed, with each cycle consisting of 30 seconds at 95°C and 1 minute at 60°C. Finally, electrophoresis analysis is performed after 10 minutes at 70°C.

[0040] (4) Species specificity of the red yeast fungus Monascus pilosus / ruber (M. pilosus / ruber) by PCR The species-specific PCR for M. pilosus / ruber is designed based on a conserved fragment of the FAS gene in the pigment biosynthesis gene cluster within the M. ruber genome, and can effectively differentiate whether or not Monascus purpurea belongs to the M. pilosus / ruber group. The total volume of the PCR reaction is 25 μL, containing 1x PCR buffer, 0.05 mM dNTPs, 5 U of SupeTherm GOLD DNA polymerase, 0.12 μM RubPil forward primer (primer RubPil F, SEQ ID NO: 7) and RubPil reverse primer (primer RubPil R, SEQ ID NO: 8), and template DNA (0.16 μg). The PCR reaction conditions are as follows: first, denaturation at 95°C for 10 minutes. Then, 35 cycles of 30 seconds at 95°C followed by 1 minute at 60°C are performed, and finally, electrophoresis analysis is performed after 10 minutes at 70°C.

[0041] (5) PCR of the pksCT gene The pksCT gene is a core gene for citrinin biosynthesis in M. purpureus, and since this gene segment in the M. pilosus / ruber genome is already deleted, the aforementioned gene can be used as an auxiliary indicator for identifying M. pilosus / ruber. The total volume of the PCR reaction is 25 μL, containing 1x PCR buffer, 0.05 mM dNTPs, 5 U of SupeTherm GOLD DNA polymerase, 80 nM of pksCT-M reverse primer (primer pksCT-M R, SEQ ID NO: 9) and pksCT-M forward primer (primer pksCT-M F, SEQ ID NO: 10), and template DNA (10 ng). The PCR reaction conditions are as follows: first, denaturation at 95°C for 10 minutes. Then, 30 cycles are performed, each consisting of 30 seconds at 95°C, 30 seconds at 54°C, and 40 seconds at 72°C. Finally, electrophoresis analysis is performed after 10 minutes at 70°C.

[0042] (6) Alignment analysis of sequences The alignment analysis of sequences is performed using Geneious 8.1.9 software (Biomatters Ltd., Auckland, New Zealand). Among them, sequence combinations are analyzed using the built-in assembler in Geneious, multiple alignment is performed using MAFFT 7.017, the construction of a phylogenetic tree showing the maximum likelihood phylogenetic relationship is performed using MEGA, the General Time Reversible (GTR) is selected as the nucleic acid substitution model, and bootstrap analysis is performed 1000 times. The phylogenetic tree showing the phylogenetic relationship by the Bayesian method and the posterior probability test are performed using MrBayes, the nucleic acid substitution model is GTR, and Aspergillus terreus is used as an outgroup.

[0043] <Results of alignment analysis of ITS sequences and β-tubulin sequences> The phylogenetic trees showing the relationships between the β-tubulin and ITS sequences of the Monascus purpurea species of the present invention are shown in Figures 2 and 3, respectively. As shown in the β-tubulin sequence alignment results, the Monascus purpurea species of the present invention belong to the same branch as M. pilosus and M. ruber, with a substitution support rate of 97% (>50%). As shown in the ITS sequence alignment results, the Monascus purpurea species of the present invention belongs to the same monophyletic group as the M. purpurerus branch, with a substitution support rate of 62% (>50%) (see Figure 3). The results in Figures 2 and 3 consistently indicate whether the Monascus purpurea species of the present invention belongs to M. pilosus or M. ruber. The results of the specificity PCR analysis of this species are consistent with the results of the phylogenetic tree analysis showing the relationships (see Figure 4, where RubPil: M. ruber / pilosus specific, Mpus: M. purpureus specific). The PCR results for the pksCT gene also indicate that the Monascus purpureus genome of the present invention does not contain the pksCT gene (see Figure 5, of which NTU 568 Monascus purpureus: positive control group) belongs to either M. pilosus or M. ruber.

[0044] In addition, the current taxonomic distinction between M. pilosus and M. ruber is based on the presence or absence of pigment in the ascocarp exocarp (M. ruber is usually brown, while M. pilosus is colorless). As shown in the results, the ascocarps of the Monascus mongolica species of the present invention are all colorless (see Figure 6), so it has become clear that the species of Monascus mongolica of the present invention is determined to be M. pilosus.

[0045] Therefore, the results of the ITS sequence alignment, β-tubulin sequence alignment, species specificity PCR, and pksCT gene PCR in Example 1 all consistently indicate whether the Monascus pilosus of the present invention belongs to the M. pilosus or M. ruber species. Subsequently, after observation of the ascocarp morphology, the Monascus pilosus of the present invention is determined to be the M. pilosus species. In addition, the alignment analysis results of the ITS sequence and β-tubulin sequence also show that the Monascus pilosus SWM-008 provided by the present invention is a novel Monascus pilosus isolate.

[0046] <Example 2: Method for culturing Monascus red fungus according to the present invention> 1. Cultivating the starter culture: (1) Weigh 2g of polished rice flour and place it in a 500mL Erlenmeyer culture flask with a straight groove at the bottom. Add 100mL of reverse osmosis water (RO water), attach a breathable silicone stopper, shake well, and sterilize at 1.25 atmospheric pressure (atm) at 121°C for 20 minutes (min). After sterilization, allow to cool at room temperature.

[0047] (2) Three small pieces of Aspergillus oryzae starter culture were scooped out from the culture dish and placed in a 500 mL shaking flask. A breathable silicone stopper was attached, and the flask was cultured in a 30°C culture box at a rotation speed of 150-200 rpm for 48-72 hours with shaking.

[0048] 2.Solid culture (1) Inoculation: 300g of polished rice is soaked in RO water overnight, the water is drained and dried, then wrapped in koji cloth and transferred to a koji tray. After sterilization (121°C, 30 min), it is cooled and inoculated with the fungus. The starter culture of red koji mold is poured into the solid substrate (10%) and mixed thoroughly and uniformly.

[0049] (2) The culture steps are as follows: a. Culture conditions: After inoculation, wrap the sample in koji cloth and culture it in a constant temperature and humidity chamber at 30°C and 60% relative humidity. b. Watering: During the rapid proliferation of Aspergillus oryzae, some of the water in the solid substrate evaporates as the temperature rises, and most of it is consumed by proliferation, causing the substrate to dry out. Therefore, it is necessary to replenish the water. From the second to the eleventh day, add approximately 30-50 mL of sterile water daily, and then continue watering every other day until the koji is harvested. c. Koji harvesting: The red koji fermented product (cultivated for 14 to 28 days) that has been cultured for 21 days is placed in a roasting oven to dry (37°C, 24 hours), and then stored after drying. This red koji fermented product is essentially red koji rice.

[0050] <Example 3: Steps for the purification and separation method of monascinol>

[0051] 1. Weigh out approximately 1000g of red yeast rice.

[0052] 2. Add approximately 10 liters (L) of 95% EtOH and perform the extraction in a 60°C water bath. Continue the extraction for 2 hours, ensuring uniform agitation every 30 minutes throughout the period. After filtering out the extract using filter paper, repeat the extraction process once more.

[0053] 3. The filtered extract is concentrated under reduced pressure until it becomes thick, and 2 to 2.5 times its weight in silica gel is added and mixed. After further concentration under reduced pressure until dry, it is freeze-dried overnight in a freeze-dryer, and then weighed.

[0054] 4. Weigh approximately 7 to 8 times the weight of the silica gel from step 3 above, mix it with Hex:siRNA (8:2) solvent, and pack it into an open chromatography column. After waiting for the chromatography column to equilibrate, spread the mixture of silica gel from step 3 and the extract evenly on top and await elution washing.

[0055] 5. The chromatography column is sequentially eluted and washed with Hex:RINKAN (8:2), Hex:RINKAN (7.5:2.5), Hex:RINKAN (7:3), and Hex:RINKAN (6:4) solvents. The Hex:RINKAN (7:3) eluent is collected.

[0056] 6. The eluent wash in Hex:siRNA (7:3) is concentrated under reduced pressure until dry, then redissolved in MeOH, and finally purified by preparative HPLC. The solvent conditions are changed to MeOH:ddH2O (83:17), the monascinol effluent is collected and concentrated under reduced pressure until dry, and then the residual water is removed by vacuum drying to obtain pure monascinol.

[0057] <Example 4: Method for conducting experiments using a non-alcoholic fatty liver animal model induced by a high-fat diet administered with the composition of the present invention>

[0058] 1. Preparation of the composition of the present invention

[0059] (1) L008 group and H008 group: The composition is the red yeast rice of the present invention obtained by fermenting polished rice with the red yeast fungus (Monascus pilosus) of the present invention, as described based on Example 2. The daily doses for adults of the L008 group and H008 group are 0.5 g / day and 2 g / day, respectively, and contain 1.5 mg / day and 6 mg / day of monascinol, respectively.

[0060] (2) Msol group: This group received monascinol, with a daily dose of 6 milligrams / day (mg / day) for adults.

[0061] 2. Experiment to evaluate the effect of improving non-alcoholic fatty liver disease

[0062] (1) Animal breeding and care for animals in experiments The 7-week-old male C57BL / 6J mice used in this experiment were purchased from the National Center for Experimental Animals. Their initial body weight was approximately 23 grams. Twelve mice were assigned to each group and kept in an environment with 60% relative humidity, a room temperature of 25±1°C, and a 12-hour light-12-hour dark cycle (light period from 8:00 to 20:00). The experiment lasted 22 weeks, during which the mice were allowed free feeding and drinking. Their body weight, food intake, and water intake were measured regularly each week.

[0063] (2) Conversion of feeding amounts for animals The method for calculating the feeding dose is based on the initial estimation method (Estimating the maximum safe starting dose in initial clinical trials for therapeutics in adult healthy volunteers) published by the U.S. Food and Drug Administration in 2005, and uses an adult weighing 60 kilograms as the standard. When conducting tests using advanced experimental animals, the dose conversion is, in principle, 12.3 times the recommended daily intake for humans per kilogram of body weight (mg / kg·bw / day) to obtain the recommended daily intake for mice per kilogram of body weight. The mouse group composition and feeding doses are summarized in Table 1. The calculation formula is expressed as shown in equation (1) below.

[0064] Dosage per kg of body weight for mice = Recommended daily intake for humans ÷ Body weight (60 kg) × 12.3 ... Equation (1)

[0065] The daily intake dose for a mouse per kilogram of body weight can be determined according to this formula (1).

[0066] (3) Grouping and induction of experimental animals Example 4 uses a non-alcoholic fatty liver animal model induced by a high-fat diet. In an animal experiment with a time limit of 22 weeks, the animal groupings and the dosage of the test substance are summarized in Table 1. The animal test feeds were formulated using D12450B and D12492, referring to the formulation tables of Research Diets (New Brunswick, NJ, USA), and the formulation tables for the general feed and high-fat feed are summarized in Table 2. The groupings are explained as follows.

[0067] i. ND group: This group consists of normal animals.

[0068] ii. HFD group: This is the control group, consisting of mice with high-fat diet-induced non-alcoholic fatty liver disease.

[0069] iii. Group L008: This group consists of mice with high-fat diet-induced non-alcoholic fatty liver disease that are administered red yeast rice powder daily via tube feeding, which corresponds to a 60-kilogram adult consuming 0.5 g of the red yeast rice of the present invention containing 1.5 mg of monascinol daily.

[0070] iv. Group H008: This group consists of mice with high-fat diet-induced non-alcoholic fatty liver disease that are administered red yeast rice powder daily via tube, which is equivalent to a 60-kilogram adult consuming 2 g of the red yeast rice of the present invention containing 6 mg of monascinol daily.

[0071] v.Msol group: This group consists of mice with high-fat diet-induced non-alcoholic fatty liver disease that receive daily enteral administration of monascinol powder, which is equivalent to a 60-kilogram adult consuming 6 mg of monascinol powder daily.

[0072] [Table 1]

[0073] [Table 2]

[0074] (4) Animal sacrifice and blood collection At the end of the 22-week experiment, mice are fasted for 12-14 hours before blood collection. All mice are euthanized using carbon dioxide, and blood samples are collected via inferior vena cava. The collected blood samples are centrifuged at 4°C and 3,000xg for 15 minutes, and the upper blood fraction is separated and packed into Eppendorf tubes, which are then frozen and stored at -80°C. The liver is weighed, and the second hepatic lobe is excised and preserved in 10% pathological formalin. The remaining liver is washed with 0.9% physiological saline, packed together with the kidneys in a zip-top bag, sealed, and frozen at -80°C.

[0075] (5) Serum vitalization analysis The detection and measurement are performed using an automated biochemical analyzer (model: Beckman-700, purchased from Fullerton, California, USA). Detectable parameters: TC (Total Cholesterol), TG (Triglyceride), AST (Aspartate Aminotransferase) activity, and ALT (Alanine Aminotransferase) activity.

[0076] (6) Extraction and measurement of liver lipids 0.1 g of liver tissue was weighed, and 1 mL of chloroform:methanol (2:1, v / v) was added. After homogenization using a tissue homogenizer, the tissue was centrifuged to obtain the supernatant. The supernatant was dried by aeration and suction to remove the solvent, and finally, dimethyl sulfoxide (DMSO) was added to redissolve the sample. The extract was stored at -20°C. The TC concentration was analyzed using a commercially available biochemical reagent (model number: BXC 0261, Fortress), while the TG concentration was analyzed using a commercially available biochemical reagent (model number: BXC 0271, Fortress). The procedure for this analysis was as described in the instructions for use with the biochemical reagent assay kit.

[0077] (7) Hematoxyline-eosin (H&E) staining of liver tissue Liver tissue, fixed with 10% formalin, was dehydrated and embedded in paraffin to prepare tissue sections. Basic staining was performed using hematoxylin and eosin.

[0078] (8) Biological statistical analysis method All experimental results are expressed as mean ± standard deviation (mean ± SD). Statistical analysis is performed using one-way ANOVA in the Statistical Package for the Social Sciences (SPSS 12.0) system, followed by a comparison of differences between groups using Duncan's test. p < 0.05 indicates a significant difference.

[0079] 3. Analysis of the intestinal flora of mice with non-alcoholic fatty liver disease

[0080] (1) Extraction of gene genomic DNA and PCR extension and purification Whole-genome DNA (QIAamp PowerFecal DNA, Qiagen) is extracted from the sample. The DNA concentration is measured using a Qubit 4.0 fluorometer (Thermo Scientific), and a template DNA is prepared at 1 ng / μL. The full-length sequence of the 16S gene (corresponding range: V1-V9 region) is extended using barcode-specific primers for the 16S gene (Forward primer 1: 5'Phos / GCATC-16-base barcode-SEQ ID NO: 11, Reverse primer 2: 5'Phos / GCATC-16-base barcode-SEQ ID NO: 12). PCR is performed at 95°C for 3 minutes using KAPA HiFi HotStart ReadyMix (Roche). This is followed by 20-27 cycles (sample-dependent), each cycle consisting of 30 seconds at 95°C, 30 seconds at 57°C, and 60 seconds at 72°C. The samples are then held at 72°C for 5 minutes and finally stored at 4°C for use. The PCR product is monitored on a 1% agarose gel. Samples with a bright main band of approximately 1500 bp are selected and purified using AMPure PB Beads to prepare the SMRTbell library.

[0081] (2) Construction and sequencing of the SMRTbell library The SMRTbell library is constructed by extending the full-length sequence of the 16S gene based on primers with barcodes used in the preparation of a multiplex SMRTbell library and the sequencing program (PacBio). Sequencing is performed using a circular consensus sequencing (CCS) model on a PacBio Sequel IIe instrument to generate HiFi reads with a prediction accuracy (Phred Scale) of 30. The test DNA is then sequenced repeatedly. By aligning the sequences obtained from these repeated sequencing attempts, corrections are made for errors that occur in the base sequencing, ultimately achieving a high accuracy of >99.9% (QV30).

[0082] (3) Biological information analysis i. Data processing for base sequence determination Based on the barcode sequence and the extended primer sequence from PCR, data for each sample is assigned from the data below. After removing the barcode sequence and primer sequence, all sample sequences are introduced into QIIME2 (v2019.7.0, https: / / qiime2.org / ) (Bolyen et al., 2019) to create a single corresponding artifact for storage. Using QIIME2 cutadapt, the extended subvariable region primers (forward primer 2: SEQ ID NO: 13, reverse primer 2: SEQ ID NO: 14) are removed, and the sequence from which the primers have been removed is used as QIIME2 DADA2 Input Reads.

[0083] ii. Denoising and species annotation for QIIME2 DADA2 Regarding the denoising and analysis of QIIME2 DADA2 (v2019.7.0, https: / / qiime2.org / ) (Callahan et al., 2016, Quin et al., 2018), first, the sequence filtering is performed by cutting each sequence to a specified length (forward reads: 280 bp, reverse reads: 220 bp) and filtering by setting the MaxEE (forward reads: 2, reverse reads: 2) parameter to the number of bases that the maximum expected error of the sequence can tolerate. Next, denoising is performed using the DADA2 core algorithm, and messages such as sequence richness, quality score, and relationships between sequences are used to correct bases where sequencing errors occurred, and the actual sequence is inferred. After denoising is completed on each of the sequences at both ends, splicing is performed (minimum overlap length of 20 bp, no mismatches are allowed in the overlap region). Finally, chimeric sequences are removed from the spliced ​​sequences. By comparing them with sequences that are relatively rich in the sample, if a sequence itself has low richness but is similar to many other sequences, it can be identified as a chimeric sequence and removed. After noise reduction and analysis, the sequences are used as representative sequences for amplicon sequence variants (ASVs), and an ASV table can be obtained to provide annotations for subsequent species. Using the QIIME2 feature-classifier (v2019.7.0, https: / / qiime2.org / ) (Bokulich et al., 2018), a machine learning-based classification method (classify-sklearn (McKinney, 2010, Pedregosa et al., 2011)) is selected, and a specific variable-region Bayes classifier (naive Bayes classifier) ​​(Wang et al., 2007) is trained with different databases.This classifier is used to annotate the species classification according to the representative sequence of ASVs to obtain classification information, and the composition of the microbial flora of each sample is statistically processed for each taxonomic rank, such as kingdom, phylum, class, order, family, genus, and species. 16S analysis uses the QIIME2 alignment MAFFT method (v2019.7.0, https: / / qiime2.org / ) (Katoh & Standley, 2013, Lane, 1991) and Core Sets (mostly representing prokaryotic taxonomic units) in the GreenGenes database (gg_13_8) (DeSantis et al., 2006, McDonald et al., 2012) or Silva database (v132, 2017.12) (Quast et al., 2012) to perform rapid sequence alignment. Annotation information (Balvociute & Huson, 2017, Gyarmati et al., 2016, Hong et al., 2016) can also be obtained by performing sequence alignment using the latest NCBI database. ITS analysis is performed using unit Annotations are provided using the database (v7.2, 2017.12.01) (Abarenkov et al., 2010, Koljalg et al., 2005, Koljalg et al., 2013, Nilsson et al., 2019). Finally, the sequence information of each sample is homogenized, and the sample with the smallest total number of tags is used as the standard for further homogenization (Schloss et al., 2009). Subsequently, the processed information is used for the subsequent Beta diversity analysis.

[0084] iii. Comparative analysis of sample grouping (beta diversity) UniFrac distances are calculated using Qiime, and a phylogenetic tree showing the similarity of each sample is constructed using the UPGMA cluster analysis method. A figure plotting the PLS-DA analysis using R (v3.3.1) is shown. The PLS-DA analysis was performed using the mixOmics and ggplot2 kits in R, and the Ternary Plot analysis was performed using the ggtern kit in R. Difference analysis between groups of the Beta diversity index is performed using the agricolae kit in R, with parameterized and parameterless validations performed for each, and Tukey validation and Kruskal validation (post-hoc test) are selected.

[0085] The experiment was conducted using the experimental method described in Example 4 above, and the results are summarized below.

[0086] 1. Effects of the composition of the present invention on liver weight and liver / body weight ratio in non-alcoholic fatty liver mice. The results in Table 3 show that, compared to the ND group, the HFD group exhibited an abnormal increase in liver weight. This type of phenomenon suggests that a long-term high-fat diet may lead to abnormalities in liver tissue due to fat accumulation. However, this condition can be significantly improved by feeding the Msol group with the respective doses of the Aspergillus red fermentation product of the present invention (L008 group and H008 group). This significantly reduces liver weight (p<0.05) and also suggests an improvement in the liver-to-body weight ratio.

[0087] [Table 3]

[0088] The experimental data are expressed as mean ± standard deviation (n=8). Mean values ​​for different letters indicate significant differences (p<0.05). The statistical significance of biochemical effects is determined using one-way analysis of variance (ANOVA) and Duncan's multiple comparison test.

[0089] 2. Effects of the composition of the present invention on serum liver function markers AST and ALT content in non-alcoholic fatty liver mice. Table 4 shows that serum AST and ALT activity in the HFD group were significantly higher than in the ND group (p<0.05). This result suggests that long-term high-fat diets may damage liver cells, raising concerns about the possibility of liver damage in non-alcoholic fatty liver disease. Feeding with the fermented products of Aspergillus oryzae of the present invention (L008 group and H008 group) and monascinol (Msol group) at various doses significantly reduced AST and ALT activity induced by long-term high-fat diets. The low dose group of Aspergillus oryzae rice (L008 group) corresponds to an adult consuming 0.5g of Aspergillus oryzae rice daily, demonstrating a significant reduction in AST and ALT activity. In addition, the dietary dose of monascinol, equivalent to an adult consuming 6mg daily, also has the effect of improving AST and ALT levels.

[0090] [Table 4]

[0091] The experimental data are expressed as mean ± standard deviation (n=8). Mean values ​​for different letters indicate significant differences (p<0.05). The statistical significance of biochemical effects is determined using one-way analysis of variance (ANOVA) and Duncan's multiple comparison test.

[0092] 3. Effects of the composition of the present invention on TG content and TC content in the liver of non-alcoholic fatty liver mice. Regarding the analysis of liver lipids, as shown in Table 5, the HFD group increased liver TC and TG concentrations (p<0.05), the low-dose group of the present invention's red yeast rice (L008 group) slightly reduced liver TG production that can occur due to a long-term high-fat diet, and significantly reduced the trend of TC, and the group fed red yeast rice powder to reach a high dose (H008 group) significantly reduced liver TC and TG content (p<0.05). Furthermore, the pure substance Msol group was also able to effectively reduce the accumulation of liver TC and TG (p<0.05). As shown in the above results, it has become clear that the red yeast rice and monascinol of the present invention have the effect of reducing the accumulation of liver TC and TG and have the potential to improve the development of fatty liver. From these results, it has also been demonstrated that monascinol is a functional component in red yeast rice that reduces liver lipids.

[0093] [Table 5]

[0094] The experimental data are expressed as mean ± standard deviation (n=8). Mean values ​​for different letters indicate significant differences (p<0.05). The statistical significance of biochemical effects is determined using one-way analysis of variance (ANOVA) and Duncan's multiple comparison test.

[0095] 4. Effects of the composition of the present invention on liver pathology tissue section images of non-alcoholic fatty liver mice. Figure 7 shows pathological tissue section images of the livers of experimental animals. As can be seen from these results, the liver tissue of the HFD group, which was induced by a long-term high-fat diet, exhibited the generation of a large amount of lipid droplets and a state of lipid infiltration. Furthermore, the pathological evaluation of the liver sections in Table 6 shows that the lipid droplet accumulation can be improved in each group fed with the red yeast rice and its monascinol of the present invention (p<0.05). Moreover, the H008 group showed a better improvement effect than the L008 group, demonstrating a dose-dependent effect. The Msol group was also able to significantly improve lipid droplet accumulation and lipid infiltration in the livers of non-alcoholic fatty liver mice induced by a high-fat diet, and formed a state that reduced pathological changes due to fatty acid formation.

[0096] [Table 6]

[0097] Lesions are ranked on a scale of 1 to 5 based on severity: 0 = no significant lesions, 1 = mild (<10%), 2 = moderate (10-33%), 3 = moderate / severe (33-66%), 4 = severe / high (66-100%). The data is presented in the format mean ± standard deviation (n=8). Significant differences (p<0.05) exist between means of different alphabetical groups. Statistical significance of biochemical effects is determined by one-way analysis of variance (ANOVA) and Duncan's multiple comparison test. Experimental data are presented as mean ± standard deviation (n=8). Significant differences (p<0.05) exist between means of different alphabetical groups. Statistical significance of biochemical effects is determined using one-way analysis of variance (ANOVA) and Duncan's multiple comparison test.

[0098] 5. Effects of feeding red yeast rice and its indicator active ingredient on the intestinal flora of mice with non-alcoholic fatty liver disease. As shown in Figure 8, the analysis of bacterial richness revealed significant changes in the types and richness of bacterial species after induction of a high-fat diet, compared to the normal group, with a particularly noticeable decrease in the Lactobacillus genus. Although there have been changes in the richness of bacterial groups at the genus classification level, the top 10 listed bacterial genera include Lachnospiraceae bacteria, Dubosiella bacteria, Lactobacillus bacteria, Bacteroides bacteria, Eubacterium coprostanoligen group, Ruminococcus bacteria, Ruminiclostridium bacteria, Blautia bacteria, Akkermansia bacteria, and Parabacteroides bacteria.

[0099] As shown in Figure 9, compared to the HFD group, feeding with the red yeast rice (L008 group and H008 group) and monascinol (Msol group) of the present invention was able to improve the microbial communities of Blautia and Intestinimonas in the intestinal tract. Blautia is one of the most abundant genera of intestinal microorganisms and is thought to be able to regulate and exert some effect on inflammatory responses and metabolic diseases, and is a commonly found beneficial bacterium. Intestinimonas, on the other hand, has the characteristic of increasing butyrate production and has anti-inflammatory and anti-obesity effects (Cai et al., 2020). In this comparison of each test group, the high-fat diet group was able to improve the richness of the Blautia and Intestinimonas microbial communities.

[0100] Using partial least squares discriminant analysis (PLS-DA), structural analysis of bacterial communities in samples from different groups was performed. As shown in Figure 10, in non-alcoholic fatty liver mice fed a high-fat diet, the distribution of bacterial communities was significantly different from that of a certain group in the normal group. Furthermore, when we analyzed whether the composition of the present invention (test sample) could effectively improve the bacterial colony composition of mice fed a high-fat diet, the results showed that the L008 and H008 groups administered with the red yeast rice of the present invention had relatively similar bacterial colony compositions and were significantly separated from the HFD group. The Msol group, administered with the active ingredient (monascinol) of the red yeast rice of the present invention, was quite similar to the HFD group in terms of bacterial community composition analysis, but it was revealed that there were significant differences in the analysis results of bacterial community richness.

[0101] Non-alcoholic fatty liver disease (NAFLD) is characterized by degeneration that can occur due to excessive accumulation of fat in the liver, leading to symptoms such as non-alcoholic steatohepatitis (NASH). In animal experiments in Example 4 of the present invention, it has already been demonstrated that the red yeast rice and its active ingredient (monascinol) of the present invention improve the TC ratio and TG ratio (p<0.05) and decrease the liver weight / body weight ratio (p<0.05) in mice with non-alcoholic fatty liver disease. Furthermore, liver section analysis has demonstrated that each test group can significantly alleviate the development of symptoms such as hepatic fat accumulation, fat vesicles, and fat infiltration that can occur due to a long-term high-fat diet, thus demonstrating that the red yeast rice and monascinol of the present invention have the function of improving NAFLD.

[0102] In addition, the ecology of the intestinal microbiota can be highly correlated with the health or disease of the host, including changes in the composition ratio of intestinal microbiota and microbial metabolites (Jiang et al., 2020). Furthermore, compared to the high-fat diet group (HFD group), the red yeast rice (L008 group and H008 group) and monascinol (Msol group) in the present invention's composition (test substance) can improve the richness of the Blautia genus. Literature suggests that Blautia content is strongly correlated with visceral fat accumulation, and its metabolic mechanism can effectively reduce visceral fat (Ozato et al., 2019). In addition, other literature explains that the symbiotic microorganisms of the Blautia genus in the intestinal tract produce Blautia producta, which produces short-chain fatty acids such as acetate, improving insulin sensitivity in the liver via the acetate-FFAR2 signaling pathway and preventing the development of NAFLD (Aoki et al., 2021). Furthermore, the Intestinimonas genus possesses the characteristic of increasing butyrate production, and previous research has demonstrated that Intestinimonas can alleviate the progression of NAFLD through butanoic acid and also possesses potential antioxidant and anti-inflammatory effects (Miao et al., 2022, Wang et al., 2021). The red yeast rice and monascinol of the present invention can also enhance the richness of the Intestinimonas microbial community, making them useful in improving the function of alleviating NAFLD.

[0103] As shown in the experimental results of Example 4 of the present invention, a long-term high-fat diet causes non-alcoholic fatty liver disease in normal mice and affects the composition ratio of their intestinal microbial community. Furthermore, it has become clear that the red yeast rice and monascinol of the present invention can regulate hepatic lipid metabolism and alleviate the progression of NAFLD by improving specific intestinal microorganisms such as Blautia and Intestinimonas, thereby possessing the function of improving NAFLD.

[0104] Furthermore, in this invention, different test substances (compositions) were used for different groups (L008 group, H008 group, and Msol group), and the effect of improving fatty liver in mice with fatty liver induced by a high-fat diet for 22 weeks was evaluated. From the above examples and results, the following conclusions can be drawn.

[0105] 1. The present invention contains monascinol in its red yeast rice (L008 group and H008 group). Monascinol has long been said to be effective in alleviating non-alcoholic fatty liver disease, albeit rarely. The red yeast rice of the present invention shows remarkable effects in improving various items such as liver weight and liver / body weight, serum liver function markers AST activity and ALT activity, liver TG content and TC content, and fatty infiltration pattern of liver tissue. Furthermore, the dosage of the red yeast rice powder of the present invention (L008 group) is 0.5 grams (g) per person per day, and the dosage of monascinol is 6 milligrams (mg) per person per day. The dosage is considerably low, meaning it has the function of preventing and improving fatty liver disease, thus demonstrating considerable inventiveness. In the present invention, monascinol is presented as a new functional ingredient that prevents and improves fatty liver disease.

[0106] 2. The red yeast rice (L008 group and H008 group) and monascinol (Msol group) of the present invention both aim to improve the changes in the composition of intestinal microbiota caused by a high-fat diet, and moreover, they can increase the richness of Blautia and Intestinimonas species in the intestinal tract compared to the HFD group. Since these species have the potential to increase short-chain fatty acid metabolism, have antioxidant and anti-inflammatory effects, and improve NAFLD, it has been demonstrated that the mechanism by which the red yeast rice and monascinol of the present invention improve NAFLD is highly correlated with changes that partially enhance specific bacterial groups in the intestines.

[0107] As described above, the novel Monascus red mold, its compositions, and their uses for the prevention or treatment of non-alcoholic fatty liver disease of the present invention have already been fully and clearly explained. It should be emphasized that the above detailed description specifically illustrates possible embodiments of the present invention, and the scope of the patent of the present invention is not limited to these embodiments. Any implementation or modification of the same effects, as long as it does not deviate from the spirit of the art of the present invention, is still included within the scope of the claims of this application.

[0108] The Monascus pilosus species of the present invention was deposited with the National Industrial, Food and Marine Bacteria Collections Ltd (NCIMB Ltd) in the United Kingdom on January 12, 2023 (date of deposit), with accession number NCIMB 44103.

Claims

1. This is a type of Monascus pilosus characterized by being deposited with the National Industrial, Food and Marine Bacteria Collections Ltd. (NCIMB Ltd.) in the United Kingdom under accession number NCIMB 44103.

2. Use of Monascus pilosus, characterized by being deposited with the National Industrial, Food and Marine Bacteria Collections Ltd. (NCIMB Ltd) in the United Kingdom under accession number NCIMB 44103, in the manufacture of compositions for the prevention or treatment of non-alcoholic fatty liver disease.

3. A composition for use in the prevention or treatment of non-alcoholic fatty liver disease, characterized in that it contains an effective amount of a functional component, the functional component being extracted from a red yeast rice ferment product that can be produced by fermenting a substrate using Monascus poultry, and the Monascus poultry being Monascus poultry deposited with the National Industrial, Food and Marine Bacteria Collection Ltd (NCIMB Ltd) in the United Kingdom under accession number NCIMB 44103.

4. The composition according to claim 3, characterized in that the non-alcoholic fatty liver is caused by at least one condition selected from the group consisting of obesity, hypertension, hyperlipidemia, and diabetes.

5. The composition according to claim 3, characterized in that it reduces serum liver function markers AST and ALT.

6. The composition according to claim 3, characterized in that it suppresses the accumulation of fat in liver tissue.

7. The composition according to claim 3, characterized in that it reduces the content of total cholesterol (TC) and triglycerides (TG) in the liver.

8. The composition according to claim 3, characterized in that it improves lipid droplet accumulation and fat infiltration in the liver.

9. The composition according to claim 3, characterized in that the composition has the function of improving the richness of beneficial intestinal bacteria and the function of changing the composition of intestinal bacteria.

10. The composition according to claim 3, characterized in that the functional component comprises at least one selected from the group consisting of monascinol, ankaflavin, and monascin.

11. The composition according to claim 3, characterized in that the substrate is a mixture of rice, yam, or related carbohydrates.

12. The composition according to claim 3, characterized in that the effective amount is such that an adult ingests at least 1.5 milligrams to 12 milligrams of the functional ingredient daily when used alone.

13. A composition for use in the prevention or treatment of non-alcoholic fatty liver disease, characterized in that it contains an effective amount of red yeast rice ferment, the red yeast rice ferment can be produced by fermenting a substrate using Monascus pylori, and the Monascus pylori is Monascus pylori deposited with the National Industrial, Food and Marine Bacteria Collection Ltd (NCIMB Ltd) in the United Kingdom under accession number NCIMB 44103.

14. The composition according to claim 13, characterized in that the non-alcoholic fatty liver is caused by at least one condition selected from the group consisting of obesity, hypertension, hyperlipidemia, and diabetes.

15. The composition according to claim 13, characterized in that it reduces serum liver function markers AST and ALT.

16. The composition according to claim 13, characterized in that it suppresses the accumulation of fat in liver tissue.

17. The composition according to claim 13, characterized in that it reduces the content of total cholesterol (TC) and triglycerides (TG) in the liver.

18. The composition according to claim 13, characterized in that it improves lipid droplet accumulation and fat infiltration in the liver.

19. The composition according to claim 13, characterized in that the composition has the function of improving the richness of beneficial intestinal bacteria and the function of changing the composition of intestinal bacteria.

20. The composition according to claim 13, characterized in that the substrate is a mixture of rice, yam, or related carbohydrates.

21. The composition according to claim 13, characterized in that the effective amount is such that an adult ingests at least 0.5 grams daily, and it contains 1.5 milligrams of monascinol.

22. The composition according to claim 13, wherein the red yeast rice ferment product contains at least one functional component, and the functional component comprises at least one selected from the group consisting of monascinol, ankaflavin, and monascin.

23. The composition according to claim 13, characterized in that the composition is a food composition, a pharmaceutical composition, a feed composition, a nutritional supplement composition, a dietary supplement composition, or a food additive composition.

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