Monascus, composition for preventing or treating non-alcoholic fatty liver disease, and use of monascus

Abstract

The main purpose of the present invention is to provide monascus and a composition for preventing or treating non-alcoholic fatty liver disease, the composition comprising the monascus and / or a monascus fermentation product thereof, or a functional component comprising the monascus fermentation product. The invention also aims to provide application of the monascus in preparation of the composition for preventing or treating the non-alcoholic fatty liver disease. The composition not only has the efficacy of reducing accumulation of total cholesterol (TC) and triglyceride (TG) in the non-alcoholic fatty liver disease caused by obesity, but also has the efficacy of reducing serum liver function indexes of asparaginic acid transaminase (AST) and alanine transaminase (ALT), so that the composition has the efficacy of preventing or treating the non-alcoholic fatty liver disease and related metabolic diseases thereof. , for example hyperlipidemia.

Description

Technical Field

[0001] This invention relates to the technical field of preventing or treating non-alcoholic fatty liver disease, and more particularly to a technical field of using a composition comprising Monascus purpureus, its fermented product, or the active ingredient of the fermented product for the prevention or treatment of non-alcoholic fatty liver disease. Background Technology

[0002] Nonalcoholic fatty liver disease (NAFLD) is one of the most common liver diseases in the Western world, affecting approximately 20% to 30% of the general adult population, with a prevalence reaching 70% to 90% in obese and diabetic patients. Research indicates that the prevalence of NAFLD is approximately 11.5%. Insulin resistance is known to be a significant cause of NAFLD, thus there is a clear correlation between NAFLD and metabolic syndrome. Increasing evidence suggests that NAFLD increases the risk of cardiovascular disease, and this risk increases with the severity of NAFLD.

[0003] Nonalcoholic fatty liver disease (NAFLD) refers to excessive fat accumulation in liver cells, not caused by alcohol. Generally, a small amount of fat in the liver is normal, but when more than 5%-10% of the liver's weight is fat, it is called fatty liver. NAFLD often occurs in overweight, obese individuals, or those with diabetes, high cholesterol, or high triglycerides. Rapid weight loss and poor dietary habits can also lead to NAFLD. The harm of NAFLD to the body should not be ignored. Over time, fatty liver can lead to cirrhosis, and even liver cancer or liver failure. Besides directly causing decompensated cirrhosis, hepatocellular carcinoma, and recurrence of transplanted liver, NAFLD can also affect the progression of other chronic liver diseases and participate in the pathogenesis of type 2 diabetes and atherosclerosis. NAFLD can also cause liver damage similar to that caused by alcoholism, and in the most severe cases, it can develop into cirrhosis or liver failure.

[0004] The gut microbiota refers to the vast community of bacteria residing in the human intestine. It has a significant and important impact on various physiological and pathological phenomena in the human body. Fatty liver and the gut microbiota have a close interaction, known as the gut-hepatic axis. The composition and metabolites of the gut microbiota affect hepatic lipid metabolism, inflammatory responses, and the degree of fibrosis. Some studies have found that patients with non-alcoholic fatty liver disease have reduced gut microbiota diversity, making them more susceptible to the adverse effects of microecological dysbiosis. Furthermore, abnormal gut microbiota can produce harmful substances such as phenylacetic acid, which enter the liver via the portal vein, promoting the formation and progression of fatty liver. Therefore, adjusting the gut microbiota ecology, such as increasing the intake of probiotics, may help improve the condition of fatty liver.

[0005] Therefore, in order to prevent or treat non-alcoholic fatty liver disease caused by overweight and obesity, the inventors of this invention have made great efforts in research and development, and have finally completed the development of the red yeast rice, the composition for the prevention or treatment of non-alcoholic fatty liver disease, and the uses of the red yeast rice. In daily life, obese patients and patients with hyperlipidemia can prevent or treat non-alcoholic fatty liver disease through the easily accessible and edible composition of this invention without causing adverse effects on their health. Summary of the Invention

[0006] The main objective of this invention is to provide a Monascus pilosus and a composition for the prevention or treatment of non-alcoholic fatty liver disease, the composition comprising Monascus pilosus and / or its fermented product, or comprising monascinol purified from the fermented product, wherein the composition for the prevention or treatment of non-alcoholic fatty liver disease can be ingested orally.

[0007] To achieve the above objectives, this invention proposes a novel Monascus pilosus, which is registered at the National Centre for Food Industries and Marine Bacteria (NCIMB Ltd) in the United Kingdom with the registration number NCIMB 44103.

[0008] Furthermore, the present invention provides a composition for the prevention or treatment of non-alcoholic fatty liver disease, comprising an effective amount of a red yeast ferment, wherein the red yeast ferment is prepared by fermenting a substrate with a red yeast strain, and the red yeast strain is Monascus pilosus registered at the National Centre for Food Industry and Marine Bacteria (NCIMB Ltd) in the United Kingdom with the registration number NCIMB 44103.

[0009] The non-alcoholic fatty liver disease (NAFLD) prevented or treated by the above-described composition of the present invention includes NAFLD caused by at least one of the following conditions: obesity, hypertension, hyperlipidemia, and diabetes. Furthermore, the composition can reduce serum liver function indices AST and ALT, and inhibit fat accumulation in liver tissue. Further, the composition can reduce the levels of total cholesterol (TC) and triglycerides (TG) in the liver, and improve the accumulation of oil droplets and fatty infiltration in the liver caused by NAFLD. In addition, the composition has the effect of increasing the abundance of beneficial intestinal flora and altering the composition of the intestinal flora.

[0010] Furthermore, the matrix in the above-mentioned composition of the present invention is rice, yam, or a mixture of related carbohydrates.

[0011] Furthermore, the effective amount in the above-mentioned composition of the present invention is at least 0.5 grams of the red yeast rice fermented product per day for an adult, which contains 1.5 mg of monascinol, wherein the red yeast rice fermented product contains at least one active ingredient, and the active ingredient includes at least one selected from the group consisting of monascinol, ankaflavin and monascin.

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

[0013] Furthermore, the present invention provides a composition for the prevention or treatment of non-alcoholic fatty liver disease, comprising an effective amount of an active ingredient extracted from a red yeast ferment, wherein the red yeast ferment is prepared by fermenting a substrate with a red yeast strain, and the red yeast strain is Monascus pilosus registered at the National Centre for Food Industry and Marine Bacteria (NCIMB Ltd) in the United Kingdom with the registration number NCIMB 44103.

[0014] The non-alcoholic fatty liver disease (NAFLD) prevented or treated by the above-described composition of the present invention includes NAFLD caused by at least one of the following conditions: obesity, hypertension, hyperlipidemia, and diabetes. Furthermore, the composition can reduce serum liver function indices AST and ALT, and inhibit fat accumulation in liver tissue. Further, the composition can reduce the levels of total cholesterol (TC) and triglycerides (TG) in the liver, and improve the accumulation of oil droplets and fatty infiltration in the liver caused by NAFLD. In addition, the composition has the effect of increasing the abundance of beneficial intestinal flora and altering the composition of the intestinal flora.

[0015] Furthermore, the active ingredient in the above-described composition of the present invention comprises at least one selected from the group consisting of Monascinol, Ankaflavin, and Monascin. The matrix in the above-described composition of the present invention is rice, yam, or a mixture of related carbohydrates, and the effective amount, when used alone, is at least 1.5 mg to 12 mg or at least 6 mg of the active ingredient per day for an adult.

[0016] Furthermore, the present invention provides the use of Monascus pilosus in the preparation of a composition for the prevention or treatment of non-alcoholic fatty liver disease, wherein the Monascus pilosus is registered at the National Centre for Food Industry and Marine Bacteria (NCIMB Ltd) in the United Kingdom with the registration number NCIMB 44103.

[0017] The term "obesity" as used in this article is defined as abnormal or excessive fat accumulation that may impair health. Irregular eating habits, excessive food intake, lack of physical activity, endocrine disorders, genetic factors, psychological factors, and medications can all contribute to obesity. Furthermore, obesity increases the risk of developing arteriosclerosis, cardiovascular disease (stroke and ischemic cardiovascular disease), hypertension, diabetes, hyperlipidemia, and fatty liver disease.

[0018] The term "hyperlipidemia" as used in this article refers to an excess of lipids or fats in the body, such as cholesterol and triglycerides. The term "hypertension" refers to an excessively high blood pressure (the pressure exerted by blood on the walls of blood vessels), and studies have shown a positive correlation between triglycerides and blood pressure (including systolic and diastolic blood pressure).

[0019] The term "hepatic steatosis" as used in this article refers to a common condition caused by excessive fat accumulation in the liver. A healthy liver contains a small amount of fat, but when fat accounts for 5% to 10% of the liver's weight, it becomes a problem, causing various diseases such as angina, myocardial infarction, stroke, arteriosclerosis, pancreatitis, or other similar conditions. Furthermore, fatty liver is divided into alcoholic fatty liver disease caused by alcohol consumption and non-alcoholic fatty liver disease (NAFLD) not caused by alcohol.

[0020] The term "non-alcoholic fatty liver disease" used in this article refers to fatty liver caused by excessive fat accumulation in the liver that is not caused by alcohol. The literature also mentions that obesity, hypertension, hyperlipidemia (such as high cholesterol or triglycerides), and diabetes are all factors that may cause fat accumulation in the liver. Attached Figure Description

[0021] Figure 1 The process for identifying Monascus species in this invention;

[0022] Figure 2 This is a phylogenetic tree of the β-tubulin sequence of Monascus purpureus according to the present invention, obtained by comparison.

[0023] Figure 3 This is a phylogenetic tree of the ITS sequence of Monascus purpureus according to the present invention, obtained by comparison.

[0024] Figure 4 The results of species-specific PCR analysis of Monascus purpureus in this invention are shown.

[0025] Figure 5 The results of PCR analysis of the pksCT gene of Monascus purpureus in this invention;

[0026] Figure 6 This describes the morphology of the ascocarp of Monascus purpureus according to the present invention;

[0027] Figure 7 The image shows the effect of red yeast rice and its monascinol on the pathological sections of liver tissue in mice on a long-term high-fat diet.

[0028] Figure 8 The effects of red yeast rice and its monascinol on the gut microbiota of mice on a long-term high-fat diet were shown by high-throughput sequencing of the relative abundance of the top 10 gut microbiota genera.

[0029] Figure 9 The abundance of bacterial communities in each experimental group (L008, H008, and Msol groups) compared to the HFD group; and

[0030] Figure 10 Partial least squares discriminant analysis (PLS-DA) was performed on the gut microbiota β diversity of each experimental group (ND, HFD, L008, H008 and Msol groups). Detailed Implementation

[0031] All technical and scientific terms used in this specification, unless otherwise defined, have the meanings commonly understood by one of ordinary knowledge in the art. The invention will be illustrated by the following embodiments, but these are illustrative only and not limiting; the invention is not limited to the embodiments described below. Unless otherwise stated, all materials used in this invention are commercially available and readily available; the following are merely examples of where they can be obtained.

[0032] This invention provides a novel *Monascus pilosus* strain registered under registration number NCIMB 44103 at the National Centre for Food Industry and Marine Bacteria (NCIMB Ltd). Furthermore, this invention provides a composition for the prevention or treatment of non-alcoholic fatty liver disease (NAFLD), comprising an effective amount of a red yeast ferment, wherein the red yeast ferment is obtained by fermentation of *Monascus pilosus* registered under registration number NCIMB 44103 at the National Centre for Food Industry and Marine Bacteria (NCIMB Ltd). Further, this invention provides another composition for the prevention or treatment of NFLD, comprising an effective amount of an active ingredient extracted from a red yeast ferment, wherein the red yeast ferment is obtained by fermenting a substrate with a red yeast strain registered under registration number NCIMB 44103 at the National Centre for Food Industry and Marine Bacteria (NCIMB Ltd). Furthermore, the active ingredient is Monascinol, and the matrix is ​​a mixture of rice, yam, or related carbohydrates. Moreover, experiments conducted on mice with non-alcoholic fatty liver disease induced by a high-fat diet confirmed that the Monascus purpureus of this invention and the compositions prepared therefrom have the function of preventing or treating non-alcoholic fatty liver disease.

[0033] The aforementioned Monascus strains of the present invention also include their progeny through subculture or mutant strains, but still possess the same characteristics, genomic features, or uses (for the prevention or treatment of non-alcoholic fatty liver disease) as those described in the present invention.

[0034] The compositions described herein may include, but are not limited to, food, beverages, health foods, animal drinking water additives, animal feed additives, animal and human pharmaceutical compositions, food additives, beverage additives, and other application forms applicable to this invention.

[0035] The terms "prevention" and "treatment" mean that, compared to compositions containing Monascus purpureus, its fermented form, or an extract thereof, the compositions of the present invention can effectively prevent or treat non-alcoholic fatty liver disease.

[0036] The term "effective dose" refers to the effective amount of an active ingredient that can effectively prevent or treat non-alcoholic fatty liver disease, and may also be called a "therapeutic effective dose" or "improving effective dose." The term "pharmaceutical acceptable" means that the substance or composition must be compatible with other components of the formulation and be harmless to the patient.

[0037] The composition of the present invention can be prepared into a dosage form suitable for the composition of the present invention by means of techniques known to those skilled in the art, by combining the aforementioned Monascus purpureus, its fermentation product, or an extract of the fermentation product with a pharmaceutically acceptable vehicle; wherein the dosage form includes, but is not limited to: solution, emulsion, suspension, powder, tablet, pill, lozenge, troche, capsule, and other similar or applicable dosage forms of the present invention.

[0038] The above composition may also contain one or more commonly used dissolving agents, buffers, preservatives, colorants, flavorings, and excipients, as needed.

[0039] In another preferred embodiment, the aforementioned composition provided by the present invention can be further added to an edible material to prepare a food product or health product; wherein the edible material includes, but is not limited to: water; liquid milk product; milk; concentrated milk; fermented milk, such as yogurt, frozen yogurt, sour milk, lactic acid bacteria fermenting beverage; 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; herbal medicine. Medicine; dietary supplements, etc.

[0040] Furthermore, the present invention also provides a method for preventing or treating non-alcoholic fatty liver disease, which involves administering an effective amount of the aforementioned composition to an individual with excessive liver fat for the prevention or treatment of non-alcoholic fatty liver disease.

[0041] 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 purpureus, Monascus purpureus fermentation product, or extract of the Monascus purpureus fermentation product.

[0042] The composition for the prevention or treatment of non-alcoholic fatty liver disease provided by the present invention can be administered via a route that can be adjusted as needed and is not particularly limited, but a preferred route of administration is oral administration of an appropriate dosage form.

[0043] Example 1: Identification of the novel Monascus pilosus strain of this invention

[0044] 1. Basis and Procedure for Strain Identification

[0045] Traditional classification and identification of Monascus purpureus mainly rely on colony morphology, with colony size and color being the primary criteria (Hawksworth and Pitt, 1983). However, some Monascus strains are difficult to classify based on colony appearance alone (e.g., Monascus pilosus and Monascus ruber). Therefore, molecular typing methods are used for Monascus species identification. Currently, molecular typing methods for Monascus purpureus primarily rely on β-tubulin and ITS sequence alignment (Park et al., 2004). However, to improve the accuracy of species identification, this invention, in addition to using β-tubulin and ITS sequence alignment, also employs a species-specific polymerase chain reaction (PCR) and pksCT gene PCR developed in the inventors' laboratory to further enhance the accuracy of species identification (see [link to invention]). Figure 1 ).

[0046] 2. Strains culture and deoxyribonucleic acid (DNA) extraction

[0047] Potato dextrose broth (PDB) medium was purchased from Difco (Becton-Dickinson Diagnostic System, Sparks, MD, USA). DNA samples were extracted from the test strain cultured in PDB medium. After 7 days of incubation at 28°C, the bacterial cells were collected, ground into powder in liquid nitrogen, and then dried in a vacuum oven. 0.1 g of the dried bacterial powder was weighed and placed into a 2 mL microcentrifuge tube, and DNA extraction was performed using the QIAamp DNA Mini Kit (purchased from QIAGEN NV, Velno, The Netherlands).

[0048] 3. ITS and β-tubulin sequence alignment analysis and species-specific PCR analysis

[0049] (1) PCR amplification of ITS sequence

[0050] The total volume of the PCR reaction was 25 μL, containing 1x PCR buffer, 0.05 mM of four deoxynucleoside triphosphates (dNTPs), 5 U ExSel highfidelity DNA polymerase (purchased from Bertec Enterprise Co., Ltd., Taipei, China), 0.2 μM primer ITS1 (Sequence ID Number 1) and primer ITS4 (Sequence ID Number 2), and 0.16 μg of template DNA. The PCR reaction conditions were: initial denaturation at 95°C for 5 minutes, followed by 35 cycles of 95°C for 30 seconds, 62°C for 30 seconds, and 70°C for 1 minute, with a final incubation at 70°C for 10 minutes, then stored at 4°C. After the product was confirmed by electrophoresis, it was sent to Kelonmix Biotechnology (Taipei, Taiwan) for DNA sequencing.

[0051] (2) PCR amplification of β-tubulin sequence

[0052] The total volume of the PCR reaction was 25 μL, containing 1x PCR buffer, 0.05 mM dNTPs, 5 U Exsel highfidelity DNA polymerase, 0.12 μM β-tubulin forward primer (primer β-tubulin F, Sequence ID Number 3) and β-tubulin reverse primer (primer β-tubulin R, Sequence ID Number 4) (Parket al., 2004), and template DNA (0.16 μg). The PCR reaction conditions were: initial denaturation at 95°C for 5 minutes, followed by 35 cycles of 95°C for 30 seconds, 55°C for 2 minutes, and 70°C for 2 minutes, with a final cycle of 70°C for 10 minutes, followed by storage at 4°C. After electrophoresis confirmation, the products were sent to Gironmix Biotechnology for DNA sequencing.

[0053] (3) Species-specific PCR of Monascus purpureus (M. purpureus)

[0054] The *M. purpureus* species-specific PCR was designed based on the residual *mokH* fragment of the *monacolin K* biosynthesis gene, unique to *M. purpureus*, and can effectively identify whether *Monascus purpureus* belongs to *M. purpureus*. The total volume of the PCR reaction was 25 μL, containing 1x PCR buffer, 0.05 mM dNTPs, 5 U DNA polymerase (SupeTherm GOLD DNA polymerase), 0.12 μM MPuS1 primer (sequence ID number 5) and MPuS2 primer (sequence ID number 6), and template DNA (0.16 μg). The PCR reaction conditions were: initial denaturation at 95°C for 10 minutes, followed by 35 cycles of 95°C for 30 seconds and 60°C for 1 minute, and finally 70°C for 10 minutes before electrophoresis analysis.

[0055] (4) Specific PCR of Monascus pilosus / ruber (M. pilosus / rube)

[0056] The *M. pilosus / ruber* species-specific PCR was designed based on a conserved fragment of the FAS gene in the pigment biosynthesis gene cluster within the *M. ruber* genome, effectively identifying whether *Monascus purpureus* belongs to *M. pilosus / ruber*. The total PCR reaction volume was 25 μL, containing 1x PCR buffer, 0.05 mM dNTPs, 5 U SupeTherm GOLD DNA polymerase, 0.12 μM RubPil forward primer (primer RubPil F, Sequence ID Number 7) and RubPil reverse primer (primer RubPil R, Sequence ID Number 8), and template DNA (0.16 μg). The PCR reaction conditions were: initial denaturation at 95°C for 10 minutes, followed by 35 cycles of 95°C for 30 seconds and 60°C for 1 minute, and finally, 70°C for 10 minutes before electrophoresis analysis.

[0057] (5) pksCT gene PCR

[0058] The pksCT gene is the core gene for the synthesis of citrinin in *M. purpureus*. This gene segment has been lost in the *M. pilosus / ruber* genome; therefore, this gene can be used as an auxiliary criterion for identifying *M. pilosus / ruber*. The total volume of the PCR reaction was 25 μL, containing 1x PCR buffer, 0.05 mM dNTPs, 5 U SupeTherm GOLD DNA polymerase, 80 nM pksCT-M reverse primer (primer pksCT-MR, Sequence ID Number 9), pksCT-M forward primer (primer pksCT-MF, Sequence ID Number 10), and template DNA (10 ng). The PCR reaction conditions were: initial denaturation at 95°C for 10 minutes, followed by 30 cycles of 95°C for 30 seconds, 54°C for 30 seconds, and 72°C for 40 seconds, and finally, electrophoresis analysis at 70°C for 10 minutes.

[0059] (6) Sequence alignment analysis

[0060] Sequence alignment analysis was performed using Geneious 8.1.9 software (Biomatters Ltd., Auckland, New Zealand). Sequence assembly was performed using Geneious's built-in assembler, multiple alignment was performed using MAFFT 7.017, the maximum likelihood phylogenetic tree was built using MEGA, the nucleotide substitution pattern was General Time Reversible (GTR), and 1000 bootstraps were performed. Bayesian phylogenetic analysis and post-hoc probability testing were performed using MrBayes, with the nucleotide substitution pattern set to GTR, and Aspergillus terreus as the outgroup.

[0061] ITS and β-tubulin sequence alignment analysis results

[0062] The phylogenetic relationships of the β-tubulin and ITS sequences of Monascus purpureus of this invention, after alignment, are as follows: Figure 2 and Figure 3As shown. β-tubulin sequence alignment results show that the *Monascus purpureus* strain of this invention belongs to the same clade as *M. pilosus* and *M. ruber*, with a substitution support of 97% (>50%). ITS sequence alignment results show that the *Monascus purpureus* strain of this invention belongs to the same monophyletic group as *M. purpurerus*, with a substitution support of 62% (>50%) (see...). Figure 3 ). Figure 2 and Figure 3 The results consistently showed that the *Monascus* species of this invention belongs to either *M. pilosus* or *M. ruber*. The results of species-specific PCR analysis were consistent with the results of the phylogenetic analysis (see [link to phylogenetic analysis]). Figure 4 Where RubPil: M. ruber / pilosus – specific, Mpus: M. purpureus specific). PCR results for the pksCT gene also showed that the pksCT gene was not present in the Monascus purpureus genome of this invention (see...). Figure 5 Among them, NTU 568 Monascus purpureus (positive control) belongs to the species of M. pilosus and M. ruber.

[0063] Furthermore, the current taxonomic distinction between *M. pilosus* and *M. ruber* lies in the presence or absence of pigment on the ascocarp shell (*M. ruber* is typically brown, while *M. pilosus* is colorless). The results show that the ascocarps of the *M. pilosus* strain of this invention are all colorless (see...). Figure 6 Therefore, the species of Monascus purpureus in this invention is determined to be M. pilosus.

[0064] Therefore, the ITS sequence alignment, β-tubulin sequence alignment, strain-specific PCR, and pksCT gene PCR results in Example 1 above all consistently show that the Monascus pilosus of this invention belongs to either M. pilosus or M. ruber. Further observation of the ascocarp morphology confirmed that the Monascus pilosus of this invention is M. pilosus. Furthermore, the ITS and β-tubulin sequence alignment analysis results also indicate that the Monascus pilosus SWM-008 provided by this invention is a novel Monascus pilosus isolate.

[0065] Example 2: Method for culturing Monascus purpureus according to the present invention

[0066] 1. Inoculum culture:

[0067] (1) Weigh 2g of rice flour and place it into a 500mL triangular culture flask with a groove in the bottom. Add 100mL of reverse osmosis water (RO water), plug with a breathable silicone stopper and shake well. Sterilize at 1.25 atm and 121℃ for 20 minutes. After sterilization, cool at room temperature for later use.

[0068] (2) Take 3 small pieces of red yeast rice culture from the culture dish and place them into a 500mL shake flask. Seal the flask with a breathable silicone stopper and incubate at 30℃ with shaking at 150-200rpm for 48-72 hours.

[0069] 2. Solid-state culture

[0070] (1) Inoculation: Soak 300g of rice in RO water overnight, drain the water, wrap it in a cloth, place it in a tray, sterilize (121℃, 30min), cool it and inoculate. Take the red yeast culture liquid and inoculate it into the solid substrate (10%), and stir it thoroughly.

[0071] (2) The cultivation steps are as follows:

[0072] a. Culture conditions: After inoculation, wrap the inoculated container in a cloth and place it in a constant temperature and humidity room at 30℃ and 60% relative humidity for culture.

[0073] b. Replenishing water: As the red yeast rapidly multiplies, some of the moisture in the solid substrate evaporates due to the rising temperature, while most is consumed by the growth process, causing the substrate to dry out and requiring replenishment. Replenish approximately 30-50 mL of sterile water daily from day 2 to day 11, then replenish water every other day until the koji is harvested.

[0074] c. Harvesting: Place the red yeast rice fermented material that has been cultured for 21 days (cultured for 14-28 days) in an oven to dry (37℃, 24hr), and store it after drying; this red yeast rice fermented material is red yeast rice.

[0075] Example 3: Monascinol Purification and Separation Method Steps

[0076] 1. Weigh out approximately 1000g of red yeast rice.

[0077] 2. Add about 10 liters (L) of 95% EtOH and extract in a 60°C water bath, shaking evenly every 30 minutes during the extraction. Continue extraction for 2 hours, filter out the extract with filter paper, and repeat the extraction once more.

[0078] 3. Concentrate the filtered extract under reduced pressure to a thick consistency, add 2 to 2.5 times its weight of silica gel, mix and concentrate under reduced pressure until dry, then freeze dry overnight in a freeze dryer and weigh.

[0079] 4. Weigh approximately 7 to 8 times the weight of the silica gel from step 3 above, mixed with Hex:EtOAc (8:2) solvent, and fill it into an open chromatography column. After the chromatography column is equilibrated, spread the mixture of silica gel from step 3 above with the extract on top and allow it to run.

[0080] 5. Elute the chromatography column sequentially with Hex:EtOAc (8:2), Hex:EtOAc (7.5:2.5), Hex:EtOAc (7:3), and Hex:EtOAc (6:4). Collect the Hex:EtOAc (7:3) eluent.

[0081] 6. The Hex:EtOAc (7:3) eluent was concentrated to dryness under reduced pressure and then reconstituted with MeOH. Finally, it was purified by preparative HPLC with the solvent conditions of MeOH:ddH2O (83:17). The monascinol eluent was collected, concentrated to dryness under reduced pressure, and then the residual water was removed by vacuum drying to obtain pure monascinol.

[0082] Example 4: Method for conducting experiments using the composition of the present invention in a non-alcoholic fatty liver animal model induced by a high-fat diet.

[0083] 1. Preparation of the composition of the present invention

[0084] (1) Groups L008 and H008: The composition is the red yeast rice of the present invention obtained by fermenting rice with the red yeast of the present invention according to Example 2. The daily dosage for adults in groups L008 and H008 is 0.5 g / day and 2 g / day, respectively, containing monascinol 1.5 mg / day and 6 mg / day.

[0085] (2) Msol group: Monascinol, the adult daily dose is 6 mg / day.

[0086] 2. An experiment evaluating the efficacy in improving non-alcoholic fatty liver disease.

[0087] (1) Animal experimental feeding and care

[0088] The male C57BL / 6J strain mice used in this experiment were purchased from a professional laboratory animal center at 7 weeks of age. Starting weight was approximately 23 grams, with 12 mice per group. They were housed at a relative humidity of 60%, a room temperature of 25±1℃, and a 12-hour light cycle (8:00–20:00). The experiment lasted 22 weeks, during which mice were allowed free access to food and water, and their weight, food intake, and water intake were measured weekly.

[0089] (2) Animal feeding dosage conversion

[0090] The dosage calculation was based on the estimation method for the initial clinical trials of therapeutics in adult healthy volunteers published by the U.S. Food and Drug Administration in 2005. Using a 60 kg adult as the baseline, the dosage conversion principle for mice using higher laboratory animals was generally 12.3 times the recommended daily intake per kilogram of body weight for humans (mg / kg bw / day). The mouse group configuration and feeding dosage are shown in Table 1. The calculation formula is as follows:

[0091] The recommended intake per kilogram of body weight for mice is calculated as follows: Recommended intake for humans ÷ body weight 60kg × 12.3. Based on this, the daily intake per kilogram of body weight for mice can be calculated.

[0092] (3) Grouping and induction of experimental animals

[0093] Example 4: A non-alcoholic fatty liver disease (NAFLD) animal model induced by a high-fat diet. The animal experiment lasted 22 weeks. Animal grouping and test substance dosages are shown in Table 1. The experimental animal diets (D12450B and D12492) were formulated according to the Research Diets (New Brunswick, NJ, USA). The formulations of the general diet and the high-fat diet are shown in Table 2. Grouping is as follows:

[0094] i.ND group: Normal animal group.

[0095] ii. HFD group: control group, mice with non-alcoholic fatty liver induced by a high-fat diet.

[0096] iii. Group L008: Non-alcoholic fatty liver mice induced by a high-fat diet were fed red yeast rice powder daily via tube feeding, equivalent to 0.5g of the red yeast rice of this invention per day for a 60kg adult, containing 1.5mg of monascinol.

[0097] iv.H008 group: Non-alcoholic fatty liver mice induced by a high-fat diet were fed red yeast rice powder daily via tube feeding, equivalent to 2g of the red yeast rice of this invention per day for a 60kg adult, containing 6mg of monascinol.

[0098] v.Msol group: Non-alcoholic fatty liver mice induced by a high-fat diet were fed monascinol powder daily via tube feeding, equivalent to 6 mg monascinol powder per day for a 60 kg adult.

[0099] Table 1. Animal grouping and experimental substances in the high-fat diet-induced non-alcoholic fatty liver injury test in mice.

[0100]

[0101] Table 2. Feed formulations for non-alcoholic mouse animal experiments induced by a high-fat diet

[0102]

[0103]

[0104] (4) Animal sacrifice and blood collection

[0105] In week 22 of the experiment, mice were fasted for 12–14 hours before blood collection. All mice were euthanized using carbon dioxide, and blood samples were collected via inferior vena cava. The blood samples were centrifuged at 4°C and 3,000 x g for 15 minutes. The supernatant was aliquoted into eppendorf tubes and frozen at -80°C. The livers were weighed, and the second largest lobe was removed and preserved in 10% formalin for pathological purposes. The remaining liver tissue was washed with 0.9% saline, and the kidneys were placed in resealable bags and frozen at -80°C.

[0106] (5) Serum biochemical analysis

[0107] The following tests were performed using an automated biochemical analyzer (Beckman-700, purchased from Fullerton, California, USA). The tests measured TC (Total Cholesterol), TG (Triglycerides), AST (Aspartate Transaminase), and ALT (Alanine Transaminase) activities.

[0108] (6) Liver Lipid Extraction and Determination: Weigh 0.1 g of liver tissue and add 1 mL of chloroform:methanol (2:1, v / v). Homogenize with a tissue homogenizer, centrifuge, and collect the supernatant. Dry the supernatant under vacuum to remove the solvent, and finally add dimethyl sulfoxide (DMSO) to reconstitute the sample. Store the extract at -20℃. Analyze the TC concentration using a commercially available biochemical reagent (model BXC 0261, Fortress) and the TG concentration using a commercially available biochemical reagent (model BXC 0271, Fortress). The operating procedure is as shown in the instructions for the biochemical reagent kit.

[0109] (7) Hematoxyline-eosin (H&E) staining of liver tissue

[0110] The liver was fixed in 10% formalin, dehydrated, and embedded in paraffin to prepare tissue sections. Routine staining with hematoxylin and eosin was performed.

[0111] (8) Biostatistical Analysis Methods

[0112] All experimental results are expressed as mean ± standard deviation (mean ± SD). Statistical analysis was performed using one-way ANOVA with the Statistical Package for the Social Sciences (SPSS 12.0) system, followed by Duncan's test for inter-group differences. p < 0.05 was considered statistically significant.

[0113] 3. Analysis of gut microbiota in mice with non-alcoholic fatty liver disease

[0114] (1) Extraction and PCR amplification and purification of genomic DNA

[0115] Total genomic DNA (QIAamp PowerFecal DNA, Qiagen) was extracted from the samples. DNA concentration was determined using a Qubit 4.0 fluorometer (Thermo Scientific) and adjusted to 1 ng / μL. The full-length 16S gene (V1-V9 region) was amplified using 16S gene-specific primers with barcodes (Forward primer 1: 5' Phos / GCATC-16-base barcode-Sequence ID Number 11, Reverse primer 2: 5' Phos / GCATC-16-base barcode-Sequence ID Number 12). PCR reactions were performed using a KAPA HiFiHotStartReadyMix (Roche): 95°C for 3 minutes; 20-27 cycles (sample-dependent): 95°C for 30 seconds, 57°C for 30 seconds, 72°C for 60 seconds; 72°C for 5 minutes, and finally stored at 4°C for later use. PCR products were monitored on a 1% agarose gel. Samples with a bright main band of approximately 1500 bp were selected and purified using AMPure PB Beads for SMRTbell library preparation.

[0116] (2) SMRTbell library construction and sequencing

[0117] SMRTbell libraries were prepared using full-length 16S gene amplification with barcoded primers for multiplex SMRTbell library preparation and sequencing (PacBio). Sequencing was performed on a PacBio Sequel IIe instrument in circular consensus sequencing (CCS) mode, generating HiFi reads with a predicted accuracy (Phred Scale) of 30. The DNA to be tested was repeatedly sequenced. By comparing the results of repeated sequencing, sequencing errors were corrected, ultimately achieving a high accuracy of >99.9% (QV30).

[0118] (3) Bioinformatics Analysis

[0119] i. Ordered data processing

[0120] Based on the barcode sequence and PCR amplification primer sequence, the sample data were separated from the PCR data. After removing the barcode and primer sequences, the data was imported into QIIME2 (v2019.7.0; https: / / qiime2.org / ) (Bolyen et al., 2019). The sequences of all samples were stored as a single Artifact. The QIIME2 cutadapt was used to remove the amplicon variant regions of the primers (forward primer 2: Sequence ID Number 13; reverse primer 2: Sequence ID Number 14). The sequences after removing the primers were used as QIIME2 DADA2 InputReads.

[0121] ii.QIIME2 DADA2 denoising and species annotation

[0122] The QIIME2 DADA2 (v2019.7.0; https: / / qiime2.org / ) (Callahan et al., 2016; Quinet al., 2018) denoising analysis first involves sequence filtering, where each sequence is pruned to a specified length (forward reads: 280bp; reverse reads: 220bp), and the MaxEE parameter (forward reads: 2; reverse reads: 2) is set as the maximum allowable number of expected erroneous bases in the sequence. Next, the DADA2 core algorithm performs denoising, using information such as sequence abundance, quality score, and relationships between sequences to correct misordered bases and infer the true sequence. After denoising of each pair of ends, splicing is performed (minimum overlap length 20bp; mismatches are not allowed in the overlap region). Finally, chimera removal is performed on the spliced ​​sequence. By comparing it with sequences of high abundance in the sample, if a sequence has low abundance and is similar to multiple sequences, it can be identified as a chimera and removed. The sequences after denoising analysis are used as representative sequences of amplicon sequence variants (ASVs), and an ASV table can be obtained for subsequent species annotation. The QIIME2 feature-classifier (v2019.7.0; https: / / qiime2.org / ) (Bokulich et al., 2018) and the machine learning-based classification method classify-sklearn (McKinney, 2010; Pedregosa et al., 2011) were selected and Bayesian classifiers were trained on different databases and for specific variant regions. Bayes classifier (Wang et al., 2007). This classifier was used for ASV (autonomous zone) sequence classification annotation, obtaining classification information and statistically analyzing the microbial community composition of each sample at each taxonomic level: kingdom, phylum, class, order, family, genus, and species. 16S analysis used the QIIME2 alignment MAFFT method (v2019.7.0; https: / / qiime2.org / ) (Katoh & Standley, 2013; Lane, 1991) and the Core Set (representing most major prokaryotic taxa) in the GreenGenes database (gg_13_8) (DeSantis et al., 2006; McDonald et al., 2012) or the Silva database (v132; 2017.12) (Quast et al., 2012) for rapid sequence alignment. Alternatively, the latest NCBI database could be used for sequence alignment to obtain annotation information. &Huson, 2017; Gyarmati et al., 2016; Hong et al., 2016), while ITS analysis used the unite database (v7.2; 2017.12.01) (Abarenkov et al., 2010); et al., 2005; Annotations were provided by (Schloss et al., 2013; Nilsson et al., 2019). Finally, the sequence data of each sample were normalized, using the sample with the smallest total number of tags as the standard (Schloss et al., 2009). The processed data were then used for subsequent Beta diversity analysis.

[0123] iii. Comparative analysis of sample groups (Beta diversity)

[0124] Qiime was used to calculate UniFrac distances and construct UPGMA sample cluster trees. PLS-DA analysis plots were generated using R (v3.3.1), with PLS-DA analysis utilizing the `mixOmics` and `ggplot2` packages in R. Ternary Plot analysis used the `ggtern` package in R. Beta diversity index inter-group difference analysis was performed using the `agricolae` package in R, with parametric and non-parametric tests performed, including the Tukey test and the Kruskal test (post-hoc test).

[0125] The experiment was conducted using the method described in Example 4 above, and the results are as follows:

[0126] 1. Effect of the composition of the present invention on liver weight and liver / body weight ratio in mice with non-alcoholic fatty liver disease.

[0127] Table 3 shows that, compared to the ND group, the HFD group showed an abnormal increase in liver weight. This phenomenon may indicate that a long-term high-fat diet will lead to abnormal liver tissue due to fat accumulation. However, this condition can be significantly improved by feeding various doses of the red yeast fermentation product of this invention (L008 and H008 groups) and Msol, which significantly reduced liver weight (p<0.05) and also improved the liver-to-body weight ratio.

[0128] Table 3. Effects of the red yeast rice and its monascinol of the present invention on liver weight and liver / body weight ratio in mice on a long-term high-fat diet.

[0129]

[0130] Experimental data are expressed as mean ± standard deviation (mean ± SD) (n = 8). Different letters in the mean indicate statistical significance (p < 0.05). The statistical significance of biochemical effects was determined using one-way variable analysis (ANOVA) and Duncan's multiple test.

[0131] 2. Effects of the composition of the present invention on serum liver function indices AST and ALT levels in mice with non-alcoholic fatty liver disease.

[0132] Table 4 shows that serum AST and ALT activities in the HFD group were significantly higher than those in the ND group (p < 0.05). This result indicates that a long-term high-fat diet may cause liver cell damage, leading to non-alcoholic fatty liver disease. Feeding patients with various doses of the fermentation product of the red yeast rice of this invention (L008 and H008 groups) and monascinol significantly reduced AST and ALT activities induced by a long-term high-fat diet. The low-dose group of red yeast rice of this invention (L008 group), equivalent to 0.5g of red yeast rice per day for an adult, showed a significant effect in reducing AST and ALT activities. Furthermore, a monascinol intake equivalent to 6mg per day for an adult also improved AST and ALT levels.

[0133] Table 4. Effects of the red yeast rice and monascinol of the present invention on serum liver function indices AST and ALT levels in mice fed a long-term high-fat diet.

[0134]

[0135] Experimental data are expressed as mean ± standard deviation (mean ± SD) (n = 8). Different letters in the mean indicate statistical significance (p < 0.05). The statistical significance of biochemical effects was determined using one-way variable analysis (ANOVA) and Duncan's multiple test.

[0136] 3. Effects of the composition of the present invention on TG and TC content in the liver of mice with non-alcoholic fatty liver disease.

[0137] The liver lipid analysis results are shown in Table 5. The HFD group showed increased TC and TG concentrations in the liver (p<0.05). The low-dose group of red yeast rice (L008 group) showed a slight reduction in liver TG production caused by a long-term high-fat diet and a significant reduction in TC. Feeding red yeast rice powder at a high dose (H008 group) significantly reduced TC and TG levels in the liver (p<0.05). The pure substance Msol group also effectively reduced TC and TG accumulation in the liver (p<0.05). These results indicate that the red yeast rice and monascinol of this invention have the effect of reducing TC and TG accumulation in the liver and have the potential to improve fatty liver disease. These results also confirm that monascinol is the active ingredient in red yeast rice that reduces liver lipids.

[0138] Table 5. Effects of the red yeast rice and its monascinol of the present invention on the TG and TC content in the liver of mice on a long-term high-fat diet.

[0139]

[0140] Experimental data are expressed as mean ± standard deviation (mean ± SD) (n = 8). Different letters in the mean indicate statistical significance (p < 0.05). The statistical significance of biochemical effects was determined using one-way variable analysis (ANOVA) and Duncan's multiple test.

[0141] 4. Effects of the composition of the present invention on pathological sections of liver tissue from mice with non-alcoholic fatty liver disease.

[0142] Figure 7The images show histopathological sections of the livers of experimental animals. The results indicate that long-term consumption of a high-fat diet causes the liver tissue of the HFD group to show a large number of fat droplets and a state of fatty infiltration. Furthermore, as shown in Table 6, the histopathological evaluation of liver sections reveals that feeding the red yeast rice and its monascinol of this invention can improve the accumulation of fat droplets (p<0.05). The H008 group showed better improvement than the L008 group, exhibiting a dose-response effect. The Msol group can also significantly improve the accumulation of fat droplets and fatty infiltration in the livers of mice with non-alcoholic fatty liver disease induced by a high-fat diet, thereby reducing the incidence of lipid oxidation lesions.

[0143] Table 6. Pathological evaluation of the liver of mice with non-alcoholic fatty liver disease by red yeast rice and monascinol of the present invention.

[0144]

[0145] Based on the severity of lesions, they were categorized from 1 to 5: 0 = no significant lesions; 1 = mild (<10%); 2 = moderate (10-33%); 3 = moderate / severe (33-66%); 4 = severe / high (66-100%). Data are presented as mean ± standard deviation (n=8). Significant differences existed between the means of different letters (p<0.05). Statistical significance of biochemical effects was determined by analysis of variance (ANOVA) and Duncan's multiple comparison test. Experimental data are expressed as mean ± standard deviation (mean ± SD) (n=8). Significant differences existed between the means of different letters (p<0.05). Statistical significance of biochemical effects was determined using one-way analysis of variance (ANOVA) and Duncan's multiple test.

[0146] 5. Effects of feeding mice with the red yeast rice and its indicator active ingredients of this invention on the gut microbiota of mice with non-alcoholic fatty liver disease.

[0147] The results of the bacterial community abundance analysis are as follows: Figure 8As shown, after induction with a high-fat diet, the types and abundance of bacterial species changed significantly compared to the normal group, especially in the genus *Lactobacillus*, where there was a significant decrease. The top 10 genera with the most changes in abundance at the genus level were *Lachnospiraceae*, *Dubosiella*, *Lactobacillus*, *Bacteroides*, *Eubacterium coprostanoligen*, *Ruminococcaceae*, *Clostridium rumenans*, *Blautia*, *Akkermansia*, and *Parabacteroides*.

[0148] like Figure 9 As shown, feeding the red yeast rice (L008 group and H008 group) and monascinol (Msol group) of this invention to the HFD group all increased the abundance of Blautia and Intestinimonas in the gut microbiota. Blautia is one of the most abundant genera of gut microbiota and plays a regulatory role in inflammatory responses and metabolic diseases; it is a common probiotic genus. Intestinimonas has the characteristic of high butyrate production and has anti-inflammatory and anti-obesity effects (Cai et al., 2020). In this study, all experimental groups showed an increased abundance of Blautia and Intestinimonas compared to the high-fat diet group.

[0149] Partial least squares discriminant analysis (PLS-DA) was used to analyze the bacterial community structure among different groups of samples. The results are as follows: Figure 10 As shown, the bacterial community distribution of non-alcoholic liver mice after a high-fat diet was significantly different from that of the normal group. Further analysis of whether the composition (test sample) of the present invention can effectively improve the bacterial community structure of a high-fat diet showed that the red yeast rice L008 group and H008 group of the present invention had similar bacterial community structures and were significantly separated from the HFD group. Although the bacterial community structure analysis of the active ingredient (monascinol) of the red yeast rice of the present invention (Msol group) was similar to that of the HFD group, there were significant differences in the bacterial community abundance analysis results.

[0150] Nonalcoholic fatty liver disease (NAFLD) is characterized by excessive fat accumulation in the liver, leading to degeneration and symptoms such as nonalcoholic fatty liver inflammatory response (NASH). In animal experiments, Example 4 of this invention has demonstrated that the red yeast rice and its active ingredient (monascinol) of this invention can improve the ratio of total cholesterol (TC) and total triglycerides (TG) in the liver of mice with NAFLD (p<0.05) and reduce the liver weight / body weight ratio (p<0.05). Furthermore, liver section analysis and interpretation also confirmed that each experimental group significantly alleviated the symptoms of liver fat accumulation, fat vacuoles, and fat infiltration caused by a long-term high-fat diet, thus confirming the efficacy of the red yeast rice and monascinol in improving NAFLD.

[0151] Furthermore, the gut microbiota ecology is highly correlated with host health or disease, including changes in the composition ratio of gut microbiota and microbial metabolites (Jiang et al., 2020). In this study, the red yeast rice (L008 group and H008 group) and monascinol (Msol group) in the composition (test substance) of this invention both increased the abundance of Blautia species compared to the high-fat diet group (HFD group). Literature indicates that Blautia content is highly negatively correlated with visceral fat accumulation, and its metabolic mechanism can effectively reduce visceral fat (Ozato et al., 2019). Other literature describes that symbiotic microorganisms in the Blautia gut produce Blautiaproducta, which generates short-chain fatty acids such as acetate, improving insulin sensitivity in the liver and preventing NAFLD through the acetate-FFAR2 signaling pathway (Aoki et al., 2021). Intestinimonas bacteria are characterized by high butyrate production. Previous studies have confirmed that Intestinimonas can alleviate the NAFLD process through butyrate and has the potential for antioxidant and anti-inflammatory effects (Miao et al., 2022; Wang et al., 2021). The red yeast rice and monascinol of this invention can also increase the abundance of Intestinimonas flora, which helps to improve the efficacy of alleviating NAFLD.

[0152] The experimental results of Example 4 of this invention show that a long-term high-fat diet can cause non-alcoholic fatty liver disease in normal mice and affect the composition ratio of their intestinal microbiota. The red yeast rice and monascinol of this invention can improve specific intestinal microorganisms, such as Blautia and Intestinimonas, thereby regulating liver lipid metabolism, slowing down the progression of NAFLD, and thus improving the efficacy of NAFLD.

[0153] Furthermore, this invention evaluated the effect of different test substances (compositions) on improving fatty liver in mice induced by a 22-week high-fat diet in different groups (L008 group, H008 group, and Msol group). The following conclusions can be drawn from the above examples and results:

[0154] 1. The red yeast rice of this invention (groups L008 and H008) contains monascinol, which has been less previously reported to have the effect of reducing non-alcoholic fatty liver disease. The red yeast rice of this invention has significant effects on improving liver weight and liver / body weight, serum liver function indices AST and ALT activity, liver TG and TC content, and the degree of fatty infiltration in liver tissue. Furthermore, the low-dose dosage of the red yeast rice powder of this invention (group L008) is 0.5 grams (g) per person per day, and the dosage of monascinol is 6 milligrams (mg) per person per day. These relatively low doses demonstrate the efficacy in preventing and improving fatty liver disease, thus representing a significant advancement. This invention also shows that monascinol is a novel active ingredient for preventing and improving fatty liver disease.

[0155] 2. The red yeast rice (L008 group and H008 group) and monascinol (Msol group) of the present invention can improve the changes in intestinal microbial composition caused by a high-fat diet. Compared with the HFD group, they can increase the abundance of Blautia and Intestinimonas in the intestine. These bacteria have the potential to increase short-chain fatty acid metabolism, anti-oxidation, anti-inflammation and improve NAFLD. This can confirm that the mechanism of action of red yeast rice and monascinol in improving NAFLD is highly correlated with the enhancement of specific intestinal flora.

[0156] The foregoing description has fully and clearly illustrated the novel Monascus purpureus, its compositions, and its use in the prevention or treatment of non-alcoholic fatty liver disease. It must be emphasized that the above detailed description is a specific illustration of feasible embodiments of the invention; however, these embodiments are not intended to limit the scope of the patent. All equivalent implementations or modifications that do not depart from the spirit of the invention should be included within the scope of this patent.

[0157] [Biomaterial Storage]

[0158] The red Monascus pilosus strain of this invention is deposited at the National Centre for Food Industry and Marine Bacteria (NCIMB Ltd) in the UK on January 12, 2023, with the deposit number NCIMB 44103.

Claims

1. A red mold, which is a red mold (Monascus pilosus) registered at the National Centre for Food Industry and Marine Bacteria (NCIMB Ltd) in the United Kingdom with the registration number NCIMB 44103.

2. Use of a red Monascus strain for the preparation of a composition for the prevention or treatment of non-alcoholic fatty liver disease, wherein the red Monascus strain may be a red Monascus strain registered at the National Centre for Food Industry and Marine Bacteria (NCIMB Ltd) with the registration number NCIMB 44103.

3. A composition for the prevention or treatment of non-alcoholic fatty liver disease, characterized in that... It contains an effective amount of an active ingredient, wherein the active ingredient is extracted from a red yeast ferment, which is prepared by fermenting a substrate with a red yeast fungus, and the red yeast fungus can be the red yeast fungus (Monascus pilosus) registered at the National Centre for Food Industries and Marine Bacteria (NCIMB Ltd) in the United Kingdom with the registration number NCIMB 44103.

4. The composition of claim 3, wherein the non-alcoholic fatty liver disease comprises non-alcoholic fatty liver disease caused by at least one of the group consisting of obesity, hypertension, hyperlipidemia, and diabetes.

5. The composition according to claim 3, wherein the composition reduces serum liver function indices AST and ALT.

6. The composition according to claim 3, wherein the composition inhibits the accumulation of fat in liver tissue.

7. The composition according to claim 3, wherein the composition reduces the levels of total cholesterol (TC) and triglycerides (TG) in the liver.

8. The composition according to claim 3, wherein the composition improves liver oil droplet accumulation and fatty infiltration.

9. The composition according to claim 3, wherein the composition has the effect of increasing the abundance of beneficial intestinal flora and altering the composition of intestinal flora.

10. The composition of claim 3, wherein the active ingredient comprises at least one selected from the group consisting of Monascinol, Ankaflavin and Monascin.

11. The composition according to claim 3, wherein the matrix is ​​rice, yam, or a mixture of related carbohydrates.

12. The composition according to claim 3, wherein the effective amount, when used alone, is an adult daily intake of at least 1.5 mg to 12 mg.

13. A composition for the prevention or treatment of non-alcoholic fatty liver disease, characterized in that... It contains an effective amount of a red yeast ferment, wherein the red yeast ferment is prepared by fermenting a substrate with a red yeast fungus, and the red yeast fungus may be the red yeast fungus (Monascus pilosus) registered at the National Centre for Food Industries and Marine Bacteria (NCIMB Ltd) in the United Kingdom with the registration number NCIMB 44103.

14. The composition of claim 13, wherein the non-alcoholic fatty liver disease comprises non-alcoholic fatty liver disease caused by at least one of the group consisting of obesity, hypertension, hyperlipidemia, and diabetes.

15. The composition of claim 13, wherein the composition reduces serum liver function indices AST and ALT.

16. The composition according to claim 13, wherein the composition inhibits the accumulation of fat in liver tissue.

17. The composition of claim 13, wherein the composition reduces the levels of total cholesterol (TC) and triglycerides (TG) in the liver.

18. The composition of claim 13, wherein the composition improves liver oil droplet accumulation and fatty infiltration.

19. The composition according to claim 13, wherein the composition has the effect of increasing the abundance of beneficial intestinal flora and altering the composition of intestinal flora.

20. The composition according to claim 3, wherein the matrix is ​​rice, yam, or a mixture of related carbohydrates.

21. The composition according to claim 13, wherein the effective amount is at least 0.5 g per day for an adult, containing 1.5 mg of monacinol.

22. The composition of claim 13, wherein the red yeast rice ferment contains at least one active ingredient, and the active ingredient comprises at least one selected from the group consisting of Monascinol, Ankaflavin and Monascin.

23. The composition of claim 13, wherein 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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