Monascus pilosus, composition for losing weight and improving gut microbiota composition and use of said monascus pilosus

Abstract

To provide Monascus pilosus, a composition for weight loss and improvement of gut microbiota composition, and uses of the Monascus pilosus.SOLUTION: The main purpose of the present invention is to provide Monascus pilosus and a composition for weight loss and improvement of gut microbiota composition, wherein the composition comprises Monascus pilosus and / or a fermented product thereof, or comprises a functional ingredient comprising the fermented product thereof. Another objective of the present invention is to provide uses of Monascus pilosus in the manufacture of a composition for weight loss and improvement of gut microbiota composition. The composition of the present invention has the ability to reduce body weight and the ability to improve gut microbiota composition, for example, to reduce a ratio of Firmicutes to Bacteroidetes.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to the technical fields of weight loss and improvement of intestinal flora, and more particularly to a composition for weight loss and improvement of intestinal flora, which comprises Monascus, a Monascus fermentation product thereof, or a functional ingredient such as the Monascus fermentation product. [Background technology]

[0002] Overweight and obesity are currently recognized as a serious and often overlooked health problem facing humans. Even in developing countries with growing economic prosperity, the number of obese people is rapidly increasing, and obesity rates are comparable to those in developed countries. The World Health Organization (WHO) defines overweight and obesity as abnormal or excessive fat accumulation that is detrimental to health. The WHO further defines overweight as a body mass index (BMI) of 25 or higher, and obesity as a body mass index of 30 or higher.

[0003] Not only are overweight and obesity not generally recognized as "diseases," but they are also considered to be significant risk factors for contracting non-communicable diseases, i.e., overweight and obesity increase the risk of contracting cardiovascular diseases (mainly heart disease and stroke), diabetes, musculoskeletal diseases (particularly osteoarthritis, a highly disabling and degenerative disease of the joints), and some cancers (including uterine cancer, breast cancer, ovarian cancer, prostate cancer, liver cancer, gallbladder cancer, kidney cancer, and colon cancer). Furthermore, as body mass index increases, the risk of developing non-communicable diseases also increases.

[0004] Currently, there are many treatments available for overweight and obesity, including exercise, calorie intake control, surgery, and medication. For example, physical training, dietary therapy, liposuction, gastrectomy, gastric bypass surgery, and metabolic stimulants, appetite suppressants, and starch blockers are all currently common obesity treatments. These obesity treatments currently primarily focus on natural health, such as controlling calorie intake through dietary therapy and burning off excess calories through physical training. However, these treatments cannot always be sustained due to individual factors of the obese patient, and therefore often lack effective results. Weight loss through surgical procedures such as liposuction, gastrectomy, and gastric bypass surgery has several drawbacks, including relatively high costs and the risk of posing uncertain risks to obese patients during the surgical process. Furthermore, the use of medications such as metabolic stimulants, appetite suppressants, and starch blockers can cause side effects for obese patients, potentially causing adverse physical effects. Summary of the Invention [Problem to be solved by the invention]

[0005] In view of the limitations and shortcomings of current treatments for overweight and obesity, the inventors of the present application have conducted extensive research and invention, and as a result have researched, developed, and perfected the Monascus mold, composition for weight loss and intestinal flora improvement, and uses of the Monascus mold of the present invention. In daily life, obese patients can easily lose weight using the composition of the present invention, which is easy to obtain and take, thereby achieving treatment for obesity without causing any adverse effects on the body.

[0006] The main object of the present invention is to provide Monascus pilosus and a composition for weight loss and for improving intestinal flora, the composition comprising Monascus pilosus and / or its red koji fermentation product, or monascinol obtained by purifying the red koji fermentation product, and the composition for weight loss and for improving intestinal flora can be provided as a composition for oral administration. [Means for solving the problem]

[0007] To achieve the above object, the present invention proposes a novel Monascus pilosus, which has been deposited at the National Collection of Industrial, Food and Marine Bacteria (NCIMB Ltd), UK, under accession number NCIMB 44103.

[0008] The present invention also provides a composition for weight loss and improving intestinal flora, which comprises an effective amount of a red koji fermentation product, wherein the red koji fermentation product can be prepared by fermenting a substrate using Monascus mold, and the red koji mold is Monascus pilosus, deposited with the National Collection of Industry, Food and Marine Bacteria (NCIMB Ltd) in the United Kingdom under accession number NCIMB 44103. In addition, the composition of the present invention may comprise a pharmaceutically acceptable carrier.

[0009] The weight loss and intestinal flora improving composition of the present invention has the ability to increase the abundance of Bacteroidales bacteria, thereby reducing the ratio of Firmicutes to Bacteroidetes.

[0010] Furthermore, the composition for weight loss and improving intestinal flora of the present invention has the ability to increase the abundance of beneficial intestinal bacteria such as Verrucomicrobiales and Bifidobacteriales, and has the function of increasing the abundance of beneficial intestinal bacteria and changing the composition of intestinal flora.

[0011] In addition, the substrate in the composition of the present invention is rice, yam, or a mixture of related carbohydrates.

[0012] In addition, the effective amount in the composition of the present invention is such that an adult takes at least 0.5 grams of the red koji fermented product daily, containing 1.5 milligrams of monascinol, wherein the red koji fermented product contains at least one functional ingredient, and the functional ingredient includes at least one selected from the group consisting of monascinol, ankaflavin, and monascin.

[0013] The composition of the present invention may be a food composition, a pharmaceutical composition, a feed composition, a nutritional supplement composition, a dietary supplement composition or a food additive composition.

[0014] The present invention also provides a composition for weight loss and improving intestinal flora, which comprises an effective amount of a functional ingredient extracted from a red koji fermentation product, wherein the red koji fermentation product can be produced by fermenting a substrate using Monascus mold, and the red koji mold is Monascus pilosus, deposited with the National Collection of Industry, Food and Marine Bacteria (NCIMB Ltd) in the United Kingdom under accession number NCIMB 44103. In addition, the composition of the present invention may comprise a pharmaceutically acceptable carrier.

[0015] The weight loss and intestinal flora improving composition of the present invention has the ability to increase the abundance of Bacteroidales bacteria, thereby reducing the ratio of Firmicutes to Bacteroidetes.

[0016] In addition, the weight loss and intestinal flora improving composition of the present invention has the ability to increase the abundance of beneficial intestinal bacteria such as Verrucomicrobiales and Bifidobacteriales, and also has the ability to increase the abundance of beneficial intestinal bacteria and change the composition of intestinal flora.

[0017] In the composition of the present invention, the functional ingredient comprises at least one selected from the group consisting of monascinol, ankaflavin, and monascin. The substrate in the composition of the present invention is a mixture of rice, yam, or related carbohydrates, and the effective amount is a daily intake of at least 0.75 milligrams to 12 milligrams of the functional ingredient for an adult, or a daily intake of at least 3 milligrams of the functional ingredient.

[0018] Furthermore, the present invention provides use of Monascus in the manufacture of a composition for weight loss and improving intestinal flora, wherein the Monascus is Monascus pilosus deposited with the National Collection of Industry, Food and Marine Bacteria (NCIMB Ltd) in the United Kingdom under accession number NCIMB 44103. [Effects of the Invention]

[0019] In addition, the present invention also provides the application of compositions for weight loss and improvement of intestinal flora. This type of application includes forms that are easy to use in daily life, such as foods, beverages, health foods, additives, and medical compositions, and can be provided to the general public to take, thereby maintaining intestinal health and achieving weight loss on a daily basis. The reduction of body fat can also help avoid symptoms and diseases caused by overweight and obesity, such as cardiovascular disease, diabetes, musculoskeletal disorders, and cancer. [Brief explanation of the drawings]

[0020] [Figure 1] FIG. 1 is a diagram showing the process for identifying and differentiating species of Monascus according to the present invention. [Figure 2] 1 is a phylogenetic tree showing the phylogenetic relationships after alignment of the β-tubulin sequences of Monascus of the present invention. [Figure 3] 1 is a phylogenetic tree showing the phylogenetic relationships after alignment of the ITS sequences of the Monascus fungi of the present invention. [Figure 4] 1 shows the results of species-specific PCR analysis of Monascus of the present invention. [Figure 5] 1 shows the results of PCR analysis of the pksCT gene of Monascus of the present invention. [Figure 6] 1 is a photograph showing the ascocarp morphology of Monascus of the present invention. [Figure 7A] 1 is a graph showing the results of an analysis of the richness of bacterial groups by phylum classification in rats fed a high-fat diet with red koji rice of the present invention (RL: 0.5 grams / day and RH: 2 grams / day) and monascinol (MSol: 3 milligrams / day). [Figure 7B] 1 is a graph showing the results of analyzing the abundance of bacterial groups by class when rats fed a high-fat diet were fed red koji rice of the present invention (RL: 0.5 g / day and RH: 2 g / day) and monascinol (MSol: 3 mg / day). [Figure 7C]1 is a graph showing the results of an analysis of the abundance of bacterial groups classified by order when rats were fed red koji rice of the present invention (RL: 0.5 g / day and RH: 2 g / day) and monascinol (MSol: 3 mg / day) on a high-fat diet. [Figure 7D] 1 is a graph showing the results of an analysis of the richness of bacterial groups by family classification in rats fed a high-fat diet with red koji rice of the present invention (RL: 0.5 g / day and RH: 2 g / day) and monascinol (MSol: 3 mg / day). [Figure 7E] 1 is a graph showing the results of an analysis of the abundance of bacterial groups by genus classification in rats fed a high-fat diet with red koji rice of the present invention (RL: 0.5 g / day and RH: 2 g / day) and monascinol (MSol: 3 mg / day). [Figure 7F] 1 is a graph showing the results of an analysis of the abundance of bacterial communities by species when rats fed a high-fat diet were fed red koji rice of the present invention (RL: 0.5 g / day and RH: 2 g / day) and monascinol (MSol: 3 mg / day). [Figure 8] FIG. 1 shows a comparison of the relative abundance of key bacterial groups between rats in each test group (NOR: normal group, RL, RH, and MSol) and rats in the high-fat diet group (HF) at the eye level classification. [Figure 9A] FIG. 1 shows the relative abundance of bacterial community enrichment in each test group (NOR, HF, RL, RH, and MSol). [Figure 9B] FIG. 1 shows the relative abundance of bacterial community enrichment in each test group (NOR, HF, RL, RH, and MSol). [Figure 9C] FIG. 1 shows the relative abundance of bacterial community enrichment in each test group (NOR, HF, RL, RH, and MSol). [Figure 9D] FIG. 1 shows the relative abundance of bacterial community enrichment in each test group (NOR, HF, RL, RH, and MSol). DETAILED DESCRIPTION OF THE INVENTION

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this invention belongs. Hereinafter, the details of the present invention will be explicitly explained using examples, but these examples are merely illustrative and not limiting, and the present invention is not limited to these examples. Unless otherwise specified, all materials used in the present invention are preferably commercially available and easily available, and the available routes shown below are merely examples.

[0022] The present invention provides a novel Monascus pilosus, which has been deposited with the National Collection of Industry, Food and Marine Bacteria (NCIMB Ltd.) in the United Kingdom under accession number NCIMB 44103. The present invention also provides a composition for weight loss and improving intestinal flora, which comprises an effective amount of a red koji fermentation product, and the red koji fermentation product is obtained by fermenting Monascus pilosus, which has been deposited with the National Collection of Industry, Food and Marine Bacteria (NCIMB Ltd.) in the United Kingdom under accession number NCIMB 44103. The present invention also provides a composition for weight loss and intestinal flora improvement, comprising an effective amount of a functional ingredient extracted from a red koji fermentation product, wherein the red koji fermentation product can be prepared by fermenting a substrate using Monascus pilosus, which is deposited with the National Collection of Industrial, Food and Marine Bacteria (NCIMB Ltd.) in the United Kingdom under accession number NCIMB 44103. The functional ingredient is monascinol, and the substrate is a mixture of rice, yam, or related carbohydrates. Furthermore, experiments using high-fat diet-induced obese rats confirmed that the Red Koji fungus of the present invention and the composition prepared as described above are effective for weight loss and intestinal flora improvement.

[0023] The Monascus strain of the present invention described above also includes its subculture progeny or mutant strains, which still have the same strain characteristics, genomics, or uses (used to reduce weight and improve intestinal flora) as the present invention.

[0024] The compositions herein may include, but are not limited to, application forms 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.

[0025] The term "weight loss" means that the composition of the present invention can reduce body weight more effectively than a composition that does not use the Red koji mold of the present invention, its Red koji fermentation product, or a composition containing an extract from the Red koji fermentation product. The term "improvement" means that the composition of the present invention can improve the intestinal flora more effectively than a composition that does not use the Red koji mold of the present invention, its Red koji fermentation product, or a composition containing an extract from the Red koji fermentation product.

[0026] The term "effective amount" means an effective amount of a functional ingredient that can effectively reduce weight and improve the intestinal flora, and is also referred to as a "therapeutically effective amount" or an "improvement effective amount." The term "pharmaceutically acceptable" means that the substance or composition must be compatible with other ingredients in the formulation and be harmless to the patient.

[0027] The composition of the present invention refers to a compound prepared by using techniques well known to those skilled in the art, in which the above-mentioned Monascus fungus, its Monascus fermentation product, or an extract of the Monascus fermentation product is combined with a pharmaceutically acceptable carrier to form a dosage form applicable to the composition of the present invention, and among these, the dosage form includes, but is not limited to, a solution, emulsion, suspension, powder, tablet, pill, orally disintegrating tablet, troche, capsule, and other dosage forms similar to or applicable to the present invention.

[0028] If necessary, one or more solubilizing agents, buffers, preservatives, coloring agents, fragrances, flavoring agents, excipients, and the like commonly used in the pharmaceutical field may be added to the above composition.

[0029] In another preferred embodiment, the composition of the present invention can be further added to edible materials to prepare food products or health maintenance products, wherein the edible materials are 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, etc. This includes, but is not limited to, dietary supplements, herbal medicines, and the like.

[0030] Furthermore, the present invention also provides a method for weight loss and improvement of gut flora, which comprises administering an effective amount of the above-described composition to an overweight or obese patient to reduce weight and improve the patient's gut flora.

[0031] In addition, the present invention also provides a method or use of the above-mentioned Monascus mold, a Monascus fermentation product, or an extract of the Monascus fermentation product for preparing a composition for improving the intestinal microbial species composition.

[0032] The administration route of the composition for weight loss and intestinal flora improvement provided by the present invention is not particularly limited as long as it can be appropriately adjusted according to requirements, and a preferred administration route includes oral administration suitable for an appropriate dosage form.

[0033] Example 1: Identification and differentiation of the novel Monascus pilosus fungus of the present invention 1. Basis for bacterial species identification and process Traditional classification and identification of Monascus fungi are based primarily on colony morphology, primarily colony size and color (Hawksworth and Pitt, 1983). However, some Monascus fungi strains are difficult to classify based on colony appearance (e.g., Monascus pilosus and Monascus ruber), so molecular typing is often used to identify Monascus fungi. Current molecular typing methods for Monascus fungi rely primarily on the alignment of β-tubulin and ITS sequences (Park et al., 2004). However, the present invention improves the accuracy of species identification by incorporating a species-specific polymerase chain reaction (PCR) developed by the inventors in their laboratory, in addition to the alignment of β-tubulin and ITS sequences, and PCR of the pksCT gene (see Figure 1).

[0034] 2. Strain Cultivation and Deoxyribonucleic Acid (DNA) Extraction Potato dextrose broth (PDB) medium was purchased from Difco (Becton-Dickinson Diagnostic Systems, Sparks, MD, USA). For DNA extraction, test strains were cultured in PDB medium at 28°C for 7 days, after which the cells were harvested, ground in liquid nitrogen, and then dried in a vacuum oven. 0.1 grams (g) of dried cell powder was weighed into a 2-milliliter (mL) microcentrifuge tube and subjected to DNA extraction using a QIAamp DNA Mini Kit (purchased from QIAGEN NV, Velno, The Netherlands).

[0035] 3. ITS sequence and β-tubulin sequence alignment analysis and species-specific PCR analysis

[0036] (1) PCR amplification of ITS sequences The total PCR reaction volume was 25 μL, containing 1x PCR buffer, 0.05 mM of four deoxynucleoside triphosphates (dNTPs), 5 U of ExSel high fidelity DNA polymerase (purchased from Bertec Enterprise Co., Ltd., Taipei), 0.2 μM of primers ITS1 (sequence number 1) and ITS4 (sequence number 2), and 0.16 μg of template DNA. The PCR reaction conditions were as follows: 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. Finally, the mixture was stored at 70°C for 10 minutes at 4°C. After confirmation via electrophoresis, the products were sent to Genomics Bioscience Technology Co., Ltd. (Taipei, Taiwan) for DNA sequencing.

[0037] (2) PCR amplification of β-tubulin sequences The total volume of the PCR reaction was 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 were as follows: 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, and finally 70°C for 10 minutes, followed by storage at 4°C. After electrophoresis, the products were sequenced by Genomics Bioscience Technology Co., Ltd.

[0038] (3) Species-specific PCR of Monascus purpureus (M. purpureus) A species-specific PCR for M. purpureus was designed based on the remaining fragment of the unique monacolin K biosynthetic gene mokH in the M. purpureus genome and can effectively identify whether Monascus species belong to M. purpureus. The total PCR volume was 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 0.16 μg of template DNA. The PCR reaction conditions were as follows: 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 a final 70°C for 10 minutes, followed by electrophoretic analysis.

[0039] (4) Species-specific PCR of Monascus pilosus / ruber (M. pilosus / ruber) A species-specific PCR for M. pilosus / ruber was designed based on a conserved fragment of the FAS gene in the pigment biosynthesis gene cluster within the M. ruber genome and can effectively identify whether Monascus species belong to M. pilosus / ruber. The total PCR volume was 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 reverse primer (primer RubPil R, SEQ ID NO: 8), and 0.16 μg of template DNA. The PCR conditions were as follows: 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 a final 70°C for 10 minutes, followed by electrophoretic analysis.

[0040] (5) PCR of the pksCT gene The pksCT gene is a core gene for citrinin biosynthesis in M. purpureus. Since the gene for this segment in the M. pilosus / ruber genome has been deleted, this gene can be used as a basis 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 of SupeTherm GOLD DNA polymerase, 80 nM 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 10 ng of template DNA. The PCR reaction conditions were as follows: 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, followed by 10 minutes at 70°C before electrophoresis.

[0041] (6) Alignment analysis of sequences The alignment analysis of sequences was performed using Geneious 8.1.9 software (Biomatters Ltd., Auckland, New Zealand). Among them, sequence combinations were analyzed using the built-in assembler in Geneious, multiple alignments were performed using MAFFT 7.017, phylogenetic tree construction showing maximum likelihood relationships was performed using MEGA, the General Time Reversible (GTR) was selected as the nucleic acid substitution model, and bootstrap analysis was performed 1000 times. Phylogenetic tree construction showing Bayesian relationships and posterior probability tests were performed using MrBayes, with the nucleic acid substitution model being GTR and Aspergillus terreus used as the out group.

[0042] <Results of alignment analysis of ITS sequences and β-tubulin sequences> After aligning the β-tubulin sequence and ITS sequence of the Monascus fungus of the present invention, phylogenetic trees showing their relatedness are shown in Figures 2 and 3, respectively. As shown by the results of the β-tubulin sequence alignment, the Monascus fungus of the present invention is in the same branch as M. pilosus and M. ruber, with a substitution support rate of 97% (>50%). As shown by the results of the ITS sequence alignment, the Monascus fungus of the present invention is in the same monophyletic group as M. purpurerus, with a substitution support rate of 62% (>50%) (see Figure 3). The results of Figures 2 and 3 indicate whether the Monascus fungus of the present invention belongs to either M. pilosus or M. ruber. The results of this species-specific PCR analysis are consistent with the results of the phylogenetic tree analysis showing the relatedness (see Figure 4, where RubPil: M. ruber / pilosus-specific, Mpus: M. purpureus-specific). The PCR results for the pksCT gene also show that the Monascus genome of the present invention does not contain the pksCT gene (see Figure 5, among which NTU 568 Monascus purpureus: positive control group) belongs to either M. pilosus or M. ruber.

[0043] Additionally, currently, M. pilosus and M. ruber are taxonomically distinguished based on the presence or absence of pigment in the ascocarp exosporium (M. ruber is usually brown, while M. pilosus is colorless). As shown in the results, all ascocarps of the Monascus of the present invention are colorless (see Figure 6), and it was therefore clear that the species of Monascus of the present invention can be determined to be M. pilosus.

[0044] Therefore, the results of the ITS sequence alignment, β-tubulin sequence alignment, species-specific PCR, and pksCT gene PCR in Example 1 all indicate whether the Monascus of the present invention belongs to M. pilosus or M. ruber, and after observing the ascocarp morphology, the Monascus of the present invention is determined to be M. pilosus. In addition, the results of the ITS sequence and β-tubulin sequence alignment analysis show that the Monascus of the present invention is a novel Monascus isolate.

[0045] Example 2: Method for culturing Monascus of the present invention 1. Seed culture: (1) Weigh out 2 g of polished rice flour and place it in a 500 mL Erlenmeyer culture flask with a straight groove at the bottom. Add 100 mL of reverse osmosis water (RO water). Attach a breathable silicone stopper and shake well. Sterilize at 1.25 atmospheres (atm) and 121°C for 20 minutes (min). After sterilization, leave to cool at room temperature.

[0046] (2) Three small pieces of Monascus seed culture were scooped out from the culture dish and placed in a 500 mL shake flask. An air-permeable silicone stopper was attached and the flask was shaken in a culture box at 30°C at a rotation speed of 150-200 rpm for 48-72 hours.

[0047] 2.Solid culture (1) Inoculation: 300g of polished rice is soaked in RO water overnight, drained dry, wrapped in koji cloth, transferred to a koji tray, sterilized (121°C, 30 minutes), cooled, and inoculated. The red koji mold starter liquid is poured into the solid substrate (10%) to inoculate, and the mixture is thoroughly and uniformly stirred.

[0048] (2) The culture steps are as follows:

[0049] a. Culture conditions: After inoculation, the mixture is wrapped in koji cloth and left to stand in a constant temperature and humidity room at a temperature of 30°C and a relative humidity of 60% for culture.

[0050] b. Rehydration: During the rapid growth of Monascus, some of the water in the solid substrate evaporates as the temperature rises, and most of it is consumed during growth, causing the substrate to dry out, so it is necessary to replenish the water. From the second to the eleventh day, approximately 30-50 mL of sterile water is replenished daily, and thereafter, water replenishment continues every other day until the koji is harvested.

[0051] c. Koji recovery: After 21 days of cultivation, the red koji fermented product (cultured for 14 to 28 days) is placed in a roasting oven to dry (37°C, 24 hours), and then stored. This red koji fermented product is then called red koji rice.

[0052] <Example 3: Steps in the method for purifying and isolating Monascinol>

[0053] 1. Weigh out approximately 1000g of red koji rice.

[0054] 2. Add approximately 10 liters (L) of 95% EtOH and perform extraction in a 60°C water bath, shaking evenly every 30 minutes over the period, for 2 hours. After filtering and removing the extract using filter paper, repeat the above extraction procedure once more.

[0055] 3. After filtration, the extract is concentrated under reduced pressure until thick, and 2 to 2.5 times the weight of silica gel is added and mixed. The concentrate is then concentrated under reduced pressure until dry, after which it is placed in a freeze-dryer and freeze-dried overnight, and the weight is then measured.

[0056] 4. Approximately 7 to 8 times the weight of the silica gel from step 3 above is weighed out and mixed with Hex:EtOAc (8:2) solvent and packed into an open chromatography column. After waiting for the chromatography column to equilibrate, the mixture of silica gel from step 3 above and the extract is spread evenly on top and allowed to eluate.

[0057] 5. Elute and wash 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) wash.

[0058] 6. The eluate of Hex: EtOAc (7:3) was concentrated under reduced pressure to dryness, then redissolved in MeOH, and finally purified by preparative HPLC. The solvent conditions were changed to MeOH: ddH2O (83:17). The red yeast yellow pigment effluent was collected and concentrated under reduced pressure to dryness, and then the residual water was removed by vacuum drying to obtain pure monascinol.

[0059] Example 4: Method for conducting experiments using high-fat diet-induced obese rats administered the composition of the present invention

[0060] 1. Preparation of the Composition of the Present Invention (1) Msol group: This group received monascinol, the daily dose for adults being 3 milligrams per day (mg / day).

[0061] (2) RL group and RH group: These groups were administered red koji rice obtained by fermenting polished rice with the Red Koji mold of the present invention. The monascinol content per gram of red koji rice was 3 milligrams per gram (mg / g). The daily dose of red koji rice for adults in the RL group and RH group was 0.5 g / day and 2 g / day, respectively.

[0062] 2. Experiments to adjust body fat composition assessment

[0063] (1) Animal husbandry and care The experimental rats used were 40 8-week-old male Sprague-Dawley (SD) rats obtained from LASCO Biotechnology Co., Ltd. (Taipei, Taiwan). Their environment was maintained at a constant room temperature (25±1°C), a relative humidity of 60%, and a 12-hour light / 12-hour dark cycle (light period: 8:00-20:00). After four days of preliminary breeding, the rats were formally introduced into five groups of eight rats each. The experiment lasted for eight weeks. They were allowed to feed ad libitum throughout the experiment, and their weight, food intake, and drinking water intake were measured weekly.

[0064] (2) Conversion of feeding dose to animals The method for calculating the feeding dose is based on the Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers published by the US Food and Drug Administration in 2005, and is based on an adult weighing 60 kg. When conducting tests using advanced laboratory animals, the dosage is generally converted to the recommended daily intake for humans per kilogram of body weight (mg / kg·bw / day) multiplied by 6.2 to obtain the recommended daily intake for rats per kilogram of body weight. The rat group composition and feeding dose are summarized in Table 1. The calculation formula is shown below (1).

[0065] Rat intake dose per kg of body weight = Recommended intake for humans ÷ 60 kg body weight × 6.2…Equation (1)

[0066] According to this formula (1), the daily intake dose for rats per kilogram of body weight can be calculated.

[0067] (3) Grouping and induction of experimental animals After grouping the experimental rats, the grouping of high-fat diet-induced obese rats is described below with reference to Table 1.

[0068] i.NOR group: normal animal group.

[0069] ii. HF group: high-fat control group and high-fat diet-induced obese rat group.

[0070] iii. RL group: High-fat diet-induced obese rats were given red yeast rice powder by gavage every day, equivalent to a 60-kilogram adult consuming 0.5g of red yeast rice containing 1.5mg of monascinol every day.

[0071] iv. RH group: High-fat diet-induced obese rats were given red yeast rice powder by gavage every day. This group is equivalent to a 60-kilogram adult consuming 2 g of red yeast rice containing 6 mg of monascinol every day.

[0072] v. Msol group: High-fat diet-induced obese rats were given monascinol powder by gavage every day, which is equivalent to a 60-kilogram adult taking 3 mg of monascinol powder every day.

[0073] [Table 1]

[0074] The normal diet consisted of 100% chow diet, and the high-fat diet consisted of 72.3% chow diet, 26.7% butter powder, and 1% cholesterol.

[0075] (4) Feeding method of test substance: The test substance was administered by oral gavage. The concentration of the test substance was adjusted using reverse osmosis water. Each group was fed the required dose daily using a 2.0 mL sterile plastic syringe equipped with a stainless steel feeding needle. Under normal conditions, the stainless steel feeding needle was immersed in a 70% alcohol solution and then rinsed several times with sterile distilled water before use. The test substance was administered to the experimental animals in each group once a day for 8 consecutive weeks.

[0076] (5) Sacrifice blood collection: In the 8-week study, rats were fasted for 12 to 14 hours before blood collection. All rats were euthanized using carbon dioxide, and blood samples were collected from the inferior vena cava. The collected blood samples were centrifuged at 4°C and 3,000 x g for 15 minutes.

[0077] (6) Feeding method of test substance The rats were kept for four days before being divided into groups for the experiment. The NOR group received reverse osmosis water as their drinking water and was fed the standard Lab Diet 5001 (chow diet) with a calorie content of 3.35 kilocalories per gram (kcal / g). The remaining four groups were used in a high-fat (obesity) induction method, and obesity was induced using a high-calorie diet (4.31 kcal / g) containing a mixture of 72.3% LabDiet 5001 powder, 26.7% butter powder, and 1% cholesterol.

[0078] (7) Evaluation of body fat composition index i. Weight change The weight of each rat was measured weekly, and after the experiment, the changes in animal weight in each group were compared.

[0079] ii. Body fat mass and body fat percentage At the time of sacrifice, the adipose tissue around the perirenal and epididymal regions of the experimental animals was removed and weighed. The formula for calculating the body fat percentage was expressed as follows:

[0080] Body fat efficiency = (fat weight / body weight) x 100%...Equation (2)

[0081] (8) Animal Sacrifice and Sample Preparation The rats were fasted for 16 hours before sacrifice and then sacrificed by carbon dioxide asphyxiation. After confirming the absence of a heartbeat, blood samples were collected and organs were harvested. Blood was collected from the inferior vena cava of each rat using a syringe. After collection, the needle was slowly removed from the vein and the sample was transferred to a 2 mL Eppendorf tube and allowed to stand. After centrifugation at 10,000 x g for 10 minutes, the upper serum fraction was collected and placed in an Eppendorf tube. The tube was then frozen and stored in a -20°C refrigerator for later analysis.

[0082] (9) Serum vitalization analysis Measurements were performed using an automated biochemistry analyzer (model number: Beckman-700, purchased from Fullerton, California, USA). Measurement items included TC (total cholesterol) and TG (triglyceride).

[0083] (10) Extraction and measurement of lipids in liver and feces 0.1 g of liver tissue or fecal powder was weighed and added to 1 mL of chloroform:methanol (2:1, v / v volume ratio), homogenized using a tissue homogenizer, centrifuged, and the supernatant was removed. The supernatant was then aerated and dried to remove the solvent. Finally, dimethyl sulfoxide (DMSO) was added to reconstitute the sample, and the extract was stored at -20°C. TC concentrations were analyzed using a commercially available biochemical reagent (product number: BXC 0261, manufactured by Fortress) while TG concentrations were analyzed using a commercially available biochemical reagent (product number: BXC 0271, manufactured by Fortress) according to the instructions in the assay kit's instruction manual.

[0084] (11) Biological statistical analysis method All experimental results are expressed as mean ± standard deviation (SD). Statistical analysis was performed using one-way ANOVA in the Statistical Package for the Social Sciences (SPSS 12.0) system, followed by Duncan's test to compare differences between groups. p<0.05 indicates a significant difference.

[0085] (12) Research methods for intestinal flora i. Fecal analysis of enterobacteriaceae Collection of fecal samples Before sacrifice, approximately 0.3 g of fresh feces was collected from each rat and transferred to a 1.5 mL Eppendorf tube, sealed with parafilm, and frozen at -80°C. The feces were then sent to Biotools Bioscience Technology Co., Ltd. (New Taipei, Taiwan) for further processing and analysis.

[0086] ii. Extraction of genomic DNA and PCR extension / purification Total genomic DNA (QIAamp PowerFecal DNA, Qiagen) was extracted from the sample. The DNA concentration was measured using a Qubit 4.0 fluorometer (Thermo Scientific), and the template DNA was adjusted to 1 ng / μL. The full-length sequence of the 16S gene (covering regions V1 to V9) was elongated using barcoded 16S gene-specific primers (Forward primer 1: 5'Phos / GCATC-16-base barcode - SEQ ID NO: 11, Reverse primer 1: 5'Phos / GCATC-16-base barcode - SEQ ID NO: 12). PCR was performed using KAPA HiFi HotStart ReadyMix (Roche) at 95°C for 3 minutes, followed by 20–27 cycles (sample dependent) of 95°C for 30 seconds, 57°C for 30 seconds, and 72°C for 60 seconds. The PCR products were then monitored on a 1% agarose gel. The samples with a bright main band of approximately 1500 bp were selected and purified using AMPure PB Beads to prepare the SMRTbell library.

[0087] iii. SMRTbell library construction and sequencing The full-length sequence of the 16S gene was extended using barcoded primers in a multiplex SMRTbell library preparation and sequencing program (Pacbio) to construct the SMRTbell library. Sequencing was performed using the circular consensus sequencing (CCS) model on a PacBio Sequel IIe instrument, generating HiFi reads with a predicted accuracy (Phred Scale) of 30. The test DNA was then sequenced repeatedly. Sequence alignments were performed to correct for sequencing errors, ultimately achieving a high accuracy of >99.9% (QV30).

[0088] iv.Biological information analysis Information analysis (sequencing data processing / noise removal and species annotation) Based on the barcode sequence and PCR-extended primer sequence, data for each sample was assigned from the data below. After removing the barcode and primer sequences, all sample sequences were imported into QIIME2 (v2019.7.0, https: / / qiime2.org / ) and stored as a single corresponding artifact. Using QIIME2 cutadapt, the extended subvariable region primers (forward primer 2: SEQ ID NO: 13, reverse primer 2: SEQ ID NO: 14) were removed, and the primer-stripped sequences were used as QIIME2 DADA2 Input Reads. For noise removal and analysis, sequences were first filtered by cutting each sequence to a specified length (forward reads: 280 bp, reverse reads: 220 bp) and setting the MaxEE (forward reads: 2, reverse reads: 2) parameter to the number of bases that allowed for the maximum expected sequence error. Next, denoising is performed using the DADA2 core algorithm, and sequences with errors are corrected based on sequence abundance, quality score, and sequence relationships, resulting in a predicted real sequence. After denoising is completed for each end of the sequence, splicing is performed (minimum overlap length of 20 bp, no mismatches allowed in the overlapping region). Finally, chimeras are removed from the spliced ​​sequence. By comparing the spliced ​​sequence with sequences with relatively high abundance in the sample, sequences with low abundance but similarity to multiple sequences are identified as chimeras and can be removed. After denoising and analysis, the sequence is used as a representative sequence for amplicon sequencing variants (ASVs), and an ASV table is generated, allowing subsequent species annotation.We used the QIIME2 feature-classifier (v2019.7.0, https: / / qiime2.org / ) to select a machine learning-based classification method and train a specific variable region Bayesian classifier with different databases. We used this classifier to annotate species classification according to the representative sequences of ASVs to obtain taxonomic information, and statistically processed the microbial flora composition of each sample at each taxonomic level, such as kingdom, phylum, class, order, family, genus, and species.

[0089] v. Statistical analysis of species differences Welch's t-test was verified using STAMP (v2.1.3) analysis, and bar graphs of species differences between groups were plotted. For metagenomeSeq analysis, permutation tests were performed between groups at each taxonomic level using the metagenomeSeq suite kit in R. p-values ​​were then obtained. The p-values ​​were then corrected using the Benjamini and Hochberg method to adjust for false discovery rates. q-values ​​were obtained, and boxplots showing the distribution of relative abundance between species groups with statistically significant differences (p / q<0.05) were plotted.

[0090] Experiments were carried out using the compositions described in Examples 2 and 3 in accordance with the experimental method described in Example 4 above, and the results are summarized below.

[0091] 1. Effect of the composition of the present invention on daily food, drinking water, calorie intake and body weight in obese rats The experimental animals were induced to become obese using a high-fat diet. The results in Table 2 below show that compared with the NOR group, the daily energy intake of each of the other groups was significantly higher (p<0.05) after being fed a high-fat diet every day. Based on the results of the weight gain of the animals during the animal experiment, when there was no difference in the average initial body weight, the weight gain of each rat after being induced by feeding HF was significantly increased (p<0.05). However, feeding different compositions (RL, RH, and Msol) showed that the weight gain could be significantly reduced (p<0.05) in each case.

[0092] [Table 2]

[0093] 2. Effect of the composition of the present invention on liver weight in obese rats The results in Table 3 show that the HF group had an abnormal increase in liver weight compared to the NOR group. This phenomenon indicates that a daily high-fat diet can lead to a tendency for liver tissue to become abnormal. However, this condition can be significantly improved by feeding the RL, RH, and Msol groups, which can significantly reduce liver weight (p<0.05) and also have an improving effect on the liver-to-body weight ratio.

[0094] [Table 3]

[0095] 3. Effect of the composition of the present invention on fat regulation in obese rats The results in Table 4 show that the fat mass and percentage of obese rats induced with a high-fat diet were significantly reduced in the RL, RH, and Msol groups (p<0.05), compared to a significant increase in the NOR group (p<0.05). These results demonstrate that both red koji rice, a product containing monascinol, and monascinol have the effect of reducing body fat.

[0096] [Table 4]

[0097] 4. Effect of the composition of the present invention on blood lipid regulation in obese rats A high-fat diet increases serum and liver TC and TG concentrations, and large amounts of accumulated lipids accumulate in the liver, causing liver damage. As shown in Table 5, the HF group had significantly higher serum TC and TG concentrations than the NOR group (p<0.05) after the high-fat diet. After feeding RL and RH, TC concentrations were significantly reduced (p<0.05). The pure substance Msol also reduced serum TC concentrations (p<0.05) caused by high fat intake. Msol is the active ingredient in red yeast rice that reduces TC concentrations. Regarding serum TG concentrations, the HF group significantly increased TG concentrations (p<0.05) compared with the NOR group. All doses of red yeast rice significantly reduced TG concentrations (p<0.05), and the Msol group also significantly reduced TG concentrations (p<0.05).

[0098] [Table 5]

[0099] 5. Effect of the composition of the present invention on hepatic lipid regulation in obese rats As shown in Table 6, the liver lipid analysis results showed that the HF group increased liver TC and TG concentrations (p<0.05), feeding low doses of red koji rice significantly reduced the accumulation of TC and TG in the liver (p<0.05) that can be caused by a high-fat diet, and RL, RH, and Msol all effectively reduced liver TC content. In terms of TG content reduction, the RL, RH, and Msol groups achieved significantly greater reductions than the HF group (p<0.05). These results demonstrate that red koji rice is effective in reducing the accumulation of TC and TG in the liver caused by obesity and has the potential to ameliorate the development of fatty liver. Msol is an active ingredient in red koji rice that reduces liver lipids.

[0100] [Table 6]

[0101] 6. Effect of the composition of the present invention on fecal lipid content in obese rats Fecal TC and TG contents were analyzed as shown in Table 7. After the high-fat diet, fecal TC and TG contents were significantly higher than those of the NOR group (p<0.05). Feeding RL and RH promoted the excretion of TC from the body, and the Msol group also significantly promoted TC excretion (p<0.05). As shown by the results of fecal TG content, RL, RH, and Msol were able to effectively reduce TG accumulation in the body. Therefore, it is speculated that red koji rice and Msol improve TG metabolism in the body, leading to reduced accumulation in the liver. These findings also demonstrate that red koji rice and Msol can promote the reduction of body fat by eliminating lipids.

[0102] [Table 7]

[0103] 7. Effect of feeding the present invention's red koji rice and monascinol on the intestinal flora of rats on a high-fat diet The most abundant phyla in the normal intestinal tract are Firmicutes and Bacteroidetes, with the sum of their abundance ratios preferably reaching 80% or greater. The abundance ratio of Firmicutes to Bacteroidetes (F / B ratio) has recently become one of the factors used in microbiome research to assess the risk of specific diseases. Early literature suggests that a relatively higher Firmicutes ratio is associated with obesity in mammals (including mice and humans). Figures 7A to 7F show the effects of feeding the red koji rice and monascinol of the present invention to rats on a high-fat diet on the intestinal bacterial community. These figures are graphs showing the relative abundance of ASVs at the phylum, class, order, family, genus, and species levels of the organism's taxonomic hierarchy. In addition, as shown in Figure 7A, the bacterial abundance analysis results show that the bacterial abundance was mainly distributed among seven phyla, including Bacteroidetes, Proteobacteria, Verrucomicrobia, Deferribacteres, Actinobacteria, Firmicutes, and Tenericutes, with Firmicutes having the highest abundance ratio, followed by Bacteroidetes. It was found that when mice were fed a high-fat diet and then the red koji rice of the present invention and monascinol, the abundance of Firmicutes decreased while the abundance of Bacteroidetes increased (i.e., a decrease in the F / B ratio), indicating that the red koji rice of the present invention can effectively reduce risk factors for obesity-related diseases.

[0104] Akkermansia muciniphila, a member of the Verrucomicrobia phylum, has been demonstrated to be a beneficial intestinal bacterium. It can improve metabolic disorders, blood glucose stabilization, and insulin sensitivity in high-fat diet-induced obese mice. It also suppresses the expression of factors related to mild intestinal inflammation and adiposity, reduces lipogenesis, and prevents hepatic steatosis, contributing to the maintenance of intestinal health. As shown in Figure 7C, feeding with red yeast rice and monascinol, both of which contain the test substance monascinol, increased the abundance of Verrucomicrobiales in the intestinal tract. As shown in Figure 7F, the test substances red yeast rice and monascinol increased A. muciniphila abundance and improved lipid metabolism in high-fat diet-induced obese rats.

[0105] The Bifidobacteriales order within the Actinobacteria phylum is recognized as an important component of beneficial intestinal bacteria in healthy humans, and its numerous beneficial properties include maintaining a balanced intestinal flora, increasing lactose digestibility, anticancer activity, reducing serum cholesterol, and promoting calcium absorption. As shown in Figure 7C, feeding animals the present red koji rice and monascinol can increase the abundance of beneficial intestinal bacteria in the Bifidobacteriales order. Furthermore, as shown in Figure 8, compared with the normal diet (NOR group), the high-fat diet (HF group) significantly increased the abundance of harmful intestinal bacteria in the Eubacteriales order (from 35.07% to 50.14%) and reduced the abundance of beneficial bacteria in the Lactobacillales order (from 36.94% to 10.66%) (p<0.05). Compared to the HF group (50.14%), feeding the red koji rice and monascinol of the present invention significantly reduced the abundance of Eubacteriales (36.91%, 37.66%, and 43.21%). Regarding the abundance of Bacteroidales, feeding low-dose (RL) or high-dose (RH) red koji fermented food significantly increased the abundance of Bacteroidales (p<0.05). Furthermore, in the analysis of the composition of the ranked intestinal bacteria, the abundance of beneficial bacteria such as Ligilactobacillus and Lactobacillales in the red koji fermented food test group was higher than that in the high-HF group (p<0.05).

[0106] Hotspot maps of the relative abundance of bacterial communities in each test group (see Figures 9A-9D) show the composition of bacterial communities between each test group. The bacterial community composition induced by the high-fat diet in the HF group was different from that in the NOR group. After attempting to feed the rats the red yeast rice of the present invention, it was discovered that red yeast rice did not restore the intestinal bacterial community composition to a similar level as the NOR group, but instead improved the abundance of beneficial bacterial communities, thereby ameliorating the changes in bacterial community composition caused by the high-fat diet. For example, feeding red yeast rice and monascinol not only increased the abundance of A. muciniphila, as described above, but also increased the abundance of Dubosiella newyorkensis in the intestinal tract. D. newyorkensis is a beneficial bacteria for which a patent application has already been filed in the United States and is used to treat metabolic syndromes such as obesity and diabetes, as well as immune-related gastrointestinal disorders. Bacteroides thetaiotaomicron is one of the beneficial bacteria that live in the human intestine. B. thetaiotaomicron is closely related to carbohydrate and starch metabolism and polysaccharide utilization. In addition to nutrient metabolism, it also has a variety of positive effects on the intestinal ecosystem, the intestinal immune system, and intestinal gene expression. Red koji rice can also improve the content of this bacterium in the intestine, with the most significant effect being on the pure substance monascinol. Monascinol is the main functional substance that regulates B. thetaiotaomicron and has been proven to have stable potential to improve the intestinal system.

[0107] Taking the above results into consideration, a high-fat diet alters the composition of intestinal flora in normal rats, thereby affecting lipid metabolism in vivo and exacerbating the risks of obesity. The red koji rice and monascinol of the present invention can improve the composition of intestinal flora, thereby reducing the ratio of the abundance of Firmicutes to Bacteroidetes, increasing the abundance of beneficial intestinal bacteria such as Verrucomicrobiales and Bifidobacteriales, and reducing obesity-related bacteria, thereby regulating lipid metabolism in vivo, reducing the accumulation of serum and liver cholesterol and triglycerides, increasing fecal lipid excretion, and reducing body fat.

[0108] Furthermore, in the present invention, RL, RH, and Msol were used as different test substances (compositions) to evaluate the effects of improving the intestinal microflora and body fat in obese rats induced by a high-fat diet for 8 weeks. From the above examples and test results, the following conclusions can be drawn.

[0109] 1. The red yeast rice group (RL, RH) of the present invention contains monascinol. Significant effects were observed on factors such as weight change, liver weight and liver / body weight, body fat weight and body fat percentage, serum and liver TC and TG concentrations. Low-dose red yeast rice powder (RL group) was administered at a daily dose of 0.5g per person. Because the dose was relatively low and effective, this substance offers significant advances in reducing body fat.

[0110] 2. The discovery of monascinol in this application has shown that it has a significant reducing effect on factors such as body weight change, liver weight and liver / body weight, body fat weight and body fat percentage, serum and liver TC activity, and TG activity, even at a low dose, and has the ability to significantly reduce body fat. The dosage is 3 mg of monascinol per person per day, which is quite low and effective, reducing body fat composition and demonstrating considerable advancement.

[0111] 3. A high-fat diet alters the composition of intestinal flora in normal rats, thereby affecting lipid metabolism in vivo and exacerbating the risks associated with obesity. The red koji rice and monascinol of the present invention can improve the composition of intestinal flora, thereby reducing the ratio of the abundance of Firmicutes to Bacteroidetes phyla, and increasing the abundance of beneficial intestinal bacteria such as Verrucomicrobiales and Bifidobacteriales, while also reducing obesity-related bacteria. This can regulate lipid metabolism in vivo, reduce the accumulation of serum cholesterol and triglycerides, and liver cholesterol and triglycerides, increase fecal lipid excretion, and reduce body fat and body weight.

[0112] As explained above, the novel Monascus mold of the present invention, the composition for weight loss and improvement of intestinal flora, and the use of the Monascus mold have been fully and clearly explained. It should be emphasized that the above detailed description specifically describes possible embodiments of the present invention, and the patent scope of the present invention is not limited to these embodiments. As long as it does not deviate from the spirit of the technology of the present invention, equivalent implementations or modifications thereof are still included in the scope of the claims of this application.

[0113] The Monascus pilosus of the present invention has been deposited with the National Collection of Industrial, Food and Marine Bacteria (NCIMB Ltd) in the UK on January 12, 2023 (date of deposit), and has accession number NCIMB 44103.

Claims

1. A Monascus pilosus fungus deposited with the National Collection of Industrial, Food and Marine Bacteria (NCIMB Ltd) in the United Kingdom under accession number NCIMB 44103.

2. Use of Monascus in the manufacture of a composition for weight loss and improving intestinal flora, characterized in that the Monascus may be Monascus pilosus deposited with the National Collection of Bacteria for Industry, Food and Marine Bacteria (NCIMB Ltd) in the United Kingdom under accession number NCIMB 44103.

3. A composition used for weight loss and improving intestinal bacterial flora, characterized in that it contains an effective amount of a functional ingredient, the functional ingredient being extracted from a red koji fermentation product that can be produced by fermenting a substrate using red koji mold, and the red koji mold may be Monascus pilosus, deposited with the National Collection of Bacteria for Industry, Food and Marine Bacteria (NCIMB Ltd) in the United Kingdom under accession number NCIMB 44103.

4. The composition of claim 3, characterized in that it has the ability to increase the abundance of the Bacteroidales bacterial community, thereby reducing the ratio of Firmicutes to Bacteroidetes.

5. 4. The composition according to claim 3, characterized in that it has the ability to increase the abundance of beneficial intestinal bacteria and to modify the composition of intestinal bacteria.

6. The composition according to claim 3, wherein the functional ingredient comprises at least one selected from the group consisting of monascinol, ankaflavin, and monascin.

7. The composition of claim 3, wherein the substrate is rice, yam, or a mixture of related carbohydrates.

8. 4. The composition of claim 3, wherein the effective amount is a daily intake of at least 0.75 milligrams to 12 milligrams of the functional ingredient for an adult when used alone.

9. A composition used for weight loss and improving intestinal bacterial flora, characterized in that it contains an effective amount of red koji fermentation product, the red koji fermentation product can be produced by fermenting a substrate using red koji mold, and the red koji mold can be Monascus pilosus, deposited with the National Collection of Bacteria for Industry, Food and Marine Bacteria (NCIMB Ltd) in the United Kingdom under accession number NCIMB 44103.

10. 10. The composition of claim 9, characterized in that it has the ability to increase the abundance of Bacteroidales bacteria, thereby reducing the ratio of Firmicutes to Bacteroidetes.

11. The composition according to claim 9, characterized in that it has the function of increasing the abundance of beneficial intestinal bacteria and of changing the composition of intestinal bacteria.

12. 10. The composition of claim 9, wherein the substrate is rice, yam, or a mixture of related carbohydrates.

13. 10. The composition of claim 9, wherein the effective amount is at least 0.5 grams per day for an adult, and contains 1.5 milligrams of monascinol.

14. 10. The composition according to claim 9, wherein the red koji fermented product contains at least one functional ingredient, and the functional ingredient includes at least one selected from the group consisting of monascinol, ankaflavin, and monascin.

15. 10. The composition of claim 9, 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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