Chylomicron generation-related gene expression regulators

Abstract

To provide a chylomicron production-related gene expression regulator.SOLUTION: A chylomicron production-related gene expression regulator contains at least one selected from the group consisting of euglena, paramylon, processed paramylon, and β-1,3-glucan.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This invention relates to a chylomicron generation-related gene expression regulator, etc. [Background technology]

[0002] Lipids in food are converted into a form suitable for absorption within the intestinal lumen and absorbed by intestinal epithelial cells. Within the intestinal epithelial cells, absorbed lipids undergo various transformations, ultimately producing chylomicrons, which are lipid transport particles. Chylomicrons are then released from the intestinal epithelial cells into the circulatory system, consisting of lymphatic vessels and blood vessels, and are responsible for the transport of lipids throughout the body. Thus, since chylomicrons play a crucial role in the initial stages of the circulation and transport of exogenous lipids in the body, it is believed that suppressing their production can fundamentally suppress the circulation and transport of lipids in the body.

[0003] Euglena is a microalga belonging to the genus Euglena and is used as a food ingredient. Euglena extract is also applied to the skin (Patent Document 1). Paramylon is a β-1,3-glucan produced by Euglena and has been reported to be useful in wound healing and allergy suppression. However, the relationship between Euglena, β-1,3-glucan, and chylomicron formation remains unknown. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Special Publication No. 2008-526954 [Overview of the project] [Problems that the invention aims to solve]

[0005] The object of this invention is to provide a gene expression regulator related to chylomicron formation. [Means for solving the problem]

[0006] In view of the above problems, the inventors conducted diligent research and found that a chylomicron-generating gene expression regulator containing at least one selected from the group consisting of Euglena, paramylon, paramylon processed products, and β-1,3-glucan can solve the above problems. Based on this finding, further research led to the completion of the present invention. That is, the present invention encompasses the following aspects.

[0007] Item 1. A chylomicron-generating gene expression regulator containing at least one selected from the group consisting of Euglena, paramylon, paramylon processed products, and β-1,3-glucan.

[0008] Item 2. A chylomicron-generating gene expression regulator according to Item 1, comprising at least one selected from the group consisting of Euglena, paramylon, and paramylon processed products.

[0009] Item 3. Contains Euglena and / or paramylon, and The Euglena is Euglena gracilis and / or the paramylon is paramylon derived from Euglena gracilis. A chylomicron generation-related gene expression regulator as described in item 1 or 2.

[0010] Item 4. Containing the aforementioned Euglena and / or paramylon, The Euglena is Euglena gracilis EOD-1 strain (accession number FERM BP-11530) and / or the paramylon is paramylon derived from Euglena gracilis EOD-1 strain (accession number FERM BP-11530), A chylomicron generation-related gene expression regulator as described in item 1 or 2.

[0011] Item 5. A chylomicron-producing gene expression regulator according to any one of items 1 to 4, for use in suppressing the expression of at least one chylomicron-producing gene selected from the group consisting of SR-B1, FABP, ACAT, MGAT, Apo-A IV, ABCA1, Lipase, CLPS, and PAP.

[0012] Item 6. The chylomicron generation-related gene expression regulator according to any one of Items 1 to 5, for use in regulating the expression of chylomicron generation-related genes in intestinal tissue.

[0013] Item 7. The chylomicron generation-related gene expression regulator according to any one of Items 1 to 6, for use in at least one selected from the group consisting of chylomicron generation suppression, lipid absorption suppression in the intestine, and lipid suppression in chylomicrons.

[0014] Item 8. The chylomicron generation-related gene expression regulator according to Item 6 or 7, wherein the intestine is the small intestine.

[0015] Item 9. The chylomicron generation-related gene expression regulator according to any one of Items 1 to 8, for use in at least one selected from the group consisting of obesity prevention and / or improvement, promotion of body fat burning, activation of fatty acid metabolism, induction of fatty acid β-oxidation, prevention and / or improvement of insulin resistance, prevention and / or improvement of diabetes, prevention and / or improvement of dyslipidemia, prevention and / or improvement of fatty liver, prevention and / or improvement of arteriosclerosis, improvement of endurance, prevention and / or improvement of atopic dermatitis, promotion of fatty acid metabolism, normalization of blood lipids, suppression of fatty liver, promotion of energy consumption, suppression of inflammation, reduction of body fat and / or visceral fat, cholesterol suppression, suppression of blood glucose level rise, suppression of overweight, and suppression of BMI.

[0016] Item 10. The chylomicron generation-related gene expression regulator according to any one of Items 1 to 9, which is a food composition, a dietary supplement, a food additive, or a medicine.

[0017] Item 11. The chylomicron generation-related gene expression regulator according to any one of Items 1 to 10, which is an oral composition.

[0018] Item 12. An inhibitor of chylomicron generation, containing at least one selected from the group consisting of Euglena, paramylon, processed paramylon, and β-1,3-glucan.

[0019] Item 13. An inhibitor of lipid absorption in the intestine, containing at least one selected from the group consisting of Euglena, paramylon, processed paramylon, and β-1,3-glucan.

[0020] Item 14. An inhibitor of lipid in chylomicrons, containing at least one selected from the group consisting of Euglena, paramylon, processed paramylon, and β-1,3-glucan.

[0021] Item 14. The inhibitor according to any one of Items 12 to 14, for use in at least one selected from the group consisting of prevention and / or improvement of obesity, promotion of body fat burning, activation of fatty acid metabolism, induction of fatty acid β-oxidation, prevention and / or improvement of insulin resistance, prevention and / or improvement of diabetes, prevention and / or improvement of dyslipidemia, prevention and / or improvement of fatty liver, prevention and / or improvement of arteriosclerosis, improvement of endurance, prevention and / or improvement of atopic dermatitis, promotion of fatty acid metabolism, normalization of blood lipids, suppression of fatty liver, promotion of energy consumption, suppression of inflammation, reduction of body fat and / or visceral fat, suppression of cholesterol, suppression of increase in blood glucose level, suppression of overweight, and suppression of BMI.

Effects of the Invention

[0022] According to the present invention, a regulator of chylomicron production-related gene expression can be provided.

Brief Description of the Drawings

[0023] [Figure 1] It is a graph showing the measurement results of the expression levels of chylomicron production-related genes (SR-B1, FABP, ACAT, MGAT, Apo-A IV, ABCA1) in Test Example 1. The control group is the group fed a high-fat diet supplemented with lard, and the test group is the group fed a high-fat diet in which paramylon was added instead of the cellulose contained in the high-fat diet. The horizontal axis indicates the measured genes. The vertical axis indicates the relative signal when the signal of the control group is set to 1 for each gene. [Figure 2]This graph shows the measurement results of chylomicron production-related gene (Lipase, CLPS, PAP) expression levels in Test Example 1. The control group was given a high-fat diet supplemented with lard, while the test group was given a high-fat diet supplemented with paramylon instead of cellulose. The horizontal axis shows the measured genes. The vertical axis shows the relative signal for each gene, with the signal of the control group set to 1. [Modes for carrying out the invention]

[0024] In this specification, the terms “contains” and “includes” include the concepts of “contains,” “includes,” “substantially consist of,” and “consist solely of.”

[0025] In one aspect, the present invention relates to a chylomicron-generating gene expression regulator (sometimes referred to as "the agent of the present invention" in this specification) containing at least one selected from the group consisting of Euglena, paramylon, paramylon processed products, and β-1,3-glucan. This is described below.

[0026] 1. Euglena Euglena is a microalga belonging to the genus Euglena, and is not particularly limited to that extent. Specifically, Euglena includes, for example, Euglena gracilis (Euglena gracilis) Euglena longa , Euglena caudata , Euglena oxyuris , Euglena tripteris , Euglena proxima , Euglena viridis , Euglena sociabilis , Euglena ehrenbergii , Euglena deses , Euglena pisciformis , Euglena spirogyra , Euglena acus , Euglena geniculata , Euglena intermedia , Euglena mutabilis , Euglena sanguinea , Euglena stellata , Euglena terricola , Euglena klebsi , Euglena rubra , Euglena cyclopicolaThese are some examples. Among these, Euglena gracilis is preferred from the viewpoint of more reliably demonstrating the effects of the present invention, and Euglena gracilis EOD-1 strain is preferred [internationally deposited on June 28, 2013, under the provisions of the Budapest Convention, with accession number FERM BP-11530 at the National Institute of Technology and Evaluation Patent Organism Depositary Center {NITE-IPOD (Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan 292-0818)}].

[0027] The form of Euglena is not particularly limited, as long as it contains the majority of the Euglena cell bodies or their components. Examples of Euglena forms include dried powder, suspension, and extract, with dried powder being preferred.

[0028] The paramylon content of Euglena in its dry state is, for example, 50% or more, preferably 60% or more, and more preferably 70% or more.

[0029] Euglena may be used alone or in combination of two or more species.

[0030] 2. β-1,3-glucan, paramylon β-1,3-glucans are not particularly limited as long as they have a single sugar chain (or sugar chain structure) as their main chain, consisting only of glucose units linked by β1,3 bonds. β-1,3-glucans are not limited to linear structures; branched structures are also included.

[0031] The weight-average molecular weight of the β-1,3-glucan derivative is not particularly limited, but for example, 1 × 10⁻⁶ 4 ~2×10 6 Preferably 5 × 10 4 ~1 × 10 6 More preferably 1 × 10 5 ~1 × 10 6 Yes, it exists. Furthermore, the weight-average molecular weight can be measured by the GPC method.

[0032] β-1,3-glucan may be obtained by chemical synthesis, but from the viewpoint of easy availability, etc., natural β-1,3-glucan produced by various organisms is preferred. Examples of natural β-1,3-glucan include paramylon, curdlan, laminaran, callose, lentinan, schizophyllan, etc. Among these, paramylon is particularly preferred. Hereinafter, paramylon will be described.

[0033] Paramylon is a β-1,3-glucan derived from Euglena and is not particularly limited in that regard.

[0034] Regarding Euglena from which paramylon is derived, it is the same as the description in the above "1. Euglena".

[0035] The mass average molecular weight of paramylon is not particularly limited, but for example, it is 1×10 4 ~5×10 6 , preferably 2×10 4 ~1×10 6 , more preferably 5×10 4 ~1×10 6 , still more preferably 1×10 5 ~5×10 5 is.

[0036] The mass average molecular weight can be measured under the following conditions by SEC-MALS analysis: Detector: Multi-angle light scattering detector (DAWN HELEOS II manufactured by Wyatt Technology), Differential refractive index detector (Optilab T-rEX manufactured by Wyatt Technology) Columns used: 2 TSKgel α-M (manufactured by Tosoh) Mobile phase: DMSO added with 0.05 M potassium bromide Flow rate: 0.5 mL / min.

[0037] Paramylon usually exists as paramylon particles in which triple helix structures formed by β-1,3-glucan chains are highly aggregated based on a certain regularity in the cells of Euglena.

[0038] The shape of paramylon particles is not particularly limited, but is usually a flattened ellipsoid.

[0039] The particle size distribution of paramylon particles is not particularly limited, but is, for example, 0.5 to 15 μm, preferably 1 to 6 μm. The average particle size of paramylon particles is also not particularly limited, but is, for example, 1 to 10, preferably 2 to 4 μm.

[0040] The form of paramylon is not particularly limited as long as it contains paramylon. Examples of Euglena forms include dried paramylon powder and paramylon suspension, with dried paramylon powder being preferred.

[0041] Paramylon may be a single type or a combination of two or more types.

[0042] 3. Method for producing Euglena and paramylon Euglena can be prepared in large quantities by a method that includes a step of culturing Euglena contained in a liquid (culturing step). The culturing step can be carried out, for example, according to a known method (for example, the method described in Japanese Patent Publication No. 5883532). In this culturing step, Euglena microalgae are typically cultivated under aerobic conditions while stirring a liquid (culture medium) containing water, Euglena, and nutrients that Euglena can utilize.

[0043] Nutrients include sugars (monosaccharides such as glucose and fructose), minerals (e.g., sodium, potassium, magnesium, calcium, iron, zinc, molybdenum, copper, phosphorus, nitrogen, sulfur, or boron), and B vitamins (e.g., vitamin B1 (thiamine), vitamin B2 (riboflavin), niacin, pantothenic acid, vitamin B6 (pyridoxine, pyridoxal, or pyridoxamine), vitamin B12 (cyanocobalamin), folic acid, biotin, etc.). The concentration of nutrients in the culture medium is not particularly limited as long as it is at a concentration that allows Euglena to survive and grow.

[0044] The light conditions for the cultivation process are not particularly limited, and the cultivation process may be carried out under either light or dark conditions. When culturing using heterotrophic methods, cultivation is performed under dark conditions. For light conditions, a normal light intensity for algal growth can be used. For dark conditions, for example, 10 μmol / m² 2 Conditions less than / s, preferably complete darkness where no light is present at all, are mentioned.

[0045] The culture temperature in the cultivation process is not particularly limited, as long as it is a temperature at which Euglena can grow. For example, a culture temperature (temperature of the culture medium) of 20°C to 35°C is used.

[0046] The pH of the liquid used in the culture process is not particularly limited, as long as it is within the range at which Euglena can grow. For example, a pH of 3.0 to 5.5 is used for Euglena growth.

[0047] After the culture process, it is preferable to concentrate the Euglena by methods such as centrifugation of the liquid or gravity separation. The obtained Euglena can be subjected to additional processing (e.g., suspension in liquid, dispersion in water or oil, extract, drying and powdering, etc.) depending on the desired form.

[0048] Paramylon particles can be produced by separating, isolating, or purifying them from Euglena according to known methods (e.g., the method described in Japanese Patent Publication No. 5883532). Paramylon particles can be easily obtained, for example, by recovering cellular contents obtained by disrupting the cell membrane of Euglena. Paramylon particles may also be purified as needed. Various methods for purifying paramylon particles are known (e.g., Japanese Patent Publication No. 5883532), and can be carried out according to these methods. Examples of purification steps include surfactant treatment and washing. The obtained Euglena can be subjected to additional processing (e.g., suspension in liquid, dispersion in water or oil, drying and powdering, etc.) depending on the desired form.

[0049] 4. Paramylon processed products Paramylon processed products are obtained by processing paramylon, such as physical treatment or chemical treatment, and are not particularly limited to that extent. Examples of paramylon processed products include fibrous paramylon and amorphous paramylon. Amorphous paramylon can be obtained by chemical treatment according to or similar to known methods, for example, using the method described in Japanese Patent Application Publication No. 2011-184592.

[0050] As the paramylon processed product, fibrous paramylon is preferred. Fiberus paramylon will be described below.

[0051] Fibrous paramylon is a β-1,3-glucan derived from Euglena, and is not particularly limited as long as it is in a fibrous form. Amorphous paramylon obtained by chemically treating paramylon particles (such as alkali treatment) has been reported, but when observed with an electron microscope, it is not recognized as fibrous and is an irregularly shaped mass, so it is not included in fibrous paramylon.

[0052] The weight-average molecular weight of fibrous paramylon is not particularly limited, but for example, 1 × 10⁻⁶ 4 ~2×10 7 Preferably 1 × 10 5 ~5×10 5 That is the case.

[0053] The weight-average molecular weight can be measured by SEC-MALS analysis using the following method: Detector: Multi-angle scattering detector (DAWN HELEOS II, Wyatt Technology) Differential refractometer detector (Optilab T-rEX, Wyatt Technology) Column: Two TSKgel α-M columns (Tosoh Corporation) Mobile phase: DMSO with 0.05M potassium bromide Flow rate: 0.5 mL / min.

[0054] The fiber diameter of fibrous paramylon is not particularly limited, but is, for example, 10 to 500 nm, preferably 20 to 300 nm, and more preferably 50 to 200 nm. The fiber diameter of fibrous paramylon can usually be measured based on electron microscope images of the fibrous paramylon.

[0055] The volume of fibrous paramylon to be deposited in water is not particularly limited, but is, for example, 30 to 300 mL / g, preferably 50 to 250 mL / g, and more preferably 70 to 200 mL / g.

[0056] The volume of sediment in water can be measured according to or in accordance with the following method: The measurement will be performed in accordance with the method described in "Dietary Fiber - Fundamentals and Applications - 3rd Edition, p.111, Daiichi Shuppan, Tokyo (supervised by the Japanese Society for Dietary Fiber Research, edited by the Editorial Committee of the Japanese Society for Dietary Fiber Research, 2008)". Specifically, it will be as follows: Weigh 125 mg of the sample slurry into a 25 mL plastic tube (based on dry mass), and shake the plastic tube vigorously by hand to mix the contents. Then, transfer the contents to a 25 mL graduated cylinder and add pure water until the volume reaches 25 mL. After stirring the liquid in the graduated cylinder, let it stand at 37°C for 24 hours. This will cause the sample to precipitate, creating two layers separated by an interface (a layer mainly containing the precipitated sample (lower layer) and a layer mainly containing water (upper layer)). Determine the volume of the lower layer from the scale on the graduated cylinder, and divide the obtained volume by the sample mass (dry mass) to calculate the volume of sediment in water (mL / g). The test will be performed 3 or 4 times, and the mean and standard deviation will be calculated.

[0057] Fibrous paramylon exhibits relatively high resistance to enzymatic degradation. For example, the amount of monomer (glucose) produced by β-glucanase degradation is, for example, 0.1 to 50 mg, preferably 1 to 10 mg, per gram of fibrillated paramylon.

[0058] This quantity can be measured according to or in accordance with the following method: Prepare a reaction solution [30 mg of test substance (dry weight), 5 mL of buffer solution (Tokyo Chemical Industries, Ltd., B0156, potassium bitopropyl phthalate-sodium hydroxide buffer (pH 4.0)), 0.1 mL of enzyme solution (Nippon Biocon Co., Ltd., endo-1,3-β-Glucanase (enzyme content: 50 units / mL)), pure water, 10 mL of reaction solution] and shake horizontally at 45 rpm at 40°C for 24 hours. Immediately after shaking, freeze and store, then freeze-dry for concentration. After freeze-drying, add 0.5 mL of pure water to each sample and stir (20-fold concentration). Repeat the centrifugation (10000G, 5 minutes, 4°C) and collection of the supernatant twice. Measure the glucose concentration in the collected supernatant using a measurement kit (Wako Pure Chemical Industries, Ltd., Glucose CII-Test Wako). Calculate the amount of glucose produced per 1 g of test substance (mg) based on the measurement value.

[0059] Fiberized paramylon has relatively low solubility in alkaline solutions. For example, fiberized paramylon does not dissolve in a 0.1-0.3 M aqueous sodium hydroxide solution. Here, "does not dissolve" means, for example, that the absorbance (660 nm) of the solution after suspending fiberized paramylon in the aqueous solution (for example, immediately after to 1 hour later) is, for example, 0.1 or higher, preferably 1.0 or higher.

[0060] Solubility can be measured according to or in accordance with the following method: 250 mg (dry weight) of the test substance is suspended in 10 mL of the test solution (pure water, 0.1 M NaOH aqueous solution, 0.3 M NaOH aqueous solution) in a vial. The vial is shaken vigorously by hand for 20 seconds, and then shaken in a shaker at 80 rpm for 1 hour. The absorbance of the solution in the vial at 660 nm is measured after each shake. The absorbance is measured using a spectrophotometer V-730 manufactured by JASCO Corporation.

[0061] The relative value of the crystallinity of fibrous paramylon to granular paramylon (crystallinity of fibrous paramylon / crystallinity of granular paramylon) is, for example, 0.60 to 0.90, preferably 0.65 to 0.80.

[0062] Crystallinity can be measured according to or in accordance with the following method: XRD measurements will be performed on the test substance. The conditions are as follows: Instrument: PANalytical X'Pert3 Powder, Tube voltage: 45kV, Tube current: 40mA, Measurement range: 5.005~50.018°, Measurement interval: 0.013°, Analysis software: HighScore. Crystallinity will be analyzed by the ratio of the intensity of the amorphous region to the intensity of the crystalline region at 2θ=5~80°. The analysis will be performed after removing the instrument's background from each measurement test (background setting Auto, venting factor 0, granularity 100), and the amorphous region will be determined by the tangent passing through 2θ=14, 29°. The conditions for the pending factor and granularity used to determine each amorphous region will be 0 / 20.

[0063] Fiberized paramylon may be dispersed in a solvent such as water, or it may be in a dry form. Even in its dry form, fiberized paramylon can be redispersed in water.

[0064] In this specification, "dried form" means that the moisture content is 15% by mass or less, preferably 10% by mass or less, and more preferably 5% by mass or less.

[0065] Preferably, as the fibrous paramylon, a defibrated product of paramylon particles obtained by physically defibrating the paramylon particles can be used. Alternatively, a defibrated product of Euglena obtained by applying this defibration treatment to Euglena can also be used as the fibrous paramylon.

[0066] The defibration treatment is not particularly limited as long as it is a treatment that can defibrate the paramylon particles without breaking most of the hydrogen bonds of the β-1,3-glucan present in the paramylon particles (for example, by breaking only 10% or less, 5% or less, 2% or less, or 1% or less of the hydrogen bonds of the β-1,3-glucan), or a treatment that can unravel some or all of the β-1,3-glucan chains present in the paramylon particles or the triple helix structure they form. Preferably, the defibration treatment is performed without breaking most of the hydrogen bonds of the β-1,3-glucan present in the paramylon particles to make them fibrous. Known treatments that can grind (shear) or crush (preferably grind (shear)) fine particles such as paramylon particles can be used as the defibration treatment.

[0067] The defibration process can be carried out using known devices such as grinders (shearers) and crushers. Examples of devices used for the defibration process include millstone grinders, jet mills, twin-screw kneaders, high-pressure homogenizers, high-pressure emulsifiers, twin-screw extruders, and bead mills. Among these, millstone grinders and bead mills are preferred.

[0068] The defibration process can be carried out wet or dry. Wet defibration is preferable because it allows for more efficient dispersion of fibrous paramylon in the solution. When using a wet method, the solvent is not particularly limited as long as it can disperse fibrous paramylon; water is a suitable choice.

[0069] The defibration treatment may be performed by a single method or by a combination of two or more methods. Furthermore, the paramylon may be partially defibrated, and as long as it contains defibrated paramylon, it is in line with the intent of the present invention.

[0070] 5.Applications At least one substance selected from the group consisting of Euglena, paramylon, paramylon processed products, and β-1,3-glucan (hereinafter sometimes referred to as "the active ingredient of the present invention") has a chylomicron-related gene expression regulatory effect and can therefore be used as an active ingredient in a chylomicron-related gene expression regulator.

[0071] Genes related to chylomicron formation include those involved in chylomicron production, such as SR-B1 (scavenger receptor class B), FABP (fatty acid binding protein 2), ACAT (acetyl-CoA acetyltransferase 1), MGAT (monoacylglycerol O-acyltransferase), Apo-A IV (apolipoprotein A4), ABCA1 (ATP binding cassette subfamily A member 1), Lipase (Phospholipase A), CLPS (colipase), and PAP (phospholipid phosphatase). The products of these genes are enzymes that convert lipids in the intestinal lumen into a form that can be absorbed into intestinal epithelial cells, receptors that are responsible for the absorption of lipids in the intestinal lumen into intestinal epithelial cells, lipid transport proteins within intestinal epithelial cells, enzymes that convert lipids within intestinal epithelial cells into a form suitable for excretion into the circulatory system, or constituent proteins of particles (chylomicrons) that excrete lipids from intestinal epithelial cells into the circulatory system. All of these have the function of promoting chylomicron formation. The active ingredient of the present invention can suppress the expression of these genes.

[0072] Since chylomicrons are produced in intestinal tissue, the tissue to which expression regulation should be performed is intestinal tissue (preferably small intestinal tissue). Specifically, for example, intestinal (preferably small intestinal) epithelial cells.

[0073] The active ingredient of the present invention has, based on its chylomicron formation-related gene expression regulatory effect, chylomicron formation inhibitory effect, lipid absorption inhibitory effect in the intestine (preferably the small intestine), lipid (preferably triglyceride) inhibitory effect in chylomicrons, etc. Therefore, the active ingredient of the present invention can be used for at least one selected from the group consisting of chylomicron formation inhibition, lipid absorption inhibition in the intestine, and lipid inhibition in chylomicrons. In one embodiment, the present invention relates to a chylomicron formation inhibitor, a lipid absorption inhibitor in the intestine, a lipid inhibitor in chylomicrons, etc., containing the active ingredient of the present invention. In this specification, these agents may also be referred to as "agents of the present invention" together with the chylomicron formation-related gene expression regulators described above.

[0074] "Inhibition of lipid absorption in the intestines" refers to suppressing the process by which exogenous lipids (lipids ingested orally) are absorbed from the intestinal lumen into the body (circulatory system) via intestinal epithelial cells. This inhibition can fundamentally suppress the circulation and transport of lipids within the body.

[0075] "Lipid suppression within chylomicrons" refers to reducing the concentration / amount of lipids within chylomicrons. This suppression can fundamentally inhibit the circulation and transport of lipids in the body.

[0076] Based on the above-mentioned effects, the active ingredients of the present invention can be used for the prevention and / or improvement of obesity, promotion of body fat burning, activation of fatty acid metabolism, induction of fatty acid β-oxidation, prevention and / or improvement of insulin resistance, prevention and / or improvement of diabetes, prevention and / or improvement of dyslipidemia, prevention and / or improvement of fatty liver, prevention and / or improvement of arteriosclerosis, improvement of endurance, prevention and / or improvement of atopic dermatitis, promotion of fatty acid metabolism, normalization of blood lipids, suppression of fatty liver, promotion of energy consumption, suppression of inflammation, reduction of body fat and / or visceral fat, cholesterol suppression, suppression of rise in blood glucose levels, suppression of overweight, and suppression of BMI. Preferably, the active ingredients of the present invention can be used in a comprehensive range of applications including multiple (two or more, more preferably three or more, even more preferably four or more, even more preferably five or more, and even more preferably six or more) of these applications.

[0077] Furthermore, "suppression" encompasses not only reducing something, but also preventing an increase or deterioration in something that is on an upward or worsening trend (reducing the rate of increase or the degree of deterioration, or preventing it from increasing or worsening).

[0078] Furthermore, the active ingredients of the present invention are used for the following purposes and targets: (a) Reduce visceral fat (b) Reduce body fat (c) Helps you lose weight (d) Improve BMI (e) Helps reduce waist circumference (f) Increase the ability to metabolize fat (g) Normalize blood cholesterol levels (h) Lowers blood triglycerides (i) Helps eliminate excess lipids from the body. (j) Suppresses the absorption of excess lipids into the body. (k) On the back with the Venus line (l) Fat Limit (m) For those concerned about belly fat (n) To flatten a protruding belly (o) Blood thinning It can also be used for purposes such as [examples of uses].

[0079] The agent of the present invention can be used in various fields, for example, as a food composition (including health foods, health enhancers, nutritional supplements), a food additive, a cosmetic, a cosmetic additive, a pharmaceutical, a reagent, or a feed. The agent of the present invention is preferably an oral composition.

[0080] The form of the agent of the present invention is not particularly limited and can take the form that is commonly used in each application, depending on the application.

[0081] Examples of the present invention's form of the agent when used in food compositions include liquid, gel, or solid foods such as juices, soft drinks, teas, soups, soy milk, salad oils, dressings, yogurts, jellies, puddings, seasonings, infant formula, cake mixes, dairy products (e.g., in powder, liquid, gel, or solid form), bread, and confectionery (e.g., cookies).

[0082] Examples of the present invention's formulations, when used in cosmetics, include emulsions, cosmetic liquids, face creams, hand creams, lotions, body soaps, shampoos, conditioners, cosmetic gels, masks, foundations, lip balms, and facial cleansers.

[0083] The forms of the preparation of the present invention include, when the use is pharmaceutical, preparations suitable for parenteral administration (especially external preparations), such as ointments, external liquids (liniments, lotions, etc.), sprays (external aerosols, pump sprays, etc.), creams, gels, patches (plasters, ointments, tapes (reservoir type, matrix type, etc.), poultices, patches, microneedles, etc.), eye drops, eye ointments, nasal drops, suppositories, semi-solid rectal preparations, and enema preparations; and preparations suitable for oral administration (oral preparations), such as tablets (including orally disintegrating tablets, chewable tablets, effervescent tablets, lozenges, jelly-like drops, etc.), pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions, syrups), and jellies.

[0084] Examples of the present invention's formulations, when used as additives, health enhancers, or nutritional supplements, include tablets (including orally disintegrating tablets, chewable tablets, effervescent tablets, lozenges, and jelly-like drops), pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including suspensions and syrups), and jellies.

[0085] The agent of the present invention may further contain other components as needed. Other components are not particularly limited as long as they can be incorporated into food compositions (including health foods, health enhancers, nutritional supplements), food additives, cosmetics, cosmetic additives, pharmaceuticals, reagents, animal feed, etc., but examples include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, colorants, fragrances, chelating agents, and the like.

[0086] The content of the active ingredient in the agent of the present invention depends on the application, manner of use, and the condition of the target, and is not limited, but can be, for example, 0.0001 to 100% by mass, preferably 0.001 to 50% by mass.

[0087] The amount of the agent of the present invention to be administered (e.g., given, ingested, or inoculated) is not particularly limited as long as it is an effective amount that produces the desired effect, and is generally 0.1 to 10,000 mg / kg body weight per day as the dry weight of the active ingredient. The above amount is preferably administered in one or more divided doses per day (e.g., 1 to 3 times), and can be increased or decreased as appropriate depending on age, condition, and symptoms.

[0088] In a preferred embodiment of the present invention, the amount of Euglena applied (dry weight) is preferably 100 to 1000 mg, more preferably 250 to 750 mg, and even more preferably 400 to 600 mg per day. It is preferably applied once a day, and preferably daily. The application period is preferably 3 days or more, more preferably 1 week or more, even more preferably 2 weeks or more, even more preferably 4 weeks or more, particularly preferably 6 weeks or more, and especially preferably 8 weeks or more. Since the active ingredient of the present invention is naturally derived and highly safe, there is no particular upper limit to the application period, but it could be, for example, 3 years, 1 year, 6 months, 3 months, or 2 months. [Examples]

[0089] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0090] Reference example 1 Paramylon particles were prepared as follows.

[0091] The prepared Euglena gracilis EOD-1 strain (in its cultured, undried state) was collected in 5 flasks, and the collected liquid was centrifuged in a centrifuge tube (500 × g, 4 minutes, room temperature). The supernatant was removed from the centrifuge tube and collected. The collected supernatant was added to the centrifuge tube to disperse the precipitate, and the entire volume was transferred to a 100 mL graduated cylinder. The collected supernatant was then added to the graduated cylinder to make up 90 mL.

[0092] [Enzyme treatment process] The liquid, which had been made up to 90 mL, was transferred to a 200 mL beaker, and the pH of the liquid was adjusted to 3 by adding hydrochloric acid solution while stirring. A proteolytic enzyme (acid protease, product name "Protease YP-SS", manufactured by Yakult Pharmaceutical Co., Ltd., optimal pH 2.5-3.0) was added to the liquid at a concentration of 5 g / L. The liquid was treated with the enzyme at 50°C for 2 hours while stirring.

[0093] [Surfactant treatment process] An aqueous solution of sodium dodecyl sulfate was added to the enzyme-treated liquid so that the concentration of sodium dodecyl sulfate was 3.0% by mass / volume (w / v). While stirring the liquid containing sodium dodecyl sulfate, the pH of the liquid was adjusted to 3 by adding an aqueous hydrochloric acid solution. Furthermore, the liquid was stirred with a propeller agitator (rotation speed 200 rpm) at 60°C for 30 minutes.

[0094] [Separation process] Paramylon was precipitated by centrifugation (1000 × g, 2 minutes, room temperature), and the paramylon was separated from the liquid after the surfactant treatment step. The surfactant treatment step was repeated in the same manner, except that the concentration of sodium dodecyl sulfate was changed to 1.0 mass / vol.% and the pH was not adjusted. Subsequently, the separation step was performed in the same manner as above. In this way, the surfactant treatment step and the separation step were each performed three times.

[0095] [Washing process] In the separation process, the paramylon precipitated by centrifugation was suspended in pure water and allowed to stand at 40°C for 10 minutes. Next, the paramylon was precipitated again by centrifugation (1000 × g, 2 minutes, room temperature). This procedure was repeated a total of three times.

[0096] [Drying process] Paramylon precipitated by centrifugation during the washing process was dried at 50°C to obtain paramylon particles. The obtained paramylon particles were used as paramylon in the following test examples.

[0097] Example 1: Analysis of the effect on the expression levels of chylomicron-related genes. Mice were fed a diet containing paramylon (Reference Example 1), and the expression levels of chylomicron-related genes were measured. Specifically, the procedure was as follows.

[0098] <1-1. Test Method> <1-1-1. Experimental Animals and Rearing Conditions> Four-week-old male C57BL / 6J mice (manufactured by Charles River Co., Ltd., Japan) were used. After a one-week preliminary rearing period on solid feed (NMF, manufactured by Oriental Yeast Co., Ltd.), the mice were divided into two groups of 10 each to ensure uniform weight distribution.

[0099] The feed used in the experiment was as follows: 20% lard was added to the feeds of both the control and test groups so that the fat energy ratio was 50%. Cellulose was added to the control group's feed so that the dietary fiber weight percentage was 5%, and paramylon was added to the test group's feed so that the dietary fiber weight percentage was 5%. The feed composition of each group is shown in Table 1. The values ​​in Table 1 represent the weight (g) of each component per 1 kg of feed.

[0100] [Table 1]

[0101] In the experiment, mice were given the above-mentioned diet and water ad libitum for 87 days. The rearing environment consisted of a temperature of 22±1℃, humidity of 50±5%, and a 12-hour light-dark cycle (light period: 8am to 8pm, dark period: 8pm to 8am). On the final day of the experiment, the mice were fasted for 8 hours and euthanized with isoflurane / carbon dioxide. The ileum was removed and preserved in RNA Later (Qiagen) to be used as a sample for RNA extraction.

[0102] <1-1-2. Measurement of chylomicron-related gene expression levels> RNA was extracted from the ileum and liver using the RNeasy mini kit (Qiagen), and the mRNA expression levels of chylomicron production-related genes were measured using a microarray method (array: SurePrint G3 Mouse GE 8x60K (Agilent), instrument: GeneChi Scanner 3000 7G system (Thermo Fisher Scientific), analysis software: Transcriptome Viewer (Kurabo)). The chylomicron production-related genes measured are as follows. ·SR-B1 (scavenger receptor class B, Gene ID:12492) ·FABP (fatty acid binding protein 2, Gene ID:14079) ·ACAT (acetyl-CoA acetyltransferase 1, Gene ID:110446) ·MGAT (monoacylglycerol O-acyltransferase, Gene ID:67800) ·Apo-A IV (apolipoprotein A4, Gene ID:11816)·ABCA1(ATP binding cassette subfamily A member 1, Gene ID:11303)·Lipase(phospholipase A2, Gene ID:18778)·CLPS(colipase, Gene ID:109791) ·PAP (phospholipid phosphatase, Gene ID:19012).

[0103] <1-2.Results> The results are shown in Figures 1 and 2. The products of the above genes are enzymes that convert lipids in the intestinal lumen into a form that can be absorbed into intestinal epithelial cells, receptors responsible for the absorption of lipids in the intestinal lumen into intestinal epithelial cells, lipid transport proteins in intestinal epithelial cells, enzymes that convert lipids in intestinal epithelial cells into a form suitable for excretion into the circulatory system, or constituent proteins of particles (chylomicrons) that excrete lipids from intestinal epithelial cells into the circulatory system, and all of these have the function of promoting chylomicron formation. As shown in Figures 1 and 2, the production of these genes was suppressed by paramylon intake, indicating that paramylon has a regulatory effect on the expression of these genes, and furthermore, it has an inhibitory effect on chylomicron formation and lipid absorption in the intestines. In addition, since Euglena contains paramylon, it was considered that Euglena also has a regulatory effect on the expression of chylomicron-related genes, an inhibitory effect on chylomicron formation, and an inhibitory effect on lipid absorption in the intestines.

[0104] Test Example 2. Analysis of the effect on chylomicrons Mice were fed a diet containing paramylon (Reference Example 1), and the triglyceride concentration in chylomicrons was measured. Specifically, the procedure was as follows.

[0105] <2-1. Test Method> The mice were reared in the same manner as in Experiment 1, except that the ad libitum period for both the control and test groups was 83 days. On the final day of the experiment, serum samples were collected from the mice and frozen at -80°C.

[0106] Lipoproteins in 4 μL of thawed serum were separated using tandem-connected Skylight PakLP1-AA gel permeation columns (Skylight Biotech Inc. 300 mm × 4.6 mm ID). Based on the Gaussian approximation, the lipoproteins were separated into 20 subclasses, and the fraction of lipoproteins with a particle size (diameter) of 90 nm was separated as the chylomicron fraction. The triglyceride concentration of the chylomicron fraction was measured. The lipoprotein particle size was determined by the retention time of each peak observed in the chromatogram using a linear calibration curve. The triglyceride concentration of the chylomicron fraction was measured by component peak analysis based on the lipoprotein particle size using the Gaussian curve fitting method.

[0107] As a result, the triglyceride concentration in the chylomicron fraction was 5.09 ± 0.50 mg / dL in the control group and 3.74 ± 0.28 mg / dL in the test group, with a p-value of less than 0.05 between the two groups. Therefore, it was found that paramylon (and Euglena containing it) has a lipid-suppressing effect in chylomicrons.

Claims

1. A chylomicron-generating gene expression regulator comprising Euglena containing paramylon and at least one selected from the group consisting of paramylon.

2. The chylomicron-generating gene expression regulator according to claim 1, wherein the Euglena and / or the paramylon are in the form of a dried powder.

3. The Euglena is Euglena gracilis and / or the paramylon is paramylon derived from Euglena gracilis, A chylomicron generation-related gene expression regulator according to claim 1 or 2.

4. The Euglena is Euglena gracilis EOD-1 strain (accession number FERM BP-11530) and / or the paramylon is paramylon derived from Euglena gracilis EOD-1 strain (accession number FERM BP-11530), A chylomicron generation-related gene expression regulator according to claim 1 or 2.

5. A chylomicron-related gene expression regulator according to any one of claims 1 to 4, for use in suppressing the expression of at least one chylomicron-related gene selected from the group consisting of SR-B1, FABP, ACAT, MGAT, Apo-A IV, ABCA1, Lipase, CLPS, and PAP.

6. A chylomicron-gene-related gene expression regulator according to any one of claims 1 to 5, for use in regulating the expression of chylomicron-gene-related genes in intestinal tissue.

7. A chylomicron-gene-related gene expression regulator according to any one of claims 1 to 6, for use in at least one selected from the group consisting of inhibition of chylomicron generation, inhibition of lipid absorption in the intestines, and inhibition of lipids in chylomicrons.

8. The chylomicron-generating gene expression regulator according to claim 6 or 7, wherein the intestine is the small intestine.

9. A chylomicron-generating gene expression regulator according to any one of claims 1 to 8, for use in at least one selected from the group consisting of prevention and / or improvement of obesity, prevention and / or improvement of insulin resistance, prevention and / or improvement of diabetes, prevention and / or improvement of dyslipidemia, prevention and / or improvement of fatty liver, prevention and / or improvement of arteriosclerosis, normalization of blood lipids, suppression of fatty liver, reduction of body fat and / or visceral fat, suppression of cholesterol, suppression of rise in blood glucose levels, suppression of overweight, and suppression of BMI.

10. A chylomicron-generating gene expression regulator according to any one of claims 1 to 9, which is a food composition, a nutritional supplement, a food additive, or a pharmaceutical product.

11. A chylomicron-generating gene expression regulator according to any one of claims 1 to 10, which is an oral composition.

12. A chylomicron formation inhibitor comprising Euglena containing paramylon and at least one selected from the group consisting of paramylon.

13. A lipid inhibitor in chylomicrons, comprising Euglena containing paramylon and at least one selected from the group consisting of paramylon.

14. An inhibitor according to claim 12 or 13, for use in at least one selected from the group consisting of preventing and / or improving obesity, preventing and / or improving insulin resistance, preventing and / or improving diabetes, preventing and / or improving dyslipidemia, preventing and / or improving fatty liver, preventing and / or improving arteriosclerosis, normalizing blood lipids, suppressing fatty liver, reducing body fat and / or visceral fat, suppressing cholesterol, suppressing the rise in blood glucose levels, suppressing overweight, and suppressing BMI.

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