WO2018159714A1 - Sugar and/or lipid metabolism-improving agent - Google Patents

Abstract

The present invention addresses the problem of providing a sugar and/or lipid metabolism-improving agent. The problem is solved by a sugar and/or lipid metabolism-improving agent that contains fibrous paramylon.

Description

Sugar and / or lipid metabolism improver

The present invention relates to a sugar and / or lipid metabolism-improving agent.

In recent years, lifestyle-related diseases have increased. This is thought to be due to changes in eating habits and lack of exercise. Lifestyle-related diseases are known to be associated with, for example, abnormal lipid metabolism and abnormal sugar metabolism. For this reason, lifestyle-related diseases can lead to various diseases such as arteriosclerosis and hypertension. Therefore, effective means for improving lipid metabolism and sugar metabolism are required to prevent such lifestyle-related diseases.

On the other hand, pharmaceuticals that improve lipid metabolism and sugar metabolism are currently known as antidiabetic drugs and obesity-improving drugs. However, many of these are synthetic low-molecular compounds, and natural components are desirable from the viewpoint that they can be ingested constantly. Further, from the viewpoint of more effectively preventing lifestyle-related diseases, it is desirable that both lipid metabolism and sugar metabolism can be improved.

Paramylon is a kind of β-1,3-glucan contained in Euglena. In recent years, it has been reported that paramylon is useful for wound treatment and allergy suppression (Patent Documents 1 and 2).

Japanese Unexamined Patent Publication No. 2011-184371 Japanese Unexamined Patent Publication No. 2014-231479

An object of the present invention is to provide a sugar and / or lipid metabolism-improving agent. Preferably, an object of the present invention is to provide a sugar and / or lipid metabolism improving agent containing a natural ingredient as an active ingredient.

As a result of intensive studies in view of the above problems, the present inventor has found that the action of improving the metabolism of sugar and / or lipid is remarkably enhanced by fiberizing the natural ingredient paramylon. As a result of further research based on this knowledge, the present inventor completed the present invention.

That is, the present invention includes the following embodiments:
Item 1. A sugar and / or lipid metabolism-improving agent comprising fibrotic paramylon.

Item 2. Item 1. The metabolism improving agent according to Item 1, which is in a dry form.

Item 3. Item 3. The metabolism improving agent according to Item 1 or 2, wherein the fiberized paramylon is a defibrated material of paramylon particles.

Item 4. Item 4. The metabolism improving agent according to any one of Items 1 to 3, wherein the fiberized paramylon is a network structure in which fibers are entangled.

Item 5. Item 5. The metabolism improving agent according to any one of Items 1 to 4, which is a food additive.

Item 6. Item 5. The metabolism improving agent according to any one of Items 1 to 4, which is a food composition.

Item 7. Item 5. The metabolism improving agent according to any one of Items 1 to 4, which is a pharmaceutical agent.

Item 8. Item 8. The metabolism according to any one of Items 1 to 7, which is used for prevention or amelioration of at least one selected from the group consisting of (1) metabolic syndrome, or (2) obesity, diabetes, dyslipidemia, and fatty liver. Improver.

Item 9. A method for producing a sugar and / or lipid metabolism-improving agent, comprising blending fiberized paramylon.

Item 10. Item 10. The method according to Item 9, wherein the fiberized paramylon is in a dry form.

Item 11. Item 11. The method according to Item 9 or 10, further comprising blending water.

Item 12. Fibrotic paramylon for use as a sugar and / or lipid metabolism-improving agent.

Item 13. A method for improving sugar and / or lipid metabolism, including applying fibrotic paramylon to a subject.

Item 14. Use of fiberized paramylon to produce sugar and / or lipid metabolism improvers.

According to the present invention, a sugar and / or lipid metabolism improving agent containing a natural component as an active ingredient can be provided. According to the sugar and / or lipid metabolism improving agent of the present invention, at least selected from the group consisting of metabolic syndrome, dyslipidemia such as hypercholesterolemia and hyperlipidemia, fatty liver, diabetes and obesity It is possible to prevent or ameliorate one kind of disease or condition. In addition, since the sugar and / or lipid metabolism-improving agent of the present invention contains a naturally-occurring polysaccharide as an active ingredient, it is considered that the risk of side effects is low. For this reason, it is suitable for long-term intake.

The microphotograph (upper stage) and vial appearance photograph (lower stage) of a paramylon particle suspension (PM granule) and a fiberized paramylon liquid (fiberized PM) are shown. An electron micrograph of fiberized paramylon (Production Example 1) is shown. The length of one scale on the lower right scale in the figure is 1 μm. The enlarged view in the square frame of the photograph of FIG. 2 is shown. The length of one graduation shown in the lower right of the figure is 100 nm. It is a graph which shows the weight change during a test. The horizontal axis indicates the number of days elapsed from the start of the test, and the vertical axis indicates the average value of body weight. It is a graph which shows the weight gain in a test. There is a significant difference between groups with different alphabets in the control group, biomass group, paramylon group, and fibrotic paramylon group (p <0.05). The display of significant differences by different alphabets in the graph is the same as in FIGS. It is a graph which shows a glucose tolerance measurement result. The horizontal axis shows the elapsed time from administration of the glucose solution, and the vertical axis shows the blood glucose level. * Indicates that there is a significant difference compared to the control group (p <0.05). In (*), p is 0.058. It is a graph which shows the measurement result of the retroabdominal wall fat tissue weight after a test. It is a graph which shows the measurement result of the testicular periphery fat tissue weight after a test. It is a graph which shows the measurement result of the mesenteric adipose tissue weight after a test. It is a graph which shows the measurement result of the liver weight after a test. It is a graph which shows the measurement result of the cecal weight after a test. It is a graph which shows the measurement result of the serum total cholesterol density | concentration after a test. It is a graph which shows the measurement result of the serum LDL cholesterol concentration after a test. It is a graph which shows the serum LDL cholesterol / HDL cholesterol ratio after a test. It is a graph which shows the measurement result of the serum free fatty acid (NEFA) density | concentration after a test. It is a graph which shows the measurement result of the serum ALT (GPT) density | concentration after a test. It is a graph which shows the measurement result of the serum CRP density | concentration after a test. It is a graph which shows the measurement result of the serum leptin density | concentration after a test. It is a graph which shows the measurement result of the serum insulin density | concentration after a test. It is a graph which shows the measurement result of the blood glucose level after a test. It is a graph which shows the measurement result of the diffusion rate of glucose. Each item shown in the graph indicates the presence or absence and the type of the test substance in the test solution. It is a graph which shows the evaluation result of the degradability by (beta) glucanase. The horizontal axis indicates the type of test substance in the test solution. It is a graph which shows the evaluation result of the solubility to an alkaline solution. 0H shows the result measured immediately after shaking, and 1H shows the result measured after standing for 1 hour after shaking. The XRD chart obtained by X-ray diffraction (XRD) analysis is shown. An electron micrograph of fiberized paramylon (Production Example 2) is shown. The length of one scale on the lower right scale in the figure is 1 μm.

In this specification, the expressions “containing” and “including” include the concepts of “containing”, “including”, “consisting essentially of”, and “consisting only of”.

In one aspect of the present invention, the sugar and / or lipid metabolism-improving agent containing fibrotic paramylon (herein referred to as “the metabolism-improving agent of the present invention” or “the agent of the present invention”). Yes.) This will be described below.

1． Fibrous paramylon Fibrous paramylon is a β-1,3-glucan derived from a microalga belonging to the genus Euglena (= genus Euglena) (also referred to as “euglena” in the present specification), and is fibrous. It is not particularly limited as long as it is in the form, and the fibrous form of paramylon is also referred to as fiberized paramylon in the present application. Until now, amorphous paramylon obtained by chemical treatment (alkali treatment, etc.) of paramylon particles has been reported, but this is not recognized as fiberized when observed with an electron microscope, and has a shape and size. Since it is an irregular mass, it is not included in the fiberized paramylon.

Euglena the fiberizing paramylon is derived is not particularly limited, 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 cyclopica , Euglena rubra ola, etc. Among these, Euglena Gracilis is preferably mentioned from the viewpoint that the effects of the present invention can be more reliably exhibited, more preferably Euglena Gracilis EOD-1 strain [product evaluation as of June 28, 2013 As the accession number FERM BP-11530 under the provisions of the Budapest Treaty at the National Institute for Biotechnology Patent Biology Center {NITE-IPOD (zip code 292-0818, room 2-5-8 Kazusa-Kamashita, Kisarazu, Chiba, Japan)} International deposits].

The weight average molecular weight of the fiberized paramylon is not particularly limited, but is, for example, 1 × 10 ⁴ to 2 × 10 ⁷ , preferably 1 × 10 ⁵ to 5 × 10 ⁵ .

The weight average molecular weight can be measured by SEC-MALS analysis by the following method: SEC apparatus: LC-10ADvp system (Shimadzu Co., Japan), column used: KD-806M (shodex., Japan) ,
MALS detector: DAWN HELEOSII (wyatt Technologies., USA), eluent: 1% LiCl / DMI,
Flow rate: 0.5 mL / min.

The diameter of the fiber of the fiberized 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 the fiberized paramylon can be usually measured based on an electron microscopic image of the fiberized paramylon.

The subsidence volume of fiberized paramylon in water is not particularly limited, but is, for example, 30 to 300 μmL / g, preferably 50 to 250 μmL / g, more preferably 70 to 200 μmL / g. The subsidence volume in water can be measured in accordance with or according to Test Example 2.

Fibrous paramylon has a relatively high resistance to enzymatic degradation. For example, the amount of monomer (glucose) produced by the degradation of β-glucanase is, for example, 0.1 to 50 mg, preferably 1 to 10 mg per 1 g of fiberized paramylon. This amount can be measured according to or according to Test Example 5.

Fibrous paramylon has relatively low solubility in alkaline solutions. For example, fiberized paramylon does not dissolve in 0.1-0.3M aqueous sodium hydroxide solution. Here, “does not dissolve” means, for example, the absorbance (660 nm) of the solution after suspending fibrotic paramylon in the aqueous solution (for example, immediately after 1 hour), for example, 0.1 or more, preferably 1.0 That means that. Solubility can be measured in accordance with or according to Test Example 6.

The relative value of the crystallinity of the fiberized paramylon relative to the granular paramylon (the crystallinity of the fiberized paramylon / the crystallinity of the granular paramylon) is, for example, 0.60 to 0.90, preferably 0.65 to 0.80. The degree of crystallinity can be measured according to Test Example 7 or according thereto.

The fiberized paramylon may be in a form dispersed in a solvent such as water, or in a dry form. Fibrotic paramylon can be redispersed in water even in dry form.

In the present specification, “dry form” indicates that the water content is 15% by mass or less, preferably 10% by mass or less, more preferably 5% by mass or less.

Paramylon usually exists in Euglena cells as paramylon particles in which triple-helical structures formed by β-1,3-glucan chains are highly accumulated based on a certain regularity. . As the fiberized paramylon, a defibrated material of paramylon particles, preferably obtained by physically defibrating the paramylon particles, can be used. Further, a euglena defibrated product obtained by applying this defibrating treatment to Euglena can also be used as fiberized paramylon.

The shape of the paramylon particles is not particularly limited, but is usually a flat spheroid.

The particle size distribution of the 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 the paramylon particles is not particularly limited, but is, for example, 1 to 10, preferably 2 to 4 μm.

Paramylon particles can be produced by separating, isolating, or purifying from Euglena according to or according to a known method (for example, the method described in Japanese Patent No. 5858832). Paramylon particles can be easily obtained, for example, by collecting cell content components obtained by destroying the cell membrane of Euglena. Moreover, you may refine | purify a paramylon particle as needed. Various types of purification of paramylon particles are known (for example, Japanese Patent No. 5858832) and can be performed according to these methods. Examples of the purification step include a surfactant treatment step and a washing step.

Defibration treatment does not substantially break the hydrogen bond of β-1,3 glucan present in paramylon particles (for example, 10% or less, 5% or less, 2% or less of β-1,3 glucan hydrogen bond) Treatment that can be defibrated (with only 1% or less being cut), or part or all of the β-1,3-glucan chain present in the paramylon particles or the triple helical structure formed by this There is no particular limitation as long as it can be processed. Preferably, it is preferable to form a fiber by fibrillating the β-1,3 glucan present in the paramylon particles without almost breaking the hydrogen bond. A known treatment that can grind (shear) or grind (preferably grind (shear)) fine particles such as paramylon particles can be employed as the defibrating treatment.

The fibrillation treatment can be performed using a known grinder (shearing machine), a crusher or the like. Examples of the apparatus used for the defibrating treatment include a stone mill, a jet mill, a twin screw kneader, a high pressure homogenizer, a high pressure emulsifier, a twin screw extruder, a bead mill, and the like. Among these, a stone mill type mill and a bead mill are preferable.

The defibrating treatment can be performed wet or dry. It is preferable to perform the defibrating treatment in a wet manner because the fiberized paramylon can be more efficiently dispersed in the solution. The solvent used in the wet process is not particularly limited as long as it is a solvent capable of dispersing fiberized paramylon, and water can be preferably used.

The defibrating treatment may be one type alone or a combination of two or more types. Further, it may be a partially defibrated paramylon, and is intended by the present invention as long as it includes a defibrated paramylon.

The various conditions of the defibrating treatment can be appropriately adjusted according to the defibrating principle, the type of apparatus used for the defibrating treatment, whether it is wet or dry. As an example, each condition (defibration treatment target liquid, clearance, grinding wheel rotation speed, number of times of defibrating treatment) when performing wet defibrating using a mortar mill (supermass colloider) manufactured by Masuyuki Sangyo is as follows: It is as follows.

Defibration target liquid: A water suspension of paramylon particles. The concentration of the paramylon particles is not particularly limited, but is, for example, 0.1 to 40% by mass, preferably 0.5 to 30% by mass, more preferably 1 to 20% by mass, and further preferably 2 to 15% by mass.

Clearance (gap between the grinding wheels): Although not particularly limited, for example, -10 to -800 μm, preferably -30 to -400 μm, more preferably -50 to -300 μm, and further preferably -80 to -150 μm.

Grinding wheel rotation speed: Although not particularly limited, it is, for example, 500 to 3000 rpm, preferably 700 to 2000 rpm, more preferably 800 to 1600 rpm.

Defibration treatment frequency: Although not particularly limited, for example, it is 1 to 30 times, preferably 3 to 25 times, more preferably about 5 to 20 times.

The conditions are appropriately changed depending on the type and manufacturer of the grindstone. For example, when a Glow Engineering millstone grinder (glow mill) is used, the clearance is preferably 10 to 100 μm, for example.

The fiberized paramylon may be used alone or in combination of two or more.

2． Uses Fibrous paramylon has an effect of improving sugar and / or lipid metabolism, and can therefore be used as an active ingredient of a sugar and / or lipid metabolism improving agent. In addition, in this specification, sugar metabolism includes each of a series of phenomena from ingestion to excretion of sugar. In this specification, lipid metabolism includes each of a series of phenomena from intake of lipid or a substance that can be converted into lipid to elimination of lipid or a substance converted from lipid.

In addition, other uses based on the metabolism improving action of sugar and / or lipid, for example, the uses listed below:
(A) Preventive or ameliorating agent such as metabolic syndrome or at least one disease or condition selected from the group consisting of dyslipidemia such as hypercholesterolemia and hyperlipidemia, diabetes, obesity, and fatty liver,
(B) weight suppressant,
(C) an inhibitor of weight gain,
(D) body fat and / or visceral fat inhibitor,
(E) an inhibitor of increase in body fat and / or visceral fat,
(F) a sugar and / or lipid absorption inhibitor,
(G) fat consumption promoter,
(H) blood lipids (eg, cholesterol, neutral fat, etc.) and / or blood sugar level inhibitors,
(I) a blood lipid (eg, cholesterol, neutral fat, etc.) and / or an inhibitor of an increase in blood glucose level, (J) a blood LDL cholesterol inhibitor,
(K) an inhibitor of blood LDL cholesterol elevation,
(L) a blood LH ratio (LDL cholesterol / HDL cholesterol) inhibitor,
(M) an inhibitor of an increase in blood LH ratio (LDL cholesterol / HDL cholesterol),
(N) Intestinal flora improving agent (O) Fecal improvement agent (P) Liver function improving agent (R) Arteriosclerosis preventing agent (S) Insulin resistance improving agent (T) Glucose absorption delaying agent (U) Blood glucose level It can be used as an active ingredient such as a rise retarder.

In addition, the uses, purposes, and subjects listed below:
(A) Reduce visceral fat (b) Reduce body fat increase, reduce body fat, reduce fat absorption (c) Reduce neutral fat (d) Easily consume fat as energy (e) Blood Gently increase the level of triglycerides and blood sugar (f) Suppress sugar absorption, suppress carbohydrate absorption (g) Decrease blood cholesterol, lower LDL cholesterol (h) Improve, improve bowel environment, improve intestinal environment (i) increase blood HDL (good) cholesterol (j) for those who are concerned about visceral fat (k) for those with higher BMI (l) neutral For those who are high in fat (m) For those who are worried about cholesterol (n) For those who are worried about blood sugar level after meals (o) For those who are concerned about liver health it can.

In addition to visceral fat obesity, metabolic syndrome is one of three: (1) high blood pressure, (2) high blood glucose level, (3) low HDL cholesterol or high neutral fat It is said that more than one applies.

The agent of the present invention can be used in various fields, for example, as a food additive, a food composition (including health promoting agents, nutritional supplements (such as supplements)), and pharmaceuticals.

The form of the agent of the present invention is not particularly limited, and can take a form usually used in each application depending on the application.

When the application is food additives, medicines, health enhancers, nutritional supplements (such as supplements), etc., for example, tablets (orally disintegrating tablets, chewable tablets, effervescent tablets, lozenges, jelly-like drops) Pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions, syrups), jelly, etc. .

As a form, when the use is a food composition, liquid, gel or solid food, for example, beverages such as juice, soft drink, tea, soup, soy milk, salad oil, dressing, yogurt, jelly, pudding, sprinkle, Examples include infant formula, cake mix, powdered or liquid dairy products, bread and cookies.

The agent of the present invention may further contain other components as necessary. The other components are not particularly limited as long as they are components that can be blended in food additives, food compositions, pharmaceuticals, health enhancers, nutritional supplements (such as supplements), etc. , Solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, colorants, fragrances, chelating agents and the like.

The agent of the present invention may be in a form in which fiberized paramylon is dispersed in a solvent such as water, or in a dry form. Fibrous paramylon can be more easily dispersed in water, even in dry form.

The agent of the present invention can be produced by a method including a step of blending fiberized paramylon. When the agent of this invention is a drink, for example, the process of mix | blending water is further included. In this case, fiberized paramylon may be blended with an object that already contains water, or water may be blended after blending fiberized paramylon. When blended, the fiberized paramylon can be dispersed in water even in a dry state.

When the agent of the present invention contains a component other than fiberized paramylon, the content of the active ingredient depends on the application, usage mode, application target state, and the like, and is not limited. For example, 0.0001 to 95 mass %, Preferably 0.001 to 50% by mass.

The amount of application (for example, administration, ingestion, inoculation, etc.) of the agent of the present invention to the target organism is not particularly limited as long as it is an effective amount that exhibits a medicinal effect, and is usually the dry weight of fibrinated paramylon that is an active ingredient. In general, it is 0.1 to 10000 mg / kg body weight per day. The above-mentioned application amount is preferably applied at least once a day (for example, 1 to 3 times), and can be appropriately increased or decreased depending on age, disease state, and symptoms.

Hereinafter, the present invention will be described in detail based on examples, but the present invention is not limited to these examples.

Reference Production Example 1: Production of paramylon particles Paramylon particles were purified as follows.

[Culture process]
Euglena Gracilis EOD-1 strain (Internationally Deposited as Accession Number FERM BP-11530 under the provisions of the Budapest Treaty under the Patent Biological Deposit Center (NITE-IPOD) of the National Institute of Technology and Evaluation) under the following conditions Cultured.

“Culture vessel”: 500 mL Sakaguchi flask “Shaking culture conditions”: 125 rpm
“Incubation temperature”: 28 ℃
“Liquid pH at the start of culture”: 4.7 (adjusted with hydrochloric acid)
“Liquid volume for culture”: about 200 mL / 1 flask “Composition of liquid for culture”: As shown in Table 1 “Light irradiation conditions”: 24 hours in the dark “Initial weight of microalgae”: 0.78 g / L (dry weight)
"Culture period": 2 days

After completion of the culture, the liquid for 5 flasks was collected, and the collected liquid was centrifuged in a centrifuge tube (500 × g, 4 minutes, room temperature). The supernatant in the centrifuge tube was once removed and collected. The collected supernatant was put into a centrifuge tube to disperse the precipitate in the centrifuge tube and transferred to a 100 mL graduated cylinder. Further, the collected supernatant was added to a measuring cylinder to make up to 90 mL.

[Enzyme treatment process]
The liquid made up to 90 mL was transferred to a 200 mL beaker, and the pH of the liquid was adjusted to 3 by adding aqueous hydrochloric acid while stirring. Proteolytic enzyme (acidic protease, product name “Protease YP-SS”, Yakult Pharmaceutical Co., Ltd., optimum pH 2.5 to 3.0) was added to the liquid to a concentration of 5 g / L. While stirring the liquid, the enzyme treatment was performed at 50 ° C. for 2 hours.

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

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

[Washing process]
Paramylon precipitated by centrifugation in the separation step was suspended in pure water and allowed to stand at 40 ° C. for 10 minutes. Next, paramylon was precipitated by centrifugation (1000 × g, 2 minutes, room temperature). Such operation was performed 3 times in total.

[Drying process]
Paramylon precipitated by centrifugation in the washing step was dried at 50 ° C. to obtain paramylon particles.

Production Example 1: Production of fiberized paramylon 1
Paramylon particles produced by repeating Reference Production Example 1 were mixed with purified water to prepare a paramylon particle suspension. The concentration of paramylon particles in the paramylon particle suspension was 5% by mass. The paramylon particle suspension was wet defibrated under the conditions shown in Table 2 below using a stone mill grinder (Supermass colloider, manufactured by Masuko Sangyo Co., Ltd.) to obtain slurry discharged from the grinder. This wet defibrating treatment was repeated 20 times in total. The finally obtained slurry was used as a fiberized paramylon solution in the test. FIG. 1 shows a microphotograph of the paramylon particle suspension, a fiberized paramylon solution, and an appearance photograph of a vial containing these solutions.
In the following charts, fiberized paramylon is sometimes referred to as fibrous paramylon.

As shown in the micrograph of FIG. 1, it was confirmed that the paramylon particles were unwound and formed into fibers by wet defibrating treatment. Further, it was confirmed that the fiberized paramylon had a branched structure (branched structure) and a network structure. In addition, as shown in the outer appearance photograph of FIG. 1, in the fiberized paramylon liquid, it was shown that the fiberized paramylon was uniformly dispersed in the liquid.

Comparative Production Example 1: Production of Chemically Treated Paramylon The paramylon particles of Reference Production Example 1 were chemically treated using the method described in Japanese Patent Application Laid-Open No. 2011-184592. Specifically, the neutralization treatment was performed by dissolving paramylon particles in 1M NaOH aqueous solution and adding hydrochloric acid aqueous solution after dissolution. A gel-like material was generated by the neutralization treatment. The supernatant obtained by the separation process by centrifugation was removed to obtain a solid content. Since the solid content contains salt (NaCl) from the neutralization treatment, a large amount of water is added to the obtained solid content to disperse the solid content, resulting in a gel-like material, and similarly centrifuged. By performing the separation process by separation, the salt contained in the gel-like material was removed. The salt removal treatment was repeated until the dry mass of NaCl contained in the gel-like material became 0.1 mass% or less per dry weight of the paramylon particles dissolved in the 1M NaOH aqueous solution, to obtain chemically treated paramylon. The dry weight of NaCl contained in the gel-like material was determined by calculating the NaCl concentration of the supernatant after centrifugation from the electrical conductivity of the supernatant. In addition, according to published literature (2014 Strategic Substrate Technology Advancement Support Project, Research and Development on Advanced Culture Production Technology and Use of Polysaccharide Paramylon (Report on Research and Development Results March 2015)), this chemical treatment As a result of observation by an electron microscope, the paramylon was not a fibrous shape but a lump having an indefinite shape or size.

Test Example 1: Analysis of the structure of fiberized paramylon The structure of the fiberized paramylon of Production Example 1 was observed with an electron microscope. Specifically, it was performed as follows. First, 1.5 times the volume of t-butanol was added to the mixture of fiberized paramylon and water, and the fiberized paramylon was dispersed by a vortex mixer. A part of the obtained dispersion was dropped on a flat plate, and the dropped test liquid was frozen. The frozen product was treated under reduced pressure to evaporate the solvent. The obtained sample was subjected to osmium plasma ion coating (thickness 20 nm) and observed with a scanning electron microscope. Observation images are shown in FIGS.

2 and 3, the fiberized paramylon observed in this test had a network structure in which the fibers were intertwined with each other.

Test example 2: Measurement of settling volume in water “Supervised by the Japan Dietary Fiber Society, edited by the Japanese Dietary Fiber Society Editorial Board (2008) Dietary fiber-Basics and applications-3rd edition, p. 111, Daiichi Shuppan, Tokyo” The measurement was performed according to the method. Specifically, it was performed as follows. Each sample test sample (paramylon particles (reference production example 1), fiberized paramylon (production example 1), or chemically treated paramylon (comparative production example 1)) is placed in a 25 mL plastic tube with a dry mass. In conversion, 250 mg (fiberized paramylon only, 125 mg) was weighed, and the contents were stirred by shaking the plastic tube vigorously by hand. Thereafter, the contents were transferred to a 25 mL graduated cylinder, and pure water was added to 25 mL. The liquid in the graduated cylinder was stirred and allowed to stand at 37 ° C. for 24 hours. This resulted in the precipitation of the sample, resulting in two layers separated through the interface (a layer mainly containing the precipitated sample (lower layer) and a layer mainly containing water (upper layer)). The volume of the lower layer was determined from the scale of the graduated cylinder, and the volume obtained was divided by the sample mass (dry mass) to calculate the settling volume in water (mL / g). The test was performed 3 or 4 times, and the average value and standard deviation were calculated. The results are shown in Table 3.

As shown in Table 3, the subsidence volume of fiberized paramylon in water was relatively high. This suggests that fiberized paramylon is excellent in water dispersibility and water retention.

Test Example 3: Analysis of effects on sugar and lipid metabolism Mice were raised with diet containing fibrotic paramylon (Production Example 1) as feed, body weight, glucose tolerance, visceral fat content, organ weight, serum biochemical values Etc. were measured. Specifically, it was performed as follows.

<3-1. Test method>
<3-1-1. Experimental animals and rearing conditions>
This experiment was conducted with the approval of the Ethics Review Committee in accordance with the “Rules Establishing Methods and Maintenance of Animal Experiment Facilities and Specific Experiment Methods” established by the Animal Experiment Committee of the Faculty of Home Economics, Otsuma Women's University. It was.

5-week-old male C57BL / 6J mice (Nippon Charles River) were used. After preliminary breeding for 1 week with solid feed (NMF, manufactured by Oriental Yeast Co., Ltd.), groups were divided into 10 groups per group so that the body weight was uniform (however, the standard feed breeding group was 5 animals).

The feed used for the test is as follows. 20% lard was added to the feed of the control group and the test group so that the fat energy ratio was 50%. To the feed of the control group, 5% cellulose was added as dietary fiber. For the feed of the test group, Euglena gracilis EOD-1 strain dry powder (hereinafter referred to as biomass group), paramylon particles (reference production example 1) (hereinafter referred to as paramylon group), or fiberized paramylon (production example 1) is doubled. The dried fiberized paramylon obtained by mixing with dextrin and freeze-dried (hereinafter referred to as fiberized paramylon group) was added. Each feed was added in consideration of the water content so that the dry weight would be 5%. In addition, since biomass contained 80.2% paramylon, it was prepared with a shortage of 9.9 g of cellulose so that the amount of dietary fiber in the feed was 5%. Paramylon particles and fiberized paramylon were each treated as dietary fiber, and for this reason, no cellulose was added to the feed of the paramylon group and the fiberized paramylon group. Moreover, since dextrin was added to the fiberized paramylon, the same amount of dextrin was added to each group. Table 4 shows the feed composition of each group.

In addition to the control group and the test group described above, a group (hereinafter referred to as a standard group) for ingesting a standard feed to which lard or the like was not added was also prepared.

In the test, mice were allowed to freely consume the above feed and water for 12 weeks, and body weight and feed intake were measured every 2 to 3 days. The breeding environment was a temperature of 22 ± 1 ° C., humidity of 50 ± 5%, and a 12 hour light / dark cycle (light period: 8 o'clock → 20 o'clock, dark period: 20 o'clock → 8 o'clock). On the last day of the test, the food intake and body weight were measured and then fasted overnight, euthanized with isoflurane / carbon dioxide, and blood was collected from the heart. Liver, caecum, retroabdominal wall fat, mesenteric fat and adipose tissue around the epididymis were removed and weighed. Thereafter, the liver was freeze-dried and crushed to obtain a sample for analysis.

<3-1-2. Glucose tolerance measurement>
After the fasting from 8 o'clock in the morning for 8 hours in the last week of breeding, a 20% glucose solution was administered into the stomach of the mouse using a gastric sonde so that the body weight was 1 g / kg body weight. Blood was collected from the tail before administration (0 minutes), and blood was similarly collected 15 minutes, 30 minutes, 60 minutes, and 120 minutes after administration. For quantification of the blood glucose level, “Small blood glucose meter Glutest Ace R” (manufactured by Sanwa Scientific Research Institute) was used.

<3-1-3. Biochemical examination of serum>
Serum or blood concentrations of AST, ALT, ALP, total cholesterol, LDL-cholesterol, HDL-cholesterol, triglyceride (neutral fat), free fatty acid (NEFA), CRP, leptin, insulin, and glucose were measured.

<3-1-4. Statistical analysis>
All statistical processing was performed using statistical software (JMP Pro.12), and one-way analysis of variance was performed. The difference between the mean values was tested using the Turkey-Kramer multiple comparison method. The measurement results are shown as mean ± standard deviation, and the significance level was 5%.

<3-2. Result>
<3-2-1. Weight>
Graphs of body weight change during the test are shown in FIGS.

In addition, there was no significant difference between the groups in the body weight before the test, and the diet of the mice fed with the high fat diet was adjusted so that the calories were aligned, and there was a significant difference in the intake of each group during the test period. I confirmed that there was no.

As shown in FIGS. 4 and 5, the biomass group and the paramylon group showed a slight suppression tendency compared to the control group, whereas the fibrotic paramylon group showed markedly suppressed weight gain compared to the control group. It was. The fibrotic paramylon group has the same level of weight gain as the group fed the standard feed without lard (standard group), despite continuing to eat the lard-added feed. Met.

<3-2-2. Glucose tolerance>
The glucose tolerance measurement results are shown in FIG.

As shown in FIG. 6, the biomass group and the paramylon group had a lower blood glucose level after glucose administration than the control group, but a significant difference was observed 120 minutes after administration. In contrast, the fibrotic paramylon group has a lower blood glucose level after glucose administration than the biomass group and the paramylon group, and it can be seen that the increase in blood glucose level is significantly suppressed compared to the control group. In addition, a significant difference from the control group was observed at an earlier stage (15 minutes after administration), and it was confirmed that the blood glucose level was significantly lower after 60 minutes after administration. Furthermore, although the fiberized paramylon group continues to ingest the feed to which lard was added, the glucose tolerance (blood glucose level after administration of glucose) was the group ingested the standard feed to which lard was not added It was the same level as (standard group).

<3-2-3. Visceral fat mass>
The results of measuring visceral fat after the test are shown in FIGS.

As shown in FIGS. 7 to 9, the biomass group and the paramylon group had the same amount of visceral fat as the control group, whereas the fibrotic paramylon group had significantly less visceral fat than the control group. . Moreover, the visceral fat amount of the fiberized paramylon group tended to be significantly less or less than the biomass group and the control group.

Specifically, regarding mesenteric fat, the paramylon group tended to be less than the control group, and the fibrotic paramylon group was significantly less than the control group.

The fiberized paramylon was significantly less than the control group, biomass group, and paramylon group for the retroabdominal wall fat and the fat around the testicles.

<3-2-4. Accumulation of fat in the liver>
The result of measuring the liver weight after the test is shown in FIG.

As shown in FIG. 10, the liver weight of the fibrotic paramylon group was significantly smaller than that of the control group. This is presumed to be due to a small amount of fat accumulated in the liver.

<3-2-5. Cecal weight>
The results of measuring the cecal weight (including contents) after the test are shown in FIG. 11 for the control group and the test group.

As shown in FIG. 11, the cecal weight of the fibrotic paramylon group was significantly higher than that of the control group, and the biomass group and paramylon group also showed a tendency to be higher than that of the control group. The increase in cecal weight suggests that fibrotic paramylon is broken down in the intestinal bacteria of the large intestine to produce short chain fatty acids. This short-chain fatty acid is known to have an anti-obesity action and the like (for example, Japanese Patent Application Laid-Open No. 06-256402).

<3-2-6. Serum biochemistry>
The results of measuring each serum biochemical value after the test are shown in FIGS.

As shown in FIG. 12, the total cholesterol is significantly less fibrotic paramylon compared to the control group, the biomass group, and the paramylon group, the paramylon group is significantly smaller than the control group, and the biomass group is smaller than the control group. The trend was low. Furthermore, as shown in FIG. 13, LDL cholesterol was also significantly less in the fibrotic paramylon group than in the control group.

It is pointed out that those with a high LDL cholesterol / HDL cholesterol ratio may have higher risk factors for arteriosclerosis than those with a low LDL cholesterol / HDL cholesterol ratio.
(Non-patent literature Ningen Dock 25 (1) 65-70,2010)
As shown in FIG. 14, the LDL cholesterol / HDL cholesterol ratio in the group in which fibrotic paramylon was administered to the high fat diet was significantly lower than that in the control group (high fat diet group). The possibility of adjusting the balance of medium cholesterol appropriately was suggested. Moreover, although there was no significant difference in the group administered biomass to the high fat diet, the LDL cholesterol / HDL cholesterol ratio tended to decrease compared to the high fat diet group.

It was suggested that fibrotic paramylon contributes to vascular health (eg, prevention of arteriosclerosis) by reducing the ratio of LDL cholesterol to HDL cholesterol. Biomass can also contribute to vascular health (eg, prevention of arteriosclerosis).

As shown in FIG. 15, the NEFA value of the fibrotic paramylon group was significantly reduced as compared with the control group (high fat diet group).

NEFA value is an abbreviation of Non-esterified fatty acid, and is a non-ester type fatty acid released into the blood when neutral fat of adipose tissue is degraded by hormone-sensitive lipase. Insulin is known as one of the inhibitors of the action of hormone-sensitive lipase. When a glucose metabolism disorder such as a decrease in insulin secretion or insulin action occurs, adipose tissue degradation by hormone-sensitive lipase increases and NEFA increases. It is considered. In addition, NEFA has a surface-active effect, and when the blood concentration is high, it dissolves the cell membrane and destroys the cells, which is considered to be one of the causes of organ dysfunction.

As shown in FIG. 15, NEFA in the fibrotic paramylon group was significantly lower than that in the control group (high-fat diet group), suggesting the possibility of inhibiting glucose metabolism disorder. Moreover, it was suggested that the risk of developing organ dysfunction may be reduced by suppressing the increase in NEFA.

Also, as shown in FIG. 16, the serum ALT concentration in the fibrotic paramylon group was significantly lower than that in the control group. The serum ALT concentration in the fibrotic paramylon group tended to be lower than that in the biomass group and the control group. ALT is considered to be a component that is released into the blood when liver cells break down, and it is thought that when fat accumulates in the liver, liver cells break down and ALT is released. It was suggested that damage can be suppressed.

Although AST and TG (triglyceride) are not shown, AST was not significantly different in all groups including standard feed, and TG was not significantly different from the control group in other groups. All showed the tendency for the value to become small.

As shown in FIG. 17, the CRP value of the fiberized paramylon group was significantly less than that of the control group, and showed a tendency to be lower than that of the biomass group and paramylon group.

CRP is an abbreviation for C-reactive protein. It is a protein that increases in serum when inflammation or tissue cell destruction occurs, and serves as an index of inflammation. It is known that when chronic inflammation occurs and the value of CRP increases, insulin resistance deteriorates and blood glucose level rises. In addition, it is said that CRP shows a mild high level in diabetes, obesity, hyperlipidemia etc. which are considered as atherosclerotic disease and its risk state, and when metabolic syndrome and hypercholesterolemia overlap in people with high CRP, It has been shown that it is prone to heart disease and stroke.
As shown in FIG. 17, in the group in which fibrotic paramylon was administered to a high-fat diet, the CRP concentration was significantly lower than that in the control group (high-fat diet group), and insulin resistance, increased blood glucose level, etc. This suggests the possibility of reducing the risk.

Also, as shown in FIG. 18, the leptin value was significantly less in the fibrotic paramylon group than in others.

Leptin is an adipocytokine (a physiologically active substance) secreted from white adipocytes, which transmits a powerful satiety signal, resulting in increased energy consumption due to increased sympathetic nerve activity, controlling obesity and controlling weight gain. Play a role.

In most obese people, leptin production increases as the adipose tissue increases, so the blood leptin level is rather high. Accordingly, obese people become so-called “leptin-resistant” states in which eating disorders are not observed regardless of high leptin levels, and obesity is increasingly promoted.
It is also considered that this leptin resistance may trigger insulin resistance. In humans, blood leptin levels are higher in hypertensive diseases than in normal individuals, and it has been reported that blood leptin levels correlate with blood pressure.

As shown in FIG. 18, in the group administered fibrotic paramylon to a high-fat diet, the leptin concentration was significantly reduced compared to the control group (high-fat diet group), suggesting the possibility of reducing the risk. It was.

As shown in FIG. 19 and FIG. 20, the insulin concentration was significantly lower in the fibrotic paramylon group than in the control group, although the blood glucose concentration was not so different between the groups. This suggests that the sensitivity to insulin is increased in the fiberized paramylon intake group, in other words, insulin resistance is improved. In addition, the biomass group and the paramylon group also show a tendency that the insulin concentration is lower than that of the control group, and the insulin resistance tends to be improved in the same manner.

The results of FIGS. 19, 20, and 6 suggest that the fibrotic paramylon group may be used as a diabetes preventive or therapeutic agent because it improves insulin resistance and suppresses an increase in blood glucose level.

Test Example 4: Sugar diffusion inhibition test 1
The amount of sugar in the solution that diffuses and permeates the semipermeable membrane was compared with and without fibrotic paramylon. This test was conducted with reference to a previously published document (J. Agric. Food Chem. 2001, 49, 1026-1029). Specifically, it was performed as follows.

<4-1. Test method>
Glucose concentration is 100 mM, and test substances (fibrinated paramylon (Production Example 1), pectin (Petulin derived from citrus, manufactured by Tokyo Chemical Industry Co., Ltd., product code: P0024-25G), resistant starch (Pine Starch RT, (Matsuya Chemical Industry Co., Ltd.) or indigestible dextrin (Fibersol 2, Matsutani Chemical Industry Co., Ltd.) concentration of 2% by mass, or 3 mL of an aqueous solution containing no test substance was prepared as a test solution. . Specifically, each component was mixed and then prepared by stirring at 37 ° C. for 30 minutes with a rotator. Put 2.5 mL of each test solution into a dialysis tube (MWCO 12000-14000, Thermofisher DIALYSIS TUBING standard grade cat # 2115215), dialyzing with 20 mL of pure water as an external solution while shaking gently (55 rpm) at 37 ° C. did. After 10, 20, 30, 60, 90, 180, and 300 minutes from the start of dialysis, a part (100 μL) of the external solution was collected as a sample solution. The glucose concentration in the sample solution was measured using Glucose CII-Test Wako (manufactured by Wako). The theoretical value of the glucose concentration in the external liquid when dialysis progresses and glucose reaches an equilibrium state is 11.1 mM. Therefore, the ratio of the glucose concentration of the sample liquid to this theoretical value [= (glucose concentration of the sample liquid (mM) /11.1 (mM)) × 100] was calculated as the diffusivity.

<4-2. Result>
The above test was repeated 3 times, and the average value of 3 times was calculated. The result is shown in FIG. As shown in FIG. 21, it was found that fibrotic paramylon suppresses sugar diffusion. Moreover, it was found that this sugar diffusion inhibitory action is not an action of all polysaccharides, and that the fiber diffusion inhibitory action of fibrotic paramylon is higher than that of pectin.

Test Example 5 Evaluation Test for Degradation Resistance by Enzyme Degradation resistance by β-glucanase was evaluated by measuring the amount of monomer produced. Specifically, it was performed as follows.

<5-1. Test method>
Reaction solution [test substance (paramylon particles (reference production example 1), fiberized paramylon (production example 1), chemically treated paramylon (comparative production example 1: dissolved in 1.0 M NaOH aqueous solution)) 30 mg (dry weight), buffer Liquid (Tokyo Chemical Industry B0156, potassium hydrogen phthalate-sodium hydroxide buffer (pH4.0)) 5 mL, enzyme solution (Japan Biocon endo-1,3-β-Glucanase (Lot 91102c) (enzyme-containing Amount: 50 units / mL)) 0.1 mL, pure water, reaction volume 10 mL] was prepared (n = 2) and shaken horizontally at 45 rpm for 24 hours at 40 ° C. Immediately after shaking, it was stored frozen and lyophilized for concentration. After freeze-drying, 0.5 mL of pure water was added to each sample and stirred (concentration 20 times). In addition, only when the chemically treated paramylon of Comparative Production Example 1 was used as a test substance, 0.5 mL was not sufficiently dissolved and suspended, so 0.8 mL of pure water was added to obtain 12.5-fold concentration. Centrifugation (10000 G, 5 minutes, 4 ° C.) and the operation of collecting the supernatant were repeated twice. The glucose concentration in the collected supernatant was measured using a measurement kit (manufactured by Wako Pure Chemical Industries, Ltd., glucose CII-Test Wako). Based on the measured value, the glucose production amount (mg) per 1 g of the test substance was calculated.

<5-2. Result>
The results are shown in FIG. As shown in FIG. 22, it was found that fibrotic paramylon has significantly higher resistance to degradation by β-glucanase than chemically treated paramylon.
In addition, when the same test was implemented about what freeze-dried the test substance once, it became the same result and it was confirmed that a tendency does not change with the presence or absence of prior drying of a test substance.

Test Example 6: Evaluation of solubility in alkaline solution The solubility in alkaline solution was evaluated. Specifically, it was performed as follows.

<6-1. Test method>
Substance to be tested (paramylon particles (reference production example 1) pulverized into powder, fiberized paramylon (production example 1), chemically treated paramylon (comparative production example: dissolved in 1.0M NaOH aqueous solution)) 250 mg ( (Dry weight) was suspended in 10 mL of a test solution (pure water, 0.1 M NaOH aqueous solution, 0.3 M NaOH aqueous solution, 1 M NaOH aqueous solution) in a vial. After the vial was shaken vigorously by hand for 20 seconds and after shaking for 1 hour at 80 rpm on a shaker, the absorbance at 660 nm of the liquid in the vial was measured. The absorbance was measured using a spectrophotometer V-730 manufactured by JASCO Corporation.

<6-2. Result>
The results are shown in FIG. As shown in FIG. 23, it was found that the fiberized paramylon had significantly lower solubility in an alkaline solution than the chemically treated paramylon.
Although not shown in the graph, it was confirmed that both the paramylon particles and the fiberized paramylon were not dissolved in the 0.1M NaOH aqueous solution but were suspended in the same manner as when suspended in pure water.
In addition, any test substance was not dissolved in the 0.5 M HCl aqueous solution, and the suspended state was maintained.

Test example 7: X-ray diffraction (XRD) analysis test substance (paramylon particles (reference production example 1), fiberized paramylon (production example 1), chemically treated paramylon (comparative production example 1: dissolved in 1.0M NaOH aqueous solution)) XRD was measured for each. The conditions are as follows. Equipment: PANalytical X'Pert3 Powder, tube voltage: 45kV, tube current: 40mA, measurement range: 5.005-50.018 °, measurement interval: 0.013 °, analysis software: HighScore. The obtained XRD chart is shown in FIG. FIG. 24 shows that the test substances have different crystallinity.

The crystallinity was analyzed by the ratio of the strength of the amorphous part to the strength of the crystal part at 2θ = 5 to 80 °. The analysis is performed after removing the background from the instrument from each measurement table (background setting Auto, venting factor 0, granularity 100), and amorphous part is determined by tangent passing through 2θ = 14, 29 ° did. The pending factor and granularity conditions for determining each amorphous part were 0/30 for paramylon particles, 0/25 for chemically treated paramylon, and 0/20 for fiberized paramylon. As a result, the crystallinity was 66.2% for paramylon particles, 37.6% for chemically treated paramylon, and 51.0% for fiberized paramylon.

Production Example 2: Production of fiberized paramylon 2
Using a bead mill, a shear force was applied to the paramylon particles (Reference Production Example 1) to fiberize the paramylon particles to produce a liquid additive (dispersion) containing fiberized paramylon. The defibrating treatment with a bead mill was performed under the general operating conditions used for submicron grinding. The raw material solution containing 10% by mass of paramylon particles was defibrated by a bead mill. The obtained fiberized paramylon was observed with an electron microscope. An observation image is shown in FIG.

As shown in FIG. 25, the fiberized paramylon obtained by the bead mill had a network structure in which the fibers were intertwined with each other.

Claims (14)

1. A sugar and / or lipid metabolism-improving agent comprising fibrotic paramylon.
2. The metabolism improving agent according to claim 1, which is in a dry form.
3. The metabolism improving agent according to claim 1 or 2, wherein the fiberized paramylon is a defibrated material of paramylon particles.
4. The metabolism improving agent according to any one of claims 1 to 3, wherein the fiberized paramylon is a network structure in which fibers are intertwined.
5. The metabolism improving agent according to any one of claims 1 to 4, which is a food additive.
6. The metabolism improving agent according to any one of claims 1 to 4, which is a food composition.
7. The metabolism improving agent according to any one of claims 1 to 4, which is a medicine.
8. The metabolism according to any one of claims 1 to 7, which is used for prevention or amelioration of at least one selected from the group consisting of (1) metabolic syndrome, or (2) obesity, diabetes, dyslipidemia and fatty liver. Improver.
9. A method for producing a sugar and / or lipid metabolism-improving agent, comprising blending fiberized paramylon.
10. The manufacturing method of Claim 9 whose said fiberized paramylon is a dry form.
11. Furthermore, the manufacturing method of Claim 9 or 10 including mix | blending water.
12. Fibrotic paramylon for use as a sugar and / or lipid metabolism improver.
13. A method for improving sugar and / or lipid metabolism, comprising applying fibrotic paramylon to a subject.
14. Use of fibrotic paramylon for producing a sugar and / or lipid metabolism-improving agent.

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