JPWO2018159714A1 - Sugar and / or lipid metabolism improver - Google Patents

Abstract

An object of the present invention is to provide a sugar and / or lipid metabolism improving agent, which is solved by a sugar and / or lipid metabolism improving agent containing fiberized paramylon.

Description

  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 glucose metabolism. Therefore, there is a possibility that various diseases such as arteriosclerosis and hypertension may be caused by lifestyle-related diseases. Therefore, effective means for improving lipid metabolism and glucose metabolism in order to prevent such lifestyle-related diseases are required.

  On the other hand, at present, medicines that improve lipid metabolism and glucose metabolism are known as antidiabetic drugs and obesity improving drugs. However, most of these are synthetic low-molecular compounds, and natural components are desirable from the viewpoint that they can be ingested constantly. From the viewpoint that lifestyle-related diseases can be more effectively prevented, it is desirable that both lipid metabolism and glucose 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, allergy suppression, and the like (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.

  The present inventor has conducted intensive studies in view of the above problems, and as a result, has found that fibrosis of paramylon, a natural component, significantly enhances the effect of improving the metabolism of sugar and / or lipid. The present inventors have further studied based on this finding, and as a result, completed the present invention.

That is, the present invention includes the following embodiments:
Item 1. An agent for improving sugar and / or lipid metabolism, comprising fibrillated paramylon.

  Item 2. Item 10. The metabolic improving agent according to Item 1, which is in a dry form.

  Item 3. Item 3. The metabolic ameliorating agent according to Item 1 or 2, wherein the fiberized paramylon is a defibrated paramylon particle.

  Item 4. Item 5. The metabolic improving agent according to any one of Items 1 to 3, wherein the fibrillated paramylon is a network-like structure in which fibers are intertwined.

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

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

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

Item 8. Item 1. The metabolism according to any one of Items 1 to 7, which is used for prevention or improvement 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 production method according to Item 9, wherein the fiberized paramylon is in a dry form.

  Item 11. Item 10. The production method according to item 9 or 10, further comprising adding water.

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

  Item 13. A method for improving metabolism of sugar and / or lipid, which comprises applying fibrillated paramylon to a subject.

  Item 14. Use of fiberized paramylon for producing a sugar and / or lipid metabolism improving agent.

  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 one selected from the group consisting of metabolic syndrome, hypercholesterolemia, dyslipidemia such as hyperlipidemia, fatty liver, diabetes, and obesity. It is possible to prevent or ameliorate one kind of disease or condition. Moreover, 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. Therefore, it is suitable for long-term intake.

The micrograph (upper part) and the vial appearance photograph (lower part) of a paramylon particle suspension (PM granule) and a fibrillated paramylon solution (fibrous PM) are shown. 3 shows an electron micrograph of fiberized paramylon (Production Example 1). The length of one scale of the scale at the lower right of the figure is 1 μm. FIG. 3 shows an enlarged view in a square frame of the photograph of FIG. 2. The length of one scale on the scale shown at the lower right of the figure is 100 nm. It is a graph which shows the weight change during a test. The horizontal axis shows the number of days elapsed from the start of the test, and the vertical axis shows the average value of body weight. It is a graph which shows the amount of weight gain in a test. There is a significant difference between the control group, biomass group, paramylon group and fibrillated paramylon group between groups with different alphabets (p <0.05). The display of the significant difference by different alphabets in the graph is the same in FIGS. It is a graph which shows the glucose tolerance measurement result. The horizontal axis shows the elapsed time from the administration of the glucose solution, and the vertical axis shows the blood glucose level. * Indicates that there is a significant difference (p <0.05) compared to the control group. (*) P is 0.058. It is a graph which shows the measurement result of the posterior abdominal wall adipose tissue weight after a test. It is a graph which shows the measurement result of adipose tissue surrounding epididymis 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 cecum weight after a test. It is a graph which shows the measurement result of the serum total cholesterol 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) concentration after a test. It is a graph which shows the measurement result of the serum CRP concentration after a test. It is a graph which shows the measurement result of the serum leptin concentration after a test. It is a graph which shows the measurement result of the serum insulin concentration after a test. It is a graph which shows the measurement result of the blood glucose concentration after a test. It is a graph which shows the measurement result of the diffusivity 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 decomposability by (beta) glucanase. The horizontal axis indicates the type of the test substance in the test solution. It is a graph which shows the evaluation result of solubility to an alkaline solution. 0H indicates the result measured immediately after shaking, and 1H indicates the result measured after standing for 1 hour after shaking. 3 shows an XRD chart obtained by X-ray diffraction (XRD) analysis. 4 shows an electron micrograph of fiberized paramylon (Production Example 2). The length of one scale of the scale at the lower right of the figure is 1 μm.

  In this specification, the expressions “contain” and “include” include the concepts of “contain”, “include”, “consist essentially”, and “consist only”.

  In one embodiment, the present invention relates to a sugar and / or lipid metabolism improving agent containing a fibrillated paramylon (hereinafter, also referred to as “the metabolic improving agent of the present invention” or “the agent of the present invention”). There is). Hereinafter, this will be described.

1． Fiberized paramylon Fiberized paramylon is β-1,3-glucan derived from a microalga belonging to the genus Euglena (= Euglena), which may be referred to as “Euglena” in the present specification. It is not particularly limited as long as it is in a form, and paramylon in a fibrous form is also referred to as fibrillated paramylon in the present application. Until now, amorphous paramylon obtained by chemically treating paramylon particles (such as alkali treatment) has been reported, but this is not recognized as fibrosis when observed with an electron microscope. Since it is an amorphous mass, it is not included in 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 rubra, like Euglena cyclopicola. Among these, from the viewpoint that the effects of the present invention can be more reliably exerted, preferably Euglena gracilis is mentioned, and more preferably Euglena gracilis EOD-1 strain (Independent administrative corporation product evaluation dated June 28, 2013) Under the provisions of the Budapest Treaty, the Patent Organism Depositary of the National Institute of Technology, NITE-IPOD (Zip code 292-0818, 2-5-8 Kazusa Kamatari, Kisarazu, Chiba, Japan) under accession number FERM BP-11530 Internationally deposited].

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 device: 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 diameter of the fiber of the fiberized paramylon can be usually measured based on an electron microscope image of the fiberized paramylon.

  The sedimentation volume of the fiberized paramylon 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. The volume of sedimentation in water can be measured according to or according to Test Example 2.

  Fiberized paramylon has a relatively high resistance to enzymatic degradation. For example, the amount of the 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.

  Fiberized paramylon has relatively low solubility in alkaline solutions. For example, fiberized paramylon does not dissolve in a 0.1 to 0.3 M aqueous sodium hydroxide solution. Here, “not dissolved” means, for example, that the absorbance (660 nm) of the solution after suspending the fibrillated paramylon in the aqueous solution (for example, immediately after 1 hour) is, for example, 0.1 or more, preferably 1.0 It means above. The solubility can be measured according to or according to Test Example 6.

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

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

  In the present specification, the “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.

  In addition, paramylon usually exists as a paramylon particle in which the triple helical structure formed by β-1,3-glucan chains is highly accumulated under a certain regularity group in Euglena cells. . As the fiberized paramylon, a fibrillated paramylon particle obtained by physically fibrillating the paramylon particles can be preferably used. In addition, a fibrillated product of Euglena obtained by applying this fibrillation treatment to Euglena can also be used as a 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, and preferably 1 to 6 μm. The average particle diameter 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 by a known method (for example, the method described in Japanese Patent No. 5835332) or according thereto. Paramylon particles can be easily obtained, for example, by recovering a cell content component obtained by disrupting the cell membrane of Euglena. Moreover, you may refine | purify paramylon particles as needed. Various methods for purifying paramylon particles are known (for example, Japanese Patent No. 5835332) and can be performed according to those methods. Examples of the purification step include a surfactant treatment step, a washing step, and the like.

  The defibration treatment hardly breaks hydrogen bonds of β-1,3 glucan present in paramylon particles (for example, 10% or less, 5% or less, 2% or less of hydrogen bonds of β-1,3 glucan) A process that can be defibrated (without cutting less than 1%), or unraveling some or all of the β-1,3-glucan chains present in paramylon particles or the triple helical structure formed by them The processing is not particularly limited as long as the processing can be performed. Preferably, it is preferable to fibrillate the β-1,3 glucan present in the paramylon particles without substantially breaking the hydrogen bonds, thereby forming a fibrous state. 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 defibrating treatment can be performed using a known device such as a grinder (shearing machine) and a crusher. Examples of an apparatus used for the defibration treatment include a stone mill type 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 grinder and a bead mill are preferably mentioned.

  The defibration treatment can be performed by a wet method or a dry method. It is preferable to perform the fibrillation treatment in a wet manner, because the fibrillated 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 fibrous paramylon, and water can be suitably used.

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

  Various conditions of the defibration process can be appropriately adjusted according to the defibration principle, the type of apparatus used for the defibration process, whether the process is a wet process or a dry process. As an example, each condition (liquid to be defibrated, clearance, number of rotations of grindstone, number of times of defibration) when performing wet defibration using a stone mill type mill (supermass colloider) manufactured by Masuyuki Sangyo is as follows. It is as follows.

  Liquid to be defibrated: aqueous 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 still more preferably 2 to 15% by mass.

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

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

  Number of defibration treatments: although not particularly limited, for example, about 1 to 30, preferably 3 to 25, and more preferably about 5 to 20 times.

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

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

2． Since fiberized paramylon has a sugar and / or lipid metabolism improving effect, it can be used as an active ingredient of a sugar and / or lipid metabolism improving agent. In addition, in this specification, glucose metabolism includes each of a series of phenomena from sugar intake to excretion. In this specification, lipid metabolism includes each of a series of phenomena from ingestion of lipids or substances that can be converted to lipids to elimination of lipids or substances converted from lipids.

In addition, other uses based on the metabolic improving effect of sugar and / or lipid, for example, the uses listed below:
(A) metabolic syndrome, or hypercholesterolemia, dyslipidemia such as hyperlipidemia, diabetes, obesity, and a preventive or ameliorating agent for at least one disease or condition selected from the group consisting of fatty liver,
(B) weight suppressants,
(C) weight gain inhibitors,
(D) body fat and / or visceral fat inhibitor,
(E) an inhibitor of increase in body fat and / or visceral fat,
(F) sugar and / or lipid absorption inhibitor,
(G) fat consumption promoter,
(H) blood lipids (for example, cholesterol, neutral fats and the like) and / or a blood sugar level inhibitor,
(I) a blood lipid (for example, cholesterol, neutral fat, etc.) and / or an inhibitor of blood glucose elevation, (J) a blood LDL cholesterol inhibitor,
(K) an inhibitor of blood LDL cholesterol elevation,
(L) 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 improver (O) Fecal bowel improver (P) Liver function improver (R) Arteriosclerosis preventive (S) Insulin resistance improver (T) Sugar absorption delayer (U) Blood glucose level It can be used as an active ingredient such as a rise retardant.

In addition, the uses, purposes, and targets listed below are:
(A) Reduce visceral fat (b) Suppress body fat increase, reduce body fat, suppress fat absorption (c) Reduce neutral fat (d) Facilitate consumption of fat as energy (e) Blood Slows rise in neutral fats and blood sugar level (f) Suppresses sugar absorption and suppresses carbohydrate absorption (g) Reduces blood cholesterol, lowers LDL cholesterol level (h) Prepare, improve bowel movements, improve intestinal environment (i) Increase blood HDL (good) cholesterol (j) For those who are worried about visceral fat (k) For those who have a high BMI (l) Neutral For those who have high fat (m) For those who are concerned about cholesterol (n) For those who have high (worry) blood sugar level after eating (o) For those who are worried about liver health it can.

  In addition, metabolic syndrome, in addition to visceral fat-type obesity, is one of three of (1) hypertension, (2) high blood sugar level, (3) low HDL cholesterol or high neutral fat. It is said that more than one condition applies.

  The agent of the present invention can be used in various fields, for example, as a food additive, a food composition (including a health enhancer, a nutritional supplement (such as a supplement)), a medicine, and the like.

  The form of the agent of the present invention is not particularly limited, and may be a form usually used in each use depending on the use.

  In the form of food additives, medicines, health enhancers, nutritional supplements (supplements and the like), for example, tablets (eg, disintegrating tablets inside the mouth, chewable tablets, effervescent tablets, lozenges, jelly-shaped drops) Preparations, pills, granules, fine granules, powders, hard capsules, soft capsules, dry syrups, liquids (including drinks, suspensions, syrups), jellies and the like. .

  As a form, when the use is a food composition, liquid, gel or solid foods, such as juices, soft drinks, tea, soups, drinks such as soy milk, salad oil, dressing, yogurt, jelly, pudding, sprinkle, Examples include infant formula, cake mix, powdered or liquid dairy products, bread, cookies and the like.

  The agent of the present invention may further contain other components as necessary. Other components are not particularly limited as long as they can be added to food additives, food compositions, medicines, health enhancers, nutritional supplements (supplements and the like), but, for example, bases, carriers , Solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, coloring agents, 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. Fiberized paramylon, even in dry form, can be more easily dispersed in water.

  The agent of the present invention can be produced by a method including a step of blending fibrillated paramylon. When the agent of the present invention is, for example, a beverage, a step of adding water is further included. In this case, fibrillated paramylon may be blended with an object that already contains water, or water may be blended after blending the fibrillated 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 use, the mode of use, the state of the application target, and is not limited, but is, for example, 0.0001 to 95 mass. %, Preferably 0.001 to 50% by mass.

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

  Hereinafter, the present invention will be described in detail with reference to 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 step]
Euglena Grasilis EOD-1 strain (deposited internationally under the provisions of the Budapest Treaty with the National Institute of Technology and Evaluation, Patent and Organism Depositary Center (NITE-IPOD) under the accession number FERM BP-11530) under the following conditions: Cultured.

"Culture container": 500 mL Sakaguchi flask "Shaking culture conditions": 125 rpm
"Culture temperature": 28 ° C
"PH of liquid at the start of culture": 4.7 (adjusted with hydrochloric acid)
"Amount of liquid for culture": Approx. 200 mL / flask "Composition of liquid for culture": As shown in Table 1 "Light irradiation conditions": 24 hours dark place "Initial weight of microalgae": 0.78 g / L (dry weight)
"Culture period": 2 days

  After the completion of the culture, the liquid in 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 all of the precipitate was transferred to a 100-mL graduated cylinder. Further, the collected supernatant was added to a measuring cylinder, and the volume was increased to 90 mL.

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

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

[Separation step]
Paramylon was precipitated by centrifugation (1000 × g, 2 minutes, room temperature), and paramylon was separated from the liquid after the surfactant treatment step. A surfactant treatment step was performed in the same manner except that the concentration of sodium dodecyl sulfate was changed to be 1.0% by mass / volume, and the pH was not adjusted. Thereafter, a separation step was performed in the same manner as described above. Thus, each of the surfactant treatment step and the separation step was 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 an operation was performed three 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 subjected to wet fibrillation treatment using a stone mill type grinder (Super Mascolloider, manufactured by Masuko Sangyo Co., Ltd.) under the conditions shown in Table 2 below to obtain a slurry discharged from the grinder. This wet defibration treatment was repeated 20 times in total. The finally obtained slurry was used for the test as a fiberized paramylon solution. FIG. 1 shows a microphotograph of the paramylon particle suspension, the fibrillated paramylon solution, and an appearance photograph of a vial containing these solutions.
In the following, the fiberized paramylon may be referred to as a fibrous paramylon in the figures and tables.

  As shown in the micrograph of FIG. 1, it was confirmed that the paramylon particles were unwound and fibrillated by the wet fibrillation treatment. In addition, it was confirmed that the fiberized paramylon had a branched structure (branched structure) and a network structure. In addition, as shown in the appearance photograph of FIG. 1, in the fibrillated paramylon liquid, it was shown that the fibrillated 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 paramylon particles were dissolved in a 1 M aqueous NaOH solution, and after the dissolution, a neutralization treatment was performed by adding an aqueous hydrochloric acid solution. A gel was produced by the neutralization treatment. The supernatant obtained by the separation treatment by centrifugation was removed to obtain a solid content. Since the solid content contains a salt (NaCl) due to the neutralization treatment, a large amount of water is added to the obtained solid content to disperse the solid content to form a gel, and similarly centrifuge. By performing the separation process in the separation, the salt contained in the gel was removed. The salt removal treatment was repeated until the dry mass of NaCl contained in the gel-like material became 0.1% by mass or less based on the dry weight of paramylon particles dissolved in a 1M aqueous solution of NaOH to obtain chemically treated paramylon. The dry weight of NaCl contained in the gel was determined by calculating the NaCl concentration of the supernatant after centrifugation from the electrical conductivity of the supernatant. In addition, according to the previously reported literature (R & D on advanced culture production technology and utilization of the polysaccharide paramylon in the 2014 strategic substrate technology advancement support project (R & D results report, March 2015)), this chemical treatment Paramylon was observed by an electron microscope and was found to be not a fibrous form but a lump having an irregular shape and size.

Test Example 1: Analysis of the structure of fibrillated paramylon The structure of the fibrillated paramylon of Production Example 1 was observed with an electron microscope. Specifically, the procedure was performed as follows. First, to a mixture of fiberized paramylon and water, 1.5 times the volume of t-butanol was added to the mixture, 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 subjected to a reduced pressure treatment to evaporate the solvent. The obtained sample was coated with an osmium plasma ion coat (thickness: 20 nm) and observed with a scanning electron microscope. The observed images are shown in FIGS.

  As shown in FIGS. 2 and 3, the fibrillated paramylon observed in this test had a network structure in which each fiber was entangled with each other.

Test Example 2: Measurement of submerged sedimentation volume Described in "Dietary Fibers-Supervision and Application-3rd Edition, p.111 First Publishing, Tokyo, supervised by the Japan Dietary Fiber Society, edited by the Editorial Committee of the Japanese Dietary Fiber Society (2008)" The measurement was performed according to the method described. Specifically, the procedure was performed as follows. Each slurry sample of a sample (paramylon particles (Reference Production Example 1), fiberized paramylon (Production Example 1), or chemically treated paramylon (Comparative Production Example 1)) was placed in a 25 mL plastic tube and dried. 250 mg (converted into fiber only, paramylon only, 125 mg) was weighed, and the contents were stirred by vigorously shaking the plastic tube by hand. Thereafter, the contents were transferred to a 25-mL graduated cylinder, and pure water was added until the volume became 25 mL. After stirring the liquid in the measuring cylinder, the liquid was allowed to stand at 37 ° C. for 24 hours. This caused the sample to precipitate, resulting in two layers separated by an 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 measuring cylinder, and the obtained volume was divided by the sample mass (dry mass) to calculate the sedimentation volume in water (mL / g). The test was performed three or four times, and the average value and standard deviation were calculated. Table 3 shows the results.

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

Test Example 3: Analysis of Effects on Sugar and Lipid Metabolism Mice were bred with feed containing fiberized paramylon (Preparation Example 1) as feed, body weight, glucose tolerance, visceral fat, organ weight, serum biochemical values Etc. were measured. Specifically, it was performed as follows.

<3-1. Test method>
<3-1-1. Experimental animals and breeding conditions>
This experiment was conducted with the approval of the ethics review committee in accordance with the "Rules for the Development and Management of Animal Experiment Facilities and Specific Experimental Methods" established by the Animal Experiment Committee of the Faculty of Home Economics at Otsuma Women's University. Was.

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

  The feed used for the test is as follows. 20% lard was added to the diets of the control group and the test group so that the fat energy ratio became 50%. The diet of the control group was supplemented with 5% cellulose as dietary fiber. 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 twice as much as the feed of the test group. Was mixed with dextrin and freeze-dried to obtain a dried fiberized paramylon (hereinafter, fiberized paramylon group). Each feed was added in consideration of the water content so that the dry weight was 5%. In addition, since biomass contained 80.2% of paramylon, a shortage of 9.9 g of cellulose was prepared so that the amount of dietary fiber in the feed was 5%. The paramylon particles and the fiberized paramylon were each treated as dietary fiber, and therefore, no cellulose was added to the feeds of the paramylon group and the fiberized paramylon group. In addition, since dextrin was added to fibrillated paramylon, the same amount of dextrin was added to each group. Table 4 shows the feed composition of each group.

  In addition, apart from the control group and the test group described above, a group (hereinafter, 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 take the above-mentioned feed and water for 12 weeks, and body weight and feed intake were measured every 2-3 days. The breeding environment was a temperature of 22 ± 1 ° C., a humidity of 50 ± 5%, and a 12-hour light / dark cycle (light period: 8: 00 → 20: 00, dark period: 20: 00 → 8: 00). On the last day of the test, the animals were fasted overnight after measuring the food intake and body weight, euthanized with isoflurane / carbon dioxide, and blood was collected from the heart. The liver, cecum, posterior abdominal wall fat, mesenteric fat, and epididymal adipose tissue were excised and weighed. Thereafter, the liver was freeze-dried and pulverized to obtain a sample for analysis.

<3-1-2. Glucose tolerance measurement>
In the last week of breeding, after 8 hours of fasting from 8:00 in the morning, a 20% glucose solution was administered into the stomach of the mouse using a gastric probe at a weight of 1 g / kg body weight. Before administration, blood was collected from the tail (0 minutes), and blood was similarly collected at 15, 30, 60, and 120 minutes after administration. For the determination of the blood glucose level, "Small blood glucose meter Glutest Ace R" (manufactured by Sanwa Kagaku Kenkyusho) was used.

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

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

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

  In addition, there was no significant difference between the groups in body weight before the test, and the diet of mice fed a high-fat diet was adjusted to have the same calorie, and there was no significant difference in the intake of each group during the test period. Confirmed that there is 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 fiberized paramylon group showed a marked suppression of weight gain compared to the control group. Was. Despite the fact that the fiberized paramylon group continued to consume lard-added diet, the weight gain was almost the same as that of the group fed the lard-free standard diet (standard group). Met.

<3-2-2. Glucose tolerance>
FIG. 6 shows the results of the glucose tolerance measurement.

  As shown in FIG. 6, the biomass group and the paramylon group tended to have lower blood glucose levels after glucose administration than the control group, but a significant difference was observed 120 minutes after administration. In contrast, the fiberized paramylon group has a lower blood glucose level after glucose administration than the biomass group and the paramylon group, indicating that the increase in blood glucose level is significantly suppressed as compared to the control group. A significant difference from the control group was also observed at an earlier stage (15 minutes after administration), and it was confirmed that the blood glucose level was significantly lower even after 60 minutes after administration. Furthermore, despite the fact that the fiberized paramylon group continued to ingest lard-added feed, the glucose tolerance (blood glucose level after glucose administration) was changed to the group in which lard-added standard diet was ingested. (Standard group).

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

  As shown in FIGS. 7 to 9, the biomass group and the paramylon group had substantially the same visceral fat amount as the control group, whereas the fibrillated paramylon group had significantly less visceral fat amount than the control group. . In addition, the visceral fat amount of the fiberized paramylon group was significantly smaller or tended to be smaller than those of the biomass group and the control group.

  Specifically, the mesenteric fat tended to be lower in the paramylon group than in the control group, and significantly lower in the fibrillated paramylon group than in the control group.

  Regarding the retro-abdominal wall fat and epididymal fat, the amount of fibrillated paramylon was significantly less than the control group, biomass group, and paramylon group.

<3-2-4. Fat accumulation 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 fibrillated paramylon group was significantly smaller than that of the control group. This is presumed to be due to the small amount of fat accumulated in the liver.

<3-2-5. Cecal weight>
The results of measuring the cecal weight (including the 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 fibrillated paramylon group was significantly higher than the control group, and the biomass group and the paramylon group also tended to be higher than the control group. The increase in cecal weight suggests that fibrillated paramylon is degraded by intestinal bacteria in the large intestine to produce short chain fatty acids. It is known that this short-chain fatty acid has an anti-obesity effect and the like (for example, Japanese Patent Application Laid-Open No. 06-256402).

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

  As shown in FIG. 12, the total cholesterol in the fibrillated paramylon was significantly less than the control, biomass and paramylon groups, the paramylon group was significantly less than the control group, and the biomass group was less than the control group. It became a low trend. In addition, as shown in FIG. 13, LDL cholesterol was significantly lower in the fibrillated paramylon group than in the control group.

It has been pointed out that those with a high LDL / HDL cholesterol ratio may have a higher risk factor for atherosclerosis than those with a low LDL / HDL cholesterol ratio.
(Non-patent literature Health screening 25 (1) 65-70,2010)
As shown in FIG. 14, the LDL cholesterol / HDL cholesterol ratio was significantly lower in the group to which fiberized paramylon was administered to the high-fat diet than in the control group (high-fat diet group). It was suggested that the balance of medium cholesterol could be adjusted appropriately. In addition, although there was no significant difference in the group to which biomass was administered to the high fat diet, the LDL cholesterol / HDL cholesterol ratio tended to be lower than that in the high fat diet group.

  It was suggested that fibrinated paramylon contributes to vascular health (such as prevention of arteriosclerosis) by reducing the ratio of LDL cholesterol to HDL cholesterol. Biomass may also contribute to vascular health (such as prevention of arteriosclerosis).

  As shown in FIG. 15, the NEFA value of the fiberized paramylon group was significantly lower than that of the control group (high fat diet group).

  The NEFA value is an abbreviation for Non-esterified fatty acid, which is a non-esterified fatty acid released into the blood when triglycerides of adipose tissue are degraded by hormone-sensitive lipase. Insulin is known as one of the inhibitors of the action of hormone-sensitive lipase, and when glucose metabolism disorders such as decreased insulin secretion and suppression of insulin action occur, adipose tissue degradation by hormone-sensitive lipase increases and NEFA increases. It is considered. In addition, NEFA has a surfactant activity, and as its concentration in blood increases, it dissolves cell membranes and destroys cells, and is considered to be one of the factors that cause organ dysfunction.

  As shown in FIG. 15, NEFA in the fiberized paramylon group was significantly lower than that in the control group (high fat diet group), suggesting that glucose metabolism disorders may be suppressed. It was also suggested that suppressing the elevation of NEFA may reduce the risk of developing organ dysfunction.

  In addition, as shown in FIG. 16, the serum ALT concentration of the fibrillated paramylon group was significantly lower than that of the control group. The serum ALT concentration in the fibrillated paramylon group tended to be lower than in the biomass group and the control group. ALT is considered to be a component 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 could be suppressed.

  Although AST and TG (triglyceride) are not shown, there was no significant difference in AST between all groups including the standard diet, and TG was not significantly different from the control group in the other groups. In all cases, the values tended to decrease.

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

CRP value is an abbreviation for C-reactive protein, a protein that increases in serum when inflammation or tissue cell destruction occurs, and serves as an indicator of inflammation. It is known that when chronic inflammation occurs and CRP levels increase, insulin resistance worsens and blood sugar levels increase. In addition, it is said that CRP shows mildly elevated values in arteriosclerotic diseases and its risk states of diabetes, obesity, hyperlipidemia, etc., and metabolic syndrome and hypercholesterolemia overlap with people with high CRP, It has been shown to be prone to heart disease and stroke.
As shown in FIG. 17, the CRP concentration was significantly reduced in the group to which fiberized paramylon was administered to the high-fat diet as compared with the control group (high-fat diet group), and insulin resistance and blood glucose level were increased. The possibility of reducing the risk of

  In addition, as shown in FIG. 18, the value of leptin was significantly smaller in the fibrillated paramylon group than in the others.

  Leptin is one of the adipocytokines (bioactive substances) secreted from white adipocytes, transmits a strong satiety signal, increases energy expenditure by increasing sympathetic nervous activity, and controls obesity and controls weight gain. Play a role.

Most of obese people have higher blood leptin levels because leptin production is increased with increase in adipose tissue. Therefore, obese people do not suffer from eating disorders regardless of high leptin levels, so-called "leptin resistance", which further promotes obesity.
It is also thought that this leptin resistance may trigger insulin resistance. It has been reported that in humans, blood leptin levels are higher in hypertensive diseases than in normal subjects, and that blood leptin levels correlate with blood pressure.

  As shown in FIG. 18, the leptin concentration was significantly reduced in the group in which fiberized paramylon was administered to the high-fat diet as compared with the control group (high-fat diet group), suggesting that the risk may be reduced. Was.

  As shown in FIGS. 19 and 20, the blood glucose concentration was not significantly different between the groups, but the insulin concentration was significantly lower in the fibrillar paramylon group than in the control group. This suggests that the group receiving fibrinated paramylon has increased sensitivity to insulin, in other words, improved insulin resistance. In addition, the biomass group and the paramylon group also tend to have a lower insulin concentration than the control group, and these also tend to have improved insulin resistance.

  The results of FIG. 19, FIG. 20, and FIG. 6 suggest that the fibrillated paramylon group can be used as an agent for preventing or treating diabetes because it improves insulin resistance and suppresses an increase in blood glucose level.

Test Example 4: Sugar diffusion suppression test 1
The amount of sugar in the solution that diffused and permeated through the semipermeable membrane was compared with and without fibrillated paramylon. This test was performed 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>
The glucose concentration is 100 mM, and the test substances (fibrinated paramylon (Production Example 1), pectin (citrus-derived pectin, 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, manufactured by Matsutani Chemical Industry Co., Ltd.)) was 2 mass%, or 3 mL of an aqueous solution containing no test substance was prepared as a test solution. . Specifically, after mixing each component, it was prepared by stirring with a rotator at 37 ° C. for 30 minutes. 2.5 mL of each test solution obtained is placed in a dialysis tube (MWCO 12000-14000, Thermofisher DIALYSIS TUBING standard grade cat # 2115215), and dialyzed while slowly shaking (55 rpm) at 37 ° C with 20 mL of pure water as an external solution. did. At 10, 20, 30, 60, 90, 180, and 300 minutes after the start of the 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 (Wako). The theoretical value of the glucose concentration in the external solution when the dialysis proceeds and the glucose is in an equilibrium state is 11.1 mM. Therefore, the ratio of the glucose concentration of the sample liquid to the theoretical value [= (glucose concentration of sample liquid (mM) /11.1 (mM)) × 100] was calculated as the diffusivity.

<4-2. Result>
The above test was repeated three times, and the average of the three tests was calculated. FIG. 21 shows the result. As shown in FIG. 21, it was found that fibrillated paramylon suppressed sugar diffusion. In addition, it was found that this sugar diffusion inhibitory action was not an action of polysaccharides in general, and that the action of fiberized paramylon was higher than that of pectin.

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

<5-1. Test method>
Reaction liquid [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 Solution (B0156, manufactured by Tokyo Chemical Industry Co., Ltd., potassium hydrogen phthalate-sodium hydroxide buffer (pH 4.0)), 5 mL, enzyme solution (endo-1,3-β-Glucanase (Lot 91102c), manufactured by Nippon Biocon Inc.) Amount: 50 units / mL)) 0.1 mL, pure water, reaction solution volume 10 mL] was prepared (n = 2) and horizontally shaken at 45 rpm at 40 ° C for 24 hours. After shaking, it was immediately frozen and stored, and lyophilized for concentration. After freeze-drying, 0.5 mL of pure water was added to each sample and stirred (20-fold concentration). Only when the chemically treated paramylon of Comparative Production Example 1 was used as the test substance, 0.5 mL did not sufficiently dissolve and suspend, so 0.8 mL of pure water was added, and the mixture was concentrated 12.5-fold. The operation of centrifuging (10,000 G, 5 minutes, 4 ° C.) and collecting the supernatant was repeated twice. The glucose concentration in the collected supernatant was measured using a measurement kit (Glucose CII-Test Wako, manufactured by Wako Pure Chemical Industries, Ltd.). Based on the measured values, the amount of glucose produced (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 fibrillated paramylon has significantly higher resistance to degradation by β-glucanase than chemically treated paramylon.
The same test was performed on the test substance once freeze-dried, and the same result was obtained. It was confirmed that the tendency was not changed by the presence or absence of the pre-drying of the test substance.

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

<6-1. Test method>
250 mg of test substance (paramylon particles (Reference Production Example 1) pulverized into powder, fibrillated paramylon (Production Example 1), chemically treated paramylon (Comparative Production Example: dissolved in 1.0 M NaOH aqueous solution)) (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 at 80 rpm for 1 hour on a shaker, the absorbance of the solution in the vial was measured at 660 nm. 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 fibrillated paramylon had a significantly lower solubility in an alkaline solution than 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 0.1 M NaOH aqueous solution and were suspended in the same manner as when suspended in pure water.
In addition, any test substance was not dissolved in a 0.5 M HCl aqueous solution and maintained in a suspended state.

Test Example 7: X-ray diffraction (XRD) analysis test substances (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)) 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. From FIG. 24, it was found that the test substances had different crystallinities.

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

Production Example 2: Production of fiberized paramylon 2
A shear force was applied to the paramylon particles (Reference Production Example 1) using a bead mill to fibrillate the paramylon particles, thereby producing a liquid additive (dispersion liquid) containing fibrillated paramylon. The defibration treatment by a bead mill was performed under general operating conditions used for submicron pulverization. A raw material liquid containing 10% by mass of paramylon particles was subjected to a defibration treatment using a bead mill. The obtained fiberized paramylon was observed with an electron microscope. The observed image is shown in FIG.

  As shown in FIG. 25, the fiberized paramylon obtained by the bead mill had a network structure in which each fiber was entangled with each other.

Claims (14)

An agent for improving sugar and / or lipid metabolism, comprising fibrillated paramylon. The metabolic improving agent according to claim 1, which is in a dry form. The metabolic ameliorating agent according to claim 1 or 2, wherein the fiberized paramylon is a fibrillated product of paramylon particles. The metabolic ameliorating agent according to any one of claims 1 to 3, wherein the fiberized paramylon is a network-like structure in which fibers are intertwined. The metabolic improving agent according to any one of claims 1 to 4, which is a food additive. The metabolic improving agent according to any one of claims 1 to 4, which is a food composition. The metabolic improving agent according to any one of claims 1 to 4, which is a medicament. The metabolism according to any one of claims 1 to 7, which is used for prevention or improvement of (1) metabolic syndrome or (2) at least one selected from the group consisting of obesity, diabetes, dyslipidemia, and fatty liver. Improver. A method for producing a sugar and / or lipid metabolism-improving agent, comprising blending fiberized paramylon. The method according to claim 9, wherein the fiberized paramylon is in a dried form. The method according to claim 9, further comprising adding water. Fiberized paramylon for use as a sugar and / or lipid metabolism improving agent. A method for improving metabolism of sugar and / or lipid, which comprises applying fibrillated paramylon to a subject. Use of fiberized paramylon for producing a sugar and / or lipid metabolism improving agent.

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