阅读说明：本技术 血糖值上升抑制剂、糖尿病抑制剂和食品组合物 (Blood sugar level increase inhibitor, diabetes inhibitor and food composition ) 是由 中岛绫香 铃木健吾 菅原达也 真锅祐树 于 2019-08-30 设计创作，主要内容包括：本发明提供一种血糖值上升抑制剂、糖尿病抑制剂和食品组合物,其成为源自微藻类的类胡萝卜素混合物的新的使用方法。本发明涉及含有源自微藻类的类胡萝卜素混合物作为有效成分、类胡萝卜素混合物以硅藻黄质作为主成分的血糖值上升抑制剂、糖尿病抑制剂和食品组合物。例如,源自微藻类的类胡萝卜素混合物是源自眼虫藻的类胡萝卜素混合物,包含硅藻黄质作为主成分,并且包含玉米黄质和异黄素。另外,例如,通过将其与高GI食品一起摄取,可以用于抑制血糖值的上升。(The invention provides a blood sugar rise inhibitor, a diabetes inhibitor and a food composition, which are new using methods of carotenoid mixtures derived from microalgae. The present invention relates to a blood glucose level increase inhibitor, a diabetes inhibitor, and a food composition containing a carotenoid mixture derived from microalgae as an active ingredient, the carotenoid mixture containing diatoxanthin as a main component. For example, the carotenoid mixture derived from microalgae is a carotenoid mixture derived from euglena, contains diatoxanthin as a main component, and contains zeaxanthin and isoflavin. In addition, for example, it can be used to suppress an increase in blood glucose level by taking it together with a high GI food.)

1. A blood sugar level increase inhibitor contains a carotenoid mixture derived from microalgae as an active ingredient, wherein the carotenoid mixture contains lutein as a main component, and fucoxanthin and astaxanthin are excluded.

2. The inhibitor for increasing blood glucose level according to claim 1, wherein the xanthophyll is diatoxanthin.

3. The inhibitor for increasing blood glucose level according to claim 1 or 2, wherein the microalgae is euglena.

4. The inhibitor for increasing blood glucose level according to any one of claims 1 to 3, wherein the carotenoid mixture contains diatoxanthin as a main component and contains one or more compounds selected from zeaxanthin and isoxanthin.

5. The inhibitor of increase in blood glucose level according to any one of claims 1 to 4, wherein the carotenoid mixture contains 5.0 to 80.0% of diatoxanthin.

6. The inhibitor of increase in blood glucose level according to any one of claims 1 to 5, wherein the carotenoid mixture contains 10.0 to 80.0% of diatoxanthin, 2.0 to 20.0% of zeaxanthin, and 0.1 to 6.0% of isoflavin.

7. The inhibitor for increasing blood glucose level according to any one of claims 1 to 6, which is used for inhibiting an increase in blood glucose level when taken together with a high GI food.

8. The inhibitor for increasing blood glucose level according to any one of claims 1 to 7, which is used for inhibiting an increase in blood glucose level by oral ingestion thereof for at least 6 consecutive weeks.

9. A diabetes inhibitor contains, as an active ingredient, a carotenoid mixture derived from microalgae, the carotenoid mixture containing lutein as a main component, excluding fucoxanthin and astaxanthin.

10. A food composition for inhibiting blood sugar level increase or for inhibiting diabetes contains, as an active ingredient, a carotenoid mixture derived from microalgae containing lutein as a main component, excluding fucoxanthin and astaxanthin.

11. A method for producing a carotenoid mixture for inhibiting an increase in blood glucose level or diabetes, the carotenoid mixture containing lutein as a main component and excluding fucoxanthin and astaxanthin,

the method comprises the following steps:

a culture step of culturing microalgae having the ability to produce the carotenoid mixture, and

a step of obtaining the carotenoid mixture from the microalgae obtained in the culture step.

Technical Field

The present invention relates to a novel blood glucose level increase inhibitor, a novel diabetes inhibitor and a novel food composition, and particularly relates to a blood glucose level increase inhibitor, a novel diabetes inhibitor and a novel food composition each containing a carotenoid mixture derived from microalgae as an active ingredient.

Background

In general, the glucose concentration in blood is regulated within a certain range in a living body by the action of insulin, and diabetes is a disease in which the glucose regulation function does not work normally and the glucose concentration in blood rises abnormally.

Diabetes mellitus is classified roughly into type 1 diabetes and type 2 diabetes, and type 1 diabetes is a disease in which β cells in the pancreas are destroyed and insulin cannot be secreted from the pancreas.

Type 2 diabetes is called a lifestyle-related disease, and is a disease in which an immune system is abnormal due to environmental factors such as obesity, overeating, lack of exercise, and stress, and insulin function is hindered, and which is likely to develop and progress in obesity patients of older ages.

More specifically, it is said that this is a disease in which abnormal insulin secretion and insulin resistance are caused due to abnormal accumulation of triglycerides in blood and organs.

Under such circumstances, many therapeutic agents have been developed for type 2 diabetes, which is said to account for 90% or more of diabetes patients, particularly japanese diabetes patients, but there are many cases where sufficient clinical results, mental symptoms, side effects on the heart, and the like are not obtained.

Therefore, there is a need for the development of a blood glucose level increase inhibitor and a diabetes inhibitor containing a natural substance as an active ingredient, which are safe and effective.

On the other hand, carotenoids are useful as natural pigments and have various effective actions such as antioxidant action, and therefore, the utility value in the fields of pharmaceuticals, foods, cosmetics, and the like is increasing.

As a diabetes inhibitor using carotenoids, for example, patent document 1 discloses a carotenoid mixture containing fucoxanthin as a main component. Specifically, it is disclosed that fucoxanthin is an extract derived from microalgae, shows anti-diabetic properties, and that fucoxanthin is desirable in combination with other carotenoids having nutritional auxiliary value.

Further, non-patent document 1 discloses an effect of preventing lifestyle-related diseases by ingesting astaxanthin derived from microalgae. Specifically, it has been reported that astaxanthin is administered to mice fed a high-fat diet, and the visceral fat mass, the blood glucose level in the fasting state, and the insulin value of the mice are improved.

Among the carotenoids, a typical carotenoid other than fucoxanthin and astaxanthin is diatom xanthin contained in microalgae such as phytoplankton, brown algae, and diatoms (Diathoxanthin, CAS registry No. 31063-73-7).

Diatomxanthin is one of xanthophylls, and is a carotenoid having a unique structure in which a triple bond is present in a polymer.

Diatomxanthin is expected to have an antioxidant effect as well as other carotenoids, but its functionality is not well understood because it is difficult to obtain in large quantities and is expensive.

Therefore, it is necessary to elucidate the mechanism of the function and the expression of the function of a carotenoid mixture containing, as a main component, diatom xanthin which is a carotenoid other than fucoxanthin and astaxanthin, and to develop a method for utilizing these substances.

Documents of the prior art

Patent document

Patent document 1: japanese Kohyo publication (Kohyo publication) No. 2018-512432

Non-patent document 1: YInhua Ni et al, "Astaxannthin precursors and recoveres di et-induced insulin resistance and stephanism in mic: a, composition with vitamin E ", Scientific Reports, 5: 17192(2015)

Disclosure of Invention

Problems to be solved by the invention

The present invention has been made in view of the above problems, and an object of the present invention is to provide a novel blood glucose level increase inhibitor, a diabetes inhibitor, and a food composition.

Another object of the present invention is to provide a blood glucose level increase inhibitor, a diabetes inhibitor, and a food composition, which are novel methods for using a carotenoid mixture derived from microalgae containing diatoxanthin as a main component.

Means for solving the problems

The inventors of the present invention have conducted extensive studies and found, as a result, that: a carotenoid mixture derived from microalgae containing xanthophyll, especially diatomaceous xanthine as main component has effect in inhibiting blood glucose level increase.

Therefore, according to the present invention, the above problems are solved by an inhibitor for the increase in blood glucose level, which comprises a carotenoid mixture derived from microalgae as an active ingredient, wherein the carotenoid mixture contains lutein (except fucoxanthin and astaxanthin) as a main component.

In this case, the xanthophyll may be xanthophyll, and the microalgae may be euglena.

The carotenoid mixture may contain diatoxanthin as a main component, and may contain one or more compounds selected from zeaxanthin and isoxanthin, and may further contain 5.0% to 80.0% of diatoxanthin.

In addition, the carotenoid mixture may comprise 10.0-80.0% of diatoxanthin, 2.0-20.0% of zeaxanthin and 0.1-6.0% of isoflavin.

Further, it can be used for inhibiting the increase in blood glucose level after ingestion of a high GI food by ingestion together with the high GI food. Further, it can be used for inhibiting the increase of blood glucose level by oral ingestion for at least 6 consecutive weeks.

As described above, by ingesting the carotenoid mixture of the present invention together with a high GI food, it is possible to effectively suppress an increase in blood glucose level when ingesting a high GI food.

Herein, "food with high GI" refers to food with high GI (Glycemic Index), and "GI" is an Index of the rate of increase in blood glucose level after intake of carbohydrates.

As GI increases, the blood glucose level rises rapidly after food intake, and when the blood glucose level rises rapidly, the pancreas secretes a large amount of insulin rapidly. The insulin converts blood glucose into energy, and on the other hand, excess sugar is stored in adipose tissue. Therefore, from the viewpoint of lifestyle-related diseases including diabetes, high GI foods should be avoided as much as possible.

In addition, the above problems are also solved by the following diabetes inhibitors: contains carotenoid mixture derived from microalgae as effective component, wherein the carotenoid mixture contains xanthophyll (except fucoxanthin and astaxanthin) as main component.

Further, the above problems are also solved by the following food compositions for inhibiting an increase in blood glucose level or for inhibiting diabetes: contains carotenoid mixture derived from microalgae as effective component, wherein the carotenoid mixture contains xanthophyll (except fucoxanthin and astaxanthin) as main component.

Further, the above problem is also solved by the following manufacturing method:

the method is used for producing a carotenoid mixture containing lutein (except fucoxanthin and astaxanthin) as a main component for inhibiting blood sugar level increase or diabetes, and comprises:

a culture step of culturing microalgae having the ability to produce the carotenoid mixture, and

and a step of obtaining the carotenoid mixture from the microalgae obtained in the culture step.

ADVANTAGEOUS EFFECTS OF INVENTION

The present invention can provide a novel blood glucose level increase inhibitor, a novel diabetes inhibitor, and a novel food composition.

Further, it is possible to provide an inhibitor of increase in blood glucose level, an inhibitor of diabetes, and a food composition which are novel methods of using a carotenoid mixture derived from microalgae containing diatoxanthin as a main component.

Drawings

FIG. 1 is a chromatogram of HPLC quantitative analysis of a carotenoid mixture derived from Euglena (item 1).

FIG. 2 is a chromatogram of HPLC quantitative analysis of a carotenoid mixture derived from Euglena (2 of).

FIG. 3 shows the results of TLC analysis of neutral lipids in carotenoid mixtures.

FIG. 4 shows the results of TLC analysis of polar lipids in carotenoid mixtures.

FIG. 5 is a graph comparing the change in body weight of mice given the respective groups of feeds for 6 weeks.

FIG. 6 is a graph comparing the change in liver weight after 6 weeks of administration of the respective groups of feeds to mice.

Fig. 7 is a graph comparing the values of total cholesterol in blood 6 weeks after the mice were given the respective groups of feeds.

Fig. 8 is a graph comparing the glucose values in blood 6 weeks after administration of each group of feeds to mice.

Detailed Description

An embodiment of the present invention will be described with reference to fig. 1 to 8.

The present embodiment relates to an inhibitor of blood glucose level increase, an inhibitor of diabetes, and a food composition, which contain, as an active ingredient, a carotenoid mixture derived from microalgae containing lutein (excluding fucoxanthin and astaxanthin) as a main ingredient.

The present invention also relates to a method for producing an agent for inhibiting an increase in blood glucose level, the agent containing, as an active ingredient, a carotenoid mixture containing lutein as a main component, the method comprising: a culture process of culturing microalgae having an ability to produce a carotenoid mixture, and

a carotenoid mixture obtaining step of obtaining a carotenoid mixture from the obtained microalgae.

< Carotenoid mixture derived from microalgae >

"microalgae" are plants of single cells or multicellular bodies of about several μm to several tens of μm in size, which live mainly by photosynthesis in water.

Examples of microalgae include blue-green algae, euglena (euglena), stonewort, codium, xanthomonas, brown algae, red algae, diatom, stonewort, dinoflagellate, euglena, chrysophyceae, blue-green algae, or codococcus.

In this embodiment, euglena (euglena) is desirably used, but is not particularly limited.

"Euglena" includes all microorganisms classified in the zoology, phytology classification as Euglena (Euglena), variants thereof, mutants thereof.

Here, the microorganism of euglena (eugulen) means: in zoology, microorganisms belonging to the order Euglenoidina (Euglenoidina) of the order Euglenida (Euglenida), which belongs to the class of the flagellates (Mastigophorea), the subclasses of the plant Trichididae (Phytomastigophora) of the phylum Protozoa (Protozoa). On the other hand, in botany, it belongs to the order Euglena (Eaglenales) belonging to the Euglenophyceae (Euglenophyceae) of the Euglenophyta phylum (Euglenophyta).

As the euglena microorganism (euglena cell), euglena gracilis (e.gracilis), in particular, euglena gracilis (e.gracilis) Z strain, euglena gracilis (e.gracilis) NIES-49 strain, and the like can be used. Also, β -1, 3-glucanase of a gene mutant strain such as a mutant strain SM-ZK strain (chloroplast deletion strain) of Euglena gracilis (e.gracilis) Z strain, a mutant strain of a mutant of a chloroplast derived from these species, or Euglena intermedia, Euglena piride, or other Euglena species may be used.

Euglena is widely distributed in fresh water such as ponds and marshes, and can be used by being separated from these, or any euglena that has been separated can be used.

In the present embodiment, the euglena gracilis is preferably used, and the euglena gracilis (e.gracilis) strain Z or NIES-49 is particularly preferably used, but is not particularly limited.

The "cultivation of euglena cells" can be performed using a culture solution. Adding carbon source including inorganic carbon source (CO) as nutrient source into the culture solution₂，NaHCO₃，Na₂CO₃Etc.) and organic carbon sources (glucose, etc.). As the culture solution used in the present embodiment, an independent nutrient medium containing no organic carbon source such as glucose as a nutrient source is preferably used, but the present invention is not limited thereto. For example, a culture solution supplemented with nutrient salts such as nitrogen source, phosphorus source, minerals, etc., such as Cramer-Myers medium, modified Cramer-Myers medium ((NH)₄)₂HPO₄ 1.0g/L、KH₂PO₄ 1.0g/L、MgSO₄·7H₂O 0.2g/l、CaCl₂·2H₂O 0.02g/l、Fe₂(SO₂)₃·7H₂O 3mg/l、MnCl₂·4H₂O 1.8mg/l、CoSO₄·7H₂O 1.5mg/l、ZnSO₄·7H₂O 0.4mg/l、Na₂MoO₄·2H₂O 0.2mg/l、CuSO₄·5H₂O0.02 g/l, thiamine hydrochloride (vitamin B1)0.1mg/l, cyanocobalamin (vitamin B12) (pH 3.5)). To describe (NH)₄)₂HPO₄May also be converted into (NH)₄)₂SO₄Or NH₃An aqueous solution.

The pH of the culture medium is preferably 2 or more, and the upper limit thereof is preferably 6 or less, more preferably 4.5 or less. By setting the pH to the acidic side, the photosynthetic microorganisms can grow more dominantly than other microorganisms, and thus contamination can be suppressed.

The cultivation of euglena cells can also be carried out in the following manner: an open cell system directly utilizing sunlight, and a light collecting system in which sunlight collected by a light collecting device is transmitted through an optical fiber or the like, irradiated into a culture tank, and utilized by photosynthesis.

The euglena cells may be cultured by, for example, a batch feeding method, but may be cultured by: flask culture, culture using a fermenter, batch culture, semibatch culture (fed-batch culture), continuous culture (perfusion culture), any liquid culture method.

The separation of euglena cells is performed, for example, by centrifugation or simple sedimentation of a culture solution.

The "carotenoid mixture" is a mixture containing one kind of lutein as a main component and, in addition, containing one or more compounds selected from other compounds in the lutein as an accessory component.

"lutein" is a generic term for pigments in carotenoids containing oxygen in the form of hydroxyl, carbonyl or epoxy groups, specifically lutein contains: epoxidized zeaxanthin, astaxanthin, canthaxanthin, poncirin, beta-cryptoxanthin, diadinoxanthin, diatoxanthin, diadinoxanthin, ranuncuxanthin, fucoxanthin, lutein, neoxanthin, rhodoxanthin, rubixanthin, violaxanthin, and zeaxanthin.

Specifically, the carotenoid mixture of the present embodiment is a mixture containing diatoxanthin as a main component and one or more compounds selected from zeaxanthin and isoflavin as an auxiliary component, and more preferably a mixture containing both zeaxanthin and isoflavin as an auxiliary component.

It is noted that the carotenoid mixture may further comprise carotenoids (lutein) other than these.

"Diatomxanthin" is a carotenoid having a structure with a triple bond in a polymer, as shown in the following formula (chemical formula 1) (Diathoxanthin, CAS registry No. 31063-73-7, chemical formula C)₄₀H₅₄O₂Molecular weight: 566.87).

The diatomaceous xanthins are contained in microalgae such as phytoplankton, brown algae, and diatoms, and are obtained from euglena in the present embodiment.

[ chemical formula 1]

"Zeaxanthin" is a carotenoid having a structure represented by the following formula (chemical formula 2) (Zeaxanthin, CAS registry number: 144-68-3, chemical formula C)₄₀H₅₄O₂Molecular weight: 568.89), it is a component that is contained in edible vegetables, bacteria, algae such as spirulina, and is ingested in large quantities in daily dietary life.

Zeaxanthin can be used for coloring food, resisting obesity and cancer, and its derivative has antioxidant effect.

[ chemical formula 2]

"Isoflavin" is a carotenoid having a structure with two triple bonds in a polymer (Alloxanthin, CAS registry No.: 28380-31-6, formula C) as shown in the following formula (formula 3)₄₀H₅₂O₂Molecular weight: 564.85) contained in Crypthecodinium sp.

[ chemical formula 3]

The carotenoid mixture of the present embodiment may contain, in terms of a ratio of peak areas in a chromatogram for quantitative analysis by liquid chromatography using a detector having a detection wavelength of 450 nm: 5.0-80.0% of diatoxanthine, 1.0-20.0% of zeaxanthin and 0.1-10.0% of isoflavin relative to 100% of the total weight of the carotenoid mixture.

In addition, the composition preferably comprises 10.0-80.0% of diatomite, 2.0-20.0% of zeaxanthin and 0.1-6.0% of isoflavin.

< method for producing Carotenoid mixture >

The carotenoid mixture of the present embodiment is produced by a production method which performs the following steps: the "culturing step" of culturing euglena under the above culturing conditions, the "separating step" of separating euglena, the "pulverizing step" of pulverizing the separated euglena, the "dispersing step" of dispersing the pulverized euglena in an extraction solvent, the "extracting step" of extracting a carotenoid extract from the dispersed euglena, and the "obtaining step" of obtaining a carotenoid fraction (carotenoid mixture) containing diatoxanthin from the carotenoid extract.

First, in the culture step (step S1), euglena is cultured under the above culture conditions. Specifically, euglena was cultured under independent culture conditions.

Next, in the separation step (step S2), euglena cultured in the above-described culture step is separated. Specifically, the separation of euglena is performed by a known separation method such as centrifugation or simple sedimentation of a culture solution, membrane filtration, or the like, but is not particularly limited.

The separated euglena is washed and then dried by a known drying method (vacuum freeze drying, spray drying, heat vacuum drying, etc.), thereby preparing a dry euglena algal body.

Next, in the pulverization step (step S3), the euglena separated in the separation step is pulverized by a known pulverization method. Specifically, there may be mentioned a method of adding an extraction solvent to the separated euglena and crushing the euglena by an ultrasonic crusher or the like, but the method is not particularly limited.

Next, in the dispersion step (step S4), the euglena cells crushed in the crushing step are dispersed in the extraction solvent to obtain a dispersion liquid.

Specifically, as the extraction solvent used in the dispersion step, a lipophilic polar solvent such as acetonitrile can be used.

Next, in the extraction step (step S5), a carotenoid extract is extracted from the euglena dispersed in the extraction solvent in the dispersion step.

The extraction conditions are not particularly limited, and the extraction of carotenoids can be promoted by heating, applying external stimulation such as ultrasonic waves, and shaking.

Finally, in the obtaining step (step S6), a carotenoid fraction (carotenoid mixture) containing diatoxanthin is obtained from the carotenoid extract obtained in the above-described extraction step.

Specific examples of the fractionation method include a method of obtaining a fraction having a high content of diatomaceous earths by fractionation using an appropriate separation means (for example, reverse-phase or normal-phase high performance liquid chromatography partition extraction, gel filtration, silica gel chromatography, or the like). Alternatively, there is a method comprising filtering a carotenoid extract containing diatoxanthin, separating into an extract and a residue, concentrating the extract, and fractionating by liquid chromatography.

< uses, methods and amounts >

The carotenoid mixture of the present embodiment can be used as a therapeutic agent for diabetes (particularly type 2 diabetes) by administering to a diabetic patient or an animal other than a human having diabetes.

In addition, the compounds can also be used as a prophylactic agent for diabetes (particularly, a prophylactic agent for type 2 diabetes), and a diabetes inhibitor (particularly, a type 2 diabetes inhibitor) for a person before onset of diabetes, a person who is a potential diabetic patient, a person with hyperglycemia in a fasting state, a person with hyperglycemia after meal, and other animals except for these persons.

In addition, it can also be used as a preventive agent for gestational diabetes or an inhibitor for gestational diabetes for a pregnant person.

In addition, it is also useful as a preventive agent for complications accompanying diabetes, a complication inhibitor, or a complication therapeutic agent.

The carotenoid mixture of the present embodiment can be used as a blood glucose level increase inhibitor.

Preferably, it can be used as a blood glucose level increase inhibitor for inhibiting an increase in blood glucose level by being taken together with a food (particularly a GI food).

Further, it is preferable that the oral ingestion is performed continuously for a certain period of time, and the composition can be used as a blood glucose level increase inhibitor for inhibiting the increase in blood glucose level.

The carotenoid mixture of the present embodiment can be used as a composition such as a pharmaceutical composition or a food composition for inhibiting an increase in blood glucose level or diabetes.

In the pharmaceutical field, a pharmaceutical composition having the above-mentioned effects is provided by compounding a carotenoid mixture in an amount effective to exert the above-mentioned effects with a pharmaceutically acceptable carrier or additive. The pharmaceutical composition can be a medicine or a quasi-medicine.

The pharmaceutical composition can be administered internally or externally, and can be used in the form of preparations such as injections for oral administration, intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection and/or intraperitoneal injection, transmucosal administration, transdermal administration, etc.

The dosage form of the pharmaceutical composition includes solid preparations such as tablets, granules, capsules, powders and powders, and semi-solid preparations such as liquids, ointments and gels.

In the field of foods, a carotenoid mixture in an amount effective for inhibiting an increase in blood glucose level or diabetes in a living body can be blended as a food material into various foods to provide a food composition having the above-mentioned effects.

That is, the present invention can provide a food composition which is labeled as having an effect of inhibiting an increase in blood glucose level in the field of foods. The food composition includes general foods, specific health foods, nutritional functional foods, hospital patient foods, supplements, and the like. In addition, it can be used as food additive.

Examples of the food composition include seasonings, processed livestock meat products, processed agricultural products, beverages (e.g., refreshing beverages, alcoholic beverages, carbonated beverages, milk beverages, fruit juice beverages, tea, coffee, and nutritional beverages), powdered beverages (e.g., fruit juice powder and soup powder), concentrated beverages, desserts (e.g., candies (throat drops), cookies, biscuits, chewing gums, soft sweets, and chocolates), bread, and grains. In the case of a specific health food, a food having a nutritional function, a food having a marked function, or the like, the food may be in the form of a capsule, a tablet, a syrup, a granule, a powder, or the like.

The "specific health food" is a food containing a health functional component affecting physiological functions and the like, and can be indicated as being suitable for a specific health use by approval of the consumer's office officer.

"nutrient functional food" is a food for supplementing nutrient components (vitamins, minerals), and indicates the function of the nutrient components.

The "food for indicating functionality" is a food for which the operator is responsible for indicating functionality according to scientific grounds. Before sale, the consumer's halls are notified of information regarding security and functional grounds, etc.

The amount of the blood glucose level increase inhibitor and the diabetes inhibitor of the present embodiment may be, for example, orally taken together with food (particularly GI food) to a predetermined intake amount for human or non-human animals.

Examples

< example 1>

A carotenoid mixture derived from euglena was prepared by the following sequence.

1) 20L of acetonitrile was added to 20kg of Euglena gracilis powder (manufactured by Euglena, Inc.) and disrupted by an ultrasonic disrupter to obtain a disrupted extract mixture.

2) Filtering the pulverized extract mixture, and separating into extractive solution and residue.

3) The extract was concentrated using a rotary evaporator and fractionated by medium pressure liquid chromatography. Fractionation was carried out using a liquid chromatography apparatus using an ODS column (InertSustain C18, manufactured by GL Science Co., Ltd.) as a column and acetonitrile as an eluent at a detection wavelength of 270 nm.

4) Using HPLC (high performance liquid chromatography), the carotenoid fraction containing diatoxanthin was recovered from each of the fractionated fractions and concentrated by an evaporator.

By the above method, 0.4g of a reddish brown powder was obtained, which is a carotenoid fraction containing diatoxanthin. The powder is used as a carotenoid mixture.

< test 1: HPLC quantitative analysis of Carotenoid mixtures >

Quantitative analysis of the carotenoid mixture of example 1 was performed using HPLC under the following conditions.

0.1g of the carotenoid mixture was dissolved in acetonitrile, and the resultant was filtered to obtain a substance as a measurement sample solution.

A high performance liquid chromatography apparatus was used, an LC-6AD (manufactured by Shimadzu corporation) was used as a pump, a PDA detector (SPD-M20A) was used as a detector, and an ODS column (TSKgel ODS-80Ts, manufactured by Tosoh corporation) was used as a column.

As the mobile phase, a mixed solvent of acetonitrile, methanol and water (a mixed solvent obtained by mixing at a volume ratio of 75: 15: 10) was used as the a liquid, and a mixed solvent of ethyl acetate and methanol (a mixed solvent obtained by mixing at a volume ratio of 70: 30) was used as the B liquid.

The measurement was carried out at a column temperature of 40 ℃ and a flow rate of 1.0ml/min at a detection wavelength of 450 nm.

As a result of the test in test 1, the chromatogram of example 1 is shown in fig. 1 and 2 (n is 2).

By the retention time in the ultraviolet-visible absorption spectrometry, the mass spectrometry, and the chromatography, it is estimated that zeaxanthin and isoxanthin are present in addition to diatoxanthine.

In the above quantitative analysis, the ratio (%) of each peak area in the chromatogram corresponding to diatoxanthine, zeaxanthin and isoxanthin was determined.

The ratio (%) of each peak area was determined relative to 100% of the total peak area of all the detected components (all the components are assumed to be carotenoids).

The chromatogram in FIG. 1 (example 1-1) is as follows.

Peak number 1: isoflavin 0.4%

Peak number 2: 13.3 percent of diatomite

Peak number 3: 2.1 percent of zeaxanthin

The chromatogram in FIG. 2 (example 1-2) is as follows.

Peak number 1: 5.6 percent of isoflavin

Peak number 2: 75.9 percent of diatomite

Peak number 3: zeaxanthin 18.4%

In the following experiments, the carotenoid mixture of example 1-1 was used.

< test 2: TLC analysis of Carotenoid mixtures (neutral lipids) >

TLC (thin layer chromatography) analysis of neutral lipids in the carotenoid mixture of example 1 was performed according to the following procedure.

The carotenoid mixture (0.01mg/ml, 0.1mg/ml, 1mg/ml) and standards (oleic acid, triolein, cholesteryl palmitate, cholesterol, diatrizine, zeaxanthin) were spotted onto silica gel plates, respectively.

Development was carried out using a mixed solvent of hexane, diethyl ether and acetic acid (mixed solvent obtained by mixing at a volume ratio of 65: 35: 1).

And (3) after taking a picture by using a digital camera, immersing the silica gel plate into a copper sulfate developing solution, and heating for 5-10 minutes at 180 ℃ by using a hot plate to develop color.

The mass of the carotenoid mixture was determined as the mass (mg/ml) of each 1L of the liquid medium of each of the diatrizine samples by comparing the amount with a known amount of the diatrizine standard.

The results of TLC analysis of run 2 are shown in FIG. 3. From the results of TLC analysis before and after the development of the color of copper sulfate in each sample, it was found that no neutral lipid was mixed in the carotenoid mixture.

< test 3: TLC analysis (polar lipids) of Carotenoid mixtures

TLC (thin layer chromatography) analysis of polar lipids in the carotenoid mixture of example 1 was performed by the following procedure.

Carotenoid mixtures (0.01mg/ml) and standards (phosphatidylcholine, monogalactosyldiacylglycerol, digalactosyldiacylglycerol, diatoxanthin, zeaxanthin) were spotted onto silica gel plates, respectively.

Development was carried out using a mixed solvent of chloroform, methanol and water (mixed solvent obtained by mixing at a volume ratio of 64: 16: 2).

And (3) after taking a picture by using a digital camera, immersing the silica gel plate into a copper sulfate developing solution, and heating for 5-10 minutes at 180 ℃ by using a hot plate to develop color.

The results of TLC analysis of run 3 are shown in FIG. 4. From the results of TLC analysis before and after the development of color of copper sulfate in each sample, it was found that the carotenoid mixture was not mixed with polar lipids.

The chromatogram of example 1 is shown in FIG. 1.

< test 4: effect of Carotenoid mixtures Using high fat diet mice >

When a carotenoid mixture having a feed composition shown in table 1 below was orally taken by a mouse, "body weight change", "liver weight", "total cholesterol amount in blood" and "glucose amount in blood" were measured and tested.

In this experiment, 18C 57BL/6J mice (6-week-old males, 20g to 23g in body weight) were used (n-6). The breeding conditions were set at room temperature of 24. + -. 2 ℃ and a light-dark period of 12 hours.

First, mice were allowed to freely ingest solid feed and water and were acclimatized to the breeding environment by 10-day preliminary breeding. After the preliminary feeding was completed, three groups of 1) AIN93G basic diet groups (hereinafter referred to as "control group"), 2) high fat diet groups containing 30% by weight of lipid in the basic diet, and 3) high fat diet + carotenoid groups containing 0.04% by weight of a carotenoid mixture were given to different feeds for each group, and the test feeding was performed for 6 weeks of free intake, according to the feed composition shown in table 1 below.

[ Table 1]

The value of totals is 1000.00 by rounding off.

The carotenoid mixture in the feed was dissolved in soybean oil in the following order and mixed.

The carotenoid mixture was placed in an eggplant-shaped flask together with ethanol and dissolved. Dissolving tert-butylhydroquinone in refined soybean oil heated to 70 deg.C, returning to normal temperature, and mixing with carotenoid mixture dissolved in ethanol. The ethanol was distilled off using a rotary evaporator, and the carotenoid adhered to the eggplant-shaped flask was redissolved with ethanol, and the procedure was repeated until the carotenoid was completely mixed with the refined soybean oil. Further, refined soybean oil was bubbled with nitrogen gas to completely remove ethanol, and the feed was mixed with the carotenoid mixture.

Daily body weights were measured during the feeding period. After the 6-week test period was terminated, overnight fasted mice were laparotomized under isoflurane anesthesia, livers were harvested and blood was collected from the inferior vena cava.

After the collected liver was washed with physiological saline, the liver weight was measured.

Furthermore, the collected blood was centrifuged (2000rpm, 15 minutes, 4 degrees), and the obtained blood was collected and stored at 80 degrees. Then, the total cholesterol value and the glucose value in blood were quantified using a quantification kit (manufactured by Wako pure chemical industries, Ltd.).

As the test results, "body weight change", "liver weight", "total cholesterol in blood value" and "glucose in blood value" of the control group, the high fat diet group and the high fat diet + carotenoid group were compared, respectively, and the obtained graphs are shown in fig. 5, 6, 7, 8. In addition, respective numerical values are shown in table 2 below.

The unit "mg/dL" means the total cholesterol value (mg) and glucose value (mg) per 1dL of blood.

[ Table 2]

Regarding the body weight during the feeding period, it was found that the body weight of the mice in the "high fat diet group" and the "high fat diet + carotenoid group" tended to increase almost all the time as compared with the "control group". In addition, a tendency was found that the body weight of the "high fat diet + carotenoid group" was lower than that of the "high fat diet group".

In addition, regarding the body weight after 6 weeks of the feeding period, it was confirmed that the body weight of the mice in the "high fat diet group" and the "high fat diet + carotenoid group" was significantly increased (P < 0.05) compared to the "control group". In addition, a tendency was found that the body weight of the "high fat diet + carotenoid group" was lower than that of the "high fat diet group".

Regarding the liver weight after 6 weeks of the feeding period, a tendency was found that the liver weight of the mice of the "high fat diet group" was significantly increased compared to the "control group". On the other hand, it was found that the liver weight of the "high fat diet + carotenoid group" was not increased as compared with the "control group" but was similarly inclined.

Regarding the total cholesterol value in blood after 6 weeks of the feeding period, it was found that the total cholesterol value in blood of the mice of the "high fat diet group" and the "high fat diet + carotenoid group" was increased more than that of the "control group". In addition, a tendency was found that the total cholesterol value in the blood was reduced in the "high fat diet + carotenoid group" as compared with the "high fat diet group".

Regarding the blood glucose values after 6 weeks of the feeding period, it was found that the blood glucose values of the mice of the "high fat diet group" were significantly increased (P < 0.05) compared to the "control group". On the other hand, the blood glucose level of the "high fat diet + carotenoid group" was considered to be equivalent to that of the "control group" without increasing (P < 0.05).

From the above, the results of the tests on the change in body weight during the feeding period, the body weight after 6 weeks of the feeding period, the weight of liver and the total cholesterol value in blood show that: by orally ingesting a high-fat diet (e.g., high GI food) and the carotenoid mixture of example 1 simultaneously, the mice exhibited the effect of suppressing the increase in body weight (particularly body fat), liver weight (particularly liver fat), and blood cholesterol level after ingesting a high-fat diet.

In addition, from the results of the test of the blood glucose value after 6 weeks of the feeding period, it was found that: by orally ingesting the high-fat diet together with the carotenoid mixture of example 1, the mice exhibited an effect of suppressing the increase in blood sugar level after the intake of the high-fat diet.

The "blood glucose level" is a glucose concentration in blood, and is a value indicating how much mg of glucose is contained in 1dl blood, and corresponds to the blood glucose level in this test.