JP6941746B1 - Immunostimulant food compositions and immunostimulants - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an immunostimulatory food composition, an immunostimulator, a food composition for promoting macrophage nitric oxide production, and a macrophage nitric oxide production promoter. An immunostimulatory food composition, an immunostimulator, and macrophage nitric oxide, which comprises at least one Euglena-derived substance selected from the group consisting of paramylon and Euglena and lactic acid bacteria as active ingredients. It is solved by a food composition for promoting nitrogen production and a macrophage nitric oxide production promoter. It is preferable that the lactic acid bacterium belongs to the genus Enterococcus and the Euglena-derived substance is paramylon. It was suggested that the synergistic effect when combined with lactic acid bacteria was greater in Euglena than in paramylon. [Selection diagram] Fig. 1

Description

The present invention relates to immunostimulatory food composition and immunostimulants.

In recent years, improvement of immunity has been attracting attention as an important factor in maintaining health. Immunity is a mechanism that protects the body from foreign substances such as bacteria and viruses that invade the body. Due to the outbreak of colds, influenza, and the new coronavirus infection (COVID-19), food compositions and agents having an immunostimulatory action, which is a function of improving immunity, are required. It is known that some lactic acid bacteria and bifidobacteria have an immunostimulatory effect, and are used in food compositions.

On the other hand, Euglena (genus name: Euglena, Japanese name: Euglena) is attracting attention as a biological resource that is expected to be used as food, feed, fuel and the like. Euglena has 59 kinds of nutrients such as vitamins, minerals, amino acids, unsaturated fatty acids, etc., which correspond to most of the nutrients necessary for human life, and as a supplement for balanced intake of many kinds of nutrients. It has been proposed to be used and used as a food supply source in poor areas where necessary nutrients cannot be obtained.

Patent Document 1 describes that a lactic acid bacterium growth promoter containing Euglena cells or Euglena cell extract as an active ingredient promotes the growth of lactic acid bacteria.

Japanese Unexamined Patent Publication No. 7-99967

There is a demand for food compositions and agents that can effectively enhance immunity. The present invention has been made in view of the above problems, and an object of the present invention is to provide an immunostimulant food composition and an immunostimulant capable of enhancing immunity. Another object of the present invention is to provide a food composition for promoting macrophage nitric oxide production and a macrophage nitric oxide production promoter.

As a result of diligent research, the present inventors have found that the combination of paramylon contained in Euglena with lactic acid bacteria effectively activates immunity and promotes the production of nitric oxide in macrophages. It came to.

Therefore, according to the immunostimulatory food composition of the present invention, the above-mentioned problem is solved by containing a substance derived from Euglena, which is paramylon, and a lactic acid bacterium as active ingredients, and the lactic acid bacterium belongs to the genus Enterococcus. To .
At this time, the immunostimulatory food composition may is lactic acid bacteria beverages.
At this time, the immunostimulatory food composition may be used for prevention of infectious diseases.
At this time, the immunostimulatory food composition may be used to promote nitric oxide production of macrophages.

Further, according to the immunostimulatory agent of the present invention, the problem is solved by containing a substance derived from Euglena, which is paramylon, and a lactic acid bacterium as active ingredients, and the lactic acid bacterium belongs to the genus Enterococcus.
At this time, the bacterium belonging to the genus Enterococcus may be Enterococcus faecalis .
At this time, the immunostimulatory agent may be used in the prevention and / or treatment of infectious diseases.
At this time, the immunostimulant may be used to promote nitric oxide production of macrophages.

The subject is characterized in that it contains at least one Euglena-derived substance selected from the group consisting of paramylon and Euglena and lactic acid bacteria as active ingredients, and is used to promote nitric oxide production of macrophages. It is solved by the food composition for promoting the production of macrophages nitric oxide.
The subject is characterized in that it contains at least one Euglena-derived substance selected from the group consisting of paramylon and Euglena and lactic acid bacteria as active ingredients, and is used to promote nitric oxide production of macrophages. It is solved by the macrophage nitric oxide production promoter.

According to the present invention, it is possible to provide an immunostimulatory food composition and an immunostimulator capable of effectively activating immunity. In addition, by ingesting the immunostimulant food composition and the immunostimulant of the present invention, it is possible to enhance immunity and prevent diseases such as infectious diseases. Further, according to the present invention, it is possible to provide a food composition for promoting macrophage nitric oxide production and a macrophage nitric oxide production promoter capable of promoting macrophage nitric oxide production.

It is a graph which shows the result when lactic acid bacterium 1 and paramylon are combined. It is a graph which shows the result when lactic acid bacterium 2 and paramylon are combined. It is a graph which shows the result when lactic acid bacterium 3 and paramylon are combined. It is a graph which shows the result when lactic acid bacterium 4 and paramylon are combined. It is a graph which shows the result when lactic acid bacterium 1 and Euglena water extract are combined. It is a graph which compared the result when lactic acid bacterium 1 and Euglena water extract were combined. It is a graph which shows the result when lactic acid bacterium 1 and Euglena hot water extract are combined. It is a graph which compared the result when lactic acid bacterium 1 and Euglena water extract were combined. It is a graph which compared the result when the lactic acid bacterium 3 and the Euglena water extract were combined. It is a graph which compared the result when the lactic acid bacterium 5 and the Euglena water extract were combined. It is a graph which compared the result when the lactic acid bacterium 6 and the Euglena water extract were combined. It is a graph which compared the result when the lactic acid bacterium 7 and the Euglena water extract were combined.

Hereinafter, embodiments of the present invention will be described with reference to FIGS. 1 to 12. This embodiment is for an immunostimulatory food composition, an immunostimulator, and a macrophage nitric oxide production promoting substance containing at least one Euglena-derived substance selected from the group consisting of paramylon and Euglena and lactic acid bacteria as active ingredients. It relates to a food composition and a macrophage nitric oxide production promoter.

<Immune activator>
The immunostimulant according to the present embodiment is an immunostimulant containing at least one Euglena-derived substance selected from the group consisting of paramylon and Euglena and lactic acid bacteria as active ingredients. The "immunity activator" is an agent having an action of activating (activating) immunity, which improves immunity. Also, when the immune system (innate immune system or acquired immune system) is activated, it is said that "immunity is activated".

Since the immunostimulatory agent according to the present embodiment can improve immunity, it can be used as a preventive agent for infectious diseases typified by viral infections caused by pathogens such as viruses, bacteria, fungi, and parasites. It can also be used as a therapeutic agent.

<Macrophage nitric oxide production promoter>
The macrophage nitric oxide production promoter according to the present embodiment contains at least one euglena-derived substance selected from the group consisting of paramylon and euglena and lactic acid bacteria as active ingredients, and promotes macrophage nitric oxide production. It is a macrophage nitric oxide production promoter, which is characterized by being used for the purpose of producing macrophages. By combining at least one Euglena-derived substance selected from the group consisting of paramylon and Euglena with lactic acid bacteria, the amount of nitric oxide (NO) produced by macrophages can be promoted.

<Euglena>
In embodiments, "Euglena" includes taxonomically classified microorganisms in the genus Euglena, varieties thereof, variants thereof and closely related species of the family Euglena. Here, the genus Euglena (Euglena) is a group of eukaryotes belonging to Excavata, Euglenozoa, Euglenidae, Euglena, and Euglena.

Specific examples of the species included in the genus Euglena include Euglena chadefaudii, Euglena deses, Euglena gracilis, Euglena granulata, Euglena mutabilis, Euglena proxima, Euglena spirogyra, Euglena viridis and the like. As Euglena, Euglena gracilis (E. gracilis), in particular Euglena gracilis (E. gracilis) Z strain can be used, but in addition, a mutant strain SM-ZK strain of Euglena gracilis (E. gracilis) Z strain can be used. (Chloroplast-deficient strain), variant E. gracilis var. Bacillaris, gene mutant strains such as chloroplast variants of these species, and other Euglena species such as Astasia longa.

The genus Euglena is widely distributed in fresh water such as ponds and swamps, and may be used separately from these, or any genus Euglena that has already been isolated may be used. The genus Euglena includes all its mutants. In addition, among these mutant strains, those obtained by genetic methods such as recombination, transduction, transformation and the like are also included.

(Euglenida algae)
In this embodiment, Euglena algae can be used as Euglena. As the Euglena algae, living Euglena cells separated by centrifugation, filtration, sedimentation or the like can be used as they are. Euglena viable cells can be used as they are after harvesting from the culture tank, but it is preferable to wash them with water or physiological saline. Further, it may be used in the state of a dispersion liquid in which Euglena algae are dispersed in a liquid such as water. In the present embodiment, it is preferable to use the dried Euglena algae (Euglena powder) obtained by freeze-drying or spray-drying the living Euglena cells as the Euglena algae.

Further, a mechanically treated product of the algae obtained by subjecting the living Euglena cells to ultrasonic irradiation treatment or mechanical treatment such as homogenization may be used as the Euglena algae. Further, the dried product obtained by subjecting the mechanically processed product to a drying treatment may be used as the Euglena algae.

(Euglena extract)
In the present embodiment, it is also possible to use Euglena extract as Euglena, and it is particularly preferable to use Euglena aqueous solvent extract. In the present embodiment, "Euglena aqueous solvent extract" means an extract extracted from Euglena using an aqueous solvent, and in particular, water is used as an aqueous solvent at 5 ° C. to 600 ° C. for several seconds to several seconds. It is preferable to use a water extract or a hot water extract of Euglena extracted for 10 hours. The water used for extraction does not necessarily have to be distilled water, pure water, or ultrapure water, and may contain, for example, tap water or impurities, but contains components that hinder the extraction of active components. No water is preferred.

In the present embodiment, the “water extract” means an extract with water at 0 to 50 ° C. (excluding 0 ° C.). Here, "water" means water at 0 to 50 ° C. (excluding 0 ° C.). The temperature of water is not particularly limited as long as it does not affect the active ingredient and is within a range in which the active ingredient can be sufficiently extracted, but is preferably 1 to 40 ° C, more preferably 5 to 35 ° C. Particularly preferably, it is 10 to 30 ° C.

In the present embodiment, the "hot water extract" means an extract with water having a temperature higher than 50 ° C., and can also be called a "hot water extract". Here, "hot water" means water having a temperature higher than 50 ° C., and is a concept including "hot water", and also includes water in a boiling state. Further, the present invention is not limited to hot water in a liquid state, but also includes hot water in a gaseous state and a supercritical state. The temperature of the hot water is not particularly limited as long as it does not affect the active ingredient and is within a range in which the active ingredient can be sufficiently extracted, but is preferably higher than 50 ° C and 120 ° C or lower, more preferably 50. It is higher than ° C and below 100 ° C.

The pH of the water used for extraction is not particularly limited as long as it does not affect the active ingredient and is within a range in which the active ingredient can be sufficiently extracted, but is preferably pH 4 to 10, more preferably pH 5 to 9. , Particularly preferably pH 6-8.

In the present embodiment, water is used alone as the aqueous solvent, but one solvent that can sufficiently extract the active ingredient without affecting the active ingredient and can usually be used for extraction is used. Alternatively, two or more types may be selected and used. For example, water, alcohols, glycols and the like can be mentioned, but the present invention is not limited thereto. Examples of alcohols include ethanol, methanol, n-propanol, isopropanol and the like. Examples of glycols include butylene glycol and propylene glycol. Examples of other aqueous solvents include acetone and the like. These solvents may be used alone or as an aqueous solution, or may be used as any two or three or more mixed solvents.

The temperature of the aqueous solvent used for extraction is, for example, 0 ° C. or higher, and is not particularly limited as long as it does not affect the active ingredient. An aqueous solvent in a boiling state or a supercritical state can be used, but it is preferable to use an aqueous solvent at 5 ° C. to 600 ° C., and more preferably an aqueous solvent at 10 ° C. to 200 ° C. Therefore, the aqueous solvent for extraction also includes an aqueous solvent in a boiling state or a supercritical state. The amount of the aqueous solvent used for extraction is preferably an amount capable of sufficiently dissolving the water-soluble active ingredient contained in Euglena.

The extraction method is also not particularly limited, and for example, extraction can be performed by the methods shown below, but the extraction method is not limited to this, and a normal extraction method can be freely selected and used. For example, a method of immersing Euglena's algae dry powder in an aqueous solvent for a predetermined time and then centrifuging or filtering, or adding Euglena's algae's dry powder to an aqueous solvent and shaking to uniformly disperse it, and then centrifuging or filtering. The method, etc. can be mentioned. It is also possible to heat the aqueous solvent after adding Euglena to facilitate the extraction.

Water extraction of Euglena can be carried out by the usual method as shown below, but is not limited thereto. For example, the Euglena tissue and water are placed in a container and allowed to stand for a predetermined time with appropriate stirring or shaking, and the obtained extract can be used as it is as a water extract. Further, for example, the supernatant obtained by centrifuging such an extract can be used as a water extract. It is also possible to concentrate and dry such an extract or supernatant to remove water and use it as a water extract. Water extraction may be carried out by adding a small amount, for example, 10% by mass or less of alcohol, preferably ethanol, to water in order to increase the extraction efficiency and shorten the extraction time. The extraction time in the case of water extraction is not particularly limited as long as the active ingredient is extracted, and can be appropriately set in the range of several seconds to several tens of hours according to the extraction temperature.

Extraction with hot water can be performed by a commonly used method as shown below, but is not limited thereto. Euglena is introduced into a commonly used extractor together with water, and then extracted by heating. When extracting using boiling water or water in a supercritical state, it is necessary to use an extractor that can withstand the vapor pressure of water. The pressure at the time of extraction can be set to 1 to 5000 atm, preferably 60 to 400 atm.

When extraction is performed under high temperature and high pressure, the active ingredient may be decomposed or a chemical reaction may occur if the extraction time is too long. Therefore, when the extraction is performed under high temperature and high pressure, the extraction time is preferably a short time, for example, 3 minutes or less, more preferably 1 minute or less, and particularly preferably 30 seconds or less.

The extracted Euglena extract can be used as it is as an active ingredient of the immunostimulatory agent and the macrophage nitrogen monoxide production promoter according to the present embodiment, but the extract can be further used as an appropriate separation means (for example, partition extraction). , Gel filtration method, silica gel chromatography, reverse phase or normal phase high performance liquid chromatography, etc.) to fractionate and use the highly active fraction. Further, the Euglena extract and its fraction can be concentrated and dried to remove the aqueous solvent, and this can be used as the aqueous solvent extract.

<Paramilon>
"Paramylon" is a polymer (β-1,3-glucan) in which about 700 glucoses are polymerized by β-1,3-bonds, is porous, and is a stored polysaccharide contained in the genus Euglena. Is. Paramylon particles are flat spheroidal particles, and are formed by spirally entwined β-1,3-glucan chains.

Paramylon exists as granules in Euglena cells of all species and varieties, and the number, shape, and uniformity of particles are characteristic of each species. Paramylon consists only of glucose, and the average degree of polymerization of paramylon obtained from the wild strain of E. gracilis Z and the chloroplast-deficient strain SM-ZK is about 700 in glucose units. Paramylon is insoluble in water and hot water, but is soluble in dilute alkalis, concentrated acids, dimethyl sulfoxide, formaldehyde, and formic acid. The average density of paramylon is 1.53 for E. gracilis Z and 1.63 for E. gracilis var. Bacillaris SM-L1.

Paramylon has a loose spiral structure in which three linear β-1,3-glucans are twisted like a right-handed rope according to X-ray analysis using the powder graphic method. Several of these glucan molecules gather to form paramylon granules. Paramylon granules have a very large crystal structure and occupy about 90%, and are compounds with the highest crystal structure ratio among polysaccharides (Euglena Physiology and Biochemistry, edited by Shozaburo Kitaoka, Academic Publishing Center). The particle size distribution of Paramylon (manufactured by Euglena Co., Ltd.) has a median diameter of 1.5 to 2.5 μm as measured by a laser diffraction / scattering type particle size distribution measuring device.

Paramylon particles are isolated from cultured Euglena cells by any suitable method and purified into fine particles, and are usually provided as a powder. For example, paramylon particles include (1) culturing Euglena cells in any suitable medium, (2) separating Euglena cells from the medium, (3) isolating paramylon from isolated Euglena cells, ( It can be obtained by 4) purification of the isolated paramylon and, if necessary, (5) cooling and subsequent freeze-drying. Isolation of paramylon is performed, for example, with a type of nonionic or anionic detergent that is largely biodegradable. Purification of paramylon occurs substantially at the same time as isolation.

(Processed paramylon)
Processed products of paramylon include water-soluble paramylon, sulfated paramylon and the like obtained by chemically or physically treating paramylon by various known methods, and paramylon derivatives.

Examples of processed paramylon products include amorphous paramylon and emulsion paramylon. Amorphous paramylon is a substance obtained by amorphizing crystalline paramylon derived from Euglena. Amorphous paramylon has a relative crystallinity of 1 to 20% with respect to crystalline paramylon produced from Euglena by a known method. However, this relative crystallinity is obtained by the method described in JP-A-2011-184592.

That is, each of amorphous paramylon and paramylon is crushed by a crusher (Ball mill MM400 manufactured by Resh) at a frequency of 20 times / second for 5 minutes, and then a tube is used by an X-ray diffractometer (H'PertPRO manufactured by Spectris). Scanning is performed in a range where 2θ is 5 ° to 30 ° at a voltage of 45 KV and a tube current of 40 mA to obtain diffraction peaks Pc and Pa near 2θ = 20 ° of paramylon and amorphous paramylon. Using the values of Pc and Pa, the relative crystallinity of amorphous paramylon is calculated by the relative crystallinity of amorphous paramylon = Pa / Pc × 100 (%).

Amorphous paramylon is prepared by subjecting crystalline paramylon powder to alkaline treatment, neutralizing with acid, and then washing and removing water, and then drying according to the method described in JP-A-2011-184592. In addition, the processed paramylon products include water-soluble paramylon, sulfated paramylon, and the like obtained by chemically or physically treating paramylon by various known methods, and paramylon derivatives.

"Emulsion paramylon" is a substance also called emulsion paramylon because its processing method and physical properties are similar to those of emulsion, and it is obtained by adding water to paramylon according to the method described in JP-A-2016-199650. It is a processed paramylon obtained by performing a collision process in which the fluid is ejected from a pore nozzle at an ultrahigh pressure to collide with an object to be collided, and is swollen by combining with water four times or more.

Emulsion paramilon is a known physical characteristic modifier (for example, Japanese Patent Application Laid-Open No. 2011-88108) in which a slurry obtained by adding a water-soluble solvent to a solid such as powder is ejected from a pore nozzle at an ultrahigh pressure to collide with an object to be collided. It can be obtained by performing collision treatment one or more times at a nozzle pressure of 245 MPa at the time of ejection in (the device described in JP-A-6-47264).

Emulsion paramilon has a median diameter of 5 times or more that of paramilon and 7 μm or more when the particle size is measured by a laser diffraction / scattering type particle size distribution measuring device. It is observed that it is attached, and it swells by binding with water four times or more as much as paramilon.

A slurry in which raw material paramylon and water are mixed is a free-flowing fluid, but in emulsion paramylon, paramylon is dispersed in water molecules to increase the viscosity and become viscous, so that it adheres to the hand when touched. It has a strong adhesiveness, elasticity, and a glue-like feel. The processed paramylon obtained is referred to as emulsion paramylon in the present specification because of its treatment method and physical properties, but it is unknown whether or not it is emulsified, and the paramylon is in a state of being swollen by binding to water. Is.

<Lactic acid bacteria>
The lactic acid bacterium contained as an active ingredient in the immunostimulatory agent and the macrophage nitrogen monoxide production promoter according to the present embodiment is not particularly limited as long as it is a bacterium having an immunostimulatory action, and is, for example, Enterococcus. Lactobacillus acidphilus, Lactobacillus gasseri, Lactobacillus plantarum, Lactobacillus plantarum, bacteria belonging to the genus Enterococcus such as faecalis and Enterococcus faecium. Lactobacillus casei, Lactobacillus paracasei, Lactobacillus helveticus, Lactobacillus brevis, Lactobacillus pentosus, Lactobacillus pentosus, Lactobacillus pentosus Bacteria belonging to the genus Lactobacillus, bacteria belonging to the genus Lactococcus such as Streptcoccus thermophilus, bacteria belonging to the genus Lactobacillus such as Lactococcus lactis, Tetragenococcus halophilus ) And other bacteria belonging to the genus Tetragenococcus, and bacteria belonging to the genus Leuconostock such as Leuconostoc mesenteroides.

From the viewpoint of immunostimulatory action when combined with Paramilon and Euglena, it is preferable to use Enterococcus faecalis, E. faecium, and other Enterococcus faecalis as lactic acid bacteria. , Enterococcus faecalis is particularly preferred. It is also preferable to use plant-derived lactic acid bacteria such as Lactobacillus plantarum, Tetragenococcus halophilus, and Leuconostoc mesenteroides.

<Use>
The immunostimulatory agent and the macrophage nitric oxide production promoter according to the embodiment are configured as a food composition such as a health food or a pharmaceutical composition, and are ingested and administered prophylactically in order to enhance immunity. Euglena-derived substances such as paramylon and euglena can be ingested and administered continuously because they can be ingested as food and have no side effects.

(Food composition)
The immunostimulatory food composition of the present embodiment and the food composition for promoting macrophage nitric oxide production are effective amounts of Euglena capable of effectively exerting an immunostimulatory effect and a macrophage nitric oxide production promoting effect in the field of food. By blending the derived substance as a food material into various foods, a food composition having the said action can be provided. That is, the present invention can provide a food composition of a food labeled as immunostimulatory utilization or the like in the field of food. Examples of the food composition include general foods, foods for specified health use, foods with nutritional function, foods with functional claims, foods for hospital patients, supplements and the like. It can also be used as a food additive.

Examples of the food composition include seasonings, processed livestock meat products, processed agricultural products, beverages (lactic acid bacteria beverages, soft beverages, alcoholic beverages, carbonated beverages, dairy beverages, fruit juice beverages, tea, coffee, nutritional drinks, etc.) and powders. Beverages (powdered juice, powdered soup, etc.), concentrated beverages, confectionery (candy (throat candy), cookies, biscuits, gum, gummy, chocolate, etc.), bread, cereals, etc. can be mentioned. Further, in the case of foods for specified health use, foods with nutritional function, foods with functional claims, etc., they may be in the form of capsules, troches, syrups, granules, powders and the like.

Here, foods for specified health use are foods containing health functional ingredients that affect physiological functions, etc., and can be labeled as suitable for specific health uses with the permission of the Commissioner of the Consumer Affairs Agency. be. In the present invention, the food is sold with a specific health use related to the improvement of immunity.

The nutritionally functional food is a food used for supplementing nutritional components (vitamins, minerals) and displays the function of the nutritional component. In order to sell as a nutritionally functional food, the amount of nutritional components contained in the daily intake guideline must be within the specified upper and lower limits, and not only nutritional function labeling but also warning labeling, etc. You also need to.

Foods with functional claims are foods with functional claims based on scientific evidence at the responsibility of the business operator. Information on the basis of safety and functionality was notified to the Commissioner of the Consumer Affairs Agency before the sale.

The functional indications displayed on the immunostimulatory food composition and the macrophage nitrogen monoxide production promoting food composition according to the present embodiment include "immunity activation", "immunity activation", and "immunity". Examples include, but are not limited to, "improvement" and "immunity enhancement".

In the food composition according to the present embodiment, in addition to the Euglena-derived substance, one or more components that can be usually used in the food composition can be freely selected and blended. For example, it can contain all additives commonly used in the food field, such as various seasonings, preservatives, emulsifiers, stabilizers, flavors, colorants, preservatives, pH regulators and the like.

(Pharmaceutical composition)
In the field of medicine, the immunostimulatory agent and the macrophage nitric oxide production promoter of the present embodiment are pharmaceutically acceptable together with an amount of Euglena-derived substance capable of effectively exerting an immunostimulatory effect and a macrophage nitric oxide production promoting effect. By blending the carrier and the additive to be prepared, a pharmaceutical composition having the said action is provided. The pharmaceutical composition may be a pharmaceutical product or a quasi-drug.
The pharmaceutical composition may be applied internally or externally. Therefore, the pharmaceutical composition is used in the form of an injection such as an oral preparation, an intravenous injection, a subcutaneous injection, an intradermal injection, an intramuscular injection and / or an intraperitoneal injection, a transmucosal application agent, a transdermal application agent and the like. be able to.
The dosage form of the pharmaceutical composition can be appropriately set depending on the form of application, and for example, solid preparations such as tablets, granules, capsules, powders and powders, liquid preparations such as liquids and suspensions, Examples thereof include a semi-solid agent such as an ointment or a gel.

In the pharmaceutical composition according to the present embodiment, one or more pharmaceutically acceptable additives can be freely selected and contained.
For example, when the pharmaceutical composition according to the present embodiment is applied to an oral preparation, for example, an excipient, a binder, a disintegrant, a surfactant, a preservative, a colorant, a flavoring agent, a fragrance, a stabilizer, and an antiseptic. It can contain all additives usually used in the field of pharmaceutical preparations, such as agents and antioxidants. In addition, a drug delivery system (DDS) can be used to prepare a sustained release preparation or the like.

Hereinafter, the present invention will be specifically described based on specific examples, but the present invention is not limited thereto.

<Euglena-derived substances>
(Paramilon)
Paramylon as a substance derived from Euglena was prepared by the following procedure.
Euglena gracilis powder (Euglena Co., Ltd.) was placed in distilled water and stirred at room temperature for 2 days. This was ultrasonically treated to destroy the cell membrane, and the crude paramylon particles were recovered by centrifugation. The recovered paramylon particles were dispersed in a 1% aqueous solution of sodium dodecyl sulfate and treated at 95 ° C. for 2 hours, and the paramylon particles recovered by centrifugation again were dispersed in an aqueous solution of 0.1% sodium dodecyl sulfate and treated at 50 ° C. for 30 minutes. bottom. Lipids and proteins were removed by this operation, followed by washing with acetone and ether, and then dried at 50 ° C. to obtain purified paramylon particles (paramylon powder).

(Euglena water extract)
Euglena water extract as a substance derived from Euglena was prepared by the following procedure.
2.5 g of Euglena powder (Euglena gracilis, manufactured by Euglena Co., Ltd.) was suspended in 10 ml of ultrapure water, and extraction treatment was performed at room temperature (25 ° C.) for 2 hours. Then, the supernatant obtained by centrifugation (5000 rpm, 5 minutes, 25 ° C.) is separated and filtered through a 0.45 μm sterile filter to obtain an Euglena water extract (Euglena water extract, 100% undiluted solution). Was prepared.

(Euglena hot water extract)
2.5 g of Euglena powder (Euglena Gracilis, manufactured by Euglena Co., Ltd.) was suspended in 10 ml of ultrapure water, and an extraction treatment was carried out at 95 ° C. using a dry heat sterilizer for 2 hours. Then, the supernatant obtained by centrifugation (5000 rpm, 5 minutes, 25 ° C.) is separated and filtered through a 0.45 μm sterile filter to obtain an Euglena hot water extract (Euglena hot water extract, undiluted solution 100). %) Was prepared.

<Lactic acid bacteria>
As lactic acid bacteria, four kinds of commercially available lactic acid bacteria 1 to 4 were used.
(Lactic acid bacteria 1)
Enterococcus faecalis EF-2001 strain (Nippon Belm Co., Ltd.), heat-treated lactic acid bacteria (heat-killed cells), as described in paragraph 0011 of JP-A-2002-249434, corresponds to refusal of acceptance. However, it is stored at the BRM Research Institute of Nippon Belm Co., Ltd. in order to respond to the request for sale.
(Lactic acid bacteria 2)
Enterococcus faecalis, heat-treated lactic acid bacteria different from lactic acid bacteria 1 (heat-killed cells)
(Lactic acid bacteria 3)
Lactobacillus paracasei, heat-treated lactic acid bacteria (heat-killed cells)
(Lactic acid bacteria 4)
Lactobacillus sporogenes, live bacteria (lactic acid bacteria 5)
Lactobacillus plantarum, heat-treated lactic acid bacteria (heat-killed cells)
(Lactic acid bacteria 6)
Plant-derived lactic acid bacteria (Tetragenococcus halophilus), heat-treated lactic acid bacteria (heat-killed cells)
(Lactic acid bacteria 7)
Plant-derived lactic acid bacteria (Leuconostoc mesenteroides), heat-treated lactic acid bacteria (heat-killed cells)

<Test 1>
Macrophages RAW264.7 involved in natural immunity were inoculated on a 24-well plate and precultured in 10% FBS, DMEM High Glucose medium for 24 hours (200,000 cells / well, 0.5 mL each), and then each lactic acid bacterium 1- 4 (12.5 μg / 0.5 mL) and paramylon (350 μg / 0.5 mL) were added alone or in combination, and after further culturing for 24 hours, the amount of nitric oxide (NO) produced in the supernatant was measured in Griess. Measured by method. NO production is one of the indicators of immune activation. Each lactic acid bacterium and paramylon were dispersed and adjusted in DMEM High Glucose medium, and DMEM High Glucose medium was used as a control.

<Result 1>
1 to 4 show the results when each of the lactic acid bacteria 1 to 4 was combined with paramylon. As shown in FIGS. 1 and 2, in lactic acid bacteria 1 and 2 belonging to the genus Enterococcus (Enterococcus faecalis), when combined with paramylon contained in Euglena, the immunostimulatory effect was synergistically enhanced.

Further, as a result of conducting the same experiment by changing the mixing ratio of lactic acid bacterium 1 and paramylon, paramylon (350 μg / mL to 1000 μg / mL) was appropriately combined with lactic acid bacterium 1 (12.5 μg / mL to 35 μg / mL). It was suggested that macrophages were strongly activated by a cooperative action as compared with the case where each of them was used alone as in Test 1.

<Test 2>
Euglena water extract or Euglena hot water extract (0.4vоl%) and lactic acid bacterium 1 were added to macrophages RAW264.7 involved in innate immunity in the same manner as in Test 1, and NO production in the supernatant the next day. By measuring the amount, the effect on immune cells was examined.

<Result 2>
5 and 7 show the results of Test 2. As shown in FIG. 5, the activity of macrophages was improved when Euglena water extract was added or when lactic acid bacterium 1 was added at 25 to 50 μg / ml. As shown in FIG. 6, no significant change was observed when the lactic acid bacteria and the Euglena water extract were added alone, but the NO production amount increased when the lactic acid bacteria and the Euglena water extract were combined. In other words, it was suggested that the combination of lactic acid bacteria and Euglena water extract can emphasize the activity of immune cells.

As shown in FIG. 7, when Euglena hot water extract was added or lactic acid bacterium 1 was added at 25 to 50 ug / ml, the activity of macrophages was improved. No significant change was observed with Euglena hot water extract alone, but when combined with lactic acid bacteria (50 μg / mL), the amount of NO produced was higher than that with lactic acid bacteria alone.

<Test 3>
In the same manner as in Test 1, Euglena water extract (0.4vоl%) and each lactic acid bacterium (lactic acid bacterium 1, lactic acid bacterium 3, lactic acid bacterium 5, lactic acid bacterium 6, lactic acid bacterium 7) were added to macrophages RAW264.7 involved in innate immunity. Then, the next day, the amount of NO produced in the supernatant was measured to examine the effect on immune cells.

<Result 3>
8 to 12 show the results of Test 3. As shown in FIGS. 8 to 12, no remarkable change was observed when the lactic acid bacteria and the Euglena water extract were added alone, but the NO production amount increased when the lactic acid bacteria and the Euglena water extract were combined. In other words, it was suggested that the combination of lactic acid bacteria and Euglena water extract can emphasize the activity of immune cells. The synergistic effect (%) of the combination of lactic acid bacteria and Euglena water extract was large in the order of lactic acid bacteria 7> lactic acid bacteria 3> lactic acid bacteria 6> lactic acid bacteria 5> lactic acid bacteria 1.

<Summary>
From the above results, by using at least one Euglena-derived substance selected from the group consisting of paramylon and Euglena in combination with lactic acid bacteria, the immune activating action and the nitric oxide production promoting action of macrophages are synergistic. It was shown to be exerted in. Since Euglena cells contain many components centered on paramylon, even when Euglena is used in combination with lactic acid bacteria, the immune activating action and macrophage nitric oxide production promoting action are synergistic. Was demonstrated. It was suggested that this synergistic effect was greater in Euglena than in paramylon.

That is, it was shown that an immunostimulatory agent or an immunostimulatory food composition in which a Euglena-derived substance and a lactic acid bacterium are combined as an active ingredient can effectively activate immunity. In addition, since it is possible to enhance immunity by ingesting the immunostimulant food composition and immunostimulant according to the present embodiment, it is possible to prevent diseases such as infectious diseases typified by viral infections. It can be treated. Furthermore, macrophage nitric oxide production promoters and macrophage nitric oxide production promoting food compositions that combine Euglena-derived substances and lactic acid bacteria as active ingredients can effectively promote macrophage nitric oxide production. It was shown to be possible.

Claims (8)

Euglena-derived substance, which is paramylon,
Contains lactic acid bacteria as an active ingredient,
An immunostimulatory food composition, wherein the lactic acid bacterium is a bacterium belonging to the genus Enterococcus. The immunostimulatory food composition according to claim 1, wherein the food composition is a lactic acid bacteria beverage. The immunostimulatory food composition according to claim 1 or 2 , which is used for prevention of infectious diseases. The immunostimulatory food composition according to any one of claims 1 to 3 , which is used for promoting nitric oxide production of macrophages. Euglena-derived substance, which is paramylon,
Contains lactic acid bacteria as an active ingredient,
An immunostimulatory agent, wherein the lactic acid bacterium is a bacterium belonging to the genus Enterococcus. The immunostimulant according to claim 5 , wherein the bacterium belonging to the genus Enterococcus is Enterococcus faecalis. The immunostimulatory agent according to claim 5 or 6 , which is used for prevention and / or treatment of infectious diseases. The immunostimulant according to any one of claims 5 to 7 , which is used for promoting nitric oxide production of macrophages.

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