JP7790653B2 - Composition for synergistically enhancing immune function - Google Patents

Description

IPOD FERM BP-08607

The present invention relates to a turmeric extract-containing composition having an immunostimulatory effect on lactic acid bacteria, preferably Lactobacillus plantarum L-137 strain (hereinafter sometimes referred to as "Lactobacillus plantarum L-137" or "L-137 strain").

Turmeric is a plant native to South Asia that belongs to the genus Curcuma, a family of plants in the Zingiberaceae family, and is known to contain physiologically active substances that are beneficial to humans and animals. For example, Patent Document 1 describes the use of turmeric oil obtained by extracting turmeric as an epilepsy and/or anticonvulsant. Furthermore, Patent Document 2 describes the administration of turmeric seed extract extracted with supercritical carbon dioxide to patients suffering from Alzheimer's disease.

However, it is not known at all that turmeric extract can enhance the immunostimulatory activity of lactic acid bacteria, preferably Lactobacillus plantarum L-137 strain.

Special Publication No. 2014-518241 Japanese Patent Application Laid-Open No. 2009-530305

The object of the present invention is to provide a composition having the immunostimulatory effect of lactic acid bacteria, preferably Lactobacillus plantarum L-137.

The inventors investigated numerous materials in search of materials that have beneficial effects on the human body or animals. As a result, they were surprised to find that turmeric extract alone does not have immunostimulating effects, but that in the presence of lactic acid bacteria, preferably Lactobacillus plantarum L-137 strain, turmeric extract improves immunostimulating effects. After further investigations, the inventors have completed the present invention.

That is, the present invention is as follows.
[1] A composition for improving the immunostimulatory effect of lactic acid bacteria, comprising turmeric extract.
[2] The composition according to claim 1, wherein the lactic acid bacteria is one or more species selected from the group consisting of Lactobacillus, Lactococcus, Bifidobacterium, and Enterococcus.
[3] A composition for enhancing the immunostimulatory activity of lactic acid bacteria Lactobacillus plantarum L-137, containing turmeric extract.
[4] The composition according to any one of [1] to [3], wherein the turmeric extract is one or more extracts selected from the group consisting of water, organic solvents, and mixtures thereof.
[5] The composition according to any one of [1] to [3], wherein the turmeric extract is a hot water extract of 90°C or higher.
[6] The composition according to any one of [1] to [3], wherein the turmeric extract is an extract of the rhizome of turmeric (one or more species selected from the group consisting of Curcuma longa, Curcuma aromatica, Curcuma zedoaria, Curcuma phaeocaulis, Curcuma kwangsiensis, Curcuma wenyujin, Curcuma xanthorrhiza, and mixtures thereof).
[7] The composition according to any one of [1] to [3], wherein the content of the turmeric extract in the entire composition is 0.0001% to 95%.
[8] The composition according to any one of [1] to [3], further comprising lactic acid bacteria or a processed product thereof.
[9] The composition according to any one of [1] to [3], further comprising lactic acid bacteria Lactobacillus plantarum L-137 or a processed product thereof.
[10] The composition described in [8], wherein the content of lactic acid bacteria or a processed product thereof relative to the total composition is 0.0001% to 20%.
[11] The composition according to [9], wherein the content of the lactic acid bacterium Lactobacillus plantarum L-137 or a processed product thereof is 0.0001% to 20% relative to the total content of the composition.
[12] The composition according to [8], wherein the content ratio of the lactic acid bacterium Lactobacillus plantarum L-137 or a processed product thereof to the turmeric extract is 1:1 to 1:1000.
[13] The composition described in [8], which is a nutritional supplement, health food, functional food, food for specified health uses, food for patients, or feed.
[14] A method for improving the immunostimulatory effect of lactic acid bacteria by administering a turmeric extract to a subject.
[15] Use of turmeric extract to enhance the immunostimulatory activity of lactic acid bacteria.

The present disclosure can provide a composition containing turmeric extract for enhancing the immunostimulatory activity of lactic acid bacteria, preferably Lactobacillus plantarum L-137. More preferably, the present disclosure can provide a composition that synergistically enhances the immune function of lactic acid bacteria, particularly preferably Lactobacillus plantarum L-137. The present disclosure can also provide a method for producing the composition.

FIG. 1 is a graph showing the evaluation of the IL-12 production ability of compositions containing lactic acid bacteria (Lactobacillus plantarum L-137 strain) and turmeric extract, and compositions containing both (dosage of L-137 strain: 5 and 10 μg). FIG. 2 is a graph showing the evaluation of the IL-12 production ability of compositions containing lactic acid bacteria (Lactobacillus plantarum L-137 strain) and turmeric extract, and compositions containing both (dosage of L-137 strain: 25 and 50 μg). FIG. 3 is a graph showing the evaluation of the IL-12 production ability of a composition containing lactic acid bacteria (Lactobacillus plantarum JCM1149 strain) and a turmeric extract, and a composition containing both (JCM1149 strain dosage: 50 μg). FIG. 4 is a graph showing the evaluation of the IL-12 production ability of a composition containing lactic acid bacteria (Lactobacillus delbrueckii subsp. lactis ATCC 7830 strain) and a turmeric extract, and a composition containing both (ATCC 7830 strain dosage: 50 μg). FIG. 5 is a graph showing the evaluation of the IL-12 production ability of a composition containing lactic acid bacteria (Enterococcus hirae ATCC 8043 strain) and a turmeric extract, and a composition containing both (ATCC 8043 strain dosage: 50 μg).

[Composition for improving the immunostimulatory effect of lactic acid bacteria, preferably Lactobacillus plantarum L-137]
The composition of the present invention is characterized by containing a turmeric extract. Preferably, the composition of the present invention is characterized by containing a turmeric extract as an active ingredient. More preferably, the composition of the present invention further contains lactic acid bacteria, including but not limited to the following lactic acid bacteria or a processed product thereof:
The lactic acid bacteria used in the present invention may be known lactic acid bacteria such as bacteria of the genera Lactobacillus, Enterococcus or Streptococcus, Lactococcus, Bifidobacterium, Pediococcus, Leuconostoc, Tetragenococcus, and Oenococcus.
These lactic acid bacteria can be easily obtained from institutions such as the International Patent Organism Depositary (IPOD), National Institute of Technology and Evaluation, American Type Culture Collection (ATCC), Japan Collection of Microorganisms (JCM), RIKEN BioResource Research Center, and Institute for Fermentation, Osaka (IFO). Commercially available strains (including pharmaceuticals and foods containing these lactic acid bacteria) can also be used as appropriate.
Examples of lactic acid bacteria belonging to the genus Lactobacillus include Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus gasseri, Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus johnsonii, Lactobacillus fermentum, and Lactobacillus delbrueckii subsp. bulgaricus. bulgaricus, Lactobacillus delbrueckii subsp. lactis, Lactobacillus paracasei subsp. paracasei, Lactobacillus reuteri, Lactobacillus salivarius, Lactobacillus rhamnosus or Lactobacillus helveticus helveticus), among which Lactobacillus plantarum and/or Lactobacillus delbrueckii subsp. lactis are preferred, and Lactobacillus plantarum L-137 strain, Lactobacillus plantarum JCM1149 strain, and/or Lactobacillus delbrueckii subsp. lactis ATCC7830 strain are more preferred, but are not limited to these.
Furthermore, examples of lactic acid bacteria belonging to the genus Lactobacillus include, in addition to those mentioned above, Lactobacillus daoliensis, Lactobacillus daowaiensis, Lactobacillus dongliensis, Lactobacillus fabifermentans, Lactobacillus herbarum, Lactobacillus modestisalitolerans, Lactobacillus mudanjiangensis, Lactobacillus nangangensis, Lactobacillus paraplantarum, Lactobacillus pentosus, Lactobacillus pingfangensis, Lactobacillus plajomi, Lactobacillus argentoratensis, Lactobacillus plantarum subsp. plantarum, Lactobacillus songbeiensis, and Lactobacillus xiangfangensis, but are not limited to these.
Examples of lactic acid bacteria belonging to the genus Enterococcus (which may be classified as the genus Streptococcus in older classifications) or the genus Streptococcus include Enterococcus hirae, Enterococcus faecalis, Enterococcus faecium, and Enterococcus thermophilus, with Enterococcus hirae being preferred and Enterococcus hirae ATCC8043 strain being more preferred, but not limited to these.
Examples of lactic acid bacteria belonging to the genus Lactococcus include, but are not limited to, Lactococcus lactis subsp. lactis and Lactococcus lactis subsp. cremoris. Examples of lactic acid bacteria belonging to the genus Bifidobacterium include, but are not limited to, Bifidobacterium bifidum, Bifidobacterium thermophilum, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, and Bifidobacterium adolescentis. Examples of lactic acid bacteria belonging to the genus Pediococcus include, but are not limited to, Pediococcus pentosaceus and Pediococcus acidilactici. Examples of lactic acid bacteria belonging to the genus Leuconostoc include, but are not limited to, Leuconostoc mesenteroides subsp. cremoris. Examples of lactic acid bacteria belonging to the genus Tetragenococcus include, but are not limited to, Tetragenococcus halophilus. Examples of lactic acid bacteria belonging to the genus Oenococcus include, but are not limited to, Oenococcus oeni.
These lactic acid bacteria cells can be easily obtained from organizations such as IPOD, ATCC, JCM, IFO, etc. Commercially available products (including pharmaceuticals, foods, etc. containing these lactic acid bacteria) can also be used as appropriate.
Of the above-mentioned lactic acid bacteria, the most preferred lactic acid bacteria is Lactobacillus plantarum L-137 strain (IPOD accession number: FERM BP-08607) or a processed product thereof.

The immunopotentiating effect preferably refers to, but is not limited to, (1) improving resistance to various diseases or (2) recovering from an immunosuppressed state or decreased immune function. Specifically, (1) for example, the composition of the present invention is preferably effective in preventing or treating infectious diseases caused by microorganisms such as viruses or bacteria, and more preferably, is effective in preventing or treating infectious enteritis caused by oral infections such as Vibrio cholerae, enterotoxigenic Escherichia coli, Shigella, Salmonella, or viruses; influenza or cold syndrome caused by respiratory tract infection; stomatitis and periodontal disease caused by oral infection; and various malignant tumors (for example, non-epithelial malignant tumors occurring in organs such as the digestive tract or respiratory mucosa, liver, or kidney). (2) For example, the composition of the present invention is also preferably suitable for recovery from immunosuppressive states or decreased immune function, recovery from tumor-induced immunosuppressive states or decreased immune function induced by anticancer drug treatment, prevention of the onset of acquired immune deficiency syndrome (AIDS), control of intracellular parasitic bacteria such as Listeria monocytogenes, Salmonella enterica, Mycobacterium tuberculosis, or Mycobacterium leprae, amelioration of stress-induced decreased Th1 immune function, and suppression of age-related decreased immune function, etc. Another preferred example of the present invention is that the composition has a protective effect against infection with the intracellular parasitic bacterium Chlamydia, which has been suggested to be involved in Chlamydia infection and therefore acts to prevent the onset of arteriosclerosis, but is not limited to this.
A preferred embodiment of the present invention is a composition that synergistically enhances the immune function of lactic acid bacteria, preferably Lactobacillus plantarum L-137.
Furthermore, in a further or alternative preferred embodiment of the composition of the present invention, the subject to which the composition of the present invention is administered has improved cytokine production capabilities such as interleukin-12 (IL-12), interferon-β (IFN-β), and interferon-γ (IFN-γ), but this is not limited to this.

[Turmeric extract]
In the present invention, turmeric extract refers to an extract (extract) obtained with an extraction solvent from a plant material derived from a plant of the genus Curcuma in the Zingiberaceae family. Turmeric extract is not limited to solvent extracts obtained by solvent extraction, but also includes solvent extracts that have been further fractionated and purified by column chromatography or the like. The turmeric extract used in the present invention may be in the form of an extract liquid after the extraction procedure (including fractionation and purification if fractionation and purification is performed), a concentrate obtained by partially removing the solvent from the extract liquid, or a dried product obtained by removing the solvent from the extract liquid. The solvent can be removed from the extract by volatilizing the solvent by heating and/or reducing the pressure, etc. The methods for heating and reducing the pressure are not particularly limited, and for example, conventionally known methods can be used.

Examples of plant raw materials for producing the turmeric extract used in the present invention include the rhizomes of plants of the genus Curcuma in the family Zingiberaceae, such as Curcuma longa, Curcuma aromatica, Curcuma zedoaria, Curcuma phaeocaulis, Curcuma kwangsiensis, Curcuma wenyujin, and/or Curcuma xanthorrhiza, with the rhizomes of Curcuma longa being particularly preferred, but not limited to these.
Rhizomes can be collected from the soil and washed before use. An appropriate portion of the rhizome can be used either intact, cut to an appropriate size or shape, or pulverized. Cutting and pulverization methods include, but are not limited to, blades such as knives and kitchen knives, lasers, mills, mixers, stamp mills, mass colloiders, comitrols, and pestle pestles. The particle size of the pulverized material can be adjusted with a sieve as needed. The plant material may also be appropriately dried.
The drying method can be, for example, hot air drying, spray drying, freeze drying, or other common methods, but is not limited to these.

At least one solvent selected from the group consisting of water and hydrophilic organic solvents can be used for extraction from plant materials. Such solvents may be water, hydrophilic organic solvents, or mixed solvents of water and hydrophilic organic solvents. The hydrophilic organic solvent may be a mixed solvent of multiple hydrophilic organic solvents. Water may be distilled water, purified water, etc., and also includes hot water. Hot water, for example, is preferably hot water at 90°C or higher, more preferably 95°C or higher, and even more preferably 97°C or higher. Examples of hydrophilic organic solvents include at least one alcohol or a mixed solvent of multiple alcohols. Preferred alcohols include ethanol, methanol, or isopropanol. Preferred organic solvents include acetone and acetonitrile, but are not limited to these. Ethanol, ethanol, or a mixed solvent thereof is more preferred. When using a mixed solvent of alcohol and water as the extraction solvent, the mixing ratio is preferably, for example, approximately 10:90 to 90:10 by weight, and more preferably, approximately 20:80 to 50:50, but is not limited to these.

Turmeric can be mixed with water and/or a hydrophilic organic solvent by immersing the turmeric in the extraction solvent, preferably at approximately 5°C to 100°C, for 3 minutes or more (e.g., 1 hour to 72 hours, preferably 18 hours to 72 hours). Shaking or stirring may be added as necessary. The shaking or stirring method can be adjusted as needed.

The method for extracting turmeric extract from plant materials is not particularly limited. In addition to extraction with the solvents described above, turmeric extract may also be produced from plants using steam distillation or supercritical carbon dioxide extraction. In the present invention, it is preferable to use a turmeric extract produced with the above extraction solvent that contains components such as turmeronol A, turmeronol B, bisacron, curcumin, ar-turmerone, α-turmerone, and β-turmerone, but the components contained in the turmeric extract of the present invention are not limited to these.

In the composition of the present invention, the turmeric extract is contained in an amount of preferably about 0.0001 to 95% by weight, more preferably about 0.001 to 97% by weight, and even more preferably about 0.01 to 99% by weight, relative to the total amount of the composition, but is not limited to these ranges.
The intake amount of turmeric extract in the composition of the present invention can be determined depending on the age and weight of the recipient, symptoms, administration time, dosage form, administration method, combination of drugs, etc. For example, it is preferable to set the intake amount of turmeric extract so that an adult (approximately 60 kg) ingests about 0.1 to 2000 mg, more preferably about 0.5 to 1000 mg, and even more preferably about 1 to 500 mg per day, but is not limited to these ranges. The number of intakes can be once a day or divided into multiple doses. The above-mentioned dosage can be administered or applied once or several times a day.

The turmeric extract used in the present invention is preferably derived from a plant material, more preferably from a plant of the genus Curcuma in the family Zingiberaceae. Examples of such plants include, but are not limited to, Curcuma longa, Curcuma aromatica, Curcuma zedoaria, Curcuma phaeocaulis, Curcuma kwangsiensis, Curcuma wenyujin, and Curcuma xanthorrhiza. Multiple plants may also be used in combination. Clones, mutants, and varieties of these plants are also included. Rhizome refers to the stem of a plant, usually found underground, with roots and shoots extending from its nodes.

Fractions of the plant extract that have been further purified may also be incorporated into the composition of the present invention. For example, the plant extract may be subjected to liquid-liquid partitioning between ethyl acetate and water, and the active compounds may be highly purified in the ethyl acetate fraction. The plant extract or a fraction thereof may also be purified by chromatography. Chromatography methods that can be used include reverse-phase column chromatography and normal-phase thin-layer chromatography. The plant extract or a fraction thereof may be further processed by conventional methods, such as drying, powdering, granulation, or solution formation.

[Lactobacillus plantarum L-137]
The lactic acid bacterium Lactobacillus plantarum L-137 strain has been deposited at the Patent Organism Depositary of the National Institute of Advanced Industrial Science and Technology (currently the Patent Organism Depositary of the National Institute of Technology and Evaluation; address: Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan, 292-0818) under accession number FERM BP-08607 (transferred from FERM P-15317 deposited on November 30, 1995). Mutant strains of Lactobacillus plantarum L-137 that have the characteristics of Lactobacillus plantarum L-137 are also included in the category of Lactobacillus plantarum L-137. Other lactic acid bacteria may be contained in the composition of the present invention together with the Lactobacillus plantarum L-137 strain.

When the composition of the present invention contains lactic acid bacteria, preferably Lactobacillus plantarum L-137 strain or a processed product thereof, the content thereof is preferably about 0.0001 to 20% by weight, more preferably about 0.001 to 15% by weight, and even more preferably about 0.05 to 10% by weight, relative to the total amount of the composition, but is not limited to these ranges.

Furthermore, when the composition of the present invention contains lactic acid bacteria, preferably Lactobacillus plantarum L-137 strain or a processed product thereof, the content ratio of the lactic acid bacteria, preferably L-137 strain, to turmeric extract is preferably 1: (1 to 10,000), more preferably 1: (2.5 to 400), and even more preferably 1: (2.5 to 50), but is not limited to these ranges.

When the lactic acid bacteria of the present invention, preferably Lactobacillus plantarum L-137 strain or a processed product thereof is contained in a composition, the intake amount can be determined depending on the age and weight of the user, symptoms, administration time, dosage form, administration method, combination of drugs, etc. For example, the intake amount of lactic acid bacteria, preferably Lactobacillus plantarum L-137 strain, in terms of dried killed cells is preferably set to about 0.5 to 200 mg, more preferably about 1 to 100 mg, and even more preferably about 2 to 50 mg per day for an adult (about 60 kg), but is not limited to these ranges. Alternatively, the dosage is set so that an adult (approximately 60 kg) ingests lactic acid bacteria, preferably Lactobacillus plantarum L-137, in terms of viable bacteria, preferably about 5 x ¹⁰ to 2 x ¹⁰ cfu (colony forming units), more preferably about 1 x ¹⁰ to 1 x ¹⁰ cfu, per day, but is not limited to this range. The number of doses can be once a day or divided into multiple doses. The above-mentioned dosage may be administered or applied once or divided into several doses per day.

[Cultivation of lactic acid bacteria]
In the present invention, Lactobacillus plantarum L-137 strain and other lactic acid bacteria may be cultured in any medium, such as a natural medium, a synthetic medium, a semi-synthetic medium, etc. In the present invention, lactic acid bacteria may be cultured according to a known method, a method known per se, or a method similar thereto.

The medium is not particularly limited, and preferably contains, for example, a nitrogen source and/or a carbon source. The nitrogen source is not particularly limited, and examples thereof include meat extract, peptone, gluten, casein, yeast extract, and amino acids. The carbon source is not particularly limited, and examples thereof include glucose, xylose, fructose, inositol, maltose, starch syrup, koji soup, starch, bagasse, bran, molasses, and glycerin. These may be used alone or in combination of two or more. In addition to the nitrogen source and/or carbon source, the medium may further contain minerals. The minerals are not particularly limited, and examples thereof include ammonium sulfate, potassium phosphate, magnesium chloride, salt, iron, manganese, molybdenum, and various vitamins. These may be used alone or in combination of two or more.

The culture temperature and culture time for Lactobacillus plantarum strain L-137 and other lactic acid bacteria are not particularly limited as long as the culture can be carried out efficiently. In one embodiment of the present invention, the culture temperature may be, for example, typically about 25 to 40 degrees (°C), preferably about 27 to 35 degrees Celsius, and the culture time may be, for example, about 12 to 48 hours. Furthermore, in one embodiment of the present invention, the lactic acid bacteria may be cultured by aeration and shaking. Furthermore, the pH of the medium is not particularly limited, but in one embodiment of the present invention, it may be typically about pH 3 to 6, preferably about pH 4 to 6.

[Processed lactic acid bacteria]
The "processed product" of the lactic acid bacteria Lactobacillus plantarum strain L-137 and other lactic acid bacteria is preferably a processed product of the L-137 strain and other lactic acid bacteria, and includes, but is not limited to, their culture solution or culture supernatant, residues obtained by filtering or centrifuging them, solutions obtained by ultrasonically disrupting bacterial cells, etc. The processed product of the present invention also includes, but is not limited to, solutions obtained by removing cell walls using enzymatic or physical treatment, protein or peptide complexes obtained by chemical or salting-out treatment, and concentrates, dried products, or dilutions thereof.
Furthermore, the L-137 strain and other lactic acid bacteria may be in the form of live cells, dried cells, centrifuged cells, disrupted cells, or killed cells, but killed cells are preferred from the viewpoints of stability, ease of handling, etc.

In the present invention, the above-mentioned processed products may be used as is, or may be prepared in powder form by freeze-drying, low-temperature drying, spray-drying, L-drying, or a combination of these. These processed products may also be diluted with an appropriate solvent (e.g., water, alcohol, organic solvent, etc.) or may be made into a gel or solid by adding appropriate additives.

The following provides a specific description of methods for preparing killed cells of Lactobacillus plantarum strain L-137 and other lactic acid bacteria. In the present invention, the method for preparing the killed cells is not particularly limited as long as it does not impair the effects of the present invention. For example, the killed cells may be prepared by any of the following methods: (I) a method in which live lactic acid bacteria cells are separated from the culture medium after the completion of culture, and then the live cells are sterilized or sterilized to kill the cells; or (II) a method in which live lactic acid bacteria cells are sterilized in the culture medium to kill the cells, and then the killed cells are separated from the culture medium. Sterilization can be carried out, for example, by filter filtration, but other known methods, such as gas sterilization using ethylene oxide or hydrogen peroxide, or heat sterilization using gamma rays, electron beam irradiation, or high frequency waves, may also be used.

The method for separating the bacterial cells from the culture medium may employ various methods commonly used in this field, and is not particularly limited. In one embodiment of the present invention, a specific example may be a method in which the culture medium and the bacterial cells are separated by removing the supernatant from the culture medium by means of centrifugation or other means. In this embodiment, distilled water is added to the culture medium, followed by centrifugation and then the supernatant is removed. If desired, the supernatant may be removed and the residue may be further centrifuged after which further distilled water may be added. This process may be repeated several times. In one embodiment of the present invention, the separation process may include a filtration step. The above-described bacterial cells may be dried using a spray dryer to obtain dried bacteria. A preferred example of the device is a spray dryer equipped with an atomizer capable of producing spray droplets of approximately 1 to 10 μm in size, but this is not limiting.

The sterilization method is not particularly limited, and examples include heating, ultraviolet irradiation, and formalin treatment. The sterilization may be performed on the collected live bacterial cells, or on the culture medium containing the live bacterial cells.

When the heat treatment is carried out, the heating temperature is not particularly limited, but may be, for example, typically about 60 to 100 degrees (°C), preferably about 70 to 90 degrees. Heating methods can be known and are not particularly limited, but may include, for example, a heater. The heating time is not particularly limited as long as the sterilization treatment is sufficiently completed, but may be, for example, typically about 5 to 40 minutes, preferably about 10 to 30 minutes, after the desired temperature is reached.

The killed bacterial cells obtained as described above may be further subjected to grinding, crushing, spray drying, low-temperature drying, or freeze-drying, or may be mixed with other raw materials (e.g., vitamins, amino acids, oligopeptides, etc.) to produce a processed product of killed bacterial cells. In the present invention, processed products of killed bacterial cells can also be suitably used as killed bacterial cells.

Preferred examples of the composition of the present invention are for use in foods and beverages and/or pharmaceuticals (including veterinary drugs). In another preferred example, the composition of the present invention is used as an additive for foods and beverages. Compositions for foods and beverages, food and beverage additives, or pharmaceuticals can be formulated by appropriately combining the above-described culture supernatant of the present invention or a processed product thereof, or the L-137 strain, other lactic acid bacteria, or a processed product thereof, with pharmaceutically acceptable carriers, additives, and the like. Since formulation methods and technologies for this purpose have been well established, these may be used. For example, in the case of pharmaceuticals, specific examples include oral preparations such as tablets, coated tablets, pills, powders, granules, capsules, liquids, suspensions, and emulsions, and parenteral preparations such as injections, infusions, suppositories, ointments, and patches. The proportion of carriers or additives may be appropriately determined based on the ranges commonly used in the fields of foods and beverages, pharmaceuticals, or veterinary medicine.

There are no particular limitations on the pharmaceutically acceptable carriers or additives, and examples of carriers include various carriers such as aqueous or oily bases. Examples of aqueous carriers include water, physiological saline, ethanol, glycerin, polyethylene glycol, propylene glycol, methylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, polyvinylpyrrolidone, polyacrylic acid, polysaccharide gum-based natural polymers, etc., and examples of oily carriers include, but are not limited to, appropriate oils and waxes such as petrolatum, squalane, and paraffin.
Examples of additives include, but are not limited to, sweeteners, acidulants, vitamins, minerals, thickeners, emulsifiers, antioxidants, enzymes, pH adjusters, preservatives, bactericides, antifungal agents, shelf life improvers, bleaching agents, glossing agents, flavorings, seasonings, bittering agents, stabilizers, gelling agents, thickening agents, excipients, binders, disintegrants, lubricants, coloring agents, flavoring agents, etc. Since the techniques relating to these have been well established in the past, they may be used in the present invention.

Sweeteners include, but are not limited to, monosaccharides and disaccharides such as glucose, fructose, sucrose, lactose, maltose, palatinose, trehalose, and xylose, isomerized sugars (glucose-fructose corn syrup, fructose-glucose corn syrup, mixed sugar isomerized sugars, etc.), sugar alcohols (erythritol, xylitol, lactitol, palatinite, sorbitol, reduced starch syrup, etc.), honey, and high-intensity sweeteners (sucralose, acesulfame potassium, thaumatin, stevia, aspartame, etc.).

Examples of acidulants include, but are not limited to, citric acid, malic acid, gluconic acid, tartaric acid, lactic acid, phosphoric acid, or salts thereof, and one or more of these may be used.

Examples of vitamins include, but are not limited to, vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin E, niacin, inositol, etc.

Minerals include, but are not limited to, calcium, magnesium, zinc, iron, etc.

Examples of thickeners include, but are not limited to, carrageenan, gellan gum, xanthan gum, gum arabic, tamarind gum, guar gum, locust bean gum, karaya gum, agar, gelatin, pectin, soybean polysaccharides, carboxymethylcellulose (CMC), etc.

Examples of emulsifiers include glycerin fatty acid esters, sucrose fatty acid esters, sorbitan fatty acid esters, lecithin, plant sterols, saponin, etc.

Antioxidants include, but are not limited to, vitamin C, tocopherol (vitamin E), enzyme-treated rutin, catechin, etc.

Furthermore, when the composition of the present invention is for use in food and beverage products, the food and beverage products include health foods, functional foods, foods for specified health uses, and foods for patients. The form of the food and beverage products is not particularly limited, but specific examples include tablets, granules, powders, drinks, and the like, which are so-called nutritional supplements or supplements. Other examples include, but are not limited to, beverages such as tea drinks, soft drinks, carbonated drinks, nutritional drinks, fruit drinks, and lactic acid drinks; noodles such as soba, udon, Chinese noodles, and instant noodles; sweets and breads such as candy, candy, gum, chocolate, snacks, biscuits, jelly, jam, cream, baked goods, and bread; processed seafood and livestock foods such as ham, sausage, fish cake, and chikuwa; dairy products such as processed milk and fermented milk; oils and fats and processed oil foods such as salad oil, tempura oil, margarine, mayonnaise, shortening, whipped cream, and dressing; seasonings such as sauces and dressings; retort pouch foods such as curry, stew, rice bowls, porridge, and rice porridge; and frozen desserts such as ice cream, sherbet, and shaved ice. Since technologies related to these have been well established, these may be used in the present invention. The foods of the present invention do not necessarily include curry, stew, or products containing these.
Furthermore, the composition of the present invention may contain any component known in the fields of medicine, pharmacology, veterinary medicine, livestock farming, food, etc., as long as the effect of the present invention is not lost.
The feed is preferably for mammals such as rats, mice, dogs, cats, rabbits, horses, cows, pigs, and monkeys; birds such as chickens, ducks, parrots, pigeons, sparrows, and owls; and seafood such as salmon, chum salmon, mackerel, saury, sardines, herring, tuna, Alaska pollock, yellowtail, eel, squid, octopus, crab, shrimp, crayfish, and sea cucumber, but is not limited to these.

[Immunostimulatory effect of lactic acid bacteria and its confirmation method]
An example of a method for confirming the effect of the composition of the present invention is a method for confirming that the composition of the present invention containing a turmeric extract is superior in the immunostimulatory effect of lactic acid bacteria, preferably Lactobacillus plantarum L-137, compared to a composition not containing a turmeric extract.
Specifically, methods for confirming the immunostimulatory activity-enhancing effect of lactic acid bacteria, preferably Lactobacillus plantarum L-137, include, but are not limited to, methods for comparing the immune cell production capacity of cytokines such as interleukin-12 (IL-12), interferon-β (IFN-β), and interferon-γ (IFN-γ) with that of other compositions. Cytokines such as IL-12, IFN-β, and IFN-γ are known to play a certain role in immune defense mechanisms against microbial infections such as bacteria, yeast, fungi, and viruses, and against tumors. For example, when a subject is administered or ingested with the composition of the present invention, the composition of the present invention can be determined to have a desired immunostimulatory activity-enhancing effect if the IL-12, IFN-β, and/or IFN-γ production capacity is significantly, markedly, or synergistically higher than that of a control group that has not been administered or ingested with the composition of the present invention.
Examples of immune cells include lymphocytes such as B cells or T cells, macrophages, natural killer (NK) cells, dendritic cells, etc. These cells are preferably obtained by known methods from organs such as the blood or spleen of animals (more preferably mammals or birds), or commercially available products can also be purchased and used. Evaluation may also be performed according to other known methods or methods well established in the art. See the Examples below for examples of these.

When the composition of the present invention is prepared into products in the form of foods and beverages, pharmaceuticals (including veterinary medicines), quasi-drugs, nutritional supplements (supplements), health foods, functional foods, foods for specified health uses, foods for patients, or feed, the product's accompanying instructions or packaging box, etc., can state that the composition of the present invention has the immunostimulatory effect of Lactobacillus plantarum L-137, taking into account its action.

[Method of producing the composition]
The present invention includes a method for producing a composition for improving immunostimulation of lactic acid bacteria, preferably Lactobacillus plantarum L-137, which comprises a step of mixing a turmeric extract with, preferably, a carrier and/or an excipient.

Preferred carriers for use in the above process have been well established in the food and pharmaceutical fields, and the present invention may also follow these. Examples include various carriers such as aqueous or oily bases. Aqueous carriers include, for example, water, saline, ethanol, glycerin, polyethylene glycol, propylene glycol, methylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, polyvinylpyrrolidone, polyacrylic acid, and polysaccharide gum-based natural polymers. Oily carriers include, but are not limited to, suitable oils and waxes such as petrolatum, squalane, and paraffin.

In addition, preferred excipients used in the above process have been well established in the food and pharmaceutical fields, and the present invention may also follow these. For example, lactose, sucrose, mannitol, corn starch, powdered cellulose, calcium hydrogen phosphate, calcium carbonate, dextrin, etc. can be preferably used, but are not limited to these.

The composition of the present invention can be appropriately processed and produced using general composition production methods, except for adding turmeric extract to the composition. Preferably, Lactobacillus plantarum L-137 strain or a processed product thereof is further added. The present invention encompasses a method for producing a composition that includes the step of mixing turmeric extract with, if desired, other ingredients.

The present invention includes various combinations of the above configurations within the technical scope of the present invention, as long as the effects of the present invention are achieved. Furthermore, appropriate modifications are possible as long as they fall within the technical scope of the present invention.

The present invention will be explained in more detail below with reference to examples and test examples, but the present invention is not limited to these.

Test Example 1 Confirmatory Test of IL-12 Production Ability of Turmeric Extract-Containing Composition 1. Production of Turmeric Extract Turmeric extract was prepared by extracting the rhizome parts of turmeric (Curcuma longa) with hot water (90°C or higher), and then removing water from the obtained extract by heating and drying under reduced pressure.
2. Production of Dried Killed Cells of Lactobacillus plantarum L-137 Lactobacillus plantarum L-137 (Accession Number: FERM BP-08607) was inoculated into an MRS (de Man, Rogosa, Sharpe) modified liquid medium and cultured at 32 ° C. for 18 hours. After culture, the cells were killed by heating at 80 ° C., the culture medium was removed, and the cells were collected. The obtained cells were thoroughly dispersed in water, and 20% by mass of the cells and 80% by mass of dextrin were mixed and spray-dried to obtain dried killed cells.
3. Method for confirming IL-12 production ability Dry killed cells of Lactobacillus plantarum L-137 were suspended in RPMI 1640 medium containing 10% FBS to a concentration of 0 to 200 μg/mL to prepare a lactic acid bacteria test solution. Turmeric extract was suspended in RPMI 1640 medium containing 10% FBS to a concentration of 0 to 1600 μg/mL to prepare a turmeric extract test solution.
J774.1 cells (cell number JCRB9108; JCRB Cell Bank, National Institutes of Biomedical Innovation, Health and Nutrition) were used as immune cells. They were suspended in 10% FBS-containing RPMI 1640 medium at 1.0 x ¹⁰ cells/mL to prepare a cell line suspension. 100 μL of the cell line suspension was added to a 96-well culture plate and cultured at 37°C for 1-3 hours in a 5% CO2 _incubator to allow the cells to adhere to the bottom. 50 μL of the turmeric extract test solution was added to each well of the 96-well culture plate and cultured at 37°C for 1 hour in a 5% CO2 _incubator . Furthermore, 50 μL of the lactic acid bacteria test solution was added to a 96-well culture plate and cultured at 37°C for 24 hours in a 5% _CO2 incubator (final lactic acid bacteria concentration: 0-50 μg/mL, final turmeric extract concentration: 0-400 μg/mL, final cell concentration: 5.0 × 10 ⁵ /mL). After the culture was completed, the IL-12p40 concentration in the supernatant was measured by ELISA after freezing the plate at -20°C and thawing it later.

The results of Test Example 1 above demonstrate that a composition containing the turmeric extract of the present invention exhibits an immunostimulatory effect on lactic acid bacteria (strain L-137) and a synergistic effect of enhancing the immune function of lactic acid bacteria L-137. This is a surprising effect, as such an effect was hardly observed with turmeric extract alone.

Test Example 2: Test to confirm the ability of a turmeric extract-containing composition to produce IL-12 A test was conducted in the same manner as in Test Example 1, except that live or killed cells of Lactobacillus plantarum JCM1149, Lactobacillus delbrueckii subsp. lactis ATCC 7830, or Enterococcus hirae ATCC 8043 were used instead of Lactobacillus plantarum L-137, and that the dose of each lactic acid bacterium was 50 μg.
The results are shown in Figures 3 to 5. These results demonstrate that the composition containing the turmeric extract of the present invention exhibits an immunostimulatory effect on various lactic acid bacteria, even those other than the L-137 strain, and an effect of synergistically enhancing the immune functions of various lactic acid bacteria.

As described above, the turmeric extract-containing composition of the present invention has the effect of improving the immunostimulatory activity of lactic acid bacteria, preferably Lactobacillus plantarum L-137. Therefore, the composition of the present invention is useful as a food or drink, pharmaceutical product, quasi-drug, nutritional supplement, health food, functional food, food for specified health uses, food for patients, or feed, etc.

Claims (15)

A composition for improving the immunostimulatory effect of lactic acid bacteria, comprising turmeric extract,
A composition, wherein the lactic acid bacteria is one or more species selected from the group consisting of Lactobacillus and Enterococcus . The composition according to claim 1 , wherein the lactic acid bacteria are of the genus Lactobacillus. A composition containing turmeric extract for enhancing the immunostimulatory activity of the lactic acid bacterium Lactobacillus plantarum L-137. The composition according to any one of claims 1 to 3, wherein the turmeric extract is one or more extracts selected from the group consisting of water, organic solvents, and mixtures thereof. The composition according to any one of claims 1 to 3, wherein the turmeric extract is a hot water extract at 90°C or higher. The composition according to any one of claims 1 to 3, wherein the turmeric extract is an extract of the rhizome of turmeric (one or more species selected from the group consisting of Curcuma longa, Curcuma aromatica, Curcuma zedoaria, Curcuma phaeocaulis, Curcuma kwangsiensis, Curcuma wenyujin, Curcuma xanthorrhiza, and mixtures thereof). The composition according to any one of claims 1 to 3, wherein the turmeric extract content is 0.0001% to 95% of the entire composition. The composition according to any one of claims 1 to 3, further comprising lactic acid bacteria or a processed product thereof. The composition according to any one of claims 1 to 3, further comprising the lactic acid bacterium Lactobacillus plantarum L-137 or a processed product thereof. The composition according to claim 8, wherein the content of lactic acid bacteria or a processed product thereof is 0.0001% to 20% of the total composition. The composition according to claim 9, wherein the content of the lactic acid bacterium Lactobacillus plantarum L-137 or a processed product thereof is 0.0001% to 20% of the entire composition. The composition according to claim 8, wherein the ratio of the lactic acid bacteria or a processed product thereof to the turmeric extract is 1:1 to 1:1000. The composition according to claim 8, which is a nutritional supplement, health food, functional food, food for specified health uses, food for medical patients, or feed. A method for improving the immunostimulatory effect of lactic acid bacteria by administering a turmeric extract to a subject, wherein the lactic acid bacteria is one or more species selected from the group consisting of the genus Lactobacillus and the genus Enterococcus (however, this method does not apply to human treatment). Use of turmeric extract for improving the immunostimulatory effect of lactic acid bacteria, wherein the lactic acid bacteria is one or more species selected from the group consisting of the genus Lactobacillus and the genus Enterococcus (excluding methods for treating humans).