Composition synergistically enhancing immune function

Abstract

The purpose of the present invention is to provide a composition for enhancing immune activating action of lactobacillus sp., preferably Lactobacillus plantarum

Description

[Technical Field]

[0001] This invention relates to a composition containing turmeric extract, which has an immune-enhancing effect of lactic acid bacteria, preferably Lactobacillus plantarum strain L-137 (hereinafter sometimes referred to as "Lactobacillus plantarum L-137" or "L-137 strain"). [Previous Technology]

[0002] Turmeric is a plant of the genus Curcuma in the family Zingiberaceae, native to South Asia, and is known to contain physiologically active substances that are beneficial to humans or animals. For example, Patent Document 1 describes the use of turmeric oil obtained from the extraction of turmeric as an epilepsy and / or anticonvulsant. Furthermore, Patent Document 2 describes the administration of turmeric seed extract obtained by supercritical carbon dioxide extraction to patients suffering from Alzheimer's disease.

[0003] However, it is completely unknown whether turmeric extract can enhance the immune activation of lactic acid bacteria, preferably Lactobacillus germinatus strain L-137.

[0003] [Previous Technical Documents]

[0003] [Patent Documents]

[0004] [Patent Document 1] Japanese Patent Publication No. 2014-518241

[0004] [Patent Document 2] Japanese Patent Application Publication No. 2009-530305

[0005] The purpose of this invention is to provide a composition having an immune-enhancing effect of lactic acid bacteria, preferably Lactobacillus germinarum L-137.

[0006] The inventors, seeking materials that have effects on humans or animals, and after reviewing numerous materials, surprisingly discovered that turmeric extract alone does not have immunomodulatory activity, but in the presence of lactic acid bacteria, preferably Lactobacillus cocciformis strain L-137, turmeric extract enhances immunomodulatory activity. The inventors further devoted themselves to further investigation, thereby completing this invention.

[0007] That is, the present invention is as follows.

[0007] [1] A composition for enhancing the immune activation of lactic acid bacteria, which contains turmeric extract.

[0007] [2] As described in [1], the lactic acid bacteria are selected from one or more species in the group consisting of Lactobacillus, Lactococcus, Bifidobacterium and Enterococcus.

[0007] [3] A composition for enhancing the immune activation of Lactobacillus germinarum L-137, which contains turmeric extract.

[0007] [4] The composition described in any 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.

[0007] [5] The composition described in any of [1] to [3], wherein the turmeric extract is a hot water extract at 90°C or higher.

[0007] [6] The composition described in any one of [1] to [3], wherein the turmeric extract is a rhizome extract selected from one or more of the group consisting of autumn turmeric (Curcuma longa), spring turmeric (Curcuma aromatic), zedoaria (Curcuma zedoaria), phaeocaulis (Curcuma phaeocaulis), kwangsiensis (Curcuma kwangsiensis), wenyujin (Curcuma wenyujin), xanthorrhiza (Curcuma xanthorrhiza) and mixtures thereof).

[0007] [7] The composition described in any of [1] to [3], wherein the content of turmeric extract relative to the total composition is 0.0001% to 95%.

[0007] [8] The composition described in any of [1] to [3] further contains lactic acid bacteria or its treatment.

[0007] [9]The composition described in any of [1] to [3] further contains Lactobacillus blastomonas L-137 or a treatment thereof.

[0007]

[10] The composition as described in [8], wherein the proportion of lactic acid bacteria or its treatment to the total composition is 0.0001% to 20%.

[0007]

[11] The composition as described in [9], wherein the content of Lactobacillus blastomonas L-137 or its treatment thereof is 0.0001% to 20% relative to the total composition.

[0007]

[12] The composition described in [8] contains Lactobacillus blastophylla L-137 or its processed form turmeric extract in a ratio of 1:1 to 1:1000.

[0007]

[13] The composition described in [8] is a nutritional supplement, health food, functional food, food for specific health care, food for patients or feed.

[0007]

[14] A method for enhancing the immune activation of lactic acid bacteria by administering turmeric extract to a subject.

[0007]

[15] The use of a turmeric extract for enhancing the immune activation of lactic acid bacteria.

[0008] According to this disclosure, a lactic acid bacteria containing turmeric extract can be provided, preferably a composition for enhancing the immune activation of Lactobacillus conogenus L-137. Furthermore, more preferably, according to this disclosure, a composition can be provided that synergistically enhances the immune function of lactic acid bacteria, particularly Lactobacillus conogenus L-137. Furthermore, according to this disclosure, a method for manufacturing this composition can also be provided. [Simplified Explanation of the Diagram]

[0009] Figure 1 is a graph showing the evaluation of IL-12 production capacity for each of the lactic acid bacteria (Lactobacillus germinarum L-137 strain) and turmeric extract and their components (L-137 strain dosage: 5, 10 μg).

[0009] Figure 2 is a graph showing the evaluation of IL-12 production capacity for each of the lactic acid bacteria (Lactobacillus germinarum L-137 strain) and turmeric extract and their components (L-137 strain dosage: 25, 50 μg).

[0009] Figure 3 is a graph showing the evaluation of IL-12 production capacity for each of the lactic acid bacteria (Lactobacillus germinarum JCM1149 strain) and turmeric extract and their composition (JCM1149 strain dosage: 50 μg).

[0009] Figure 4 is a graph showing the evaluation of IL-12 production capacity for a combination of lactic acid bacteria (Lactobacillus delbrueckii subsp.lactis ATCC7830 strain) and turmeric extract, as well as the composition of both (ATCC7830 strain dosage: 50 μg).

[0009] Figure 5 is a graph showing the ability to evaluate IL-12 production by including each of the lactic acid bacteria (Enterococcus hirae (ATCC8043 strain)) and turmeric extract and their components (ATCC8043 strain dosage: 50 μg).

Implementation Method

[0010] [Lactic acid bacteria, preferably Lactobacillus cocciformis L-137, an immune-enhancing component]

[0010] The composition of the present invention is characterized by containing turmeric extract. Preferably, the composition of the present invention is characterized by containing turmeric extract as an active ingredient. More preferably, the composition of the present invention further contains lactic acid bacteria, which is not particularly limited, but may be the following lactic acid bacteria or their treatments.

[0010] The lactic acid bacteria used in this invention may be, for example, bacteria of the genera *Lactobacillus*, *Enterococcus*, *Streptococcus*, *Lactococcus*, *Bifidobacterium*, *Pediococcus*, *Leuconostoc*, *Tetragenococcus*, and *Oenococcus*, or other known lactic acid bacteria.

[0010] The bacterial cells of these lactic acid bacteria can be readily obtained, for example, from organizations such as the International Patent Organism Depositary (IPOD), the American Type Culture Collection (ATCC), the Japan Collection of Microorganisms (JCM), and the Institute for Fermentation (Osaka: IFO). Furthermore, commercially available products (including pharmaceuticals and food products containing these lactic acid bacteria) can also be used appropriately.

[0010] 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*, *Lactobacillus delbrueckii subsp. bulgaricus*, *Lactobacillus delbrueckii subsp. lactis*, *Lactobacillus paracasei subsp. paracasei*, and *Lactobacillus lode*. Lactobacillus reuteri, Lactobacillus salivarius, Lactobacillus rhamnosus, or Lactobacillus helveticus, preferably Lactobacillus germinatus and / or Lactobacillus delbrueckii subsp. lactic acid bacteria, more preferably Lactobacillus germinatus strain L-137, Lactobacillus germinatus strain JCM1149, and / or Lactobacillus delbrueckii subsp. lactic acid bacteria ATCC7830, but not limited to these.

[0010] Furthermore, in addition to the above, other lactic acid bacteria belonging to the genus *Lactobacillus* can also be listed as follows: *Lactobacillus daoliensis*, *Lactobacillus daowaiensis*, *Lactobacillus dongliensis*, *Lactobacillus fabifermentans*, *Lactobacillus herbarum*, *Lactobacillus modestisalitolerans*, *Lactobacillus mudanjiangensis*, *Lactobacillus nangangensis*, *Lactobacillus paraplantarum*, *Lactobacillus pentosus*, *Lactobacillus pingfangensis*, *Lactobacillus plajomi*, and *Lactobacillus spp.* Examples include *Lactobacillus argentoratensis*, *Lactobacillus plantarum subsp. plantarum*, *Lactobacillus songbeiensis*, and *Lactobacillus xiangfangensis*, but are not limited to these species.

[0010] Lactic acid bacteria belonging to the genus Enterococcus (in the old classification, there were also cases classified as the genus Streptococcus) or Streptococcus can be listed as such as: Enterococcus hirae, Enterococcus faecalis, Enterococcus faecium, Enterococcus thermophilus, etc., preferably Enterococcus hirae, more preferably Enterococcus hirae ATCC8043 strain, but not limited to these.

[0010] Examples of lactic acid bacteria belonging to the genus *Lactococcus* include: *Lactococcus lactis* subsp. *lactis*, *Lactococcus lactis* subsp. *cremoris*, etc., but are not limited to these. Examples of lactic acid bacteria belonging to the genus *Bifidobacterium* include: *Bifidobacterium bifidum*, *Bifidobacterium thermophilum*, *Bifidobacterium longum*, *Bifidobacterium breve*, *Bifidobacterium infantis*, *Bifidobacterium adolescentis*, etc., but are not limited to these. Examples of lactic acid bacteria belonging to the genus *Pediococcus* include *Pediococcus pentosaceus* and *Pediococcus acidilactici*, but are not limited to these. Examples of lactic acid bacteria belonging to the genus *Leuconostoc* include *Leuconostoc mesenteroides* subsp. *cremoris*, but are not limited to these. Examples of lactic acid bacteria belonging to the genus *Tetragenococcus* include *Tetragenococcus halophilus*, but are not limited to these. Examples of lactic acid bacteria belonging to the genus *Oenococcus* include *Oenococcus oeni*, but are not limited to these.

[0010] The bacteria of these lactic acid bacteria can be easily obtained, for example, from organizations such as iPod, ATCC, JCM, and IFO. Furthermore, commercially available products (including pharmaceuticals and food containing these lactic acid bacteria) can also be used appropriately.

[0010] Among the above-mentioned lactic acid bacteria, the best lactic acid bacteria is Lactobacillus germinatus strain L-137 (IPOD accession number: FERM BP-08607) or its processed form.

[0011] Immune-enhancing effect preferably means (1) an increase in resistance to various diseases, or (2) a recovery from an immunosuppressed state or a decrease in immune function, but is not limited to these. Specifically, (1) for example, the composition of the present invention is preferably effective in the prevention and treatment of infections caused by microorganisms such as viruses or bacteria, and more preferably effective in the prevention and treatment of infectious enteritis caused by oral infections such as cholera bacteria, toxin-producing Escherichia coli, Shigella, Salmonella or viruses; influenza or cold syndrome caused by respiratory infections; stomatitis and periodontal disease caused by oral infections; and various malignant tumors (e.g., non-epithelial malignant tumors occurring in the mucosa of the digestive tract or respiratory tract, liver / kidneys, etc.), but is not limited to these. Furthermore, (2) for example, the components of the present invention are also preferably suitable for recovery from immunosuppression and decreased immune function; recovery from immunosuppression induced by tumors or decreased immune function induced by anticancer drug treatment; prevention of acquired immunodeficiency syndrome (AIDS); prevention and treatment of intracellular parasitic bacteria such as Listeria, Salmonella, Mycobacterium tuberculosis, or Leukobacterium tumefaciens; improvement of Th1-type immune function decline caused by stress; and suppression of age-related immune function decline. Furthermore, while the protective effect against infection by Chlamydia trachomatis, an intracellular parasitic bacterium, suggests a preventive effect against the pathogenesis of arteriosclerosis associated with Chlamydia trachomatis infection, this is another preferred embodiment of the present invention, but is not limited to such applications.

[0011] Furthermore, a preferred embodiment of the present invention may be a component that synergistically enhances the immune function of lactic acid bacteria, preferably Lactobacillus germinarum L-137.

[0011] Further, other or other preferred embodiments of the composition of the present invention may include the present invention enhancing the production capacity of cytokines such as interleukin-12 (IL-12), interferon-β (IFN-β) or interferon-γ (IFN-γ) in the object to which it is applied, but are not limited to such.

[0012] [Turmeric Extract (Concentrate)]

[0012] The turmeric extract in this invention refers to an extract (extract) obtained from plant raw materials of the genus Curcuma in the ginger family through an extraction solvent. The turmeric extract is not limited to solvent extracts obtained through solvent extraction, but also includes solvent extracts that have been further purified by fractionation, such as column chromatography. The turmeric extract used in this invention can also be an extract that has undergone extraction (including fractionation purification), a concentrate from which some solvent has been removed, or a dried product from which solvent has been removed. Solvent removal from the extract can be carried out by evaporating the solvent using methods such as heating and / or reducing pressure. These heating and reducing pressure methods are not particularly limited; for example, conventional methods can be used.

[0013] The plant materials used to manufacture the turmeric extract used in this invention may include: the rhizomes of plants belonging to the genus Curcuma of the ginger family, such as autumn turmeric (Curcuma longa), spring turmeric (Curcuma aromatica), turmeric (Curcuma zedoaria), turmeric (Curcuma phaeocaulis), turmeric (Curcuma kwangsiensis), turmeric wenyujin, and / or Javanese turmeric (Curcuma xanthorrhiza), with turmeric rhizomes being particularly preferred, but not limited to these.

[0013] Rhizomes can be used after being washed from the soil, or can be used directly from the appropriate part of the rhizome, or cut into appropriate sizes or shapes, or can be made into pulverized form. Examples of cutting and pulverizing methods include: knives, kitchen knives, etc.; lasers, grinders, mixers, stamping mills, block colliders, ultra-high-speed cutting pulverizers (Comitrol), mortars and pestles, etc. The particle size of the pulverized material can be adjusted as needed using a sieve, but is not limited to these methods. Furthermore, plant materials can also be appropriately dried.

[0013] The drying method may be, for example, hot air drying, spray drying, freeze drying and other general methods, but is not limited to these.

[0014] The extraction from plant raw materials may use at least one solvent selected from the group consisting of water and hydrophilic organic solvents. These solvents may be any one of water, a hydrophilic organic solvent, or a mixture of water and a hydrophilic organic solvent; the hydrophilic organic solvent may also be a mixture of several hydrophilic organic solvents. Water may be distilled water, purified water, etc., and also includes hot water. Hot water may be preferably at a temperature of 90°C or higher, more preferably at 95°C or higher, and even more preferably at 97°C or higher. The hydrophilic organic solvent may include at least one alcohol or a mixture of several alcohols, preferably ethanol, methanol, or isopropanol; other organic solvents are preferably acetone, acetonitrile, etc., but are not limited to these. Ethanol or a mixture of these is more preferred. When using a mixture of alcohol and water as an extraction solvent, the mixing ratio is preferably in the range of about 10:90 to 90:10 by weight, more preferably in the range of about 20:80 to 50:50, but is not limited to these.

[0015] A mixture of turmeric and water and / or a hydrophilic organic solvent may be used to immerse the turmeric in the extraction solvent, preferably at a temperature of about 5°C to 100°C for at least 3 minutes (e.g., 1 hour to 72 hours, preferably 1 hour to 72 hours). Shaking or stirring may also be applied as needed. The method of shaking or stirring may be adjusted as appropriate.

[0016] The method for extracting turmeric extract from plant raw materials is not particularly limited. In addition to extraction using the solvents described above, turmeric extract can also be produced from plants by steam distillation or supercritical carbon dioxide extraction. In this invention, it is preferred to use turmeric extract obtained by the aforementioned solvents, which contains turmerol A, turmerol B, bisacurone, curcumin, ar-turmerone, α-turmerone, β-turmerone, etc., but the components contained in the turmeric extract of this invention are not limited to these.

[0017] In the composition of the present invention, the turmeric extract preferably comprises about 0.0001 to 95% by weight, more preferably about 0.001 to 97% by weight, and even more preferably 0.01 to 99% by weight, but is not limited to such ranges, relative to the total amount of the composition.

[0017] Furthermore, the intake amount of turmeric extract in the composition of the present invention may be determined based on the age and weight of the user, symptoms, administration time, dosage form, administration method, and combination of drugs. For example, for an adult (approximately 60 kg), the preferred daily intake of turmeric extract is approximately 0.1 to 2000 mg, more preferably approximately 0.5 to 1000 mg, and even more preferably approximately 1 to 500 mg, but is not limited to these ranges. The intake may be once daily or divided into multiple administrations. The aforementioned dosage may be divided into one to multiple administrations per day.

[0018] The turmeric extract used in this invention is preferably derived from plant materials, and more preferably from plants of the genus *Curcuma* in the family Zingiberaceae. Examples of such plants include: *Curcuma longa*, *Curcuma aromatica*, *Curcuma zedoaria*, *Curcuma phaeocaulis*, *Curcuma kwangsiensis*, *Curcuma wenyujin*, *Curcuma xanthorrhiza*, etc., but are not limited to these. Furthermore, multiple uses of these plants may be used. Furthermore, pure lines, variants, and varieties of these plants are also included. Rhizome refers to the stem of a plant that is usually visible below the ground surface and has roots and buds extending from its nodes.

[0019] Furthermore, the fractionated portion of the plant extract, after further purification, can also be formulated into the composition of the present invention. For example, the plant extract is provided in a liquid-liquid partition of ethyl acetate / water, and the active compound can be purified in the ethyl acetate fractionation portion. Furthermore, the plant extract or its fractionated portion can be purified by chromatography. Chromatography can use reverse-phase column chromatography, normal-phase thin-layer chromatography, etc. The plant extract or its fractionated portion can also be further processed by conventional methods such as drying, pulverizing, granulation, and solidification.

[0020] [Lactobacillus plantarum strain L-137]

[0020] Lactobacillus plantarum strain L-137 is stored at the Licensed Biological Depository Center of the National Institute of Advanced Industrial Science and Technology (now the Licensed Biological Depository Center of the Technical Base for Product Evaluation; address: Room 120, 2-5-8 Kazusa-Kamazu, Kisarazu City, Chiba Prefecture, Japan, postal code 292-0818), under deposit number FERM BP-08607 (from transfer tube FERM P-15317 deposited on November 30, 2018). Furthermore, even variants of Lactobacillus plantarum L-137, as long as they possess the characteristics of Lactobacillus plantarum L-137, are also considered part of Lactobacillus plantarum L-137. Additionally, Lactobacillus plantarum strain L-137 may be included in the composition of this invention along with other lactic acid bacteria.

[0021] The composition of the present invention contains lactic acid bacteria, preferably Lactobacillus germinarum L-137 strain or its treatment, and its content relative to the total amount of the composition 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, but is not limited to such range.

[0022] Furthermore, the composition of the present invention includes lactic acid bacteria, preferably Lactobacillus germinarum strain L-137 or a treatment thereof, and the ratio of the lactic acid bacteria, preferably L-137 strain, to turmeric extract is preferably 1:(1 to 10000), more preferably 1:(2.5 to 400), and even more preferably 1:(2.5 to 50), but is not limited to these ranges.

[0023] The lactic acid bacteria of the present invention are preferably Lactobacillus plantarum strain L-137 or its processed form included in the composition. The intake amount can be determined according to the age and weight of the ingestor, symptoms, administration time, dosage form, administration method, combination of drugs, etc. For example, for an adult (about 60 kg), the intake of lactic acid bacteria, preferably Lactobacillus plantarum strain L-137 as dried dead bacteria, is preferably set at about 0.5 to 200 mg per day, more preferably about 1 to 100 mg, and even more preferably about 2 to 50 mg, but is not limited to these ranges. Furthermore, for an adult (approximately 60 kg), the recommended daily intake of lactic acid bacteria, preferably *Lactobacillus plantarum* L-137 (converted to live bacteria), is approximately 5 × 10⁸ to 2 × 10¹¹ cfu (colony forming units), more preferably approximately 1 × 10⁹ to 1 × 10¹¹ cfu, but is not limited to this range. Intake can be once daily or divided into multiple doses. The aforementioned dosage can be divided into one or more daily doses.

[0024] [Cultivation of Lactic Acid Bacteria]

[0024] In this invention, *Lactobacillus germinarum* strain L-137 and other lactic acid bacteria can be cultured on any of the following media: natural culture medium, synthetic culture medium, and semi-synthetic culture medium. In this invention, the cultivation of lactic acid bacteria can be carried out according to conventional methods, methods known to the present invention, or methods based on such methods.

[0025] The aforementioned culture medium is not particularly limited; however, it is preferred to use a medium containing a nitrogen source and / or a carbon source. The aforementioned nitrogen source is not particularly limited and may include, for example, meat extract, protein, gluten, casein, yeast extract, or amino acids. The aforementioned carbon source is not particularly limited and may include, for example, glucose, xylose, fructose, inositol, maltose, starch syrup, yeast extract, starch, bagasse, bran, molasses, or glycerol. One or more of these may be used. In addition to the aforementioned nitrogen source and / or carbon source, inorganic substances may also be added to the aforementioned culture medium. The aforementioned inorganic substances are not particularly limited and may include, for example, ammonium sulfate, potassium phosphate, calcium chloride, salt, iron, manganese, molybdenum, or various vitamins. One or more of these may be used.

[0026] The culture temperature and culture time of *Lactobacillus cocciformis* strain L-137 and other lactic acid bacteria are not particularly limited, as long as efficient culture can be carried out. In one sample of the present invention, the culture temperature is typically about 25 to 40 degrees Celsius (°C), preferably about 27 to 35 degrees Celsius, and the culture time is, for example, about 12 to 48 hours. Furthermore, in one sample of the present invention, the culture of lactic acid bacteria can also be carried out by aeration and shaking. Furthermore, the pH of the culture medium is not particularly limited. In one sample of the present invention, it is typically about pH 3 to 6, preferably about pH 4 to 6.

[0027] [Lactic acid bacteria treatment product]

[0027] The "processed products" of *Lactobacillus cocciformis* strain L-137 and other lactic acid bacteria may preferably include processed L-137 strain and other lactic acid bacteria, their culture medium or culture supernatant, the residues obtained by filtration or centrifugation, and ultrasonic disruption fluid of bacterial cells, but are not limited to these. Furthermore, processing solutions in which cell walls are removed by enzyme or physical treatment, complexes of proteins or peptides obtained by pharmaceutical or salting-out treatment, concentrates, dried products, or dilutions of these are also included in the processed products of the present invention, but are not limited to these.

[0027] Furthermore, L-137 strain and other lactic acid bacteria can be live cells, dried cells, centrifuged cells, broken cells, etc., or dead cells. From the point of view of stability and ease of handling, dead cells are preferred.

[0028] In this invention, the above-mentioned processed products can be used directly, or they can be freeze-dried, low-temperature dried, spray-dried, or L-dried, or a combination thereof, to form a powder for use. Furthermore, these processed products can be used after being diluted with a suitable solvent (e.g., water, alcohol, organic solvent, etc.), or suitable additives can be added to form a gel or solidifying agent for use.

[0029] Hereinafter, a method for preparing dead bacterial bodies of Lactobacillus cocci strain L-137 and other lactic acid bacteria will be specifically described. In this invention, the method for preparing the aforementioned dead bacterial bodies is not particularly limited to any method that does not impair the effects of this invention, and can be performed by, for example, any of the following methods: (I) a method of sterilizing or disinfecting the lactic acid bacteria cells after separation from the culture medium to prepare dead bacterial bodies; (II) a method of sterilizing the lactic acid bacteria cells in the culture medium to prepare dead bacterial bodies, and then separating the dead bacterial bodies from the culture medium, etc. Sterilization can be performed, for example, by filtration, or by conventional methods, such as gas sterilization using ethylene oxide or hydrogen peroxide, or heat sterilization using gamma rays, electron beams, or high-frequency waves.

[0030] The method for separating bacterial cells from the culture medium can employ various methods commonly used in the relevant technical field and is not particularly limited. Specifically, in one embodiment of the present invention, for example, a method for separating the culture medium and bacterial cells can be used to remove the supernatant from the culture medium by means of centrifugation. Furthermore, in this embodiment, after adding distilled water to the culture medium and centrifuging to remove the supernatant, the operation of further adding distilled water to the residue after removing the supernatant and centrifuging can be repeated several times as desired. In one embodiment of the present invention, the separation operation may also include a filtration step. Regarding the aforementioned bacterial cells, dried bacteria can be obtained by supplying them to a spray drying apparatus for drying. Preferred examples of such apparatus include spray drying apparatuses equipped with a microparticle-forming device capable of forming spray droplets of approximately 1 to 10 μm, but this is not a limitation.

[0031] The aforementioned sterilization method is not particularly limited, and examples include: heating, ultraviolet irradiation, formalin treatment, etc. Furthermore, the aforementioned sterilization treatment can be performed on the live bacterial cells or on the culture medium containing the live bacterial cells.

[0032] When performing the aforementioned heat treatment, the heating temperature is not particularly limited. For example, it is usually set to about 60 to 100 degrees Celsius, and preferably about 70 to 90 degrees Celsius. The heating method can be a conventional method and is not particularly limited, for example, a heater or the like. The heating time is not particularly limited as long as the sterilization treatment can be sufficiently completed. For example, after the desired temperature is reached, the heating time is usually about 5 to 40 minutes, and preferably about 10 to 30 minutes.

[0033] The aforementioned dead bacterial cells obtained by the above method may also be further subjected to grinding, crushing, spray drying, low-temperature drying, or freeze-drying, or mixed with other raw materials (e.g., vitamins, amino acids, oligopeptides, etc.) to produce a treated product of dead bacterial cells. In this invention, the treated product of dead bacterial cells can also be suitably used as dead bacterial cells.

[0034] Preferred examples of the composition of the present invention are for food and / or pharmaceutical use (including veterinary medicine). In other preferred examples, the composition of the present invention can be used as an additive for food and beverages. As a composition for food and beverages, food and beverage additives, or pharmaceutical use, the culture supernatant or its processed form of the present invention, or L-137 strain, other lactic acid bacteria or their processed form, can be further suitably formulated with pharmaceutically permissible carriers, additives, etc. Since formulation methods or formulation techniques for such purposes are well established, they can also be used. For example, in the case of pharmaceutical use, specifically, it can be made into oral dosage forms such as tablets, coated tablets, pills, powders, granules, capsules, liquids, suspensions, emulsions, etc., and non-oral dosage forms such as injections, infusions, suppositories, ointments, and wet dressings. The proportions of carriers or additives can be suitably set based on the ranges commonly used in the fields of food and beverages, pharmaceuticals, or veterinary medicine.

[0035] There are no particular restrictions on pharmaceutically permissible carriers or additives. 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, hydroxypropyl methylcellulose, hydroxypropyl cellulose, polyvinylpyrrolidone, polyacrylic acid, polysaccharide gums, natural polymers, etc. Examples of oily carriers include: petrolatum, squalane, paraffin, and other suitable oils or waxes, but are not limited to these.

[0035] Examples of additives include: sweeteners, acidulants, vitamins, minerals, thickeners, emulsifiers, antioxidants, enzymes, pH adjusters, preservatives, bactericides, antifungal agents, shelf-life extenders, bleaching agents, gloss agents, flavorings, seasonings, bittering agents, stabilizers, gelling agents, pastes, excipients, binders, disintegrants, lubricants, colorants, and flavoring agents, but are not limited to these. Since related technologies have been well established previously, this invention can be based on these established technologies.

[0036] Sweeteners may include, for example: monosaccharides or disaccharides such as glucose, fructose, sucrose, lactose, maltose, barragin, trehalose, and xylose; isomerized sugars (glucose-fructose syrup, fructose-glucose syrup, mixed isomerized sugars, etc.), sugar alcohols (erythritol, xylitol, lactitol, Palatinit®, sorbitol, reduced starch syrup, etc.), honey, and high-sweetness sweeteners (sucralose, ethoxylate potassium, sematriol, stevioside, aspartame, etc.), but are not limited to these.

[0037] Acidic ingredients include, for example, citric acid, malic acid, gluconic acid, tartaric acid, lactic acid, phosphoric acid, or salts thereof, and one or more of these may be used, but are not limited to these.

[0038] Vitamins may include, for example, vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin E, nicotinic acid, inositol, etc., but are not limited to these.

[0039] Minerals may include, for example, calcium, magnesium, zinc, iron, etc., but are not limited to these.

[0040] Examples of thickeners include, but are not limited to, carrageenan, gellan gum, xanthan gum, gum arabic, tamarind gum, guar gum, locust bean gum, ebony gum, agar, gelatin, pectin, soybean polysaccharides, carboxymethyl cellulose (CMC), etc.

[0041] Examples of emulsifiers include: glycerol fatty acid esters, sucrose fatty acid esters, sorbitol fatty acid esters, lecithin, phytosterols, saponins, etc.

[0042] Antioxidants may include: vitamin C, tocopherol (vitamin E), enzyme-treated progesterone, catechins, etc., but are not limited to these.

[0043] Furthermore, when the components of the present invention are food products, the food products include health foods, functionally labeled foods, foods for specific health care, and foods for patients. The form of the food products is not particularly limited, and specific examples include tablets, granules, powders, oral preparations, etc., which are so-called nutritional supplements or nutritional aids. Examples of beverages not included in this category include: tea drinks, soft drinks, carbonated drinks, nutritional drinks, fruit drinks, lactic acid drinks, etc.; noodles such as buckwheat noodles, udon noodles, Chinese noodles, instant noodles, etc.; foods such as maltose, candy, chewing gum, chocolate, snacks, biscuits, jelly, jam, cream, baked goods, bread, etc.; processed aquatic / livestock products such as ham, sausage, fish cake, fish cake, etc.; dairy products such as processed milk and fermented milk; oils and fats such as salad oil, frying oil, margarine, mayonnaise, ghee, whipped cream, and sauces, and processed oils and fats; seasonings such as sauces and broths; sterilized packaged foods such as curry, stews, donburi, porridge, and zasui; and cold foods such as ice cream, yogurt, and shaved ice, etc., but are not limited to these categories. Since these categories have been well established previously, this invention can be based on them. Furthermore, the food products of this invention may not contain curry, stews, or products containing these categories.

[0043] Furthermore, without diminishing the effects of the present invention, the composition of the present invention may also contain, for example, any ingredient known in the fields of medicine, pharmacy, veterinary medicine, animal products or food.

[0043] The feed is preferably for mammals such as rats, mice, dogs, cats, rabbits, horses, cattle, pigs, and monkeys; birds such as chickens, ducks, parrots, pigeons, sparrows, and owls; and seafood such as Atlantic salmon, salmon, saury, sardines, herring, tuna, cod, yellowtail, eel, squid, octopus, crab, shrimp, crayfish, and sea cucumber, but is not limited to these.

[0044] [The immune-enhancing effect of lactic acid bacteria and its confirmation method]

[0044] Methods for confirming the effects of the composition of the present invention may include, for example, comparing the composition of the present invention containing turmeric extract with the composition not containing turmeric extract to confirm the superior immune-enhancing effect of lactic acid bacteria, preferably Lactobacillus germination L-137.

[0044] Specifically, methods for confirming the immune-enhancing effect of lactic acid bacteria, preferably Lactobacillus germinarum L-137, include, for example, comparing the production capacity of cytokines such as interleukin-12 (IL-12), interferon-β (IFN-β), and interferon-γ (IFN-γ) of immune cells with other components, but are not limited to such methods. It is known that cytokines such as IL-12, IFN-β, and IFN-γ play a certain role in the immune defense mechanism against microbial infections and tumors caused by bacteria, yeast, fungi, viruses, etc. For example, when a subject takes in or is given the components of the present invention, compared with a control group that does not take in or is given the components of the present invention, if the production capacity of IL-12, IFN-β, and / or IFN-γ is intentionally, significantly, or synergistically higher, it can be determined that the components of the present invention have the desired immune-enhancing effect.

[0044] Immune cells may include, for example, lymphocytes such as B cells or T cells; or macrophages, natural killer (NK) cells, dendritic cells, etc. These cells are preferably obtained from animal (more preferably, mammalian or avian) blood or organs such as the spleen through known methods, or commercially available products may be used. Furthermore, evaluation can be performed using methods well-established in the art, such as those described above. An example of this can be found in the embodiments described later.

[0045] When the composition of the present invention is formulated into products in the form of food and beverage, medicine (including veterinary medicine), quasi-medicine, nutritional supplement, health food, functional food, food for specific health care, food for patients, or feed, the accompanying instructions or packaging of such products may, in view of the function of the composition of the present invention, indicate that it has the essential point of having the immune-enhancing effect of Lactobacillus germinarum L-137.

[0046] [Method for manufacturing the composition]

[0046] The present invention includes a method for manufacturing an immune-enhancing composition of lactic acid bacteria, preferably Lactobacillus germinatus L-137, characterized by a step of mixing turmeric extract, preferably with a carrier and / or excipients.

[0047] The preferred carriers used in the above steps have been well established in the past, in the food or pharmaceutical fields. The present invention can also be based on such carriers, for example, various carriers such as aqueous or oily bases. Examples of aqueous carriers include water, physiological saline, ethanol, glycerin, polyethylene glycol, propylene glycol, methylcellulose, hydroxypropyl methylcellulose, hydroxypropyl cellulose, polyvinylpyrrolidone, polyacrylic acid, polysaccharide gum-based natural polymers, etc. Examples of oily carriers include petrolatum, squalane, paraffin, and other suitable oils or waxes, but are not limited to these.

[0048] Furthermore, the preferred excipients used in the above steps have been well established in the past, food or pharmaceutical fields, and the present invention can also be based on such excipients. For example, lactose, white sugar, mannitol, corn starch, powdered cellulose, dicalcium phosphate, calcium carbonate, dextrin, etc. are preferred, but are not limited to such excipients.

[0049] The composition of the present invention, except for the addition of turmeric extract, can be suitably processed and manufactured using general composition manufacturing methods. Preferably, Lactobacillus plantarum strain L-137 or a treatment thereof is further added. The present invention includes a method for manufacturing the composition, which comprises the step of mixing turmeric extract with other desired ingredients.

[0050] This invention is limited to demonstrating the effects of the invention, and within the technical scope of this invention, it includes various combinations of the above-described structures. Furthermore, variations are possible as long as they fall within the technical scope of this invention.

[0050] [Example]

[0051] The following examples and test cases further illustrate the present invention in detail, but the present invention is not limited to these examples.

[0052] [Experimental Example 1] Confirmation test on the ability of a composition containing turmeric extract to produce IL-12.

[0052] 1. Manufacturing of turmeric extract

[0052] The turmeric extract is prepared by extracting the rhizome of turmeric (Curcuma longa) with hot water (above 90°C), and then drying the resulting extract under reduced pressure to remove moisture.

[0052] 2. Preparation of dried dead Lactobacillus cocci L-137 cells

[0052] Lactobacillus germinatus L-137 (accession number: FERM BP-08607) strain was inoculated into MRS (de Man, Rogosa, Sharpe) modified liquid medium and cultured at 32°C for 18 hours. After culture, the culture medium was removed and the bacterial cells were collected. The obtained bacterial cells were uniformly dispersed in water, and 20% by weight of the bacterial cells and 80% by weight of dextrin were mixed and spray-dried to obtain dried dead bacterial cells.

[0052] 3. Methods for confirming IL-12 production capability

[0052] Dried dead cells of *Lactobacillus germinatus* L-137, a lactic acid bacterium, were suspended at a concentration of 0 to 200 μg / mL in RPMI 1640 medium containing 10% FBS to prepare a lactic acid bacteria detection solution. Turmeric extract was suspended at a concentration of 0 to 1600 μg / mL in RPMI 1640 medium containing 10% FBS to prepare a turmeric extract detection solution.

[0052] J774.1 cells (cell number JCRB9108; JCRB Cell Bank, National Research and Development Corporation, Pharmaceutical Foundation, Health and Nutrition Institute) used as a macrophage cell line were suspended at a concentration of 1.0 × 10⁶ cells / mL in RPMI 1640 medium containing 10% FBS to prepare a cell line suspension. 100 μL of the cell line suspension was added to each well of a 96-well culture dish and incubated at 37°C for 1 to 3 hours in a 5% CO₂ incubator to allow the cells to adhere to the bottom surface. 50 μL of turmeric extract detection solution was added to each well of the 96-well culture dish and incubated at 37°C for 1 hour in a 5% CO₂ incubator. Furthermore, 50 μL of lactic acid bacteria detection solution was added to each well of the 96-well culture dish, and the dish was incubated at 37°C for 24 hours in a 5% CO2 incubator (final concentration of lactic acid bacteria: 0 to 50 μg / mL, final concentration of turmeric extract: 0 to 400 μg / mL, final concentration of cells: 5.0 × 10⁵ / mL). The concentration of IL-12p40 in the supernatant after incubation was determined by ELISA after the dish was frozen at -20°C and then thawed.

[0053] The results of Test Example 1 above clearly show that the composition of the present invention containing turmeric extract exhibits an immune-enhancing effect on lactic acid bacteria (L-137 strain) and a synergistic enhancement of the immune function of lactic acid bacteria L-137. Since such effects are almost impossible to observe with turmeric extract alone, this result is a surprising effect.

[0054] [Experimental Example 2] Confirmation Test on the Ability of Composition Containing Curcuma Extract to Produce IL-12

[0054] Except for replacing Lactobacillus plantarum L-137, the experiment was conducted in the same manner as in Experimental Example 1, using live or dead Lactobacillus plantarum strain JCM1149, Lactobacillus delbrueckii subsp. Lactis ATCC 7830, or Enterococcus hirae ATCC 8043 as lactic acid bacteria, and setting the dosage of each lactic acid bacteria to 50 μg.

[0054] The results are shown in Figures 3 to 5. These results clearly demonstrate that the turmeric extract-containing composition of the present invention, except for strain L-137, exhibits immune-enhancing effects on various lactic acid bacteria and a synergistic enhancement of the immune function of various lactic acid bacteria.

[0054] [Industry-level availability]

[0055] As described above, the composition of the present invention containing turmeric extract has an immune-enhancing effect on lactic acid bacteria, preferably Lactobacillus germinarum L-137. Therefore, the composition of the present invention can be used as food, medicine, quasi-medicine, nutritional supplement, health food, functionally labeled food, food for specific health purposes, food for patients, or feed, etc.

Claims

1. A composition for enhancing the immune activation of lactic acid bacteria, comprising turmeric extract.

2. The immune-enhancing composition as described in claim 1, wherein, The lactic acid bacteria group consists of one or more species selected from the group composed of Lactobacillus, Lactococcus, Bifidobacterium, and Enterococcus.

3. A composition for enhancing the immune activation of Lactobacillus cocciformis L-137, comprising turmeric extract.

4. An immune-enhancing composition as described in any one of claims 1 to 3, wherein, The turmeric extract is selected from one or more extracts of the group consisting of water, organic solvents and mixtures thereof.

5. An immune-enhancing composition as described in any one of claims 1 to 3, wherein, This turmeric extract is a hot water extract at temperatures above 90°C.

6. An immune-enhancing composition as described in any one of claims 1 to 3, wherein, The turmeric extract is a rhizome extract of turmeric, and the turmeric is selected from one or more species selected from the group consisting of autumn turmeric (Curcuma longa), spring turmeric (Curcuma aromatic), turmeric (Curcuma zedoaria), turmeric (Curcuma phaeocaulis), turmeric (Curcuma kwangsiensis), turmeric (Curcuma wenyujin), turmeric (Curcuma xanthorrhiza), and mixtures thereof.

7. An immune-enhancing composition as described in any one of claims 1 to 3, wherein, The content of turmeric extract relative to the total composition ranges from 0.0001% to 95%.

8. The immune-enhancing composition as described in any one of claims 1 to 3 further contains lactic acid bacteria or a treatment thereof.

9. The immune-enhancing composition as described in any one of claims 1 to 3 further contains Lactobacillus cocciformis L-137 or a treatment thereof.

10. The immune-enhancing composition as described in claim 8, wherein, The proportion of lactic acid bacteria or their processed products relative to the total composition is 0.0001% to 20%.

11. The immune-enhancing composition as described in claim 9, wherein, The content of Lactobacillus germinarum L-137 or its treatment thereof is 0.0001% to 20% relative to the total composition.

12. The immune-enhancing composition as described in claim 8, wherein, The ratio of lactic acid bacteria or its processed products to turmeric extract is 1:1 to 1:1000.

13. The immune-enhancing composition as described in claim 8 is a nutritional supplement, health food, functionally labeled food, food for specific health purposes, food for patients, or feed.

14. A method for enhancing the immune-activating effect of lactic acid bacteria by administering turmeric extract to a subject.

15. An application of turmeric extract for enhancing the immune-activating effect of lactic acid bacteria.

Images