JP6916042B2 - Composition for promoting antigen-specific interferon gamma production - Google Patents

Description

The present invention relates to an antigen-specific interferon gamma production promoting composition.

In the past, "fucoidan oligo" has been used as a compound that can be used in foods and drinks, health foods, functional foods, pharmaceuticals, and cosmetics (hereinafter referred to as "cell-mediated immunity activation composition") for the purpose of activating cell-mediated immunity. It has been proposed to use "sugar" (see, for example, Japanese Patent Application Laid-Open No. 2007-039341).

Japanese Unexamined Patent Publication No. 2007-039341

However, in order to obtain fucoidan oligosaccharides, it is necessary to extract fucoidan from algae and then hydrolyze the fucoidan using an acid or an enzyme.

As described above, in order to obtain fucoidan oligosaccharide, it is necessary to go through at least two steps of extraction and hydrolysis, and there is a high possibility that the running cost and labor cost at the time of production will increase. Therefore, it is fully assumed that the production cost of the cell-mediated immunity activation composition using fucoidan oligosaccharide will be high.

An object of the present invention is to provide a composition for promoting antigen-specific interferon gamma production, which can be expected to suppress production costs.

The composition for promoting antigen-specific interferon gamma production according to the first aspect of the present invention is an extracellular polysaccharide produced by the Lactobacillus delbrueckii subsp. Bulgaricus OLL1073R-1 strain. The body is the active ingredient. That is, a cell-mediated immune activation composition of exopolysaccharide produced by Lactobacillus delbrueckii subsp. Bulgaricus OLL1073R-1 strain (accession number: FERM BP-10741). Used as or as a component thereof . Also, here referred to "composition" (including processed foods and drinks.) Pharmaceutical, formulations of such supplements and food additives, food and drink (except for animals and plants themselves.) As well as food and beverage compositions, such as Includes foods that can be ingested by animals (including humans).

As a result of diligent studies by the inventors of the present application, the extracellular polysaccharide according to the first aspect of the present invention can promote the production of interferon gamma in an antigen-specific manner and suppress pathogen infection and tumor development. Was clarified .

Then, this exopolysaccharide can be produced, for example, by adding a specific lactic acid bacterium capable of producing an exopolysaccharide into milk and fermenting the milk. That is, this exopolysaccharide can be basically produced in one step. Therefore, it can be expected that the production cost of the antigen-specific interferon gamma production promoting composition utilizing exopolysaccharide can be suppressed.

Addition of antigens and exopolysaccharides of Examples / Comparative Examples (Example 1, Example 2, Comparative Example 1, Comparative Example 2, Comparative Example 3, and Comparative Example 4) using mice immunized with a melanoma antigen. It is a graph which summarized the condition and the result obtained from the condition. Obtained from the conditions for adding the antigen / exopolysaccharide to the medium and the conditions of Examples / Comparative Examples (Example 3, Comparative Example 5, Comparative Example 6, and Comparative Example 7) using mice immunized with influenza virus antigen. It is a graph which summarizes the result. The conditions for adding the antigen / extracellular polysaccharide of Examples / Comparative Examples (Comparative Example 8, Comparative Example 9, Comparative Example 10 / Comparative Example 11) using non-immune mice and the results obtained from the conditions are summarized. It is a graph diagram. Conditions for adding the antigen / exopolysaccharide medium of Examples / Comparative Examples (Example 4, Comparative Example 12, Comparative Example 13, and Comparative Example 14) using mice immunized with human hepatitis B virus antigen, and the conditions thereof. It is a graph which summarizes the result obtained from.

Hereinafter, the present invention will be described in detail by showing embodiments of the present invention, but the present invention is not limited to the individual embodiments described below.

The cell-mediated immune activation composition according to the embodiment of the present invention contains exopolysaccharide as an active ingredient. Here, exopolysaccharides can be produced when a specific lactic acid bacterium having the ability to produce exopolysaccharide (hereinafter referred to as “exopolysaccharide-producing lactic acid bacterium”) ferments milk. That is, exopolysaccharides can be contained in cultures, metabolites, etc. when exopolysaccharide-producing lactic acid bacteria are cultured in a medium containing milk. Milk is obtained from mammals. Here, the type of mammal is not particularly limited, but for example, primate animals such as humans, monkeys, gorillas, hamadryas baboons, and chimpanzees, and domestic animals such as horses, cows, dogs, sheep, goats, pigs, camels, and deer. Examples include pet animals such as dogs and cats. The milk is preferably raw milk, but the processed products such as sterilized milk, skim milk, full-fat milk powder, partial non-fat milk, non-fat powder milk, full-fat concentrated milk, skim concentrated milk, cream, butter, butter milk. , Whey, whey protein concentrate (WPC), whey protein isolate (WPI) and the like.

Examples of exopolysaccharide-producing lactic acid bacteria include Lactobacillus delbrueckii subsp. Bulgaricus. Among these lactic acid bacteria, Lactobacillus delbrucky subspecies bulgaricus OLL1073R-1 strain (accession number: FERM BP-10741) is particularly preferable. Here, Lactobacillus del Brooky Subspecies Bulgarics OLL1073R-1 strain was dated November 29, 2006 (consignment date), and the Patent Organism Depositary Center of the National Institute of Advanced Industrial Science and Technology (Higashi 1, Tsukuba City, Ibaraki Prefecture) -1-1 Tsukuba Center Central 6) is a lactic acid bacterium that has been internationally deposited under the Butapest Treaty under the accession number FERM BP-10741. The patented microorganism deposit business of the Patent Organism Depositary Center of the National Institute of Advanced Industrial Science and Technology was taken over by the National Institute of Technology and Evaluation as of April 1, 2012, and the patent of the National Institute of Technology and Evaluation. As of April 1, 2013, the Biological Depositary Center has moved to the National Institute of Technology and Evaluation Office, 2-5-8 Kazusakamatari, Kisarazu City, Chiba Prefecture, Japan.

The cell-mediated immunity activation composition according to the embodiment of the present invention exerts its function when ingested by an animal, particularly a human. The term "human" as used herein may be a person of a wide range of ages, from infants to the elderly, regardless of age or sex. That is, this cell-mediated immunity activation composition can be used, for example, for cell-mediated immunization activation in children and cell-mediated immunity activation in adults and the elderly. Further, the term "ingestion" as used herein is not limited to an ingestion route as long as it enters the human body, and can be realized by all known ingestion methods such as oral ingestion, tube ingestion, and intestinal ingestion. At this time, typical examples include oral ingestion via the digestive tract and enteral ingestion, but oral ingestion is preferable, and oral ingestion by eating and drinking is more preferable.

The cell-mediated immunity activation composition according to the embodiment of the present invention may contain exopolysaccharide, and the content thereof is not particularly limited, but the cell-mediated immunity activation composition is in liquid form. In the case of a body, the exopolysaccharide is preferably contained in an amount of 30 μg / mL or more, more preferably 60 μg / mL or more, further preferably 90 μg / mL or more, and 120 μg / mL or more. It is more preferably contained, and particularly preferably 150 μg / mL or more. It is expected that the higher the content of exopolysaccharide, the higher the effect, but the upper limit is, for example, 0.5 mg / mL.

The mass per unit package of the cell-mediated immunity activating composition according to the embodiment of the present invention is not particularly limited, but 50 g or more from the viewpoint that the effect can be sufficiently obtained and it is easy to ingest at one time. It is preferably in the range of 500 g or less, more preferably in the range of 60 g or more and 200 g or less, further preferably in the range of 80 g or more and 150 g or less, and more preferably in the range of 100 g or more and 120 g or less. Most preferred. Further, the above-mentioned unit wrapping may be not only the unit wrapping per bag, box, or container, but also the unit wrapping per time included in them, or the unit wrapping per day. In addition, it may be packaged in a plurality of days, for example, a quantity suitable for ingestion for one week, or may include a plurality of individual packages.

In the embodiment of the present invention, it is desirable that the cell-mediated immune activation composition is continuously ingested for 1 week or longer, preferably 2 weeks or longer, more preferably 4 weeks or longer. Since the cell-mediated immunity activation composition according to the embodiment of the present invention can be safely ingested, the ingestion period is not particularly limited and can be continued forever. In addition, the cell-mediated immunity activation composition may be continuously ingested only for a part of the period, or may be ingested intermittently for an arbitrary period.

The cell-mediated immunity activation composition according to the embodiment of the present invention can be used as a pharmaceutical product or a food or drink. The drug or food or drink is useful in that it has a cell-mediated immune activation effect. When the cell-mediated immune activation composition according to the embodiment of the present invention is used as a pharmaceutical product or a food or drink, even if an exopolysaccharide obtained from a single exopolysaccharide-producing lactic acid bacterium is used. Well, or a combination of exopolysaccharides obtained from two or more types of exopolysaccharide-producing lactic acid bacteria may be used.

When the cell-mediated immune activation composition according to the embodiment of the present invention is used as a pharmaceutical substance or a food or drink, the state of the cell-mediated immune activation composition is not limited, and a paste product, a spray-dried product, a freeze-dried product, etc. Vacuum-dried products, drum-dried products, liquid products dispersed in a medium, diluted products diluted with a diluent, and crushed products obtained by crushing a dried product with a mill or the like can be used.

Furthermore, the cell-mediated immunity activation composition according to the embodiment of the present invention can also be a food for health function or a food for the sick (cell-mediated immunity activation food, etc.). The health functional food system was established not only for ordinary foods but also for foods in the shape of tablets, capsules, etc., based on internal and external trends and consistency with the conventional food system for specified health use. .. The system stipulates two types of foods for specified health use (individual permission type) and nutritionally functional foods (standard standard type). By administering the cell-mediated immunity activation composition according to the embodiment of the present invention to an animal such as a human as a special-purpose food such as a food for specified health use or a nutritionally functional food, for example, activation of cell-mediated immunity. Is possible.

It is preferable that the cell-mediated immunity activation composition according to the embodiment of the present invention is labeled with a description of its use, efficacy, function, type of active ingredient, type of functional ingredient, ingestion method and the like. The "label" referred to here should be suitable for each of pharmaceuticals, non-pharmaceutical products, foods with health claims, foods for specified health uses, general foods, health supplements, health foods and supplements. Further, the "display" referred to here includes all the displays for informing the consumer of the above explanation. This display may be any display that can recall or infer the above-mentioned display contents, and may include all displays regardless of the purpose of the display, the contents of the display, the object / medium to be displayed, and the like. For example, displaying the above explanation on the packaging / container of the product, displaying or distributing the above explanation on the advertisement / price list or transaction documents related to the product, or electromagnetically displaying the information containing these (Internet, etc.) ) Providing by method.

When the product obtained by packaging the cell-mediated immunity activation composition according to the embodiment of the present invention is, for example, a food or drink, the food or drink may be labeled with, for example, "cell-mediated immunity activation" or "cells". It is preferable that the label "activates sexual immunity" is attached.

The wording used to perform the above display is not limited to the above example, and may be a wording synonymous with such a meaning. As such words, for example, various words such as "cell-mediated immunity is activated", "helps to suppress the infection of pathogens", or "helps to suppress the development of tumors" are allowed for consumers. obtain.

When the cell-mediated immune activation composition according to the embodiment of the present invention is used as a food or drink, the type of the food or drink is not particularly limited. Foods and drinks include, for example, milk, processed milk, soft drinks, fermented milk, yogurt, cheese, other dairy products, bread, biscuits, crackers, pizza crust, prepared milk powder, liquid food, food for the sick, milk powder for infants, etc. It may be a food, a food such as milk powder for pregnant women / lactating women, a nutritional food, or the like. In the production of such foods and drinks, exopolysaccharide which is an active ingredient of the cell-mediated immunity activation composition according to the embodiment of the present invention can be used as it is or mixed with other foods and drinks or food components. It is possible to use the manufacturing method in a normal food composition, such as shaving. Further, the shape of the food and drink is not particularly limited, and the shape of the food and drink that is usually used may be used. For example, it may be in any form such as solid form (including powder and granular form), paste form, liquid form, and suspension form, and is not limited thereto. At this time, milk drinks, fermented milk, soft drinks, jelly drinks, tablets, powdered foods are more preferable, and yogurt is even more preferable.

The extracellular polysaccharides, which are the active ingredients of the cell-mediated immune activation composition according to the embodiment of the present invention, include water, proteins, sugars, lipids, vitamins, minerals, organic acids, organic bases, and fruit juices. , Flavors, functional ingredients, food additives, and other ingredients contained in ordinary foods can be added without problems. In the production of the above foods and drinks, as a protein source, for example, animal and vegetable proteins such as soybean protein, milk protein, chicken egg protein, meat protein, and hydrolysates thereof are used as proteins or protein-containing raw materials usually used in food production. can do. Examples of sources of sugar include modified starch (in addition to textrin, soluble starch, British starch, oxidized starch, starch ester, starch ether, etc.), dietary fiber and the like. Examples of the lipid source include animal fats and oils such as lard, fish oil, and other separated oils, hydrogenated oils, and ester exchange oils; palm oil, safflower oil, corn oil, rapeseed oil, palm oil, and these separated oils. , Vegetable oils and fats such as hydrogenated oils and ester exchange oils. Examples of vitamins include vitamin A, carotene, vitamin B group, vitamin C, vitamin D group, vitamin E, vitamin K group, vitamin P, vitamin Q, niacin, nicotinic acid, pantothenic acid, biotin, inositol, and choline. , Folic acid and the like, and examples of minerals include calcium, potassium, magnesium, sodium, copper, iron, manganese, zinc, selenium and the like. Examples of the organic acid include malic acid, citric acid, lactic acid, tartaric acid and the like. Examples of the functional component include oligosaccharides, glucosamine, collagen, ceramide, royal jelly, polyphenols and the like. Examples of food additives include emulsifiers, stabilizers, thickeners, gelling agents, sweeteners, acidulants, preservatives, antioxidants, pH adjusters, colorants, flavors and the like. Various milk-derived components such as butter, milky minerals, cream, whey, non-protein nitrogen, sialic acid, phospholipids, and lactose are components that can be suitably used for producing foods and drinks according to the embodiment of the present invention. Is an example of. In addition, any component having a cell-mediated immunity activating effect, such as fucoidan oligosaccharide, may be added to the cell-mediated immunity activation composition according to the embodiment of the present invention.

These components can be used in combination of two or more. Further, as the raw material, any of natural products, processed natural products, synthetic products and / or foods containing a large amount thereof may be used.

By the way, the inventors of the present application have confirmed that the above-mentioned cell-mediated immune activation composition significantly enhances the activation of cytotoxic T cells. Therefore, the cell-mediated immune activation composition according to the embodiment of the present invention can be said to be a cytotoxic T cell activation composition. Further, since it is known that activation of cytotoxic T cells suppresses pathogen infection and tumor development, the cell-mediated immunity activation composition according to the embodiment of the present invention suppresses pathogen infection. It can also be said to be a composition or a tumor development inhibitory composition. That is, when the product obtained by packaging the cell-mediated immune activation composition according to the embodiment of the present invention is, for example, a food or drink, the food or drink is referred to as, for example, "cytotoxic T cell activation". Indications, indications such as "inhibition of pathogen infection", "inhibition of tumor development", and other indications similar to these indications may be added.

In addition, the inventors of the present application have confirmed that the production of antigen-specific interferon gamma is strongly promoted as a specific action and effect of the above-mentioned cell-mediated immunity activation composition. At this time, the antigen is not particularly limited, and examples thereof include a melanoma antigen, an influenza virus antigen, and a human hepatitis B virus antigen. Therefore, the cell-mediated immunity activation composition according to the embodiment of the present invention can be said to be an antigen-specific interferon gamma production promoting composition.

Extracellular polysaccharides, which are the active ingredients of the cell-mediated immunity activation composition according to the embodiment of the present invention, can also be used as pathogen infection protection drugs or tumor shrinkage drugs. When producing such pharmaceuticals, exopolysaccharides can be used as crushed or uncrushed processed products. Further, the exopolysaccharide used at this time may be an exopolysaccharide obtained from a single exopolysaccharide-producing lactic acid bacterium, or two or more types of exopolysaccharide-producing lactic acid bacteria. It may be a combination of extracellular polysaccharides obtained from.

The content of exopolysaccharide in the above-mentioned drug can be arbitrarily determined according to its purpose and use. An example of the content is that it occupies 150 μg / mL, but it is not limited to this in the embodiment of the present invention.

The dose of the drug containing the above-mentioned extracellular polysaccharide as an active ingredient can be appropriately set in consideration of various factors such as the administration route, the age, body weight, and symptoms of the administration target animal including humans. As an example of an appropriate dose, 0.1 g to 1000 g / kg / day can be mentioned as an active ingredient, but the embodiment of the present invention is not limited to this. For example, when ingested for a long period of time for the purpose of infection protection or tumor shrinkage, the amount may be smaller than the above range. In addition, since this active ingredient has no safety problems, it may be used in a larger amount than the above range.

The dosage form of the above-mentioned drug is preferably a dosage form that can be orally administered in order to allow extracellular polysaccharides to reach the intestine. Examples of preferred dosage forms of pharmaceuticals according to the embodiments of the present invention include tablets, pills, coated tablets, capsules, granules, powders, liquids, suspending agents, emulsions, syrups, troches and the like. be able to. These various preparations are added to the extracellular polysaccharide, which is the main drug, according to the conventional method, as an excipient, a binder, a disintegrant, a lubricant, a stabilizer, a colorant, a flavoring agent, a solubilizing agent, and a suspending agent. It can be formulated by mixing an auxiliary agent that can be usually used in the field of pharmaceutical formulation technology, such as a surfactant and a coating agent.

When exopolysaccharide is used as a drug, for example, in the case of oral administration, the exopolysaccharide can be ingested as it is, but for example, tablets and granules are prepared according to the general manufacturing method of a drug. , Powders, capsules, powders.

The content of exopolysaccharide in the above-mentioned drug can be appropriately set depending on the dosage form, usage, age, sex, degree of brain function, other conditions and the like.

The above drugs are preferably administered orally. Administration of this drug protects against pathogen infections and shrinks tumors. Therefore, this drug is useful for improving or preventing various symptoms caused by cell mutation. Further, as long as the cell-mediated immunity activating effect according to the embodiment of the present invention is not impaired, the present drug containing exopolysaccharide as an active ingredient may be used in combination with another drug.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to the following Examples.

(Preparation example)
Extracellular polysaccharides in a 10 mass% defatted milk powder culture medium of Lactobacillus delbrueckii subsp. Bulgaricus OLL1073R-1 were purified. Specifically, exopolysaccharide was purified as shown below.

After sterilizing 10% by mass defatted milk powder medium at 90 ° C. and cooling to 45 ° C., Lactobacillus del Brooky Subspecies Bulgaricus OLL1073R-1 (FERM BP-10741) as a lactic acid bacterium starter was added to the medium. 1% by mass was inoculated. After culturing at 37 ° C. for 18 hours, the medium was cooled to about 4 ° C. to stop culturing the lactic acid bacterium starter. Then, 100% by mass of trichloroacetic acid is added to and mixed with the obtained culture so that the final concentration becomes 10% by mass, and the mixture is centrifuged at 4 ° C. and 12,000 × g for 20 minutes. did. The supernatant obtained by centrifugation was taken, twice the amount of cold ethanol was added to the supernatant, and the ethanol-added supernatant was allowed to stand overnight at 4 ° C. Then, the ethanol-added supernatant was centrifuged at 12,000 × g at 4 ° C. for 20 minutes, and the obtained precipitate was dissolved in ultrapure water. This aqueous solution is dialyzed against ultrapure water, and then DNase (EC 31.21.1, manufactured by Sigma-Aldrich) and RNase (type I-AS, EC 3.1.27) are added to the dialysis solution. .5, Sigma-Aldrich) and Proteinase K (EC 3.4.21.64, Sigma-Aldrich) were added and the dialysis solution was enzymatically treated at 37 ° C. After stopping the enzyme treatment by heating the enzyme-treated solution at 90 ° C. for 10 minutes, twice the amount of ethanol was added to the enzyme-treated solution, and the ethanol-added enzyme-treated solution was allowed to stand overnight at 4 ° C. .. Then, the ethanol-added enzyme-treated solution was centrifuged at 12,000 × g at 4 ° C. for 20 minutes, and the obtained precipitate was dissolved in ultrapure water. This aqueous solution was dialyzed against ultrapure water, and the aqueous solution after dialysis was freeze-dried to obtain a purified product of exopolysaccharide. For dialysis, a dialysis membrane (molecular weight cutoff 6,000-8,000) manufactured by Spectrum Laboratories was used.

(Example 1)
100μL of Complete Freund's Adjuvant (Wako Pure Chemical Industries, Ltd.), 10 [mu] L of melanoma antigen solution ^{(H-2D b human gp100 Peptide} KVPRNQDWL, MBL Co., antigen concentration: 10 mg / mL), phosphate buffer 80 [mu] L, Then, 10 μL of human hepatitis B virus antigen (IA ^d HBc helper peptide TPPAYRPPNAPIL, manufactured by MBL, antigen concentration: 10 mg / mL) was weighed and mixed, and then this mixed solution was sufficiently emulsified to 200 μL. Antigen emulsion was prepared. The antigen emulsified solution contains 100 μg of melanoma antigen. Then, after preparing four 200 μL of this antigen emulsion, each antigen emulsion was injected into the abdominal cavity of each of four 8-week-old female C57BL / 6J mice (manufactured by Charles River Japan). That is, 100 μg of melanoma antigen was injected into each mouse. After injection of the melanoma antigen into each mouse, CD8 + T cells specific for the melanoma antigen proliferate in each mouse and eventually become functionally mature cytotoxic T cells that circulate in the mouse body.

One week after the injection of the melanoma antigen, the above-mentioned mice were euthanized. Then, the spleen was taken out from each mouse and the spleen cells of each mouse were pooled. Then, the splenocytes were cultured at 5 × 10 ⁶ cells / mL for 5 wells. The above-mentioned melanoma antigen solution and exopolysaccharide (above) were added to the medium so that the melanoma antigen was 10 μg / mL and the exopolysaccharide was 150 μg / mL during the splenocyte culture. (Prepared in Preparation Example) was added. After 72 hours from the start of the culture, the supernatant concentration of the antigen-specifically produced interferon γ was measured using BD OptEIA Mouse IFN-γ ELISA Set (Becton, manufactured by Dickinson and Company), and the measured value was measured. When the average and the standard error were calculated, the average was 891 pg / mL, and the standard error was 204 pg / mL.

(Example 2)
During spleen cell culture, an antigenic emulsion was prepared in the same manner as in Example 1 except that the melanoma antigen solution was added to the medium so that the melanoma antigen was 0.1 μg / mL, and the antigen was injected into the abdominal cavity of the mouse. Spleen cells were cultured for 5 wells in the same manner as in Example 1, and the supernatant concentration of interferon γ was measured in the same manner as in Example 1 to obtain the average and standard error of the measured values. As a result, the average was 256 pg / mL, and the standard error was 85 pg / mL.

(Example 3)
At the time of preparing the antigen emulsion and adding the antigen to the medium, the melanoma antigen solution (H-2D ^b human gp100 Peptide KVPRNQDWL, manufactured by MBL, antigen concentration: 10 mg / mL) was ^{added to the influenza virus antigen solution (H-2D b} Influenza NP peptide). An antigen emulsified solution was prepared in the same manner as in Example 1 and injected into the abdominal cavity of a mouse, and splenocytes were cultured for 5 wells in the same manner as in Example 1 except that they were replaced with ASNENMDTM (manufactured by MBL). In the same manner as above, the supernatant concentration of interferon γ was measured, and the average and standard error of the measured values were obtained. As a result, the average was 13240 pg / mL, and the standard error was 3818 pg / mL.

(Example 4)
50 μL of Complete Fluid's Adjuvant (Wako Pure Chemical Industries, Ltd.), 5 μL of human hepatitis B virus antigen solution (I-Ad HBc helper peptide TPPAYRPPNAPIL, MBL, antigen concentration: 10 mg / mL), 45 μL of phosphoric acid After weighing and mixing the buffer solution, the mixture was sufficiently emulsified to prepare 100 μL of the antigen emulsified solution. The antigen emulsified solution contains 50 μg of human hepatitis B virus antigen. Then, after preparing three 100 μL of this antigen emulsified solution, each antigen emulsified solution was injected subcutaneously into the ridge of each of three 8-week-old female C57BL / 6J mice (manufactured by Charles River Japan). That is, 50 μg of human hepatitis B virus antigen was injected into each mouse. After injecting human hepatitis B virus antigen into each mouse, CD4 + T cells specific for human hepatitis B virus antigen proliferate in each mouse, and eventually functionally mature and strongly induce cell-mediated immunity. Becomes Th1 cells and circulates in the mouse body.

One week after the injection of the human hepatitis B virus antigen, the above-mentioned mice were euthanized. Then, the spleen was taken out from each mouse and the spleen cells of each mouse were pooled. Then, the splenocytes were cultured at 2 × 10 ⁶ cells / mL for 8 wells. When the splenocytes were cultured, the above-mentioned human hepatitis B virus antigen solution and the above-mentioned human hepatitis B virus antigen solution were placed in a medium so that the human hepatitis B virus antigen was 10 μg / mL and the exopolysaccharide was 150 μg / mL. Extracellular polysaccharides were added. After 48 hours from the start of the culture, the supernatant concentration of the antigen-specifically produced interferon γ was measured using BD OptEIA Mouse IFN-γ ELISA Set (Becton, manufactured by Dickinson and Company), and the measured value was measured. When the average and the standard error were calculated, the average was 19709 pg / mL, and the standard error was 712 pg / mL.

(Comparative Example 1)
An antigenic emulsion was prepared in the same manner as in Example 1 and injected subcutaneously into the ridge of the mouse, except that extracellular polysaccharides were not added to the medium during spleen cell culture. Spleen cells were cultured in 5 wells, and the supernatant concentration of interferon γ was measured in the same manner as in Example 1 to obtain the average and standard error of the measured values. As a result, the average was 168 pg / mL, and the standard error was 48 pg / mL.

(Comparative Example 2)
An antigenic emulsion was prepared in the same manner as in Example 1 and injected subcutaneously into the ridge of the mouse, except that extracellular polysaccharides were not added to the medium during culturing of splenocytes, and the same procedure as in Example 2 was performed. Spleen cells were cultured in 5 wells, and the supernatant concentration of interferon γ was measured in the same manner as in Example 1 to obtain the average and standard error of the measured values. As a result, the average was 74 pg / mL, and the standard error was 13 pg / mL.

(Comparative Example 3)
When the antigen was added to the medium, the melanoma antigen solution ^{was replaced with the influenza virus antigen solution (H-2D b} Influenza NP peptide ASNENMDDTM, manufactured by MBL), except that extracellular polysaccharides were not added to the medium during splenocyte culture. Prepares an antigenic emulsion in the same manner as in Example 1, injects it subcutaneously into the ridge of a mouse, cultures splenocytes in 5 wells in the same manner as in Example 1, and on interferon γ in the same manner as in Example 1. The clear concentration was measured and the average and standard error of the measured values were calculated. As a result, the average was 37 pg / mL, and the standard error was 12 pg / mL.

(Comparative Example 4)
An antigen emulsion was prepared in the same manner as in Example 1 except that the melanoma antigen solution was replaced with an influenza virus antigen solution (H-2D ^{b Influenza NP peptide ASNENMDTM, manufactured by MBL) when the antigen was added to the medium.} The spleen cells were cultured in 5 wells in the same manner as in Example 1 and the supernatant concentration of interferon γ was measured in the same manner as in Example 1 to obtain the average and standard error of the measured values. .. As a result, the average was 137 pg / mL, and the standard error was 58 pg / mL.

(Comparative Example 5)
Except that the melanoma antigen solution was replaced with the influenza virus antigen solution (H-2D ^b Influenza NP peptide ASNENMDTM, manufactured by MBL) at the time of preparing the antigen emulsion, and extracellular polysaccharides were not added to the medium during splenocyte culture. , An antigen emulsified solution was prepared in the same manner as in Example 1, injected subcutaneously into the ridge of a mouse, splenocytes were cultured in 5 wells in the same manner as in Example 1, and the supernatant of interferon γ was prepared in the same manner as in Example 1. The concentration was measured and the average and standard error of the measured values were calculated. As a result, the average was 169 pg / mL, and the standard error was 23 pg / mL.

(Comparative Example 6)
An antigen emulsion was prepared in the same manner as in Example 1 except that the melanoma antigen solution was replaced with an influenza virus antigen solution (H-2D ^b Influenza NP peptide ASNENMDTM, manufactured by MBL) at the time of preparing the antigen emulsion. The ridge was injected subcutaneously, splenocytes were cultured for 5 wells in the same manner as in Example 1, and the supernatant concentration of interferon γ was measured in the same manner as in Example 1 to obtain the average and standard error of the measured values. As a result, the average was 569 pg / mL, and the standard error was 146 pg / mL.

(Comparative Example 7)
An antigenic emulsion was prepared in the same manner as in Example 3 except that extracellular polysaccharides were not added to the medium during spleen cell culture, and injected subcutaneously into the ridge of the mouse, in the same manner as in Example 3. Spleen cells were cultured in 5 wells, and the supernatant concentration of interferon γ was measured in the same manner as in Example 1 to obtain the average and standard error of the measured values. As a result, the average was 2538 pg / mL, and the standard error was 327 pg / mL.

(Comparative Example 8)
50 μL of Complete Friend's Adjuvant (Wako Pure Chemical Industries, Ltd.) and 50 μL of phosphate buffer were weighed and mixed, and then the mixed solution was sufficiently emulsified to prepare 100 μL of an antigen-free solution. Then, after preparing three 100 μL of this antigen-free solution, each antigen-free solution was injected subcutaneously into the ridge of each of three 8-week-old female C57BL / 6J mice (manufactured by Charles River Japan).

One week after the injection of the antigen-free solution, the above-mentioned mice were euthanized. Then, the spleen was taken out from each mouse and the spleen cells of each mouse were pooled. Then, the splenocytes were cultured at 2 × 10 ⁶ cells / mL for 8 wells. At the time of culturing the splenocytes, no antigen or exopolysaccharide was added to the medium. After 48 hours from the start of culturing, the supernatant concentration of interferon gamma was measured using BD OptEIA Mouse IFN-γ ELISA Set (manufactured by Becton, Dickinson and Company) to determine the average and standard error of the measured values. The average was 77 pg / mL and the standard error was 3 pg / mL.

(Comparative Example 9)
Same as in Comparative Example 8 except that exopolysaccharide (the same as the one prepared in Example 1) was added to the medium so that the amount of exopolysaccharide (the same as that prepared in Example 1) was 150 μg / mL during spleen cell culture. An antigen-free solution was prepared and injected subcutaneously into the ridge of the mouse, spleen cells were cultured for 8 wells in the same manner as in Comparative Example 8, and the supernatant concentration of interferon γ was measured in the same manner as in Comparative Example 8. The average of the measured values and the standard error were calculated. As a result, the average was 84 pg / mL, and the standard error was 6 pg / mL.

(Comparative Example 10)
When culturing splenocytes, add a human hepatitis B virus antigen solution (I-Ad HBc helper pepper TPPAYRPPNAPIL, MBL, antigen concentration: 10 mg / mL) to the medium so that the human hepatitis B virus antigen becomes 10 μg / mL. An antigen-free solution was prepared in the same manner as in Comparative Example 8 and injected subcutaneously into the ridge of the mouse. The supernatant concentration of γ was measured, and the average and standard error of the measured values were obtained. As a result, the average was 79 pg / mL, and the standard error was 4 pg / mL.

(Comparative Example 11)
When culturing splenocytes, the human hepatitis B virus antigen should be 10 μg / mL, and the extracellular polysaccharide (the same as the one prepared in Example 1) should be 150 μg / mL. Antigen-free solution in the same manner as in Comparative Example 8 except that a human hepatitis B virus antigen solution (I-Ad HBc helper peptide TPPAYRPPNAPIL, manufactured by MBL, antigen concentration: 10 mg / mL) and an extracellular polysaccharide were added. Was prepared and injected subcutaneously into the ridge of the mouse, splenocytes were cultured for 8 wells in the same manner as in Comparative Example 8, the supernatant concentration of interferon γ was measured in the same manner as in Comparative Example 8, and the average of the measured values was measured. The standard error was calculated. As a result, the average was 76 pg / mL, and the standard error was 3 pg / mL.

(Comparative Example 12)
An antigen emulsified solution was prepared in the same manner as in Example 4 and injected subcutaneously into the ridge of the mouse, except that the human hepatitis B virus antigen solution and the extracellular polysaccharide were not added to the medium during spleen cell culture. , 8 well culture of splenocytes was carried out in the same manner as in Example 4, and the supernatant concentration of interferon γ was measured in the same manner as in Example 1 to obtain the average and standard error of the measured values. As a result, the average was 74 pg / mL, and the standard error was 2 pg / mL.

(Comparative Example 13)
An antigenic emulsion was prepared in the same manner as in Example 4 except that the human hepatitis B virus antigen solution was not added to the medium during spleen cell culture, and injected subcutaneously into the ridge of the mouse, as in Example 4. The splenocytes were cultured in 8 wells, and the supernatant concentration of interferon γ was measured in the same manner as in Example 1 to obtain the average and standard error of the measured values. As a result, the average was 161 pg / mL, and the standard error was 55 pg / mL.

(Comparative Example 14)
An antigenic emulsion was prepared in the same manner as in Example 4 and injected subcutaneously into the ridge of the mouse, except that extracellular polysaccharides were not added to the medium during spleen cell culture. The splenocytes were cultured in 8 wells, and the supernatant concentration of interferon γ was measured in the same manner as in Example 1 to obtain the average and standard error of the measured values. As a result, the average was 10056 pg / mL, and the standard error was 998 pg / mL.

(summary)
The conditions and results described in the above Examples and Comparative Examples are summarized in Table 1 below. In Table 1, "gp100" indicates "melanoma antigen", "NP" indicates "influenza virus antigen", "HBc" indicates "human hepatitis B virus antigen", and "EPS" indicates "bacteria". "In vitro polysaccharide" is indicated, "IFL-γ" indicates "interferon γ", and "-" indicates "not added".

Further, the graphs of Table 1 are shown in FIGS. 1 to 4. In FIG. 1, the white bar on the left side shows the result of Comparative Example 1, the black bar on the right side shows the result of Example 1, and the white bar on the center shows the result of Comparative Example 2, and the white bar on the right side thereof shows the result. The black bar on the right side shows the result of Example 2, the white bar on the right side shows the result of Comparative Example 3, and the black bar on the right side shows the result of Comparative Example 4. Further, in FIG. 2, the white bar on the left side shows the result of Comparative Example 5, the black bar on the right side shows the result of Comparative Example 6, and the white bar on the right side shows the result of Comparative Example 7, and the white bar on the right side shows the result of Comparative Example 7. The black bar shows the result of Example 3. Further, in FIG. 3, the white bar on the left side shows the result of Comparative Example 8, the black bar on the right side shows the result of Comparative Example 9, and the white bar on the right side shows the result of Comparative Example 10, and the white bar on the right side shows the result of Comparative Example 10. The black bar shows the result of Comparative Example 11. Further, in FIG. 4, the white bar on the left side shows the result of Comparative Example 12, the black bar on the right side shows the result of Comparative Example 13, and the white bar on the right side shows the result of Comparative Example 14, and the white bar on the right side shows the result of Comparative Example 14. The black bar shows the result of Example 4.

(Discussion)
From the results shown in the graph of FIG. 1, the extracellular polysaccharide obtained from Lactobacillus delbrueckii subsp. Bulgaricus OLL1073R-1 was found in the splenocytes of melanoma antigen-immunized mice. It was clarified that it has an effect of promoting the production of interferon γ regardless of the type of antigen added to the medium during culturing. When the antigen added to the medium during splenocyte culture of melanoma antigen-immunized mice is melanoma antigen, which is the same antigen as during mouse immunization, the extracellular polysaccharide is an antigen-specific interferon γ produced by CD8 + T cells. It has been clarified that the effect of promoting the production of sardine can be further enhanced, and that the higher the concentration of the added antigen, the higher the effect.

From the results shown in the graph of FIG. 2, the extracellular polysaccharide obtained from Lactobacillus delbrueckii subsp. Bulgaricus OLL1073R-1 was found in the spleen of influenza virus antigen-immunized mice. It was clarified that it has an effect of promoting the production of interferon γ regardless of the type of antigen added to the medium during cell culture. In addition, when the antigen added to the medium during splenocyte culture of influenza virus antigen-immunized mice is the influenza virus antigen, which is the same antigen as during mouse immunization, the extracellular polysaccharide is antigen-specific produced by CD8 + T cells. It was clarified that the effect of promoting the production of interferon γ can be further enhanced.

From the results shown in the graph of FIG. 3, extracellular polysaccharides obtained from Lactobacillus delbrueckii subsp. Bulgaricus OLL1073R-1 were added to the splenocytes of non-immune mice. On the other hand, it was clarified that the effect of promoting the production of interferon γ was not exhibited.

From the results shown in the graph of FIG. 4, the extracellular polysaccharide obtained from Lactobacillus delbrueckii subsp. Bulgaricus OLL1073R-1 is immunized with human hepatitis B virus antigen. When the antigen added to the medium during mouse splenocyte culture is the human hepatitis B virus antigen, which is the same antigen as during mouse immunization, it has the effect of promoting the production of antigen-specific interferon γ produced by CD4 + T cells. It became clear that there was.

From the above results, the extracellular polysaccharide obtained from Lactobacillus delbrueckii subsp. Bulgaricus OLL1073R-1 has the effect of promoting the production of interferon gamma, and is particularly immune. Since the effect was even more remarkable for the same antigen as at the time, it can be said that the extracellular polysaccharide has an effect of strongly promoting the production of antigen-specific interferon gamma produced by T cells.

The cell-mediated immunity activation composition according to the present invention can be expected to be able to keep the production cost low, and can contribute to the promotion of human health. Specifically, foods such as exopolysaccharides and yogurt containing a large amount of exopolysaccharides are expected to activate cell-mediated immunity and contribute to infection protection and tumor suppression. In addition, ingestion of foods containing extracellular polysaccharides can be expected to activate cell-mediated immunity via T cells, prevent viral infections such as influenza, and suppress tumors such as melanoma.

FERM BP-10741

Claims (1)

Lactobacillus delbrueckii subsp. Bulgaricus OLL1073R-1 strain (accession number: FERM BP-10741) is an antigen-specific interferon gamma containing an extracellular polysaccharide as an active ingredient. Composition for promoting production.

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