JP2018050488A - Immunostimulatory composition - Google Patents

Abstract

PROBLEM TO BE SOLVED: To synergistically enhance immunostimulating effects in β-glucan and lactic acid bacteria.SOLUTION: An immunostimulatory composition contains β-glucan and Lactobacillus crispatus KT-11 strain (FERM BP-11332) cells as active ingredients.SELECTED DRAWING: None

Description

  The present invention relates to an immunostimulatory composition having an immunostimulatory effect on humans, domestic animals or pet animals, its use, a method for imparting an immunostimulatory effect to food or feed, and a method for producing a food or feed having an immunostimulatory effect. .

  Immunity is a mechanism that integrates many mechanisms that protect the body from diseases by recognizing and eliminating foreign cells that are non-self substances such as pathogens and abnormal cells such as cancer cells in the body. is there. That is, it shows an action mechanism that eliminates pathogenic bacteria and viruses that have entered the body, or an action mechanism that removes biological waste products such as cancer cells that appear in the body, mutated genes, denatured proteins, and lipids. This immune function is an indispensable function for living organisms, particularly higher organisms, to maintain health and survive in the environment. However, various environmental factors such as aging, changes in dietary habits, environmental pollution, etc. cause a decrease in immune function and immunodeficiency, leading to the development of malignant tumors and infectious diseases. Therefore, it is necessary to maintain normal immune function on a daily basis in maintaining a healthy state.

  β-glucan is a polysaccharide in which glucose is linked, and is widely distributed in nature such as plants, fungi, and bacteria. Β1,4-glucan in which glucose is linked by a β1-4 type bond is widely known as the name of cellulose regardless of its origin. Β glucan is a term used as a general term for β1,3-glucan, β1,4-glucan, and β1,6-glucan. Regarding β-glucan, in recent years, its excellent bioregulatory functions and bioactive functions, such as lipid metabolism improving action, intestinal regulating action, blood sugar level rise inhibiting action, cholesterol lowering action, blood sugar level lowering action, antitumor action, immunostimulation Effects and lifestyle-related disease improvement effects have been confirmed. In particular, β-glucan has an excellent immunostimulatory effect, and plants and bacteria containing a large amount of β-glucan, or extracts thereof, are used as immunostimulants and health supplements. The contained food and drink are widely used as health foods.

  As materials having a high β-glucan content, fruit bodies of basidiomycetes, their hyphae, barley, Aureobasidium pullulans and the like are known. For example, fruit bodies of basidiomycetes, their hyphae, or barley may be used as a raw material for food as they are, but in order to concentrate β-glucan, these are crushed, extracted with hot water, and dried to be a food material And has been put to practical use as a cosmetic material (see Patent Documents 1 and 2). Also known is a product obtained by extracting and drying β-glucan from a culture solution obtained by fermenting Aureobasidium pullulans (see Patent Document 3).

  However, in any case, β-glucan must be consumed in a large amount in order to exert an immunostimulatory effect, and the effect is not fully satisfactory.

  On the other hand, as immunostimulants using lactic acid bacteria, for example, lactic acid bacteria belonging to the genus Lactobacillus, particularly those using Lactobacillus plantarum, are known. Specifically, the lactic acid bacterium is cultured, and the bacterium is immediately killed when the pH of the culture solution does not substantially decrease. A lactic acid bacterium-containing immunostimulating composition having IL-12 production-inducing activity is produced by adding dead cells thus obtained to food and drink (see Patent Document 4). However, in order for lactic acid bacteria to exert an immunostimulatory effect, they must be ingested in large quantities, and the effect is not fully satisfactory.

  In addition, it has also been proposed to use β-glucan and lactic acid bacteria together for the purpose of synergistically enhancing the immunostimulatory action. For example, an immunostimulant containing a culture containing β-glucan obtained by culturing Aureobasidium pullulans and lactic acid bacteria (see Patent Document 5), and a material containing β-glucan A composition for enhancing immunity containing lactic acid bacteria (see Patent Document 6) has been proposed. However, in any case, β-glucan and lactic acid bacteria do not exhibit a synergistic effect, and the effect is not fully satisfactory.

Japanese Patent Publication No. 2008-228676 Japanese Patent Publication No. 2005-307150 Japanese Patent Publication No. 2004-049013 Japanese Patent Publication No. 2010-95465 Japanese Patent Publication No. 2005-220065 Japanese Patent Publication 2001-323001

  As described above, although β-glucan and lactic acid bacteria were each known to have an immunostimulatory effect, there was a problem that the effect was not fully satisfactory.

  Therefore, in view of the above-described circumstances, the present invention can synergistically enhance the immunostimulatory effect in β-glucan and lactic acid bacteria, and exhibits an immunostimulatory composition that exhibits a sufficient effect even when ingested in a small amount. Food, feed and pharmaceuticals containing composition, method for imparting immunostimulatory effect to food or feed using said immunostimulatory composition, and food or feed having immunostimulatory effect using said immunostimulatory composition An object is to provide a manufacturing method.

  As a result of intensive studies by the present inventors in order to achieve the above-mentioned object, it has been found that when β-glucan and Lactobacillus crisptus KT-11 strain are combined, the immunostimulatory effect is synergistically enhanced. The invention has been completed.

The present invention includes the following.
(1) An immunostimulatory composition comprising as an active ingredient β-glucan and cells of Lactobacillus crispatus KT-11 strain (FERM BP-11332).
(2) The immunostimulatory composition according to (1), wherein the β-glucan is a β-glucan derived from Saccharomyces cerevisiae.
(3) The immunostimulatory composition according to (1), which has an immunostimulatory effect on humans, domestic animals or pet animals.
(4) A food containing the immunostimulatory composition according to any one of (1) to (3) above.
(5) A feed comprising the immunostimulatory composition according to any one of (1) to (3) above.
(6) A pharmaceutical comprising the immunostimulatory composition according to any one of (1) to (3) above.
(7) A method of imparting an immunostimulatory effect to food or feed, comprising adding the immunostimulatory composition according to any of (1) to (3) to the food or feed.
(8) A method for producing a food or feed having an immunostimulatory effect, comprising adding the immunostimulatory composition according to any one of (1) to (3) to the food or feed.

The immunostimulatory composition according to the present invention exhibits an excellent immunostimulatory effect in which the immunostimulatory effect by β-glucan and Lactobacillus crispatas KT-11 strain is synergistically enhanced. Therefore, according to the immunostimulatory composition according to the present invention, an immunostimulatory effect can be obtained in a small amount, and can be easily developed in foods, feeds, and the like.
As described above, foods, feeds and pharmaceuticals containing the immunostimulatory composition according to the present invention exhibit an excellent immunostimulatory effect of the immunostimulatory composition, and thus are very effective in improving immune function.

Hereinafter, the immunostimulatory composition according to the present invention will be described in detail.
The immunostimulatory composition according to the present invention is a composition containing β-glucan and Lactobacillus crisptus KT-11 strain as active ingredients. That is, the composition exhibits an immunostimulatory effect, in other words, an effect of enhancing a lowered immune response. That is, when the composition for immunostimulation is taken orally into a living body, for example, it stimulates immune cells such as dendritic cells and macrophages in the intestine and secretes cytokines such as interleukin 12. Interleukin 12 leads to an anticancer effect and an infectious disease prevention and improvement effect by activating natural killer cells and the like. Thus, the mechanism of the immunostimulatory action is an action in which the induction of the production of interleukin 12 (IL-12) is most deeply involved. Therefore, the immunostimulatory effect can be determined based on the production amount of IL-12.

  The immunostimulatory composition according to the present invention exhibits a particularly remarkable immunostimulatory effect because the immunostimulatory effect by β-glucan and the immunostimulatory effect by Lactobacillus crispatas KT-11 strain act synergistically. That is, the immunostimulatory effect by the immunostimulatory composition according to the present invention has an immunostimulatory effect that cannot be predicted from the immunostimulatory effect of β-glucan alone and the immunostimulatory effect of Lactobacillus crispatas KT-11 strain alone. Can do.

  In the immunostimulatory composition according to the present invention, the content ratio of β-glucan and Lactobacillus crisptus KT-11 strain is not particularly limited, but the preferable content ratio is as follows. β-glucan 0.1-99.9% by mass and Lactobacillus kryspatus KT-11 strain 99.9-0.1% by mass, with the total content of β-glucan and Lactobacillus kryspatus KT-11 strain being 100% by mass Particularly preferred are 50-99% by mass of β-glucan and 50-1% by mass of Lactobacillus crisptus KT-11 strain.

  Here, β-glucan is also called βD-glucan and is known as a polysaccharide constituting a cell wall. Β-glucan is composed of a main chain in which glucose is linked by β1,3 bonds and / or β1,4 bonds and a side chain in which glucose is bound by β1,6 bonds. Although it does not specifically limit as beta-glucan which comprises an immunostimulation composition, For example, it is preferable to use what can be used for a foodstuff. Specifically, as the β-glucan, those derived from the fruiting bodies of basidiomycetes and their mycelia, yeast, straw, barley, aureobasidium pullulans and the like can be used.

  Although it does not specifically limit as beta-glucan which comprises an immunostimulation composition, It is preferable to use the thing of the following characteristics. For example, the β-glucan used in the present invention may be a polysaccharide having any structure as long as glucose is bound in a β-type structure among the polysaccharides composed of glucose, such as β1,3-bonded glucose and β1,4 bond. Β-glucan composed of any one or more of glucose and β1,6-linked glucose, or any one or more of β1,3-glucan, β1,4-glucan and β1,6-glucan It may be a mixture of

  Among them, it is preferable to use β-glucan derived from Saccharomyces cerevisiae as β-glucan. As the β-glucan derived from Saccharomyces cerevisiae, for example, the culture solution of Saccharomyces cerevisiae, bacterial cells or cell wall fractions thereof may be used as they are, or high-concentration β-glucan obtained by hot water extraction of these is used. You may do it. Furthermore, after obtaining a hot water extract of the Saccharomyces cerevisiae culture solution, fungus body or cell wall fraction thereof, it was treated with alkali to wash the insoluble fraction with water, then neutralized with acid, then washed with water. A higher concentration of β-glucan obtained by a method of drying and alcohol treatment and then drying may be used.

  Saccharomyces cerevisiae-derived β-glucan has the following characteristics, for example, and is distinguished from other organism-derived β-glucans in this respect. That is, β-glucan derived from Saccharomyces cerevisiae is mainly composed of β1,3 and β1,6-linked D-glucan having a complex type glucan structure, and many of them are contained in the cell wall. In particular, β-glucan, which forms the skeleton of the cell wall of Saccharomyces cerevisiae, has a structure in which a number of relatively long β1,3-glucan side chains branched at position 3 appear in the main chain of β1,6-glucan. It has a feature.

  Furthermore, since the cell surface layer of Saccharomyces cerevisiae is covered with other polysaccharides such as mannan, it is preferable that other polysaccharides such as mannan are sufficiently removed as β-glucan derived from Saccharomyces cerevisiae. . By removing other polysaccharides such as mannan, even when the Saccharomyces cerevisiae culture solution, fungus body, or cell wall fraction thereof is used as β-glucan, the effect of β-glucan can be sufficiently exerted.

  Further, the β-glucan derived from Saccharomyces cerevisiae is not particularly limited. For example, the molecular weight range is preferably 1,000 to 1,000,000, and more preferably 5,000 to 700,000. 20,000 to 500,000 is most preferable. By setting the molecular weight of β-glucan within this range, the immunostimulatory effect can be further enhanced.

  On the other hand, the Lactobacillus crispatas KT-11 strain constituting the immunostimulatory composition according to the present invention is a lactic acid bacterium having a higher antiallergic effect than the conventional lactic acid bacterium disclosed in Japanese Patent No. 4921499. Lactobacillus crispatas KT-11 strain was deposited at the National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center under the accession number: FERM BP-11332, and the National Center for Biotechnology Center Biotechnology Center Patent Biological Depository (incorporated administrative agency) NITE-IPOD) is stored for sale.

The bacteriological (morphological, cultural, physiological) properties of the strain are as follows as described in Japanese Patent No. 4921499.
[1] Morphological properties
In observation after culturing at 37 ° C. for 72 hours on MRS agar medium (Difco), the cells are gonococci with a size of 0.5 to 1 × 3 to 5 μm and do not move. There is no sporulation and Gram staining is positive.
[2] Culture characteristics
The colonies after culturing at 37 ° C. for 72 hours on MRS agar medium (Difco) have a diameter of 2 to 3 mm, a circular shape and a full edge. The color of the colony is yellowish white and translucent.
[3] Physiological properties Gas production: Negative glucose utilization: Positive catalase activity: Negative gelatin liquefaction: Negative nitrate reduction: Negative indole productivity: Negative hydrogen sulfide productivity: Attitude toward negative oxygen: Facultative anaerobic optimal Growth temperature: 37-40 ° C
Optimal growth pH: pH 5.5-5.8

  In the immunostimulatory composition according to the present invention, the Lactobacillus crispatas KT-11 strain may be blended as it is in the culture, may be blended by concentrating the culture, or as a cell recovered from the culture You may mix | blend, You may mix | blend, after drying the collect | recovered microbial cell. Moreover, in the immunostimulation composition according to the present invention, an animal or plant material may be lactic acid fermented using Lactobacillus crispatas KT-11 strain, and the obtained fermentation product may be blended. Moreover, in the immunostimulatory composition according to the present invention, the Lactobacillus crispatas KT-11 strain may be in a viable state, or may be a dead cell sterilized by heat treatment or the like.

  Furthermore, the immunostimulatory composition of the present invention, in addition to the β-glucan and Lactobacillus crispatas KT-11 strain, various carriers, excipients, and other additives that are acceptable as food, if necessary, Other ingredients may be blended to prepare a formulation.

  The dosage form of the immunostimulating composition of the present invention may be an oral preparation such as a tablet, powder, granule or capsule, and can be formulated by a conventional method. In addition, as other components, other components having immunostimulatory action, pharmaceuticals, various vitamins, herbal medicines, minerals, and the like can be appropriately blended.

  The intake of the immunostimulatory composition of the present invention is preferably contained in the food so that the daily intake per individual is 1 to 10,000 mg, particularly 10 to 2,000 mg. The immunostimulatory composition according to the present invention can be used as a food additive or a feed additive.

<Food>
The present invention further provides a food containing the immunostimulatory composition, preferably a functional food containing the immunostimulatory composition. In other words, by adding the above-described immunostimulatory composition to food, an immunostimulatory effect can be imparted to the food, and a food having an immunostimulatory effect can be produced.

  Foods containing the immunostimulating composition include breads, noodles, tablets, confectionery such as jelly and candy, supplements, health supplements, soft drinks, juices, drinks such as energy drinks, ice, ice confectionery, smoothies, etc. However, it is not limited to these. In particular, the food containing the immunostimulatory composition may be, for example, a tablet or powder supplement. The supplement has an advantage that the immunostimulating composition according to the present invention is easily taken quantitatively. Moreover, as a foodstuff containing an immunostimulatory composition, the drink which added the said immunostimulatory composition to the juice (green juice) which squeezed green leaf vegetables, such as a kale, can be mentioned.

  In addition, in the food containing the immunostimulatory composition, the timing of adding the immunostimulatory composition to the food is not particularly limited, and may be added during the manufacturing process of the food. It may be added. Alternatively, as the food containing the immunostimulatory composition, the part containing the Lactobacillus crisptus KT-11 strain and the part containing β-glucan in the immunostimulatory composition are individually packaged and added at the time of eating. You may do it. For example, a lactic acid bacterium food using Lactobacillus crispatas KT-11 strain and a seasoning for use in the lactic acid bacterium food separately packaged with a seasoning containing β-glucan are also used in the immunity according to the present invention. Included in food containing the utilization composition.

  Since the food containing the composition for immunostimulation according to the present invention has an immunostimulatory effect, it can display uses for immunostimulation, immune function improvement, immune recovery, or suppression of immune function decline. Here, the term “display” means to include all actions for informing these uses. Specifically, a food containing the immunostimulatory composition according to the present invention can be provided in a package describing the above-mentioned use, and can be provided together with a printed material describing the above-mentioned use.

  In addition, the display is preferably a display permitted by the government or the like (for example, a display which is approved based on various systems determined by the government and is performed in a mode based on such approval). For example, health food, functional food, enteral nutrition food, special purpose food, functional nutrition food, quasi-drugs, etc. can be exemplified, and other indications approved by the administrative agency, Specific health foods, and labeling approved under a similar system can be exemplified. Examples of the latter include a display as a food for specified health use, a display as a food for specified specific health use, a display that affects the structure and function of the body, a display for reducing disease risk, and the like. In more detail, the indication as food for specified health (especially the indication of the use of health) stipulated in the Enforcement Regulation of the Health Promotion Act (Ministry of Health, Labor and Welfare Ordinance No. 86 of April 30, 2003) Similar displays can be exemplified.

<Feed>
The present invention further provides a feed comprising the immunostimulatory composition. In other words, an immunostimulatory effect can be imparted to the feed by adding the above-described immunostimulatory composition to the feed, and a feed having an immunostimulatory effect can be produced.

  Here, the feed means food provided to animals other than humans. Animals to be fed are vertebrates, specifically mammals other than humans, such as primates (monkeys, chimpanzees, etc.), livestock animals (cow, horses, pigs, sheep, poultry, etc.), Pet animals (also called “love cancer animals”) (dogs, cats, etc.), laboratory animals (mouse, rats, etc.), competition animals (horses, etc.), reptiles, birds (chicken, etc.), etc. Mammals, birds and pet animals are preferred, and livestock animals and pet animals are more preferred.

  In addition to the above immunostimulatory composition, the feed is corn flour, rice flour, cereal flour, potatoes, moss, fish meal, bone meal, fats and oils, skimmed milk powder, whey, mineral feed, yeasts, It may contain inorganic substances, amino acids, proteins, vitamins, lipids and the like. Specifically, ingredients as described in the Japanese standard feed ingredient table (2009 edition, edited by National Agriculture and Food Research Organization) may be included.

  Furthermore, the feed according to the present invention may be used as a supplement for animals, particularly for pet animals, for example, in the form of tablets or powders. Alternatively, the feed according to the present invention may be a silage in which a feed crop such as a green-cutting crop or raw grass is packed in a silo and lactic acid fermented with Lactobacillus crisptus KT-11.

<Pharmaceuticals>
The present invention further provides a pharmaceutical comprising the immunostimulatory composition. Here, the drug means a drug given for diagnosing, treating or preventing human or animal diseases. The usage form of the pharmaceutical product according to the present invention is not particularly limited, and may be any dosage form such as an internal medicine, an external medicine, an injection. In addition, the drug according to the present invention is meant to include both ethical drugs and over-the-counter drugs.

  Specifically, the pharmaceutical dosage form according to the present invention includes, for example, tablets, pills, powders, solutions, suspensions, emulsions, granules, capsules, syrups, suppositories, injections, ointments, Examples thereof include patches, eye drops, and nasal drops. In formulating, additives such as excipients, binders, disintegrants, lubricants, stabilizers, flavoring agents, diluents, surfactants, or injectable solvents that are commonly used as pharmaceutical carriers are used. Can be used.

  In the pharmaceutical product according to the present invention, the content of the immunostimulatory composition (or β-glucan and Lactobacillus crispatas KT-11 strain) is the dosage form, usage, patient age, sex, disease type, disease level, And other conditions.

  In addition, the drug according to the present invention may be used in combination with other drugs such as an IL-12 production promoter and an immunostimulator. The administration time of the pharmaceutical agent according to the present invention is not particularly limited, and the administration time can be appropriately selected according to the treatment method for the target disease. It may be administered prophylactically or used for maintenance therapy. The dosage form is preferably determined according to the dosage form, the patient's age, sex, other conditions, the degree of symptoms of the patient, and the like. In any case, the pharmaceutical of the present invention can be administered once or a plurality of times a day, or can be administered once every several days or weeks.

  The pharmaceutical agent according to the present invention contains β-glucan and Lactobacillus crispatas KT-11 strain as active ingredients, and can significantly promote IL-12 production in vivo. Increased IL-12 can be used as an immunostimulator because it activates in vivo immunity. Therefore, the pharmaceutical agent according to the present invention can be used for prevention or treatment of diseases that can be prevented or treated by stimulating immunity. “Treatment” also includes improvement.

  Specifically, as an application of the pharmaceutical according to the present invention, for example, allergic symptoms such as food allergy, bronchial asthma, urticaria, rhinitis, hay fever, anaphylactic shock, etc., as well as increased resistance to infectious diseases, Examples include prevention of cancer and prevention of progression. In addition, the pharmaceutical agent according to the present invention can be widely used as an antitumor cancer agent, a prophylactic or therapeutic agent for opportunistic infections, an allergic disease preventive agent, or a therapeutic agent.

  EXAMPLES The present invention will be specifically described below with reference to examples. However, the technical scope of the present invention is not limited to these examples, and various embodiments are possible. The present invention is disclosed in this specification. It should be understood that all embodiments are included so long as they are in accordance with the idea.

(Evaluation of immunostimulatory action of β-glucan and lactic acid bacteria)
The immunostimulatory action when Lactobacillus crispatas KT-11 strain or Lactobacillus plantarum JCM1149 ^T was added to each β-glucan was evaluated as follows.

(Β-glucan used)
β-glucan is derived from Saccharomyces cerevisiae, Agaricus subrufensens as Agaricus, Sparassis crispa as Hanabiratake and Hordeum bulgare as barley.
Specifically, Saccharomyces cerevisiae-derived β-glucan and barley-derived β-glucan were purchased from Sigma-Aldrich (MO, USA). Agaricus-derived β-glucan and Hanabiratake-derived β-glucan were prepared as follows. First, the dried fruit body was cut into small pieces, and water was added first, followed by boiling at 100 ° C. for 1 hour, followed by hot water extraction. Thereafter, the hot water extract is immersed in 1M sodium hydroxide and allowed to stand overnight, and then the supernatant obtained by centrifugation is further dialyzed overnight. The concentrated solution is lyophilized and tested as β-glucan. Provided.

(Preparation of cell sample solution)
Lactobacillus chryspatus KT-11 strain or Lactobacillus plantarum JCM1149 ^T was inoculated in an MRS liquid medium and cultured at 37 ° C. for 20 hours. After culturing, the cells were collected by centrifugation and washed 3 times with purified water. The washed cells are freeze-dried, suspended in sterilized 0.15 M sodium chloride-0.01 M phosphate buffer (PBS, pH 7.2) and heat-treated (110 ° C., 15 minutes). It was.

(Culture of macrophage cells)
J774.1 cells, which are macrophage cells, were suspended in RPMI-1640 medium containing 5% FBS, 100 U / mL penicillin G sodium and 100 μg / mL streptomycin sulfate, and then 37 ° C., 5% CO ₂ in a sterile plastic petri dish. What was cultured to the state of confluence in the presence was used. A 48-well flat-bottomed microplate containing 800 μL of J774.1 cell suspension prepared to 1 × 10 ⁵ cells / mL was added to a Lactobacillus crisptus KT-11 strain or lactate prepared to a final concentration of 0 to 100 μg / mL. A cell sample solution of Bacillus plantarum JCM1149 ^T and 100 μL of a β-glucan solution suspended in PBS and adjusted to a final concentration of 0 to 100 μg / mL were added to each well, and at 37 ° C. in the presence of 5% CO ₂ . Cultured for 48 hours. After culture, the mixture was centrifuged at 7,000 rpm for 1 minute at 4 ° C., and the culture supernatant was collected.

(Measurement of IL-12)
The amount of IL-12 in the culture supernatant was measured using enzyme immunoassay (ELISA). That is, 100 μL of 0.1 M carbonate buffer (pH 9.6) containing anti-mouse IL-12 antibody and 1% bovine serum albumin (BSA) was dispensed into each well of a 96-well microplate and allowed to stand overnight at 4 ° C. I put it. After washing with PBS containing 0.05% Tween 20 (PBS-T), 200 μL of PBS containing 1% BSA was added and allowed to stand at 25 ° C. for 120 minutes. After washing again with PBS-T, 100 μL of the culture supernatant diluted to the optimal magnification with PBS containing 1% BSA was dispensed into each well and reacted at 25 ° C. for 120 minutes. Further, after washing with PBS-T, 100 μL of a biotin-labeled anti-mouse IL-12 antibody solution diluted to an optimal magnification with PBS containing 1% BSA was dispensed into each well and reacted at 25 ° C. for 60 minutes. Next, after washing with PBS-T, 100 μL of a horseradish peroxidase (HRP) -labeled streptavidin solution diluted to an optimal magnification with PBS containing 1% BSA was dispensed to each well and reacted at 25 ° C. for 30 minutes. . Dispense 100 μL of TMB solution to each well, react completely at 25 ° C. for 30 minutes with light shielding, stop 100 μL of 4N sulfuric acid into each well, and immediately stop the reaction. Bio-Rad model 550 microplate Absorbance at 450 nm was measured using a reader. The amount of IL-12 was calculated from a calibration curve obtained from a known concentration of IL-12.

[Example 1] (IL-12 production by combination of β-glucan and Lactobacillus crispatas KT-11)
Table 1 shows the amount of IL-12 in the culture supernatant of J774.1 cells cultured by adding 1 μg / mL of Lactobacillus crispatas KT-11 strain to 99 μg / mL of each β-glucan.

  As shown in Table 1, the amount of IL-12 when the Lactobacillus crispatas KT-11 strain was added to each β-glucan was significantly increased as compared with the case where the Lactobacillus crispatas KT-11 strain was not added. The value exceeded the amount of IL-12 produced when each was used alone. From this, it was shown by this test that Lactobacillus crispatas KT-11 strain synergistically enhances the immunostimulatory action of β-glucan. In particular, it was revealed that the synergistic effect was particularly remarkable when the β-glucan derived from Saccharomyces cerevisiae was combined with the Lactobacillus crisptus KT-11 strain.

[Example 2] (IL-12 production amount by combination of β-glucan derived from Saccharomyces cerevisiae and lactic acid bacteria)
Table 2 shows J774.1 cells cultured by adding Lactobacillus crispatas KT-11 strain or Lactobacillus plantarum JCM1149 ^T to 1 μg / mL to Saccharomyces cerevisiae-derived β-glucan 99 μg / mL. This shows the amount of IL-12 in the culture supernatant.

As shown in Table 2, the synergistic effect on IL-12 production observed in the combination of Saccharomyces cerevisiae-derived β-glucan and Lactobacillus kristpatus KT-11 strain was compared with that of Lactobacillus plantarum JCM1149 ^T. it was high.

[Example 3] (Synergism of IL-12 production due to difference in addition ratio)
Table 3 shows the results of examining the synergistic effect of the immunostimulatory effect due to the difference in the addition ratio of the composition containing Saccharomyces cerevisiae-derived β-glucan and Lactobacillus crispatas KT-11 strain.

  As shown in Table 3, when Lactobacillus crispatas KT-11 strain was added at 50% by mass with respect to 50% by mass of β-glucan derived from Saccharomyces cerevisiae, the amount of IL-12 produced was significantly increased, and derived from Saccharomyces cerevisiae It was shown to be higher than when β-glucan was used alone.

Claims (8)

  An immunostimulatory composition comprising β-glucan and cells of Lactobacillus crispatus KT-11 strain (FERM BP-11332) as active ingredients.   2. The immunostimulatory composition according to claim 1, wherein the β-glucan is a β-glucan derived from Saccharomyces cerevisiae.   The composition for immunostimulation according to claim 1, which has an immunostimulatory effect on humans, livestock animals or pet animals.   The foodstuff containing the immunostimulation composition as described in any one of Claims 1-3.   The feed containing the immunostimulation composition as described in any one of Claims 1-3.   The pharmaceutical containing the immunostimulation composition as described in any one of Claims 1-3.   The method to provide the immunostimulatory effect to food or feed including adding the immunostimulation composition as described in any one of Claims 1-3 to food or feed.   The manufacturing method of the foodstuff or feed which has an immunostimulatory effect including adding the immunostimulation composition as described in any one of Claims 1-3 to a foodstuff or feed.