JP7248878B2 - Immunomodulator - Google Patents

Description

TECHNICAL FIELD The present invention relates to an immunomodulator, a method for producing the same, and the like.

With the progress of aging, the awareness of health is increasing more and more, and there is a strong demand for the development of foods and medicines that can normally maintain the immune function, which is extremely important for the prevention and improvement of diseases.

So far, as an immunomodulator, an immunomodulator characterized by containing the lactic acid bacterium Lactococcus lactis subspecies cremoris H-61 strain (NITE AP-92) (Patent Document 1), and catechins as active ingredients (Patent Document 2) and an immunomodulator containing coenzyme Q10 and processed pomegranate (Patent Document 3).

Japanese Patent No. 4604207 JP 2008-63318 A JP 2015-17081 A

The present invention provides novel immunomodulators derived from natural products.

The inventors have found that a strain of Bacillus subtilis lacking the ability to form spores has an excellent immunoregulatory effect, and have completed the present invention.

That is, the present invention relates to the following aspects [1] to [7].
[1] An immunomodulator containing a strain of Bacillus subtilis deficient in spore formation as an active ingredient.
[2] The immunoregulator of [1], which is for promoting spleen cell proliferation, interferon-γ production, or interleukin-10 production.
[3] An anti-allergic agent containing, as an active ingredient, a spore-forming-deficient strain of Bacillus subtilis.
[4] An immunostimulant containing a strain of Bacillus subtilis deficient in spore formation as an active ingredient.
[5] A food, drink, cosmetic, drug or feed containing the immunomodulator of [1] or [2].
[6] Food, cosmetics, pharmaceuticals or feed containing the antiallergic agent of [3].
[7] Food, cosmetics, pharmaceuticals or feed containing the immunostimulant according to [4].

INDUSTRIAL APPLICABILITY The present invention can provide novel immunoregulators derived from natural products. In addition, since the dead bacteria after sterilization have an immunoregulatory effect, the use of dead bacteria facilitates hygiene management of manufacturing facilities and quality control of products, enabling efficient production of immunomodulators. . In addition, since the active ingredient is not a chemically synthesized product but a fungus that has been eaten, it is most suitable as an immunomodulator whose continuous long-term intake is desirable. Furthermore, since it is a spore-forming defective strain, it can be sterilized under mild conditions of 100 ° C. or less like general microorganisms, and contamination of manufacturing facilities due to insufficient sterilization, which is a problem with spore-forming bacteria, can be prevented. Therefore, it is easy to add to various foods and formulate.

Fig. 3 shows the effect of addition of Bacillus subtilis on the growth ability of spleen cells. 1 shows the effect of addition of Bacillus subtilis on the ability to produce interferon-γ or interleukin-10.

The present invention relates to an immunomodulator containing a strain deficient in sporulation as an active ingredient, which can be used for promoting spleen cell proliferation, interferon-γ production, or interleukin 10 production. It has at least one of, preferably, all of the effects of interferon-γ production promotion and interleukin-10 production promotion, and is also effective as an anti-allergic agent or an immunostimulant.

The sporulation-deficient strain according to the present invention is not particularly limited as long as it is a Bacillus subtilis sporulation-deficient strain that is an active ingredient of an immunomodulator. Subspecies subtilis (B.subtilis.subsp.subtilis) is preferable, Bacillus subtilis natto such as Bacillus subtilis NBRC3009, Bacillus subtilis NBRC3013, Bacillus subtilis NBRC13169 is more preferable, obtained from National Institute of Technology and Evaluation, etc. be able to.

Spore-forming ability-deficient strains can be obtained by mutating the above-described wild strains by genetic recombination, mutation, or the like, and spontaneous mutation is preferred. The method for obtaining the sporulation-deficient strain by spontaneous mutation is not particularly limited, and includes a high-temperature culture method, a random method for distinguishing by coloring the melanin pigment of colonies of the wild strain and the defective strain, and catabolite repression. A method using a catabolite-like phenomenon (JF Michel, B. Cami, P. Schaeffer: Ann. Inst. Pasteur, 114, 11; 21 (1968)) can be exemplified. A method is preferred. The sporulation-deficient strain obtained by the method utilizing the catabolite suppression-like phenomenon lacks sporulation ability as well as lysozyme activity and transforming ability, and thus can be passaged without problems due to bacteriolysis. , can maintain the trait of lack of sporulation ability. If a strain lacking the ability to form spores is used, it can be used as an active ingredient of the immunoregulator of the present invention. It is possible to prevent contamination of manufacturing equipment due to insufficient sterilization, which is a problem, and it becomes easy to add to various foods and formulate. In addition, although both live and dead bacteria have an immunomodulatory effect, it is preferable to use dead bacteria. The regulator can be efficiently produced.

For culturing strains lacking the ability to form spores, normal bacterial culture media can be used. Synthetic media, natural Any medium can be used. Glucose, sucrose, dextrin, starch, glycerin, molasses and the like can be used as the carbon source. Nitrogen sources include inorganic salts such as ammonium chloride, ammonium nitrate, ammonium sulfate, and ammonium phosphate, amino acids such as DL-alanine and L-glutamic acid, peptone, meat extract, yeast extract, malt extract, and nitrogen-containing corn steep liquor. Natural products can be used. As the inorganic substance, monosodium phosphate, disodium phosphate, monopotassium phosphate, dipotassium phosphate, magnesium sulfate, ferric chloride and the like can be used.

The culture conditions can be set as appropriate, but it is preferable to carry out aerobic liquid culture by aeration, shaking, stirring, etc. The culture temperature is, for example, 20 to 50°C, preferably 30 to 45°C, and the culture time is, for example, Examples include 2 to 72 hours, preferably 4 to 48 hours, more preferably 6 to 36 hours, and the pH of the medium is, for example, 5.0 to 9.0, preferably 5.5 to 8.5.

It may be sterilized after culturing, and the sterilization conditions are not particularly limited as long as it is a general method. For example, the heating temperature is 70 to 150° C. 1 to 60 minutes. In addition, since the sporulation-deficient strain according to the present invention does not form spores, it can be sterilized under mild conditions at 100° C. or lower, for example, heating at 70 to 100° C. for 5 to 20 minutes. The spore-forming-deficient strain according to the present invention can prevent contamination of production equipment due to insufficient sterilization, which is a problem with spore-forming bacteria, and thus can be easily added to various foods and formulated. The cells can be collected by removing the culture medium with a centrifuge or the like, washing the cells with a buffer solution, physiological saline solution, sterilized water or the like, and separating the solid and the liquid with a centrifuge or the like. Further, air drying, spray drying, vacuum and/or freeze drying, etc. may be performed to powderize.

The immunomodulator, anti-allergic agent, or immunostimulant of the present invention has an active ingredient derived from a natural product and is easy to produce. It may be added in a refrigerated, frozen or dried state, and foods and drinks, cosmetics, pharmaceuticals, feeds, etc. having immunoregulatory, anti-allergic, or immunostimulatory effects can be prepared. The content of Bacillus subtilis in various products is not particularly limited as long as it is an amount that is effective when ingested, but is preferably 0.1 to 20% by weight, more preferably 0.2 to 10% by weight, and 0.5 to 10% by weight. 5% by weight is more preferred.

EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to the following examples. In addition, in the present invention, all percentages are percentages by weight unless otherwise specified.

利用した自然突然変異は、特許第６０１９５２８号公報の実施例に記載の方法で行い、該公報に記載の方法で芽胞形成能が欠損した株であることを確認した。(Preparation of dead cell powder of sporulation-deficient strain)
A sporulation-deficient strain, which lacks sporulation ability, was obtained from Bacillus subtilis NBRC13169, which is a kind of Bacillus natto, by spontaneous mutation using a catabolite suppression-like phenomenon.

The natural mutation used was performed by the method described in Examples of Japanese Patent No. 6019528, and it was confirmed that the strain lacked sporulation ability by the method described in the publication.

The Bacillus subtilis was inoculated into a liquid medium (yeast extract: 2%, glucose: 5%, tap water: 93%), cultured with aeration and stirring at 37°C for 24 hours, and then heat sterilized at 90°C for 10 minutes ( general viable count: less than 10/g). Subsequently, the culture medium was removed using a centrifuge, and the collected cells were washed with tap water, and then subjected to solid-liquid separation using a centrifuge to collect the cells. The collected cells were dried with a spray drier to prepare a dead cell powder of a Bacillus subtilis strain lacking spore formation ability (practical product 1, general viable cell count: less than 10 ² cells/g).

[Comparative Example 1]
(Preparation of dead cell powder of spore-forming strain)
The Bacillus subtilis NBRC13169 described in Example 1 was used without mutation, cultured in the same manner as in Example 1, and heat sterilized at 121° C. for 15 minutes (general viable cell count: less than 10/g). Then, the treatment was carried out in the same manner as in Example 1 to prepare a dead cell powder of a spore-forming strain of Bacillus subtilis (comparative product 1, general viable cell count: less than 10 ² cells/g).
In addition, since the unmutated strain is a spore-forming strain, it could not be sterilized under the same sterilization conditions of 90 ° C. for ¹⁰ minutes as in Example 1. The processing time was 1 minute.

[Evaluation Test 1]
(Spleen cell proliferation promoting effect)
Spleen cells collected from the spleen of BALB/c mice were plated on a cell culture plate, and 50 μg/ml of the bacterial cell powder of Experimental Product 1 or Comparative Product 1 was added, or no bacterial cell powder was added, and incubated at 37°C. After 30 hours of culture at , bromodeoxyuridine (BrdU) was added. After culturing for an additional 18 hours, the proliferative capacity of the spleen cells was evaluated by developing the color using a labeled anti-BrdU antibody and measuring the absorbance at 450 nm (OD450). Each group was tested 3 wells, and the OD450 (mean value) and standard deviation of each group were calculated and shown in Table 1 and FIG. Table 1 also shows the relative values of Example Product 1 when the value of Comparative Product 1 is set to 1.

From Table 1 and FIG. 1, it was found that the cells to which Example Product 1 was added exhibited a higher absorbance than those without addition and Comparative Product 1. In other words, it was found that the addition of the spore-forming ability-deficient strain of Bacillus subtilis promoted the proliferation of spleen cells more than the addition of the Bacillus subtilis spore-forming strain. , was found to be promoted about 1.2-fold compared to the addition of the sporulation strain.

Therefore, the spore-forming ability-deficient strain of Bacillus subtilis was found to promote the growth of spleen cells, and thus can be expected to have an immunostimulatory effect that strengthens immunity.

[Evaluation Test 2]
(Cytokine production promoting effect)
Spleen cells collected from the spleen of BALB/c mice were seeded on a cell culture plate previously treated with 0 to 2 μg/mL of an anti-CD3 antibody, which is a T cell activating antibody, and the fungus powder of Example 1 or Comparative product 1 was obtained. was added to 50 μg / ml, or no bacterial powder was added, and cultured at 37 ° C. for 48 hours, then interferon-γ (IFN-γ) concentration or interleukin-10 (IL-10) Concentration was measured. Three wells for each group were tested, and the average value and standard deviation for each group of cell culture plates without anti-CD3 antibody treatment (T cell activation was not performed) were calculated and shown in Table 2. Table 2 shows the results of calculating each relative value of the practical product 1 when the value of the comparative product 1 is set to 1. Furthermore, the average value and standard deviation of the IFN-γ concentration or IL-10 concentration for each group of cell culture plates at each anti-CD3 antibody concentration were calculated and shown in FIG.

From Table 2, in the cells not treated with anti-CD3 antibody, almost no IFN-γ and IL-10 production was observed in the cells to which the bacterial powder was not added, whereas the comparative product 1 and the test product Both IFN-γ and IL-10 were produced in the cells to which the microbial cell powder No. 1 was added. Furthermore, in cells to which Working Product 1 was added, IFN-γ production was 2.8 times higher and IL-10 production was 1.1 times higher than cells to which Comparative Product 1 was added. production was further promoted. In other words, IFN-γ and IL-10, which are normally produced by activating T cells, are produced by the addition of dead Bacillus subtilis powder without activating T cells. It was found that the addition of the dead cell powder of the forming ability-deficient strain further promoted the growth.
Moreover, from FIG. 2, the production amounts of IFN-γ and IL-10 increased in proportion to the concentration of the anti-CD3 antibody. A more pronounced increase was observed in cells to which Example 1 was added.

Promoting the production of IFN-γ activates macrophages and natural killer cells, and is thought to exhibit cell-mediated immunostimulatory effects such as antitumor action and protection against infection. Furthermore, the production of IFN-γ, which is a type 1 helper T cell (Th1), is promoted, and the type 2 helper T cell (Th2) is suppressed, so that antiallergic effects are expected to be exhibited. In addition, by promoting the production of IL-10, which is an immunosuppressive cytokine, Th2 is suppressed, so it is thought that excessive inflammatory reactions are suppressed and anti-allergic effects are exhibited.

From the results of Evaluation Tests 1 and 2, the addition of the powdered dead cells of the Bacillus subtilis spore-forming-deficient strain showed strong spleen cell growth-promoting action, IFN-γ production-promoting action, and IL-10 production-promoting action. Therefore, the sporulation-deficient strain of Bacillus subtilis can be expected to have immunostimulatory and anti-allergic effects. Alternatively, it is thought to be useful as an immunoregulator that can be used as an improver, an immunostimulant, or the like.

Claims (5)

An anti-allergic agent comprising, as an active ingredient, a sporulation-deficient strain of Bacillus subtilis NBRC13169 . An immunostimulant comprising, as an active ingredient, a sporulation-deficient strain of Bacillus subtilis NBRC13169 . Antiallergic food and drink, cosmetics, pharmaceuticals or feed containing the antiallergic agent according to claim 1 . Food and drink for immunostimulation , cosmetics, pharmaceuticals, or feed, comprising the immunostimulant according to claim 2 . A method for selecting a spore-forming-deficient strain of Bacillus natto that is a candidate strain for preventing or ameliorating allergic diseases or stimulating immunity,
A step of measuring the effect of at least one sporulation-deficient strain of Bacillus natto with respect to the effect of promoting interleukin-10 production, the effect of promoting interferon-γ production, or the effect of promoting spleen cell proliferation, and
A method of selection comprising the step of selecting at least one candidate strain having the effect of promoting the production of interleukin-10 or interferon-γ or the effect of promoting spleen cell proliferation.

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