JP2022158771A - Agent for inhibiting deterioration of brain functions - Google Patents

Abstract

To provide a novel agent for inhibiting a deterioration of brain functions, the agent derived from natural products, and provide an affective disorder improver.SOLUTION: This invention has been completed with the findings that Bacillus subtilis has the effects of inhibiting a deterioration of brain functions and alleviating affective disorder.SELECTED DRAWING: Figure 1

Description

TECHNICAL FIELD The present invention relates to an agent for suppressing brain function decline, an agent for improving affective disorders, and foods and drinks containing them.

With the progress of aging, there is concern that the number of patients with brain diseases caused by age-related decline in brain function, such as memory impairment, dementia, and Alzheimer's syndrome, will increase, and food that can suppress the decline in brain function. There is a strong demand for the development of drugs and pharmaceuticals.

Patent Documents 1 and 2 reported preventive, ameliorating, and therapeutic agents for Alzheimer's dementia containing cultures and/or bacterial cells obtained by culturing lactic acid bacteria as active ingredients. In addition, Patent Document 3 discloses an anti-aging agent containing Bacillus subtilis as an active ingredient. It is described that it is considered that the effect of suppressing the change of hair, which is the aging change of the appearance, was exhibited.

Japanese Patent No. 5155960 Japanese Patent No. 5155961 Japanese Patent Application Laid-Open No. 2020-80856

The present invention provides an agent for suppressing brain function decline and an agent for improving affective disorders derived from natural products.

The inventors have found that Bacillus subtilis has an effect of suppressing brain function deterioration and an effect of improving affective disorders, and completed the present invention.

That is, the present invention relates to the following aspects [1] to [10].
[1] A brain function depression inhibitor containing Bacillus subtilis as an active ingredient.
[2] The inhibitor of brain function deterioration according to [1], which contains cells obtained by liquid culturing Bacillus subtilis as an active ingredient.
[3] The inhibitor of brain function decline according to [1] or [2], which contains dead cells as an active ingredient.
[4] The inhibitor of brain function decline according to any one of [1] to [3], wherein Bacillus subtilis is a spore-forming strain.
[5] A food, drink, drug or feed containing the inhibitor of brain function decline according to any one of [1] to [4].
[6] An affective disorder improving agent containing Bacillus subtilis as an active ingredient.
[7] The agent for improving affective disorders according to [6], which contains, as an active ingredient, cells obtained by liquid culturing Bacillus subtilis.
[8] The agent for improving affective disorders according to [6] or [7], which contains dead cells as an active ingredient.
[9] The agent for improving affective disorders according to any one of [6] to [8], wherein Bacillus subtilis is a spore-forming strain.
[10] Food, drink, medicine or feed containing the affective disorder improving agent according to any one of [6] to [9].

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide an agent for suppressing brain function deterioration and an agent for improving affective disorders derived from natural products. In addition, since dead bacteria after sterilization are effective in suppressing brain function deterioration and improving emotional disorders, the use of killed Bacillus subtilis facilitates hygiene management of manufacturing facilities and quality control of products. , the inhibitor of brain function decline can be produced efficiently. In addition, since the active ingredient is not a chemically synthesized product but a fungus that has been eaten, it is most suitable as a brain function declining inhibitor that should be taken continuously over a long period of time. Furthermore, by using strains lacking the ability to form spores, sterilization can be performed under mild conditions of 100° C. or less, as with 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.

1 shows the number of invasions in area A of a control group and a Bacillus subtilis-administered group (0.1% and 1.0%) in an open field test. Fig. 2 shows locomotion in area A of a control group and a Bacillus subtilis-administered group (0.1% and 1.0%) in an open field test. 1 shows the residence time in area A of a control group and a Bacillus subtilis-administered group (0.1% and 1.0%) in an open field test. In the light-dark box test, the number of rising times in each light-dark area of the control group and the Bacillus subtilis-administered group (0.1% and 1.0%) is shown. In the TTR immunostaining of the hippocampus of the control group and the Bacillus subtilis-administered group (0.1% and 1.0%), the number of plaques with a diameter of 50 μm or less (50 μm ≦ D) D) Shows the number of deposits and their total (All).

The agent for suppressing deterioration of brain function and the agent for improving affective disorder of the present invention contain Bacillus subtilis as an active ingredient, and when ingested, can suppress transthyretin (TTR) deposition in the hippocampus. By protecting cells, it is possible to exert an effect of suppressing deterioration of brain function due to aging and an effect of improving affective disorders caused by aging.

The Bacillus subtilis according to the present invention may be either live or dead as long as it is Bacillus subtilis, which is an active ingredient of the functional preparation, and is not particularly limited. B. subtilis.subsp.subtilis) is preferable, and Bacillus subtilis natto such as Bacillus subtilis NBRC3009, Bacillus subtilis NBRC3013, Bacillus subtilis NBRC3335, Bacillus subtilis NBRC13169 is more preferable, and obtained from National Institute of Technology and Evaluation, etc. be able to. Even dead bacteria have the effect of suppressing brain function deterioration and improving emotional disorders. An affective disorder improving agent can be efficiently produced. In addition, if a strain lacking the ability to form spores is used, dead cells can be prepared under mild sterilization conditions at 100°C or less, which prevents contamination of manufacturing facilities due to insufficient sterilization, which is a problem with spore-forming bacteria. , addition to various foods and formulation become easy.

Bacillus subtilis used in the present invention is not particularly limited, but strains deficient in sporulation ability are preferred, and examples of methods for obtaining the strain deficient in sporulation ability include genetic recombination and mutation. Methods by spontaneous mutation are 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 transformation ability, and thus can be passaged without problems due to bacteriolysis. , can maintain the trait of lack of sporulation ability.

For culturing Bacillus subtilis, a normal bacterial culture medium can be used. Any medium, synthetic medium or natural medium, can be used as long as it contains carbon sources, nitrogen sources, inorganic substances, and other micronutrients required by Bacillus subtilis. is also usable. 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 conditions for culturing Bacillus subtilis can be set as appropriate, but it is preferable to carry out aerobic liquid culture by aeration, shaking, stirring, or the like. The time can be exemplified, for example, from 2 to 72 hours, preferably from 4 to 48 hours, more preferably from 6 to 36 hours. preferable.

Bacillus subtilis may be sterilized after culturing, and sterilization conditions are not particularly limited as long as it is a general method. is usually 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 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 agent for suppressing brain function decline and the agent for improving affective disorders of the present invention has an active ingredient derived from a natural product and is easy to manufacture, so that it can be widely used, can be added to various products, and can be added in liquid form. It may be added in a refrigerated, frozen or dried state, and can be used to prepare foods, beverages, pharmaceuticals, feeds, etc. having an effect of suppressing brain function decline and improving affective disorders. The content of Bacillus subtilis in various products is not particularly limited as long as it is an amount in which the effect of suppressing brain function deterioration and an agent for improving emotional disorders can be observed by ingestion, but it is preferably 0.01 to 20% by weight, and 0.02 to 10% by weight. %, more preferably 0.05 to 5% by weight.

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。(1. Preparation of Bacillus subtilis cells)
Spore-forming ability-deficient strain Bacillus subtilis NBRC13169, which is a type of Bacillus subtilis, has been deficient in sporulation ability 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 heat sterilized at 90°C for 10 minutes. 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 dryer to prepare a powder of dead Bacillus subtilis cells (cell count: 6.4×10 ¹¹ cells/g, viable cell count: less than 100 cfu/g).

(2. Confirmation of the effect of administration of Bacillus subtilis in mice)
9-week-old senescence-accelerated mice (SAMP8) were divided into 3 groups. MF feed containing 0.1% of the bacterial cell powder obtained in 1 above, and the 1.0% Bacillus subtilis-administered group (8 animals) received MF feed containing 1% of the bacterial cell powder obtained in 1 above. The rats were fed and kept until they reached 34 weeks of age. Feed and water were freely available during the test period.

(2-1. Behavior analysis)
An open field test was performed at 25 weeks of age, and a light-dark box test was performed at 20 and 28 weeks of age. In the open field test, one mouse was released into the field of a small animal behavior analysis device (SCANET, manufactured by Melquest) and allowed to explore freely for 60 minutes, followed by observation and measurement. The field has an area A of 10.8 x 10.8 cm in the center, an area B of 26.4 x 26.4 cm around it, and an area C of 45.0 x 45.0 cm in the periphery facing the wall. 1 to 3 show the amount of exercise (a value proportional to the distance traveled), the staying time (seconds), and the number of times of intrusion in Area A. In the light/dark box test, a light/dark cage was installed in the same apparatus, and the number of rising times in each light/dark area was measured. The results are shown in FIG.

Normally, when animals are exposed to a wide and novel environment, they repeatedly stand up and walk for exploratory behavior, but at this time, they touch walls and prefer to walk around the periphery. indicates This is due to anxiety-like behavior in innate and latent emotional behavior, and it is known that the proportion of exploratory behavior in the central part of the field is extremely small. On the other hand, it is known that the aging-accelerated model mouse SAMP8 strain has an affective disorder in which anxiety-like behavior declines with aging (Nihon Pharmacology Journal, 2010, Vol. 136, No. 1, pp. 6-10). .

In the control group, the number of times of entry into area A in the center of the field, the amount of exercise, and the staying time were higher than in the Bacillus subtilis-administered group, and there was a significant difference from the 1% Bacillus subtilis-administered group (Figures 1 to 3). , indicating that administration of Bacillus subtilis maintained anxiety-like behavior. In addition, at 20 weeks of age, the number of rising times (exploratory behavior) in the light box and the dark box was the same behavior pattern in all three groups, in which the number of rising times, which is an exploratory behavior, was superior in the light box than in the dark box. At 28 weeks of age, when aging progressed, only the 1% Bacillus subtilis administration group had a significant difference in the number of times of standing up in the light box and the dark box (exploratory behavior), as at 20 weeks of age, and the difference disappeared in the other groups ( Figure 4). This indicates that there was a difference in brain function due to aging depending on the presence or absence of Bacillus subtilis administration. In other words, it is considered that the Bacillus subtilis-administered group was improved in affective disorder, in which anxiety-like behavior declines with aging, compared to the control group.

(2-2. Confirmation of TTR deposition in brain tissue)
The mice were sacrificed at 34 weeks of age, and the brain tissue was stained with a specific antibody to assess the state of deposition of transthyretin (TTR), one of the amyloidogenic proteins, in the brain. Specifically, in each of the control group and the 0.1% and 1.0% Bacillus subtilis administration groups, hippocampal sections were prepared, immunostained with an anti-TTR antibody, and the incidence of TTR deposits (plaque burden) was measured. Shown in Table 1, the number of TTR deposition per area (1.00 × 0.75 mm) (diameter of 50 µm or less (50 µm ≤ D), more than 50 µm (50 µm > D), total (All)) was calculated and 5.

TTR deposition is caused by aggregation of TTR, and since it is known that aggregated TTR in the brain activates microglia and causes neuroinflammation, reducing TTR deposition may prevent neuroinflammation. It is thought that brain cells are protected (Bioscience, Biotechnology, and Biochemistry 2018, Vol. 82, No. 4, p. 647-653).

5 and Table 1, the number of TTR deposition per area in hippocampal brain cells was smaller in the Bacillus subtilis-administered group than in the control group. 05), it is considered that administration of Bacillus subtilis suppressed TTR deposition in the hippocampus, prevented neuroinflammation, and protected brain cells.

Based on the above, it is considered that the affective disorder caused by age-related decline in brain function was suppressed and ameliorated by the protection of hippocampal brain cells, which are responsible for emotional memory. Therefore, it is considered that administration of Bacillus subtilis exerted an effect of suppressing TTR deposition in the hippocampus, leading to an effect of preventing neuroinflammation and a protective effect on brain cells, thereby suppressing deterioration of brain function and improving affective disorders.

Claims (10)

An agent for suppressing brain function deterioration containing Bacillus subtilis as an active ingredient. 2. The agent for suppressing brain function deterioration according to claim 1, which contains cells obtained by liquid culturing Bacillus subtilis as an active ingredient. 3. The agent for suppressing brain function decline according to claim 1 or 2, which contains dead cells as an active ingredient. The agent for suppressing brain function decline according to any one of claims 1 to 3, wherein Bacillus subtilis is a spore-forming strain. A food, drink, drug or feed comprising the agent for suppressing brain function decline according to any one of claims 1 to 4. An affective disorder improving agent containing Bacillus subtilis as an active ingredient. 7. The agent for improving affective disorders according to claim 6, wherein the active ingredient is cells obtained by liquid culturing Bacillus subtilis. 8. The affective disorder improving agent according to claim 6 or 7, which contains dead cells as an active ingredient. The affective disorder ameliorating agent according to any one of claims 6 to 8, wherein Bacillus subtilis is a spore-forming strain. Food, drink, medicine or feed containing the agent for improving affective disorders according to any one of claims 6 to 9.

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