Transthyretin (TTR) deposition inhibitor

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]

[0001] The present invention relates to an agent for suppressing brain function decline and an agent for improving emotional disorders, as well as foods, beverages, etc. containing them. [Background technology]

[0002] As the aging population continues to grow, there are concerns that the number of patients with brain diseases caused by age-related decline in brain function, such as memory disorders, dementia, and Alzheimer's syndrome, will increase. Therefore, there is a strong demand for the development of foods and pharmaceuticals that can suppress the decline in brain function.

[0003] Patent Documents 1 and 2 report agents for preventing, ameliorating, and treating Alzheimer's dementia, which contain as active ingredients a culture and / or bacterial cells obtained by culturing lactic acid bacteria. Patent Document 3 discloses an anti-aging agent containing Bacillus subtilis as an active ingredient, and describes that oral administration of Bacillus subtilis is thought to have the effect of suppressing the dulling of tactile sensitivity or decline in reflex behavior caused by aging, as well as the effect of suppressing changes in hair, which are age-related changes in appearance. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 5155960 [Patent Document 2] Patent No. 5155961 [Patent Document 3] JP 2020-80856 A Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention provides a naturally occurring agent for suppressing brain function decline and an agent for improving emotional disorders. [Means for solving the problem]

[0006] The inventors have discovered that Bacillus subtilis has the effect of suppressing brain function decline and improving emotional disorders, and have completed the present invention.

[0007] That is, the present invention relates to the following aspects [1] to

[10] . [1] A drug that suppresses brain function decline, with Bacillus subtilis as its active ingredient. [2] The agent for suppressing brain function decline according to [1], wherein the active ingredient is a bacterium obtained by liquid culture of Bacillus subtilis. [3] The agent for suppressing brain function decline according to [1] or [2], which contains killed bacteria as an active ingredient. [4] The agent for suppressing brain function decline according to any one of [1] to [3], wherein the Bacillus subtilis is a strain deficient in sporulation ability. [5] A food, drink, medicine, or feed containing the brain function decline inhibitor according to any one of [1] to [4]. [6] A drug for improving emotional disorders, containing Bacillus subtilis as its active ingredient. [7] The agent for improving affective disorders according to [6], wherein the active ingredient is a bacterium obtained by culturing Bacillus subtilis in a liquid. [8] The agent for improving affective disorders according to [6] or [7], which contains killed bacteria as an active ingredient. [9] The agent for improving affective disorders according to any one of [6] to [8], wherein the Bacillus subtilis is a strain deficient in sporulation ability.

[10] A food, drink, medicine, or feed containing the agent for improving affective disorders according to any one of [6] to [9]. [Effects of the Invention]

[0008] The present invention provides a brain function decline inhibitor and an emotional disorder ameliorator derived from natural products. Furthermore, since the effects of inhibiting brain function decline and improving emotional disorders are observed in killed cells after sterilization, the use of killed Bacillus subtilis cells facilitates hygiene management of manufacturing facilities and product quality control, allowing for efficient production of brain function decline inhibitors. Furthermore, because the active ingredient is not a chemically synthesized product but a bacterium that has been eaten, it is ideal as a brain function decline inhibitor for which continuous long-term ingestion is desirable. Furthermore, if a spore-forming deficient strain is used, sterilization can be performed under mild conditions of 100°C or below, similar to common microorganisms, preventing contamination of manufacturing facilities due to insufficient sterilization, which is a problem with spore-forming bacteria. This allows for easy addition to various foods and formulation. [Brief explanation of the drawings]

[0009] [Figure 1] The figures show the number of entries into area A in the control group and the Bacillus subtilis-administered groups (0.1% and 1.0%) in the open field test. [Figure 2] The figures show the amount of locomotion in area A in the control group and the Bacillus subtilis-administered groups (0.1% and 1.0%) in the open field test. [Figure 3] In the open field test, the time spent in area A in the control group and the Bacillus subtilis-administered groups (0.1% and 1.0%) is shown. [Figure 4] In the light-dark box test, the number of rearings in the light and dark areas for the control group and the Bacillus subtilis-administered groups (0.1% and 1.0%) is shown. [Figure 5] In TTR immunostaining of the hippocampus from the control group and the Bacillus subtilis-treated groups (0.1% and 1.0%), the number of deposits with plaque diameters of 50 μm or less (50 μm≦D), the number of deposits with diameters of more than 50 μm (50 μm>D), and the total number of these (All) are shown. DETAILED DESCRIPTION OF THE INVENTION

[0010] The agent for suppressing brain function decline and improving emotional disorders of the present invention contains Bacillus subtilis as an active ingredient, and when ingested, it can suppress transthyretin (TTR) deposition in the hippocampus, thereby preventing neuroinflammation and protecting brain cells, thereby suppressing the decline in brain function caused by aging and improving emotional disorders caused by aging.

[0011] The Bacillus subtilis described in the present invention may be live or killed, as long as it is Bacillus subtilis, which serves as an active ingredient in the functional formulation, and is not particularly limited. However, Bacillus subtilis subsp. subtilis is preferred, and natto bacteria such as Bacillus subtilis NBRC3009, Bacillus subtilis NBRC3013, Bacillus subtilis NBRC3335, and Bacillus subtilis NBRC13169 are more preferred, and these can be obtained from the National Institute of Technology and Evaluation, etc. Even killed Bacillus subtilis bacteria have the effect of suppressing brain function decline and ameliorating emotional disorders. Therefore, using killed Bacillus subtilis bacteria facilitates sanitation management of manufacturing facilities and quality control of products, allowing for the efficient production of agents for suppressing brain function decline and ameliorating emotional disorders. Furthermore, by using a strain that is deficient in spore formation, killed bacteria can be prepared under mild sterilization conditions of below 100°C, which prevents contamination of manufacturing equipment due to insufficient sterilization, a problem that occurs with spore-forming bacteria, and makes it easier to add the bacteria to various foods and to formulate them into food products.

[0012] The Bacillus subtilis used in the present invention is not particularly limited, but a sporulation-deficient strain is preferred. Methods for obtaining sporulation-deficient strains include genetic recombination and mutation, with spontaneous mutation being preferred. The method for obtaining sporulation-deficient strains by spontaneous mutation is not particularly limited, and examples include high-temperature culture, a random method in which wild-type and defective strain colonies are distinguished by melanin pigment coloration, and a method utilizing catabolite repression-like phenomenon (J.F. Michel, B. Cami, P. Schaeffer: Ann. Inst. Pasteur, 114, 11; 21 (1968)). The method utilizing catabolite repression-like phenomenon is preferred. Because sporulation-deficient strains obtained by the method utilizing catabolite repression-like phenomenon lack sporulation ability, lysozyme activity, and transformability, they can be passaged without problems due to bacteriolysis and maintain the trait of sporulation deficiency.

[0013] Conventional bacterial culture media can be used to culture Bacillus subtilis, including synthetic and natural media, as long as they contain a carbon source, a nitrogen source, inorganic substances, and other trace nutrients required by Bacillus subtilis. Examples of carbon sources that can be used include glucose, sucrose, dextrin, starch, glycerin, and molasses. Examples of nitrogen sources that can be used include inorganic salts such as ammonium chloride, ammonium nitrate, ammonium sulfate, and ammonium phosphate; amino acids such as DL-alanine and L-glutamic acid; and nitrogen-containing natural products such as peptone, meat extract, yeast extract, malt extract, and corn steep liquor. Examples of inorganic substances that can be used include monosodium phosphate, disodium phosphate, monopotassium phosphate, dipotassium phosphate, magnesium sulfate, and ferric chloride.

[0014] The culture conditions for Bacillus subtilis can be set as appropriate, but aerobically culturing in liquid form using aeration, shaking, stirring, etc. is preferred; the culture temperature can be, for example, 20 to 50°C, and preferably 30 to 45°C; the culture time can be, for example, 2 to 72 hours, and preferably 4 to 48 hours, and more preferably 6 to 36 hours; and the pH of the medium can be, for example, 5.0 to 9.0, and preferably 5.5 to 8.5.

[0015] Bacillus subtilis may be sterilized after cultivation. Sterilization conditions are not particularly limited as long as they are common methods, but for example, the heating temperature is 70 to 150°C, and the heating time may be determined depending on the temperature, but is usually 1 to 60 minutes. Furthermore, since the sporulation-deficient strain described in the present invention does not form spores, sterilization can be performed under mild conditions of 100°C or less, such as heating at 70 to 100°C for 5 to 20 minutes. The bacterial cells can be collected by removing the medium using a centrifuge or the like, washing the bacterial cells with a buffer solution, physiological saline, sterilized water, or the like, and then performing solid-liquid separation using a centrifuge or the like to collect the bacterial cells. Furthermore, the bacterial cells may be powdered by air drying, spray drying, vacuum drying, and / or freeze drying, etc.

[0016] The active ingredients of the agent for suppressing brain function decline and the agent for improving emotional disorders of the present invention are derived from natural products and are easy to produce, making them widely usable and addable to various products, whether in liquid, refrigerated, frozen, or dried form, allowing the preparation of foods, beverages, pharmaceuticals, feed, etc. that have the effect of suppressing brain function decline and improving emotional disorders. The content of Bacillus subtilis in various products is not particularly limited as long as it is an amount that is recognized as having the effect of suppressing brain function decline and improving emotional disorders when ingested, but is preferably 0.01 to 20% by weight, more preferably 0.02 to 10% by weight, and even more preferably 0.05 to 5% by weight. [Example]

[0017] The present invention will be specifically described below with reference to examples, but the present invention is not limited to the following examples. In the present invention, all % is by weight unless otherwise specified.

[0018] (1. Preparation of Bacillus subtilis Cells) A sporulation-deficient strain, Bacillus subtilis NBRC13169, was isolated from Bacillus subtilis by spontaneous mutation utilizing a catabolite repression-like phenomenon. The spontaneous mutation used was carried out by the method described in the Examples of Japanese Patent No. 6019528, and it was confirmed that the strain was deficient in sporulation ability by the method described in that publication.

[0019] The Bacillus subtilis was inoculated into a liquid medium (yeast extract: 2%, glucose: 5%, tap water: 93%) and cultured at 37°C for 24 hours with aeration and agitation, followed by heat sterilization at 90°C for 10 minutes. The medium was then removed using a centrifuge, and the collected bacterial cells were washed with tap water and then further collected by solid-liquid separation using a centrifuge. The collected bacterial cells were dried using a spray dryer to obtain a killed Bacillus subtilis cell powder (cell count: 6.4 x 10 11 The number of live bacteria was less than 100 cfu / g.

[0020] (2. Confirmation of the effect of Bacillus subtilis administration in mice) Nine-week-old senescence-accelerated mice (SAMP8) were divided into three groups: the control group (10 mice) was fed MF feed (manufactured by Oriental Yeast Co., Ltd.), the 0.1% Bacillus subtilis administration group (8 mice) was fed MF feed containing 0.1% of the bacterial cell powder obtained in 1 above, and the 1.0% Bacillus subtilis administration group (8 mice) was fed MF feed containing 1% of the bacterial cell powder obtained in 1 above, and they were raised until they reached 34 weeks of age. Feed and water were available ad libitum during the test period.

[0021] (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. For the open field test, a single mouse was released into the field of a small animal behavior analyzer (SCANET, Melquest) and allowed to explore freely for 60 minutes. Observations and measurements were then taken. The field consisted of a central area (Area A: 10.8 × 10.8 cm), a surrounding area (Area B: 26.4 × 26.4 cm), and a peripheral area (Area C: 45.0 × 45.0 cm) facing the wall. The locomotion (proportional to the distance traveled), time spent (seconds), and number of entries in Area A are shown in Figures 1–3. For the light-dark box test, a light-dark cage was placed inside the same apparatus, and the number of rearings in each area was measured. The results are shown in Figure 4.

[0022] Normally, when animals are exposed to a large, novel environment, they explore by repeatedly standing and walking. However, they also exhibit a behavioral pattern known as "thigmotaxis," in which they touch the walls and prefer to walk around the periphery. This is due to anxiety-like behavior in innate and latent emotional behavior, and it is known that the rate of exploration toward the center of the field is extremely low. On the other hand, the SAMP8 strain of senescence-accelerated mice is known to exhibit emotional disorders, in which anxiety-like behavior declines with age (Japanese Pharmacological Journal, 2010, Vol. 136, No. 1, pp. 6-10).

[0023] The control group showed more entries, locomotion, and time spent in Area A (the center of the field) than the B. subtilis-treated group, and there were significant differences between these groups and the 1% B. subtilis-treated group (Figures 1-3), indicating that B. subtilis treatment maintained anxiety-like behavior. Furthermore, at 20 weeks of age, all three groups showed a similar behavioral pattern of rearing (exploratory behavior) in the light and dark compartments, with the number of rearings in the light compartment being significantly greater than in the dark compartment. However, at 28 weeks of age, as aging progressed, only the 1% B. subtilis-treated group showed a significant difference in the number of rearings (exploratory behavior) in the light and dark compartments, as in the 20-week-old group, while the differences disappeared in the other groups (Figure 4). This suggests that B. subtilis treatment led to differences in brain function due to aging. In other words, the B. subtilis-treated group may have improved emotional disorders, which are associated with a decline in anxiety-like behavior with age, compared to the control group.

[0024] (2-2. Confirmation of TTR deposition in brain tissue) At 34 weeks of age, the rats were sacrificed and dissected. Brain tissue was stained with a specific antibody to evaluate the deposition of transthyretin (TTR), an amyloidogenic protein. Hippocampal sections were prepared from the control group and the 0.1% and 1.0% B. subtilis groups and immunostained with an anti-TTR antibody. The incidence of TTR plaque burden is shown in Table 1. The number of TTR deposits per area (1.00 × 0.75 mm) (diameters ≤ 50 μm (50 μm ≤ D), > 50 μm (50 μm > D), and total) was calculated and shown in Figure 5.

[0025] [Table 1]

[0026] TTR deposition occurs due to the aggregation of TTR, and it is known that aggregated TTR in the brain activates microglia and causes neuroinflammation. Therefore, it is thought that reducing TTR deposition can prevent neuroinflammation and protect brain cells (Bioscience, Biotechnology, and Biochemistry 2018, Vol. 82, No. 4, pp. 647-653).

[0027] As shown in Figure 5 and Table 1, the number of TTR deposits per area in hippocampal brain cells was lower in the Bacillus subtilis-treated group compared to the control group, and in particular, the 1% Bacillus subtilis-treated group had significantly (p<0.05) fewer TTR deposits than the control group. This suggests that Bacillus subtilis administration suppressed TTR deposition in the hippocampus, prevented neuroinflammation, and protected brain cells.

[0028] These findings suggest that emotional disorders, which are thought to be caused by age-related decline in brain function, are suppressed and improved by protecting brain cells, particularly in the hippocampus, which is responsible for emotional memory. Therefore, administration of Bacillus subtilis inhibits TTR deposition in the hippocampus, leading to preventive effects on neuroinflammation and protective effects on brain cells, thereby suppressing decline in brain function and improving emotional disorders.

Claims

1. An agent for inhibiting transthyretin (TTR) deposition in the hippocampus, the active ingredient of which is a spore-forming deficient strain of Bacillus subtilis obtained by genetic recombination or mutation.

2. 2. The transthyretin (TTR) deposition inhibitor according to claim 1, wherein the active ingredient is a bacterium obtained by liquid culture of Bacillus subtilis.

3. 3. The transthyretin (TTR) deposition inhibitor according to claim 1 or 2, which contains killed bacteria as an active ingredient.

4. A food, drink, pharmaceutical, or feed for inhibiting transthyretin (TTR) deposition, comprising the transthyretin (TTR) deposition inhibitor according to any one of claims 1 to 3.

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