Anti-stress composition

Abstract

To provide a novel anti-stress composition that utilizes a natural-origin component as an active ingredient.SOLUTION: The present invention was completed based on finding that Bacillus subtilis exhibits superior anti-stress effects.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to an anti-stress composition and a food or drink containing the same. [Background technology]

[0002] The continuous stress load in today's stressful society affects autonomic nervous function, causing stress symptoms such as fatigue, anxiety, poor sleep, decreased sociability, decreased motivation, decreased communication skills, and decreased thinking ability, and can lead to mental illnesses such as depression and anxiety disorders.

[0003] Therefore, there is a strong demand for the development of foods and pharmaceuticals that can alleviate such continuous stress, and various studies have been conducted. For example, an anti-stress composition containing Lactobacillus helveticus as an active ingredient (Patent Document 1), an anti-stress composition containing killed Bifidobacterium breve cells (Patent Document 2), and an anti-stress composition containing at least one active ingredient selected from the group consisting of Bifidobacterium bifidum cells, bacterial components, cultures, and processed products thereof (Patent Document 3) are known. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2019 / 188868 Brochure [Patent Document 2] Patent Publication No. 2021-153446 [Patent Document 3] Patent Publication No. 2022-118901 Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention provides a novel anti-stress composition containing a component derived from a natural product as an active ingredient. [Means for solving the problem]

[0006] The inventors have discovered that Bacillus subtilis has an excellent anti-stress effect, and have completed the present invention.

[0007] That is, the present invention relates to the following aspects [1] to [5]. [1] An anti-stress composition containing Bacillus subtilis as an active ingredient. [2] The anti-stress composition according to [1], wherein the Bacillus subtilis is a strain deficient in sporulation. [3] The anti-stress composition according to [1] or [2], wherein the anti-stress use is for reducing chronic social defeat stress or improving vitality. [4] The anti-stress composition according to [3], wherein the anti-stress use is for suppressing a decline in sociability, a rise in anxiety, a decline in motivation, a depression, or an increase in stress hormones, or for improving a negative mood state caused by stress. [5] A food, drink, cosmetic, pharmaceutical, or feed containing the anti-stress composition according to any one of [1] to [4]. [Effects of the Invention]

[0008] The present invention provides a novel anti-stress composition containing a naturally occurring component as an active ingredient. Furthermore, because the anti-stress effect is observed in killed bacteria after sterilization, the use of killed bacteria facilitates hygiene control of production facilities and product quality control, allowing for efficient production of anti-stress compositions. Furthermore, because the active ingredient is not a chemically synthesized product but a bacteria that has been used as a food, it is ideal as an anti-stress composition for which continuous, long-term ingestion is desired. [Brief explanation of the drawings]

[0009] [Figure 1] This shows a social avoidance test device having area (a) (a mesh cage placed upside down), area (b) (interaction zone), and area (c) (corner zone). [Figure 2]As the results of the social avoidance test for Evaluation 1, the difference (seconds) in the time spent in the Interaction Zone and Corner Zone (Measurement 2 - Measurement 1) is shown for the three groups: the non-stress / control diet group (Comparative Example 1-1), the stress / control diet group (Comparative Example 1-2), and the stress / test diet group (Example 1). [Figure 3] As the results of the elevated plus maze test for Evaluation 2, the number of entries (times) into the wall-less passage and the time spent there (seconds) are shown for the three groups: the non-stress / control diet group (Comparative Example 1-1), the stress / control diet group (Comparative Example 1-2), and the stress / test diet group (Example 1). [Figure 4] As the results of the tail suspension test for Evaluation 3, the idle time (seconds) when suspended for 6 minutes is shown for the three groups: the non-stress / control diet group (Comparative Example 1-1), the stress / control diet group (Comparative Example 1-2), and the stress / test diet group (Example 1). [Figure 5] As the measurement results of serum corticosterone concentration in Evaluation 4, the serum corticosterone concentration (ng / mL) of mice in three groups: non-stress / control diet group (Comparative Example 1-1), stress / control diet group (Comparative Example 1-2), and stress / test diet group (Example 1) is shown. [Figure 6] The POMS2 measurement results show the average values ​​for each item in the test food group before and after 8 weeks of intake. DETAILED DESCRIPTION OF THE INVENTION

[0010] The present invention relates to an anti-stress composition containing Bacillus subtilis as an active ingredient. Oral administration of the composition can reduce chronic social defeat stress, specifically, suppressing a decline in sociability, increased anxiety, decreased motivation, depressed states, or increased stress hormones. Furthermore, the composition has the effect of improving vitality, thereby improving negative mood states caused by stress. The composition of the present invention can be used to prevent or reduce stress, and is particularly effective against chronic stress. Reducing chronic social defeat stress can reduce mental stress, such as the sense of defeat felt due to harassment, such as power harassment. The composition can be administered to any animal susceptible to stress, including humans, livestock, companion animals, and aquaculture animals, but is particularly applicable to mammals such as humans.

[0011] In the present invention, the prevention or reduction of stress can be confirmed by assessing stress states using, for example, POMS (Registered Trademark) (Profile of Mood States) or POMS2 (Profile of Mood States 2nd Edition). POMS measures six scales (anger / hostility, confusion, depression / depression, fatigue, tension / anxiety, and vitality) and overall mood state, while POMS2 measures seven scales (anger / hostility, confusion / bewilderment, depression / depression, fatigue / apathy, tension / anxiety, vitality / energy, and friendliness) and overall mood state. The overall mood state is calculated by subtracting the vitality score from the sum of the scores for the anger / hostility, confusion, depression / depression, fatigue, and tension / anxiety items in POMS, or by subtracting the vitality score from the sum of the scores for the anger / hostility, confusion / bewilderment, depression / depression, fatigue / apathy, and tension / anxiety items in POMS2.

[0012] In the present invention, "improvement of negative mood states caused by stress" means improvement of mood states such as anger, hostility, confusion, embarrassment, depression, downcast, fatigue, lethargy, tension, and anxiety caused by stress, and specifically means a reduction in the score of negative mood states assessed by POMS or POMS2, etc.

[0013] The Bacillus subtilis of the present invention may be live or killed, as long as it is a Bacillus subtilis that serves as an active ingredient in an anti-stress composition, and is not particularly limited. However, B. subtilis subsp. subtilis is preferred, and natto bacteria such as B. subtilis NBRC3009, B. subtilis NBRC3013, B. subtilis NBRC3335, and B. subtilis NBRC13169 are more preferred, and these are available from the National Institute of Technology and Evaluation, etc. Furthermore, if a spore-forming deficient strain is used, killed cells can be prepared under mild sterilization conditions of 100°C or below, which prevents contamination of manufacturing equipment due to insufficient sterilization, a problem associated with spore-forming bacteria, and facilitates addition to various foods and formulation.

[0014] The Bacillus subtilis used in the present invention is not particularly limited, but is preferably a sporulation-deficient strain. Examples of methods for obtaining sporulation-deficient strains include genetic recombination and mutation. Spontaneous mutation is preferred, and genetic sporulation-deficient strains obtained by genetically abolishing sporulation are most 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 strains are distinguished by melanin pigment coloration of colonies, and a method utilizing catabolite repression-like phenomenon (J.F. Michel, B. Cami, P. Schaeffer: Ann. Inst. Pasteur, 114, 11; 21 (1968)). Catabolite repression-like phenomenon is preferred. The sporulation-deficient strains obtained by the method utilizing the catabolite repression-like phenomenon lack not only sporulation ability but also lysozyme activity and transformability, and therefore can be passaged without problems due to bacteriolysis and can maintain the trait of sporulation deficiency.

[0015] Conventional bacterial culture media can be used to culture Bacillus subtilis, including synthetic and natural media, as long as they contain carbon sources, nitrogen sources, inorganic substances, and other micronutrients required by the bacteria. Examples of carbon sources that can be used include glucose, sucrose, dextrin, starch, glycerin, sorbitol, citric acid, 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.

[0016] Culture conditions can be set appropriately, but aerobic liquid culture using aeration, shaking, stirring, etc. is preferred. The culture temperature can be, for example, 10 to 50°C, and preferably 20 to 40°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. The pH of the medium can be, for example, 4.0 to 9.0, and preferably 5.0 to 7.5.

[0017] Sterilization may be performed after the culture, and the 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. Both live and dead bacteria have an anti-stress effect, but it is preferable to use dead bacteria, as the use of dead bacteria facilitates hygiene control of production facilities and quality control of products, and enables the efficient production of anti-stress compositions, etc.

[0018] 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 collecting the bacterial cells by solid-liquid separation using a centrifuge or the like. Furthermore, the bacterial cells may be powdered by air drying, spray drying, vacuum and / or freeze drying, or the like.

[0019] The anti-stress composition of the present invention has a wide range of uses, as its active ingredient is derived from natural products and is easy to produce. It can be added to various products, whether in liquid, refrigerated, frozen, or dried form, and can be used to prepare foods and beverages, cosmetics, pharmaceuticals, feed, etc. that have anti-stress effects. The bacterial cell content in various products is not particularly limited as long as the effect is observed when ingested, but is preferably 0.01 to 20% by mass, more preferably 0.05 to 10% by mass, even more preferably 0.1 to 5% by mass, preferably 1 to 200 billion cells, more preferably 2 to 100 billion cells, and even more preferably 5 to 50 billion cells. [Example]

[0020] 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 % means % by mass unless otherwise specified.

[0021] (Preparation 1: Preparation of killed cell powder of sporulation-deficient strain of Bacillus subtilis) QOL Natto Bacillus subtilis (registered trademark) (manufactured by Ikeda Tohka Kogyo Co., Ltd.), a sporulation-deficient strain, was inoculated into a liquid medium (yeast extract: 2%, glucose: 5%, tap water: 93%) and cultured at 37°C for 24 hours with agitation and aeration, followed by heat sterilization at 90°C for 10 minutes (general viable cell count: less than 10 cells / g). The medium was then removed using a centrifuge, and the collected cells were washed with tap water and then further collected by solid-liquid separation using a centrifuge. The collected cells were dried using a spray dryer, and a killed cell powder (1.1 x 10 6 QOL Natto Bacillus (registered trademark) is a spore-forming deficient strain that was prepared from Bacillus subtilis NBRC13169, a type of Bacillus subtilis, by spontaneous mutation utilizing a catabolite repression-like phenomenon.

[0022] Example 1: Effect of Bacillus subtilis on social defeat stress (Social defeat stress load) We used an experimental model in which physically inferior male mice (evaluation mice) were placed in an environment where they were intimidated and attacked by physically superior, aggressive male mice (attack mice), causing the evaluation mice to experience social defeat (Science. 2006 Feb 10; 311(5762): 864-8. doi: 10.1126 / science.1120972.). After 10 days of social defeat stress, the evaluation mice were subjected to behavioral tests (evaluation 1: social avoidance test, evaluation 2: elevated plus maze test, and evaluation 3: tail suspension test) and serum corticosterone levels were measured (evaluation 4), to evaluate the anti-stress effect of Bacillus subtilis administration.

[0023] Five-week-old C57BL / 6J male mice were used as evaluation mice, and after one week of acclimation, they were divided into three groups: a non-stress / control diet group (12 mice) (Comparative Example 1-1), a stress / control diet group (12 mice) (Comparative Example 1-2), and a stress / test diet group (12 mice) (Example 1). 20-week-old ICR male mice were used as attacking mice, and 24 mice were selected in a five-day screening test using their aggression toward 7-week-old C57BL / 6J male mice as an index. The non-stress / control diet group and the stress / control diet group were given MF feed (manufactured by Oriental Yeast Co., Ltd.), and the stress / test diet group was given MF feed (manufactured by Oriental Yeast Co., Ltd.) containing 1% Bacillus subtilis cells obtained in Preparation 1, which was given ad libitum for 27 days from 14 days before the social defeat stress was imposed until the end of the behavioral test.

[0024] The stress / control diet and stress / test diet groups were subjected to continuous social defeat stress for 10 days. Social defeat stress was administered to one evaluation mouse and one attacking mouse in a cage designed specifically for social defeat stress administration, with a floor area of ​​225 × 338 mm2. The cage was divided into two halves by a transparent partition with a small hole that prevented the mice from passing through: an evaluation mouse living area and an attacking mouse living area.

[0025] On the first day of social defeat stress, the evaluation mice were placed in the attack mouse living area where the attack mouse had been residing for 3 days prior to the start of social defeat stress. They were then intimidated and attacked for 300 seconds. The evaluation mice were then transferred to the evaluation mouse living area and allowed to behave freely under conditions that allowed both mice to recognize each other until the next day's social defeat stress. From the second day onward, the time the evaluation mice spent in the attack mouse living area was gradually reduced by 30 seconds each day. On the 10th day of social defeat stress, the evaluation mice were placed in the attack mouse living area for 30 seconds, intimidated and attacked, and then transferred to standard cages (one mouse per cage) instead of the evaluation mouse living area and allowed to behave freely. From then on, the evaluation mice were transferred to standard cages (one mouse per cage) and allowed to behave freely until the end of the evaluation.

[0026] (Rating 1: Social Avoidance Test) One day after the social defeat stress test (for 10 days), a social avoidance test was conducted using the apparatus shown in Figure 1 to evaluate the effect on sociality. An empty mesh cage (7.2 cm wide x 7.2 cm deep x 11 cm high) was placed face down in position (A) of the apparatus. The mouse was then released into the apparatus and allowed to freely explore the apparatus for 150 seconds. Its behavior was recorded with a video camera, and the time spent in area (B) (interaction zone) and area (C) (corner zone) was measured (Measurement 1; habituation). Next, a mesh cage containing an attacking mouse, which the mouse had never encountered before, was placed face down in position (A) of the apparatus. The mouse was then released into the apparatus and allowed to freely explore the apparatus for 150 seconds. Its behavior was recorded with a video camera, and the time spent in area (B) (interaction zone) and area (C) (corner zone) was measured (Measurement 2; interaction). Longer time spent in the interaction zone and shorter time spent in the corner zone are considered to indicate higher sociability. The sociality of the mice can be evaluated by subtracting the values ​​from Measurement 1 from the values ​​from Measurement 2. The results are shown in Figure 2. All values ​​represent mean values ​​± standard error.

[0027] As a result of the test, the difference in the time spent in the interaction zone for the stress / control diet group (measurement 2 - measurement 1) was significantly smaller than that for the non-stress / control diet group (p<0.05), and the difference in the time spent in the corner zone for the stress / control diet group (measurement 2 - measurement 1) was greater than that for the non-stress / control diet group, confirming that social defeat stress reduces the sociality of mice. The difference in time spent in the interaction zone (measurement 2 - measurement 1) for the stress / test food group was smaller than that for the non-stress / control food group, but larger than that for the stress / control food group. Also, the difference in time spent in the corner zone (measurement 2 - measurement 1) for the stress / test food group was larger than that for the non-stress / control food group, but smaller than that for the stress / control food group. From the above, it is thought that the ingestion of Bacillus subtilis cells reduced the social defeat stress load and suppressed the decline in sociality.

[0028] (Evaluation 2: Elevated Plus Maze Test) Two days after the social defeat stress (10 days), an elevated plus maze test was performed using an elevated plus maze apparatus to evaluate the effect on anxiety-like behavior. The elevated plus maze apparatus (Horikawa Seisakusho Co., Ltd.) was installed at a height above the floor under conditions of 200 lux illumination. The maze, consisting of walled and unwalled passageways, was free-standing, with no walls in the central area where the passageways intersected. Mice were released into the walled passageway within the apparatus and allowed to freely explore the apparatus for 300 s. Their behavior was recorded with a video camera, and four parameters were measured: the number of entries into the unwalled passageway, the time spent in the unwalled passageway, the number of entries into the walled passageway, and the time spent in the walled passageway. The number of entries was determined as the point when all four limbs entered the passageway from the central area. Since a higher number of entries into the unwalled passageway and a longer time spent in the unwalled passageway are considered to indicate less open-space-induced anxiety, these two parameters were compared. The results are shown in Figure 3. All values ​​represent the mean ± standard error.

[0029] The results of the test showed that the stress / control diet group entered the open corridor less frequently than the non-stress / control diet group, and spent less time in the open corridor than the non-stress / control diet group, confirming that social defeat stress increases anxiety in the mice. The stressed / test food group entered the open aisle more frequently than the non-stressed / control food group or the stressed / control food group, and spent more time in the open aisle than the non-stressed / control food group or the stressed / control food group. From the above, it is thought that the ingestion of Bacillus subtilis bodies reduced the social defeat stress load and suppressed the increase in anxiety.

[0030] (Evaluation 3: Tail suspension test) Three days after the social defeat stress load (10 days), a tail suspension test was performed using a tail suspension test apparatus (15 cm wide x 28 cm deep x 50 cm high) to evaluate the effect on depressive-like behavior. Mice were fitted with a silicone tube to prevent tail climbing, and the tails of the mice were fixed to the device and hung for 6 minutes. Their behavior was recorded with a video camera, and the time spent immobile was measured, with the mouse's limbs remaining motionless. This time is considered to indicate a loss of motivation and the mouse's abandonment of escape attempts, and can therefore be used to evaluate the mouse's depressive state. The results are shown in Figure 4. All values ​​represent the mean ± standard error.

[0031] The results of the test showed that the immobility time in the stress / control diet group was longer than that in the non-stress / control diet group, confirming that social defeat stress reduces the motivation of the mice and puts them into a depression-like state. The immobility time in the stress / test diet group was significantly shorter than that in the non-stress / control diet group or the stress / control diet group (p<0.05). From the above, it is considered that the ingestion of Bacillus subtilis bodies reduced the social defeat stress load, suppressed the decline in motivation, and prevented the occurrence of a depressive state.

[0032] (Assessment 4: Serum corticosterone concentration) Three days after the social defeat stress (10 days), serum samples were collected from the mice, and serum corticosterone concentrations were measured using an ELISA kit (Revvity). Corticosterone is a stress hormone, and its secretion is known to increase with stress. The results are shown in Figure 5. All values ​​represent the mean ± standard error. Furthermore, serum corticosterone concentrations were below the detection limit in five of the 12 mice in the non-stress / control diet group and seven of the 12 mice in the stress / test diet group; therefore, data were excluded. The final N numbers are shown in Figure 5.

[0033] The results of the test showed that serum corticosterone levels in the stress / control diet group were significantly higher than those in the non-stress / control diet group (p<0.05), confirming that social defeat stress increases serum corticosterone levels. Serum corticosterone levels in the stress / test diet group were lower than those in the non-stress / control diet group, and were significantly lower than those in the stress / control diet group (p<0.01). From the above, it is considered that the ingestion of Bacillus subtilis cells reduced the social defeat stress load and suppressed the increase in serum corticosterone concentration.

[0034] [Example 2. Effect of Bacillus subtilis on mental stress] The study involved 108 healthy men and women aged 24 to 89 who are prone to fatigue and stress from daily desk work. They were divided into two groups, a test food group with 54 participants each and a placebo group, and a double-blind, randomized, placebo-controlled, parallel-group comparative study to evaluate the stress-reducing effects of Bacillus subtilis on humans. The test food group took one capsule containing 10 billion QOL Natto Bacillus (registered trademark), and the placebo group took one capsule containing dextrin per day for eight consecutive weeks. Mood states before and after 8 weeks of intake were assessed using the POMS2 test. POMS2 is a test that evaluates mood states and measures seven scales (anger / hostility (AH), confusion / embarrassment (CB), depression / depression (DD), fatigue / lethargy (FI), tension / anxiety (TA), vitality / energy (VA), and friendliness (F)) and the general mood state (TMD). Lower scores for TMD, AH, CB, DD, FI, and TA, which evaluate negative mood states, indicate a better state, while higher scores for VA and F, which evaluate positive mood states, indicate a better state. To test for significant differences between the test food group and the placebo group, Welch's t-test was used before ingestion, and analysis of covariance (ANCOVA) was used after 8 weeks of ingestion, using the pre-ingestion measurement values ​​as covariates. The results are shown in Table 1 and Figure 6.

[0035] [Table 1]

[0036] As shown in Table 1, the results of the study showed that the vitality / energy scores of the test food group after 8 weeks were significantly higher than those of the placebo group (p<0.05), and the overall mood state scores of the test food group after 8 weeks were significantly lower than those of the placebo group (p<0.05). Furthermore, as shown in Figure 6, when comparing the average values ​​before and after 8 weeks of taking the test food group, all negative mood state items (overall mood state, anger / hostility, confusion / embarrassment, depression / depression, fatigue / lethargy, tension / anxiety) decreased, while all positive mood state items (liveliness / energy, friendliness) increased. From the above, it is thought that the intake of Bacillus subtilis bodies increased vitality, and the recovery of vitality improved negative mood states caused by stress and reduced mental stress.

[0037] The results of Examples 1 and 2 revealed that Bacillus subtilis has the effect of reducing chronic social defeat stress, specifically, it can suppress a decline in sociability, an increase in anxiety, a decrease in motivation, a depressed state, or an increase in stress hormones, and furthermore, it has the effect of improving vitality, thereby making it possible to improve negative mood states caused by stress, and it was shown that ingestion of a composition containing Bacillus subtilis as an active ingredient can prevent or reduce stress.

Claims

1. An anti-stress composition containing Bacillus subtilis as an active ingredient.

2. 2. The anti-stress composition according to claim 1, wherein the Bacillus subtilis is a strain deficient in sporulation ability.

3. 2. The anti-stress composition according to claim 1, wherein the anti-stress use is for alleviating chronic social defeat stress or for improving vitality.

4. The anti-stress composition according to claim 3, wherein the anti-stress use is for suppressing a decline in sociability, a rise in anxiety, a drop in motivation, a depression, or an increase in stress hormones, or for improving a negative mood state caused by stress.

5. A food, drink, cosmetic, pharmaceutical, or feed comprising the anti-stress composition according to any one of claims 1 to 4.

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