Sleep aids

Abstract

To provide a novel sleep-improving agent derived from a natural product.SOLUTION: The present invention has been completed after discovering that Bacillus subtilis has a sleep-improving effect.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a sleep improvement agent containing Bacillus subtilis as an active ingredient.

Background Art

[0002] Sleep is essential for humans. Lack of sleep or poor-quality sleep can cause stress to the body and may lead to sleep disorders such as insomnia. Also, sleep is known to affect human memory and cognitive functions, and there is an increasing interest in sleep. Currently, it is said that about 40% of people feel sleep deprivation or daytime sleepiness, and there is an active movement to improve the quality of sleep through foods that are particularly easy to incorporate into daily life.

[0003] For example, a composition for improving sleep quality (Patent Document 1) containing at least one of lactic acid bacteria such as Lactobacillus delbrueckii and its metabolites, and a composition for promoting sleep (Patent Document 2) containing one or more lactic acid bacteria whose optical activity of the produced lactic acid is classified other than the L-type are known.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0005] The present invention provides a sleep improvement agent derived from natural products.

Means for Solving the Problems

[0006] The inventors have found that Bacillus subtilis has a sleep improvement effect and completed the present invention.

[0007] In other words, the present invention relates to the following embodiments [1] to [5]. [1] A sleep aid containing Bacillus subtilis as an active ingredient. [2] A sleep aid as described in [1], comprising dead Bacillus subtilis cells as the active ingredient. [3] The sleep aid according to [1] or [2], wherein the Bacillus subtilis strain is a spore-forming-deficient strain. [4] A sleep aid according to [1] or [2], wherein the sleep improvement is one or more of the following: improvement of drowsiness upon waking, improvement of the feeling of fatigue recovery through sleep, or improvement of the feeling of sleep deprivation. [5] A sleep aid as described in [3], wherein the sleep improvement is one or more of the following: improvement of drowsiness upon waking, improvement of the feeling of fatigue recovery through sleep, or improvement of the feeling of sleep deprivation. [Effects of the Invention]

[0008] This invention provides a sleep aid derived from natural products. Furthermore, since sleep-improving effects are observed in dead bacteria after sterilization, using dead Bacillus subtilis cells simplifies hygiene management of manufacturing facilities and quality control of products, enabling efficient production of sleep aids. In addition, because the active ingredient is not a chemically synthesized product but a bacterium with a history of being consumed, it is ideal as a sleep aid that is desirable for continuous, long-term intake. Moreover, by using a spore-forming-deficient strain, sterilization can be performed under mild conditions below 100°C, similar to general microorganisms, thus preventing contamination of manufacturing facilities due to insufficient sterilization, which is a problem with spore-forming bacteria, and making it easy to add to various foods and formulate them. [Modes for carrying out the invention]

[0009] The sleep aid of the present invention contains Bacillus subtilis as an active ingredient, and upon ingestion, it can improve morning drowsiness, enhance the feeling of fatigue recovery through sleep, or alleviate feelings of sleep deprivation.

[0010] The Bacillus subtilis described in this invention may be live or dead, as long as it is the active ingredient of the functional preparation, and is not particularly limited. However, Bacillus subtilis subsp. subtilis is preferred, and Bacillus subtilis NBRC3009, Bacillus subtilis NBRC3013, Bacillus subtilis NBRC3335, Bacillus subtilis NBRC13169, and other natto bacteria are more preferred and can be obtained from the National Institute of Technology and Evaluation, etc. Since dead bacteria also have a sleep-improving effect, using dead Bacillus subtilis cells makes it easier to manage hygiene in manufacturing facilities and quality control of products, and allows for the efficient production of sleep-improving agents. Furthermore, by using a spore-forming-deficient strain, dead cells can be prepared under mild sterilization conditions of 100°C or less, thus preventing contamination of manufacturing facilities due to insufficient sterilization, which is a problem with spore-forming bacteria, and facilitating addition to various foods and formulation.

[0011] The Bacillus subtilis used in the present invention is not particularly limited, but a spore-forming ability-deficient strain is preferred. Examples of methods for obtaining a spore-forming ability-deficient strain include genetic recombination and spontaneous mutation, but spontaneous mutation is preferred. The method for obtaining a spore-forming ability-deficient strain by spontaneous mutation is not particularly limited, and examples include high-temperature culture, a random method for distinguishing wild-type and deficient strain colonies by the coloration of melanin pigment, and a method utilizing a catabolite repression-like phenomenon (JFMichel, B.Cami, P.Schaeffer: Ann.Inst.Pasteur, 114, 11; 21 (1968)), but the method utilizing a catabolite repression-like phenomenon is preferred. Spore-forming ability-deficient strains obtained by a method utilizing a catabolite repression-like phenomenon lack spore-forming ability, as well as lysozyme activity and transformation ability. Therefore, they can be subcultured without problems due to lysis and the trait of spore-forming ability-deficient can be maintained.

[0012] For culturing Bacillus subtilis, standard bacterial culture media can be used. Both synthetic and natural media are acceptable, as long as they contain a carbon source, nitrogen source, inorganic substances, and other micronutrients required by Bacillus subtilis. Suitable carbon sources include glucose, sucrose, dextrin, starch, glycerin, and molasses. Suitable 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; and nitrogen-containing natural products such as peptone, meat extract, yeast extract, malt extract, and corn steep liquor. Suitable inorganic substances include monosodium phosphate, disodium phosphate, monopotassium phosphate, dipotassium phosphate, magnesium sulfate, and ferric chloride.

[0013] The culture conditions for Bacillus subtilis can be set as appropriate, but it is preferable to culture it aerobically in liquid by aeration, shaking, stirring, etc. The culture temperature can be exemplified as 20 to 50°C, preferably 30 to 45°C, the culture time can be exemplified as 2 to 72 hours, preferably 4 to 48 hours, and more preferably 6 to 36 hours, and the pH of the culture medium can be exemplified as 5.0 to 9.0, preferably 5.5 to 8.5.

[0014] Bacillus subtilis can be sterilized after culturing, and the sterilization conditions are not particularly limited as long as they are general methods. For example, the heating temperature is 70-150°C, and the heating time can be determined according to the temperature, but is usually 1-60 minutes. Furthermore, the spore-forming ability-deficient strain described in the present invention does not form spores, so it can be sterilized under mild conditions of 100°C or less, for example, heating at 70-100°C for 5-20 minutes. The bacterial cells can be collected by removing the culture medium with a centrifuge, washing the cells with buffer solution, physiological saline, sterile water, etc., and then separating the solid and liquid using a centrifuge. In addition, the cells may be powdered by air drying, spray drying, vacuum and / or freeze-drying.

[0015] The sleep-improving agent of the present invention has an active ingredient derived from natural products and is easy to manufacture, making it widely applicable and suitable for addition to various products. It may be added in liquid form, or in a refrigerated, frozen, or dried state, allowing for the preparation of foods, beverages, pharmaceuticals, animal feed, etc., that have sleep-improving effects. The amount of Bacillus subtilis contained in various products is not particularly limited as long as the amount that exhibits sleep-improving effects upon ingestion is sufficient, but 1 to 200 billion cells is preferred, 2 to 100 billion cells is more preferred, and 5 to 50 billion cells is even more preferred. [Examples]

[0016] 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 percentages are by weight unless otherwise specified.

[0017] (1. Preparation of Bacillus subtilis cells) By spontaneous mutation utilizing a phenomenon similar to catabolic suppression, a spore-forming-deficient strain, Bacillus subtilis NBRC13169, a type of Bacillus subtilis, was developed, resulting in a strain lacking spore-forming ability: The spontaneous mutation using TIFF0007851590000001.tif8170 was performed using the method described in the examples of Japanese Patent Publication No. 6019528, and it was confirmed that the strain lacked spore-forming ability using the method described in the said publication.

[0018] The Bacillus subtilis strain was inoculated into a liquid culture medium (yeast extract: 2%, glucose: 5%, tap water: 93%) and cultured at 37°C for 24 hours with aeration and stirring, then heat-sterilized at 90°C for 10 minutes. Next, the culture medium was removed using a centrifuge, the recovered cells were washed with tap water, and then the cells were recovered by solid-liquid separation using a centrifuge. The recovered cells were dried with a spray dryer to prepare a powder of dead Bacillus subtilis cells. Using this Bacillus subtilis strain, QOL Natto Bacteria (registered trademark) (1.0 × 10⁻⁶) 11 A sample (individuals / g) was prepared, and the following sleep evaluation test was conducted.

[0019] (2. Sleep evaluation test) Sixty healthy adults with sleep problems were divided into two groups: a test diet intake group and a control diet intake group. They were each given one capsule once a day for eight consecutive weeks. The breakdown of the contents of the test diet capsules was as follows: Bacillus subtilis: 200 mg, Pindex #100: 147 mg, and calcium stearate: 3 mg (total: 250 mg). The breakdown of the contents of the control diet capsules was as follows: Sandex #300: 200 mg, Pindex #100: 147 mg, and calcium stearate: 3 mg (total: 250 mg). The subjects in each group were surveyed about their sleep using the OSA Sleep Survey Form MA version (Yamamoto Yurika et al.: Development and Standardization of the OSA Sleep Survey Form (MA version) for Middle-Aged and Elderly Persons. Brain and Psychiatry, vol. 10, p. 401-409, 1999) before ingestion, at the 4th week of ingestion, and at the 8th week of ingestion. Since one subject was excluded from the analysis in each group, the final number of subjects for analysis was 29 in each group.

[0020] The OSA Sleep Survey Form MA version is a psychological scale that evaluates sleep introspection upon waking up from five factors: the first factor (morning sleepiness), the second factor (falling asleep and maintaining sleep), the third factor (dreaming), the fourth factor (recovery from fatigue), and the fifth factor (sleep duration). The five factors consist of 16 question items. The score for each factor is calculated from the selected psychological scale, and the higher the score, the better the sleep. For the scores of each factor obtained from the results of this survey, a one-sample t-test was performed in each group to test whether there was a difference between before ingestion, at the 4th week of ingestion, and at the 8th week of ingestion. The results are shown in Table 1.

[0021]

Table 1

[0023] Based on the above, significant improvements were observed in Factor 1 (morning drowsiness), Factor 4 (fatigue recovery), and Factor 5 (sleep duration). In the case of Factor 5 (sleep duration), it was considered that the effects of Bacillus subtilis become apparent with long-term intake. Since it was confirmed that Bacillus subtilis is effective in improving morning drowsiness, improving the feeling of fatigue recovery through sleep, and improving the feeling of sleep deprivation, it became clear that ingesting Bacillus subtilis improves the feeling of sleepiness upon waking and improves the quality of sleep.

Claims

1. A sleep-improving agent comprising dead Bacillus subtilis cells as an active ingredient, obtained by culturing in a liquid medium with aeration and stirring, and drying the cells recovered after a heat sterilization process, for the purpose of improving morning drowsiness, improving the feeling of fatigue recovery through sleep, or improving the feeling of sleep deprivation.

2. The sleep aid according to claim 1, wherein the Bacillus subtilis strain is a spore-forming ability-deficient strain.

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