JP2024161120A - Sleep-promoting composition and food, medicine, and feed containing the composition - Google Patents

Abstract

To provide a sleep-encouraging composition capable of encouraging inducing sleep at night and superior in stability and safety; and a food, a pharmaceutical, and feed including the sleep-encouraging composition.SOLUTION: A sleep-encouraging composition contains as active principle microorganisms that belong to any one of Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, Weissella, and Bifidobacterium and culture thereof.SELECTED DRAWING: Figure 1

Description

The present invention relates to a sleep-promoting composition that is capable of promoting sleep and has excellent stability and safety. The present invention also relates to foods, medicines, and feeds that contain the sleep-promoting composition.

Sleep is a necessary behavior for animals to live, and sleep or sleep-like states exist not only in mammals but also in non-mammals such as fish, flies, and nematodes. In recent years, changes in lifestyle have disrupted daily rhythms, and the prevalence of sleep disorders such as insomnia has increased. According to a survey by the Ministry of Health, Labor and Welfare, one in five people suffers from some kind of sleep-related problem (Non-Patent Document 1).
It has become clear that sleep disorders are closely related not only to reduced labor productivity due to drowsiness, but also to mental illnesses such as dementia and depression, and lifestyle-related diseases such as obesity and diabetes. It is estimated that the economic loss due to lack of sleep is about 3.5 trillion yen (Non-Patent Document 2).

For people with sleep disorders such as insomnia, the use of pharmaceutical sleeping pills is considered. Barbiturate sleeping pills, benzodiazepine sleeping pills, and non-benzodiazepine sleeping pills are the most commonly used sleeping pills. However, barbiturate sleeping pills have strong side effects such as dependency, and benzodiazepine sleeping pills induce sleep that shows an abnormal frequency range called benzodiazepine fast waves, so they are known not to lead to physiological, good quality sleep.

For this reason, there has been active development of materials that can be taken daily instead of medicines and that can induce physiological sleep, and various materials made from natural ingredients and food ingredients have been proposed. For example, a deep sleep disorder improving agent using the amino acid glycine (Patent Document 1
) and a sleep-promoting composition using theanine (Patent Document 2). Lactic acid bacteria, which have been used for a long time in fermented foods and are highly safe for living organisms, include non-pathogenic lactic acid bacteria Lactobacillus acidophilus, Lactobacillus helveticus, and Streptococcus thermophilus, which are specific strains that have hypnotic activity (improving quality of sleep by increasing REM sleep and decreasing non-REM sleep) (Patent Document 3), sleep quality improvers using Lactobacillus casei (Patent Document 4), and circadian rhythm improvers using Lactobacillus brevis (Patent Document 5).

However, it cannot be said that there are enough options available to satisfy the diverse needs of consumers.

The fruit fly Drosophila melanogaster (hereinafter referred to as Drosophila) is used as a model organism in various fields of biology.
In 2000, the entire genome sequence was determined, and it is estimated that about 75% of genes related to human diseases are also present in Drosophila. In recent years, Drosophila disease model strains have been developed for Alzheimer's disease, Parkinson's disease, metabolic syndrome, and other diseases, and are actually used to elucidate the mechanisms of disease onset and screen therapeutic drugs (Non-Patent Documents 3 and 4).
).

In the field of sleep research, many studies using Drosophila have been conducted, and it has been revealed that Drosophila not only exhibits sleep-like behavior similar to that of humans, but also that many of its characteristics and molecular basis are common to mammalian sleep (Non-Patent Documents 5 and 6). Specific examples include age-dependent changes in sleep patterns (Non-Patent Document 7), homeostatic mechanisms of sleep (sleep behavior compensating for lost sleep (Non-Patent Document 8), the awakening effect of caffeine (Non-Patent Document 9), the sleep-promoting effect of glycine (Non-Patent Document 10), neurotransmitters such as dopamine, serotonin, and GABA, and the sleep-wake control effect of EGF (Non-Patent Documents 11, 12, and 13), which have been reported to be common between Drosophila and mammals. Furthermore, the pars intercerebralis of Drosophila, which is developmentally and functionally similar to the hypothalamus of mammals, has been reported to be common to mammals.
It is known that sleep-related neural circuits are evolutionarily conserved despite differences in nervous system structure, e.g., Drosophila cerebralis plays an important role in sleep, and wake-promoting neural circuits and sleep-promoting neural circuits are interconnected in Drosophila just as in mammals (Non-Patent Documents 5 and 6).

For these reasons, Drosophila is considered to be a suitable model organism for sleep research and is also useful for discovering substances involved in sleep regulation (Non-Patent Documents 5 and 6).

JP 2013-121961 A Japanese Patent Publication No. WO01/074352 Patent No. 4527922 Patent No. 6279714 Patent No. 5943342

FY2017 National Health and Nutrition Survey, Ministry of Health, Labour and Welfare <https://www.mhlw.go.jp/content/000451755.pdf> Makoto Uchiyama, Journal of the Japanese Psychiatric Hospital Association 31(11), 1163-1169, 2012-11, 2012 Pandey, U. B. & Nichols, C. D. Human Disease Models in Drosophila melanogaster and the Role of the Fly in Therapeutic Drug Discovery. Drug Deliv. 63, 411-436 (2011) Ugur, B., Chen, K. & Bellen, H. J. Drosophila tools and assays for the study of human diseases. Dis. Model. Mech. 9, 235-244 (2016). Zimmerman, J. E., Naidoo, N., Raizen, D. M. & Pack, A. I. Conservation of sleep: insights from non-mammalian model systems. Trends Neurosci. 31, 371-376 (2008). Ly, S., Pack, A. I. & Naidoo, N. The neurobiological basis of sleep: Insights from Drosophila. Neurosci. Biobehav. Rev. 87, 67-86 (2018). Koh, K., Evans, J. M., Hendricks, J. C. & Sehgal, A. A Drosophila model for age-associated changes in sleep:wake cycles. Proc. Natl. Acad. Sci. U. S. A. 103(37), 13843-13847 (2006 ) Huber, R. et al. Sleep homeostasis in Drosophila melanogaster. Sleep 27, 628-639 (2004) Wu, M. N. et al. The effects of caffeine on sleep in Drosophila require PKA activity, but not the adenosine receptor. J Neurosci 29(35), 11029-11037 (2009) Kazuhiko Kume, Search for a new sleep control mechanism using Drosophila melanogaster, Uehara Memorial Foundation Research Reports, 30 (2016) Ueno, T. et al. Identification of a dopamine pathway that regulates sleep and arousal in Drosophila. Nat. Neurosci. 15, 1516-1523 (2012) Sehgal, A. & Mignot, E. Genetics of sleep and sleep disorders. Cell. 146, 194-207 (2011). Foltenyi, K et al. Neurohormonal and neuromodulatory control of sleep in Drosophila. Cold Spring Harb Symp Quant Biol. 72, 565-571 (2007)

An object of the present invention is to provide a sleep-promoting composition that is capable of promoting sleep and has excellent stability and safety. Another object of the present invention is to provide a food, medicine, or feed containing the sleep-promoting composition.

The present inventors have conducted extensive research to solve the above problems and have found that a variety of microorganisms promote sleep.
That is, the present invention includes the following aspects.
(1) A sleep-promoting composition comprising one or more microorganisms belonging to the genera Lactobacillus, Lactococcus, or Bifidobacterium.
(2) The sleep-promoting composition according to (1), wherein the sleep-promoting composition is a composition for promoting sleep induction.
(3) The Lactobacillus genus is Lactobacillus paracasei subspecies tolerans (Lac
tolerans species, Lactobacillus paracasei subsp. tolerans species, Lactobacillus rhamnosus species,
Lactobacillus salivarius subspecies salicinius (Lactobacillus salivarius subspecies salicinius)
The sleep-promoting composition according to any one of (1) and (2), wherein the sleep-promoting composition is one or more selected from the species Lactobacillus livarius subsp. salicinius.
(4) The genus Lactococcus is Lactococcus lactis subsp. lactis (Lac
Lactococcus lactis subsp. lactis
The sleep-promoting composition according to (1) or (2), wherein the lactobacillus species is one or more selected from the Lactococcus raffinolactis species.
(5) The genus Bifidobacterium is Bifidobacterium faecalis
obacterium faecalis species, Bifidobacterium longum subsp. longum
) species, Bifidobacterium longum subsp. infantis (Bifidobact
Bifidobacterium longum subsp. infantis species, Bifidobacterium pseudocatenulatum
ulatum species, Bifidobacterium pseudolongum subsp. globosum (Bi
fidobacterium pseudolongum subsp. globos
um species, Bifidobacterium thermophilum
The sleep-promoting composition according to claim 1, wherein the sleep-promoting composition is one or more selected from the group consisting of Thermophilum thermophilum species.
(6) A sleep-promoting food, a sleep-promoting medicine, or a sleep-promoting feed, comprising the sleep-promoting composition according to any one of (1) to (5).

The present invention provides a sleep-promoting composition capable of promoting sleep, and in this application, "promoting sleep" refers to promoting sleep onset (increasing the amount of sleep in the early part of the night). That is, in one aspect, the present invention provides a sleep-promoting composition.

FIG. 13 is a graph comparing the relative values (rates of increase in sleep amount) of the median amount of sleep in each test group to the median amount of sleep in the control group, depending on the genus of microorganism. FIG. 13 shows the sleep onset latency in the groups administered with each microorganism.

The sleep-promoting composition of the present invention will be described. The active ingredient of the present invention is a microorganism belonging to the genera Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, Weissella, and Bifidobacterium, and any of these can be used as long as they have the effect of promoting sleep induction.

The species is not particularly limited as long as it is of the genus Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, Weissella, or Bifidobacterium, but a preferred example is Lactobacillus acidophilus.
Lactobacillus amylovorus species, Lactobacillus amylovorus
vorus species, Lactobacillus delbrueckii subsp. bulgaricus (Lactobacillus
Bulgaricus species, Lactobacillus delbrueckii subsp. Bulgaricus species, Lactobacillus fermentum species, Lactobacillus gasseri species, Lactobacillus helveticus species,
Lactobacillus johnsonii species, Lactobacillus mucosae species, Lactobacillus oris species, Lactobacillus
Lactobacillus parabuchneri species, Lactobacillus paracasei subspecies tolerance
Tolerans species, Lactobacillus plantarum (Lact
obacillus plantarum species, Lactobacillus reuteri (Lact
Obacillus reuteri species, Lactobacillus rhamnosus (Lacto
bacillus rhamnosus species, Lactobacillus sakei
cillus sakei species, Lactobacillus salivarius subsp. salicinius (L
actobacillus salivarius subsp. salicinius
) species, Lactobacillus vaginalis
) species, Lactococcus lactis subsp. lactis (Lactococcus lac
Lactis subsp. Lactis species, Lactococcus raffinolactis (La
Leuconostoc raffinolactis species, Leuconostoc citreum species, Leuconostoc mesenteroides subsp. dextranicum
bsp. dextranicum species, Leuconostoc pseudomesenteroides species, Pediococcus acidilactici species, Pediococcus pentosaceus species
Pediococcus stilesi
i) species, Weissella cibaria species, Weissella confusa species, Weissella paramesenteroides species, Bifidobacterium adolescentis
) species, Bifidobacterium faecalis
ale species, Bifidobacterium longum subsp. longum
Bifidobacterium longum subsp. Longum, Bifidobacterium longum subsp. infantis
bsp. infantis species, Bifidobacterium pseudocatenulatum (Bi
Bifidobacterium pseudocatenulatum species, Bifidobacterium pseudolongum subspecies globosum
seudolongum subsp. Globosum species, and Bifidobacterium thermophilum species.

These microorganisms may be used alone or in combination of two or more kinds thereof.

Microorganisms can be cultured under conditions suitable for each microorganism. For example, various media can be used, such as milk media or media containing milk components, semi-synthetic media not containing milk components, and chemically defined synthetic media. Examples of such media include reduced skim milk and MRS media.

The cells separated from the culture by a collection means such as centrifugation can be used as the active ingredient of the present invention as is. Live cells are preferred, but the cells may be subjected to some treatment, such as concentrated, dried, or freeze-dried cells, or may be killed by heating and drying, and the treatment method is not particularly limited.
Not only purely isolated bacteria, but also cultures, suspensions, and other substances containing bacteria,
Cytoplasm or cell wall fractions obtained by treating the cells with enzymes or physical means can also be used.
Examples of the form of the culture include, but are not limited to, cultures using media that are generally used for culturing lactic acid bacteria, such as synthetic medium MRS medium (manufactured by DIFCO) and reduced skim milk medium, as well as dairy products such as cheese, fermented milk, and dairy lactic acid bacteria drinks.

The sleep-promoting composition of the present invention can be added to foods, medicines, feeds, etc.
The sleep-promoting composition of the present invention may be used as is, but since it can be used together with other raw materials that are normally contained in foods, medicines, and feeds, it can be formulated into powders, granules, tablets, or the like in the usual manner.
It can also be used in capsules, drinks, etc. It can also be added to food and drink products such as yogurt, milk drinks, and wafers, as well as feed.

When the sleep-promoting composition of the present invention is incorporated into foods, medicines, feeds, etc., the ratio of the microorganisms to be incorporated is not particularly limited, and may be appropriately adjusted according to the ease of production and the preferred daily dosage. The ratio is determined individually taking into consideration the symptoms and age of the recipient, but for adults, the culture of the microorganisms is usually added in amounts of 10 to 200 g, 20 to 200 g, 50 to 200 g, 100 to 200 g, 150 to 200 g, 200 to 200 g, 300 to 300 g, 400 to 400 g, 50 to 500 g, 60 to 700 g, 80 to 900 g, 90 to 100 g, 100 to 100 g, 150 to 200 g, 100 to 100 g, 150
g, 150-200 g, 20-150 g, 50-150 g, 100-150 g, or 50
0.1 to 5,000 mg, 0.2 to 5,000 mg of the fungus itself,
0.5-5,000mg, 1-5,000mg, 2-5,000mg, 5-5,000m
g, 10-5,000mg, 20-5,000mg, 50-5,000mg, 100-5
,000mg, 200-5,000mg, 500-5,000mg, 1,000-5,0
00mg, 2,000-5,000mg, 0.2-2,000mg, 0.5-2,000
mg, 1-2,000mg, 2-2,000mg, 5-2,000mg, 10-2,00
0mg, 20-2,000mg, 50-2,000mg, 100-2,000mg, 20
0-2,000mg, 500-2,000mg, 1,000-2,000mg, 0.5-
1,000mg, 1-1,000mg, 2-1,000mg, 5-1,000mg, 10
～1,000mg, 20～1,000mg, 50～1,000mg, 100～1,000
mg, 200-1,000mg, 500-1,000mg, 1-500mg, 2-500
mg, 5-500mg, 10-500mg, 20-500mg, 50-500mg, 10
0-500mg, 200-500mg, 2-200mg, 5-200mg, 10-200
mg, 20-200mg, 50-200mg, 100-200mg, 5-100mg, 1
0-100 mg, 20-100 mg, 50-100 mg, 10-50 mg, or 20-5
By adjusting the amount of the compound so that the intake is 0 mg, the desired effect can be achieved.

(Method of evaluating the sleep-promoting effect of a sleep-promoting composition)
The sleep-promoting effect of the sleep-promoting composition of the present invention can be confirmed by the animal test using Drosophila melanogaster described in the Examples.

The present invention will be described in detail below with reference to examples and test examples. However, these are merely illustrative and the present invention is not limited to these in any way.

(Test Example 1) Sleep-promoting effect of microorganisms Regarding various microorganisms belonging to the genera Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, Streptococcus, Weissella, and Bifidobacterium,
The sleep-promoting effect was evaluated using Drosophila.
1. Test method (1) Donor bacteria One strain each of the 21 species of the genus Lactobacillus, two species of the genus Lactococcus, four species of the genus Leuconostoc, three species of the genus Pediococcus, four species of the genus Streptococcus, three species of the genus Weissella, and nine species of the genus Bifidobacterium shown in Table 1 was used as donor bacteria.
(2) Preparation of donor cells Each donor cell in (1) above was cultured under the conditions shown in Table 1 (OD600 = 0.7 or more),
The cells were separated by centrifugation (7,000×g, 4° C., 20 min). The cells were washed twice with physiological saline and then suspended in 12.5% trehalose solution in an amount equal to 1/10 of the culture volume. The suspension was further mixed with equal amounts of 2% agar and 10% sucrose solution to prepare a feed containing donor cells.
(3) Test procedure Wild-type Drosophila melanogaster CS ^2202u strain was reared at 25°C under a 12-hour light/dark cycle. Unmated female individuals 5 days after emergence were used for the evaluation.
The above-mentioned (2) diet (test group) or diet consisting of 1% agar and 5% sucrose without lactobacillus (control group) was placed in a glass tube with a diameter of 5 mm, and the behavior of the fruit flies was measured by Drosophila activity monitor.
Behavior was measured for three days using the ng system (manufactured by Trikinetics). In Drosophila, "sleep" is defined as an immobility period (period without movement) of 5 minutes or more. Therefore, the amount of sleep during the first three hours of the night (ZT12-15 in circadian time) on the third day of measurement was compared between the test group and the control group. Each group was evaluated using approximately 96 Drosophila, and after Wilcoxon rank sum test and correction by Holm's method, it was determined that there was a significant difference at p<0.05.

2. From the test results table 2, Lactobacillus acidophilus
philus species, Lactobacillus amylovorus
ylovorus species, Lactobacillus delbrueckii subsp. bulgaricus (Lact
obacillus delbrueckii subsp. Bulgaricus)
Species, Lactobacillus fermentum
m) species, Lactobacillus gasseri species,
Lactobacillus helveticus
) species, Lactobacillus johnsonii
i) species, Lactobacillus mucosae
species, Lactobacillus oris species, Lactobacillus parabuchneri species,
Lactobacillus paracasei subspecies tolerance
casei subsp. Tolerans species, Lactobacillus plantarum (L
Actobacillus plantarum species, Lactobacillus reuteri (L
Actobacillus reuteri species, Lactobacillus rhamnosus (La
Lactobacillus rhamnosus species, Lactobacillus sakei
obacillus sakei species, Lactobacillus salivarius subsp. salicinius (Lactobacillus salivarius subsp. salicin
Lactobacillus vaginalis species, Lactobacillus vaginalis
lis species, Lactococcus lactis subsp. lactis
lactis subsp. Lactis species, Lactococcus raffinolactis species, Leuconostoc citreum species, Leuconostoc mesenteroides subsp. dextranicum
subsp. dextranicum species, Leuconostoc pseudomesenteroides species, Pediococcus acidilactici
i) species, Pediococcus pentosaceus
ceus species, Pediococcus stillisii
esi species, Weissella cibaria species, Weissella confusa species, Weissella paramesenteroides species, Bifidobacterium adolescentis
tis species, Bifidobacterium faecalis
aecale species, Bifidobacterium longum subsp. longum (Bifidoba
Bifidobacterium longum subsp. Longum, Bifidobacterium longum subsp. infantis
subsp. infantis species, Bifidobacterium pseudocatenulatum species, Bifidobacterium pseudolongum subsp. globosum
pseudolongum subsp. Globosum species, Bifidobacterium thermophilum
The amount of sleep during the early part of the night was significantly increased in the groups treated with the 1,2-dichlorophenyl 3-propanediol (1,2-dichlorophenyl) species compared to the control group. Similarly, the sleep onset latency was significantly decreased in most of the species tested (see also Figure 2).

The relative value of the median amount of sleep in each test group to the median amount of sleep in the control group was taken as the sleep increase rate, and the results of comparison between different genera are shown in Figure 1.
Since Thermophilus is said to have hypnotic activity, the sleep-increasing rate of each genera was compared to that of Streptococcus, and it was found that Bifidobacterium had a tendency to increase sleep, while Lactobacillus, Leuconostoc, and Pediococcus had significantly increased sleep. In other words, it was shown that Bifidobacterium, Lactobacillus, Leuconostoc, and Pediococcus have particularly remarkable sleep-promoting effects.

(Example 1) Production of sleep-promoting agent (granules) Lactobacillus plantarum M
The SN16 strain was inoculated at 5% by weight into an edible synthetic medium (containing 0.5% yeast extract and 0.1% trypticase peptone), and after culturing at 38°C for 15 hours, the cells were collected by centrifugation. The collected cells were freeze-dried to obtain a freeze-dried powder of the prophase cells. 1 g of this freeze-dried powder was mixed with 5 g of lactose and diluted with 100 ml of ethanol.
g and molded into granules to obtain the sleep-promoting agent of the present invention.

(Example 2) Production of sleep-promoting agent (powder) In accordance with the provisions of the General Provisions for Preparations in the 13th Edition Japanese Pharmacopoeia Commentary, "Powder," 400 g of lactose (JP) and 600 g of potato starch (JP) were added to 10 g of the lyophilized powder of MSN16 strain obtained in Example 1 above, and mixed uniformly to produce the sleep-promoting agent of the present invention.

(Example 3) Production of a nutritional composition for promoting sleep Lactobacillus plantarum M
The SN16 strain was inoculated at 5% by weight into an edible synthetic medium (containing 0.5% yeast extract and 0.1% trypticase peptone) and cultured at 38°C for 15 hours. The cells were then harvested by centrifugation and lyophilized.
40 g of the freeze-dried powder was added to 100 g of granulated sugar, 60 g of a mixture of equal amounts of cornstarch and lactose, and mixed. The mixture was packed into a bag to produce the nutritional composition for promoting sleep of the present invention.

(Example 4) Production of a sleep-promoting beverage 1 g of the MSN16 strain obtained in Example 3 above was dissolved in 699 g of deionized water, and then heated at 40° C.
After heating to 100° C., the mixture was stirred and mixed for 20 minutes at 9,500 rpm using an Ultra Disperser (ULTRA-TURRAX T-25; manufactured by IKA Japan Co., Ltd.).
Add 2g of acidulant, 20g of reduced starch syrup, 2g of flavoring, and 176g of deionized water, and then add 100ml
The beverage was filled into a glass bottle of 100 ml, sterilized at 95° C. for 15 seconds, and then sealed.
We produced 100 ml of this product.

(Example 5) Production of sleep-promoting feed Soybean meal 12 kg, skim milk powder 14 kg, soybean oil 4 kg, corn oil 2 kg, palm oil 23.2 kg
kg, corn starch 14 kg, wheat flour 9 kg, bran 2 kg, vitamin mixture 5 kg
2.8 kg of cellulose and 2 kg of mineral mixture were mixed and sterilized at 120°C for 4 minutes.
The feed of the present invention was produced by blending 10 kg of the MSN16 strain obtained in Example 3.

(Example 6) Production of fermented milk Lactobacillus plantarum M
SN16 strain was inoculated at 5% by weight into an edible synthetic medium (0.5% yeast extract, 0.1% trypticase peptone added), cultured at 37 ° C for 17 hours, and then the cells were collected by centrifugation and freeze-dried. 5 g of the freeze-dried cells, 1700 g of skim milk powder, 300 g of glucose, and 7695 g of deionized water were mixed and heated and sterilized by holding at 95 ° C for 2 hours. This was cooled to 37 ° C, and 300 g of lactic acid bacteria starter (Lb. casei) was inoculated, stirred and mixed, and fermented to pH 4.0 in an incubator held at 37 ° C. After reaching pH 4.0, it was cooled to 10 ° C or less to produce 10 kg of fermented milk of the present invention.

The present invention provides a sleep-promoting composition, which is a microorganism belonging to the genera Lactobacillus, Lactococcus, Leuconostoc, Pediococcus, Weissella, and Bifidobacterium, a composition containing the microorganism, and a food, medicine, and feed containing the composition. Sleep can be promoted by ingesting the composition of the present invention.

Claims (3)

A sleep-promoting composition containing one or more microorganisms belonging to the species Lactobacillus salivarius subsp. salicinius as an active ingredient. The sleep-promoting composition according to claim 1, wherein the sleep-promoting composition is a sleep-inducing composition. A sleep-promoting food, sleep-promoting medicine, or sleep-promoting feed comprising the sleep-promoting composition according to claim 1 or 2.

Images