Composition for improving sleep or maintaining sound sleep comprising limosilactobacillus reuteri lm1063 strain as active ingredient, and use thereof

Abstract

A composition according to the present invention not only improves sleep or maintains sound sleep but also restores the balance of intestinal microorganisms disrupted by sleep disorders, and thus can help in managing health conditions such as metabolic syndrome.

Description

Composition for improving sleep or maintaining deep sleep comprising the strain Rimosyl Lactobacillus reuteri LM1063 as an active ingredient and use thereof

[0001] The present invention relates to a composition for improving sleep or maintaining deep sleep, comprising the strain Rimosyl Lactobacillus reuteri LM1063 as an active ingredient, and its use.

[0002] Recently, as various studies have made it clear that there is a two-way communication between the gut and the brain, the activity of gut microbes, the response of the autonomic nervous system connected to the gut, and the regulation of brain function by metabolites or neurotransmitters produced by gut microbes are being studied through the internal connection pathway called the Microbiota-Gut-Brain Axis. In addition, research is being actively pursued on the prevention, treatment, and improvement of various emotional diseases such as depression, Alzheimer's, and sleep disorders using gut microbes. Patent No. 10-2149404 provides a composition for preventing, improving, or treating sleep disorders containing Bacillus subtilis fermentation culture and vitamin B6 as active ingredients, and Japanese Patent No. 6127169 provides a sleep improvement agent containing lactic acid bacteria including Lactobacillus helveticus MIKI-020 strain, a fermented product by the lactic acid bacteria, and a cell lysate of the lactic acid bacteria. Inventions combining probiotics and sleep improvement are being applied for both domestically and internationally.

[0003] Representative neurotransmitters produced by intestinal microorganisms include acetylcholine and GABA produced by Limosilactobacillus, serotonin and dopamine produced by Escherichia, and serotonin produced by Streptococcus and Enterococcus.

[0004] Clinical studies (in humans with jet lag) and animal models (in mice with forced disruption of the circadian rhythm) have reported that sleep deprivation-induced gut microbiota imbalances lead to increased inflammation, weight gain, and elevated blood sugar levels, leading to diseases such as obesity, diabetes, and metabolic syndrome.

[0005] Therefore, sleep-enhancing products utilizing intestinal microorganisms, such as probiotics and lactic acid bacteria, not only improve sleep quality but also restore the intestinal microbiota balance caused by sleep disorders, thereby helping manage health conditions such as metabolic syndrome. With this potential for health management in mind, various sleep aids using probiotics are being developed.

[0006] An analysis of 14 clinical trials on the effects of probiotics or heat-treated lactic acid bacteria on sleep improvement confirmed that consuming probiotics or heat-treated lactic acid bacteria for 8 weeks or more significantly improved the Pittsburgh Sleep Quality Index (PSQI).

[0007] Various neurotransmitter precursors produced by the gut microbiota can pass through the blood-brain barrier (BBB) ​​and be converted into neurotransmitters, helping improve brain function for healthy sleep and playing an important role in the formation of neural networks.

[0008] Although research results on the effects of probiotics on ‘gut health - sleep health - brain function’ have been published, there are currently no domestic cases of individually approved raw materials for probiotics to improve sleep health from the Ministry of Food and Drug Safety, so it is expected that the commercialization value will be very high in the future.

[0009] The challenge we are trying to solve is to develop probiotics that can prevent or treat sleep disorders, one of the modern diseases.

[0010] The above task is an example and there may be additional tasks that are within the scope of what a person of ordinary skill can understand.

[0011] To solve the above problem, the present invention provides a pharmaceutical composition for improving sleep or maintaining deep sleep, which comprises at least one of the following as an active ingredient: the strain Rimosylactobacillus reuteri LM1063 (KCTC13232BP), a culture of the strain, a lysate of the strain, and an extract of the strain.

[0012] The present invention provides a health functional food containing at least one of the following as an active ingredient: the strain Rimosyl Lactobacillus reuteri LM1063 (KCTC13232BP), a culture of the strain, a lysate of the strain, and an extract of the strain.

[0013] The present invention provides a food raw material comprising at least one of the following as an effective ingredient: the strain Rimosyl Lactobacillus reuteri LM1063 (KCTC13232BP), a culture of the strain, a lysate of the strain, and an extract of the strain.

[0014] The above food raw material may include a food composition, and may preferably be a food raw material for a health functional food, a functional raw material for a health functional food, or a food composition for a health functional food, but is not necessarily limited thereto.

[0015] The above means are examples and new means may exist within the scope understandable to a person skilled in the art.

[0016] The composition according to the present invention not only improves or maintains sleep, but also restores the intestinal microbial balance caused by sleep disorders, thereby helping manage health conditions such as metabolic syndrome. The strain according to the present invention can be used not only in health functional foods but also as a raw material or auxiliary material for food products, depending on the evolving state of probiotic research.

[0017] The above effects are examples, and additional effects may exist within the scope understandable to a person skilled in the art.

[0018] Figure 1 is a schematic diagram of a sleep-accelerated animal model induced with pentobarbital.

[0019] Figure 2 is a schematic diagram of an animal model in which sleep is induced with pentobarbital at a concentration below the sleep induction threshold.

[0020] Figure 3 is a schematic diagram showing melatonin production according to the circadian rhythm.

[0021] Figure 4 shows the results of comparing sleep latency in three groups of animal models in which sleep was induced with pentobarbital.

[0022] Figure 5 shows the results of comparing sleep times for three groups of animal models in which sleep was induced with pentobarbital.

[0023] Figure 6 shows the results of comparing sleep latency in three animal models in which sleep was induced with pentobarbital below the sleep induction threshold.

[0024] Figure 7 shows the results of comparing sleep times for three animal models in which sleep was induced with pentobarbital below the sleep induction threshold.

[0025] Figure 8 shows the results of comparing the blood melatonin concentrations of three animal models in which sleep was induced with pentobarbital.

[0026] Figure 9 shows the results of comparing the blood GABA concentrations of three animal models in which sleep was induced with pentobarbital.

[0027] Below, with reference to the attached drawings, embodiments of the present invention are described in detail to facilitate easy implementation by those skilled in the art. However, the present invention can be implemented in various different forms and is not limited to the embodiments described herein. In the drawings, irrelevant parts have been omitted for clarity, and similar reference numerals have been used throughout the specification to indicate similar elements.

[0028] Throughout this specification, whenever a part is said to "include" a component, this means that it may include other components, but not to the exclusion of other components, unless otherwise stated.

[0029] Throughout this specification, the term "combination(s) thereof" included in the expressions in the Makushi format means one or more mixtures or combinations selected from the group consisting of the components described in the expressions in the Makushi format, and means including one or more selected from the group consisting of said components.

[0030] The strain Limosilactobacillus reuteriLM1063 described herein was deposited with the Korean Collection for Type Cultures (KCTC) on April 4, 2017 under the accession number KCTC 13232BP.

[0031] Throughout this specification, "pentobarbital" means (RS)-5-Ethyl-5-(1-methylbutyl)-2,4,6(1H,3H,5H)-pyrimidinetrione, a substance used for short-term sedation, hypnosis, and seizure control in preanesthesia or emergency situations. It is also used as an anesthetic by veterinarians.

[0032] Throughout this specification, "Diazepam" refers to 7-chloro-1-methyl-5-phenyl-3H-1,4-benzodiazepin-2-one, a benzodiazepine tranquilizer developed by Roche, Switzerland. It is commonly used to treat various conditions, including anxiety disorders, alcohol withdrawal syndrome, benzodiazepine withdrawal syndrome, convulsions, seizures, insomnia, and restless leg syndrome, and is therefore classified as a psychotropic drug with a risk of abuse, including dependence.

[0033] Throughout this specification, ×g represents the unit of G-force. G-force is a unit of gravity acceleration, and is a unit commonly used to express acceleration generated by centrifugal force, etc. during centrifugation, as a ratio.

[0034] Hereinafter, implementation examples and embodiments of the present invention will be described in detail with reference to the attached drawings. However, the present invention may not be limited to these implementation examples and embodiments and drawings.

[0035] 1. Breeding and feeding of laboratory animals

[0036] The experimental animals were 30 4-week-old ICR male mice fed Lab-chaw (Harlan, Madison, WI, USA) diet for mice for 1 week and allowed to adapt for 1 week. After that, they were divided into 3 groups by the egg block method according to their body weight: control group (CON, sleep-inducing group), diazepam administration group (DIZ), and Limosilactobacillus reuteri LM1063 feeding group (S). They were fed a 10% kcal fat diet based on the AIN-76 diet for 3 weeks. The composition according to the present invention was dissolved in distilled water and 1x10 9 CFU / d, the normal control group and the diazepam-administered group were watered with distilled water once a day. During the rearing period, the temperature in the rearing room was maintained at 22±3℃, the humidity was maintained at 55±5%, and the lighting time was 07:00~19:00 (12-hour cycle).

[0037] Pentobarbital-induced sleep health function improvement animal model design group Pentobarbital oral administration (3 weeks) CON control group (sleep induction group) 45 mg / kg * or 30 mg / kg * Distilled water DIZ diazepam administration group Distilled water / 2 mg / kg * SLimosilactobacillus reuteriLM1063 intake group 1x10 9 CFU* administered only when measuring sleep latency and sleep time

[0038] 2. Pentobarbital-induced sleep test using an animal model of accelerated sleep

[0039] The pentobarbital-induced sleep test using rodents is known as a simple method for evaluating the efficacy of sleep-promoting drugs in animals, with results obtained quickly.

[0040] As shown in Table 2 below, we evaluated whether the test substance could further enhance sleep induced by the anesthetic pentobarbital. If there was a significant effect in two of these methods and there was no direct sleep effect, it was recognized as having sleep-improving functionality.

[0041] Method: Sleep improvement functional indicators 1. Decrease in sleep latency in rodents induced with pentobarbital 2. Increase in sleep time in rodents induced with pentobarbital 3. Sleep induction (decrease in latency and increase in sleep time) by pentobarbital at concentrations below the sleep induction threshold

[0042] (1) Sleep acceleration animal model induced with pentobarbital (Fig. 1)

[0043] 1) Experimental groups were established with 10 ICR mice per group and acclimated to the laboratory environment for one week. To eliminate factors that could affect sleep during breeding, conditions such as environmental noise (40 dB or less), lighting (200–300 LUX 12 h / day), constant temperature (22±3°C), and humidity (55±5%) were maintained.

[0044] 2) 1x10 of the effective strains to the mice 9 It was dissolved in distilled water at a concentration of CFU / 100uL and administered daily for two weeks using a sonde.

[0045] 3) After fasting for 24 hours prior to the experiment, pentobarbital was injected intraperitoneally at a concentration of 45 mg / kg (hypnotic dosage, sleep-inducing threshold concentration), diazepam (2 mg / kg) was administered to the positive control group, and distilled water was administered to the negative control group.

[0046] 4) After pentobarbital treatment, each individual was moved to an independent space and sleep latency and sleep duration were measured.

[0047] 5) Sleep latency was considered as the time elapsed from intraperitoneal injection of pentobarbital until the righting reflex was lost for more than 1 minute, and sleep time was set as the time until the righting reflex was recovered.

[0048] (2) Sleep acceleration animal model induced with subthreshold concentrations of pentobarbital (Fig. 2)

[0049] 1) Experimental groups were established with 10 ICR mice per group and acclimated to the laboratory environment for one week. To eliminate factors that could affect sleep during breeding, conditions such as environmental noise (40 dB or less), lighting (200–300 LUX 12 h / day), constant temperature (22±3°C), and humidity (55±5%) were maintained.

[0050] 2) 1x10 of the effective strains to the mice 9 It was dissolved in distilled water at a concentration of CFU / 100uL and administered daily for two weeks using a sonde.

[0051] 3) After fasting for 24 hours prior to the experiment, pentobarbital, a subthreshold concentration, was administered intraperitoneally at 30 mg / kg, diazepam (2 mg / kg) was administered to the positive control group, and distilled water was administered to the negative control group. The experiments were conducted at regular intervals to avoid the influence of the sleep-inducing threshold concentration experiment.

[0052] 4) After pentobarbital treatment, each individual was moved to an independent space and sleep latency and sleep duration were measured.

[0053] 5) Sleep latency was considered as the time elapsed from intraperitoneal injection of pentobarbital until the righting reflex was lost for more than 1 minute, and sleep time was set as the time until the righting reflex was recovered.

[0054] 3. Melatonin analysis in blood

[0055] It is known that the concentration of melatonin in the human body increases due to changes in the composition of intestinal microorganisms when sleep health improves, and melatonin measurement is necessary to confirm the mechanism of improving sleep health.

[0056] Blood isolated from experimental animals was centrifuged at 3000×g for 30 minutes to obtain serum. The obtained serum was analyzed for the relative expression level of melatonin in vivo using a mouse melatonin ELISA kit at room temperature.

[0057] Serum was applied to the plate and reacted for 90 minutes at 37°C. After the reaction was complete, the plate was washed, treated with biotinylated antibody, and reacted for 60 minutes at 37°C. After the reaction was complete, the plate was washed, treated with enzyme conjugate, and reacted for 30 minutes at 37°C. Afterwards, the reaction was stopped, and the melatonin content in the blood was measured at 450 nm (Fig. 3).

[0058] 4. Measurement of GABA in blood

[0059] γ-aminobutyric acid (GABA) is an inhibitory neurotransmitter that acts in the central nervous system and plays a role in regulating nerve excitation in the nervous system.

[0060] GABA binds to receptors to inhibit neural activity. It is released presynaptly from inhibitory neurons and binds to GABA receptors located on the surface of postsynaptic neurons. GABA receptors bound to GABA inhibit the excitability of neurons. GABA functions as a substance that helps stabilize sleep by preventing and alleviating stress, anxiety, nervousness, and panic disorder. Therefore, measuring GABA content in sleeping animals can confirm its effectiveness in improving sleep.

[0061] Blood isolated from experimental animals was centrifuged at 3000×g for 30 minutes to obtain serum. The obtained serum was treated with serum and biotin-labeled antibody together on a washed plate and reacted at 37℃ for 45 minutes. After the reaction was completed, the plate was washed, treated with HRP-Streptavidin Conjugate (SABC), and incubated at 37℃ for 30 minutes. After the reaction was stopped, the GABA content in the blood was measured at 450 nm.

[0062] 5. Statistical processing

[0063] Statistical analysis of all data (Tables 3 to 9) was performed using SPSS Statistics 20, and the results are expressed as the mean ± standard error. The significance of the means was verified at a 95% confidence level (p < 0.05). Each item was subjected to a one-way ANOVA to obtain the F value, and the significant difference between each interval was verified using Duncan's multiple range test. In each table or figure, the meaning of numbers using different letters such as a, b, c, ab as superscripts or regular letters means that each number is statistically significantly different from each other. For example, in Table 3, the sleep latency values ​​of the DIZ group and the S group both use a, so there is no statistically significant difference. However, the CON group and the other two groups use different letters such as b and a, which means that there is a statistically significant difference in sleep latency. The same applies to Tables 3 to 9 below.

[0064] 6. Results

[0065] 1) Results of analysis of the effects of ingestion of the composition according to this invention on sleep latency and sleep duration in an animal model of sleep acceleration induced with pentobarbital

[0066] The composition according to the present invention is 1x10 9 The composition was dissolved in a concentration of CFU / 100uL and administered orally, the control group was dissolved in distilled water, and diazepam was dissolved in a concentration of 2 mg / kg and administered orally. 45 minutes after the sample administration, 45 mg / kg of pentobarbital was injected intraperitoneally to analyze the sleep acceleration effect of the composition according to the present invention.

[0067] Each animal was placed in a transparent cage, and the sleep latency was defined as the time from the time pentobarbital was injected to the time sleep began, and the sleep time was defined as the time from the time sleep began to the time sleep ended. These were measured and recorded in seconds.

[0068] The sleep latency evaluation criterion was considered to be the time until the righting reflex was lost for more than 1 minute after intraperitoneal injection of pentobarbital, and sleep time was set as the time until the righting reflex was recovered.

[0069] In a sleep experiment, if sleep latency decreases and sleep time increases compared to the control group, the composition according to the present invention is considered to have a sleep-inducing effect. Furthermore, if sleep latency decreases and sleep time increases to the level of the drug control group, the composition according to the present invention can be considered to have a very high sleep-accelerating effect.

[0070] In terms of sleep latency, the S group showed a significant decrease of 25.0% in sleep latency compared to the CON group, and the S group showed a decrease in sleep latency to the DIZ level.

[0071] From the above results, it was confirmed that the composition according to the present invention is effective in reducing sleep latency (Table 3, Fig. 4).

[0072] Effect of ingestion of the composition according to the present invention on pentobarbital-induced sleep latency (unit: min) Group Sleep latency CON Control group (sleep-induced group) 4.8±0.5 b DIZ diazepam administration group 3.2±0.2 a SLimosilactobacillus reuteriRLM1063 intake group 3.6±0.3 a

[0073] In terms of sleep time, the DIZ group showed a significant increase in sleep time compared to the CON group, and the S group showed a significant increase in sleep time compared to the CON group, and this effect was very high, comparable to that of the DIZ group. In addition, the S group showed a sleep acceleration effect, with sleep time significantly increasing by 60.0% compared to the CON group (Table 4, Fig. 5).

[0074] Effect of ingestion of the composition according to this invention on pentobarbital-induced sleep duration (unit: min) Group Sleep duration CON Control group (sleep-induced group) 57.5±4.7a DIZ diazepam administration group 104.4±10.1 b SLimosilactobacillus reuteriLM1063 intake group 92.0±13.7 b

[0075] From the above results, it was confirmed that the composition according to the present invention has the effect of improving sleep health, as the S group affected the reduction of sleep latency and the increase of sleep time in the sleep acceleration animal model administered with 45 mg / kg pentobarbital.

[0076] 2) Results of analysis of the effects of ingestion of the composition according to the present invention on sleep latency and sleep duration in an animal model of sleep acceleration induced with subthreshold concentrations of pentobarbital.

[0077] The composition according to the present invention is 1x10 9 The composition was dissolved in a concentration of CFU / 100uL and administered orally, the control group was dissolved in distilled water, and diazepam was dissolved in a concentration of 2 mg / kg and administered orally. 45 minutes after the sample administration, 30 mg / kg of pentobarbital, a subthreshold concentration, was injected intraperitoneally to analyze the sleep acceleration effect of the composition according to the present invention.

[0078] The CON group (16.8±3.2 minutes) administered with 30 mg / kg of subthreshold pentobarbital showed an increase in sleep latency of approximately 12 minutes (3.5 times) compared to the group administered with 45 mg / kg of pentobarbital (4.8±0.5 minutes), and the sleep latency of the DIZ group administered with diazepam also showed an increase from 3.2±0.2 minutes to 6.0±0.7 minutes (Table 3, Table 5).

[0079] The S group showed a significantly reduced sleep latency compared to the CON group, and this effect was very high, comparable to the DIZ group (Table 5).

[0080] Group S showed a sleep acceleration effect with a significant 56.0% decrease in sleep latency compared to the CON group (Table 5, Fig. 6).

[0081] Effect of ingestion of the composition according to the present invention on sleep latency induced by pentobarbital at concentrations below the sleep-inducing threshold (unit: min) Group Sleep latency CON Control group (sleep-inducing group) 16.8±3.2 b DIZ diazepam administration group 6.0±0.7 a SLimosilactobacillus reuteriLM1063 intake group 7.4±0.7 a

[0082] The CON group (22.1±4.4 minutes) administered subthreshold pentobarbital (30 mg / kg) showed a decrease in sleep time of approximately 35.4 minutes compared to when 45 mg / kg of pentobarbital was administered (57.5±4.7 minutes), and the DIZ group administered diazepam also showed a decrease in sleep time from 104.4±10.1 minutes to 82.4±7.7 minutes (Table 4, Table 6).

[0083] Group S showed a sleep acceleration effect, with sleep time significantly increasing by 154.8% compared to the CON group (Table 6, Fig. 7).

[0084] Effect of ingestion of the composition according to the present invention on sleep duration induced by pentobarbital at concentrations below the sleep-inducing threshold (unit: min) Group Sleep duration CON Normal control group 22.1±4.4 a DIZ diazepam administration group 82.4±7.7 c SLimosilactobacillus reuteriLM1063 intake group 56.3±10.5 b

[0085] From the above results, it was confirmed that the composition according to the present invention has a sleep health improvement effect, as the S group affected a decrease in sleep latency and an increase in sleep time in an animal model of sleep acceleration administered at a subthreshold concentration of 30 mg / kg pentobarbital.

[0086] In addition, the sleep acceleration effect due to the intake of the composition according to this invention was more effective when administered at 30 mg / kg than when administered at 45 mg / kg of pentobarbital, so it is thought that a high sleep health improvement functionality can be expected.

[0087] 3) Results of analysis on the effect of ingestion of the composition according to this invention on blood melatonin concentration

[0088] It is known that the concentration of melatonin in the human body increases due to changes in the composition of intestinal microorganisms when sleep health improves, and melatonin measurement is necessary to confirm the mechanism of improving sleep health.

[0089] Melatonin participates in the 24-hour circadian rhythm and regulates its secretion, altering physiological functions. Melatonin's physiological function is as a hormone that promotes relaxation and sleep induction. Increased melatonin levels in the blood are believed to accelerate sleep onset.

[0090] As a result of measuring the blood melatonin concentration after ingesting the composition according to this invention, the blood melatonin content of group S was significantly higher by 49.8% than that of group CON (Table 7, Fig. 8).

[0091] Effect of intake of composition according to this invention on blood melatonin concentration Group Melatonin (pg / mL) CON Control group (sleep-inducing group) 77.5±8.5 a DIZ diazepam administration group 92.7±5.3 ab SLimosilactobacillus reuteriLM1063 intake group 116.4±7.1 b

[0092] From the above results, it was confirmed that the composition according to the present invention is effective in reducing sleep latency and increasing sleep time by increasing the blood melatonin concentration.

[0093] 4) Results of analysis on the effect of ingestion of the composition according to this invention on blood GABA concentration

[0094] GABA binds to receptors and inhibits neural activity. Released presynaptly from inhibitory neurons, it binds to GABA receptors located on the surface of postsynaptic neurons. GABA receptors bound to GABA inhibit the excitability of neurons. Increased GABA secretion can act as a substance that helps stabilize sleep.

[0095] The DIZ group showed a significant increase in blood GABA compared to the CON group, and the blood GABA content of the S group was 25.5% higher than that of the CON group (Table 8, Fig. 9).

[0096] From the above results, it was confirmed that the composition according to the present invention is effective in improving sleep by increasing blood GABA.

[0097] Effect of intake of composition according to this invention on blood GABA concentration Group GABA (pg / mL) CON Control group (sleep-inducing group) 1869.4±357.4 a DIZ diazepam administration group 3134.2±402.3 b SLimosilactobacillus reuteriLM1063 intake group2345.2±242.6 ab

[0098]

Claims

1. A pharmaceutical composition for improving sleep or maintaining deep sleep, comprising at least one of the following as an active ingredient: Limosylactobacillus reuteri LM1063 strain (KCTC13232BP), a culture of the strain, a lysate of the strain, or an extract of the strain.

2. In paragraph 1, A composition wherein the above sleep improvement or sleep maintenance includes reducing sleep latency or increasing sleep time.

3. In paragraph 1, The composition above is a composition that increases melatonin formation.

4. In paragraph 1, The composition above is a composition that increases the formation of GABA (γ-aminobutyric acid).

5. A health functional food comprising at least one of the following as an active ingredient: Limosylactobacillus reuteri LM1063 strain (KCTC13232BP), a culture of the strain, a lysate of the strain, or an extract of the strain.

6. A food raw material comprising at least one of the following as an effective ingredient: Limosylactobacillus reuteri LM1063 strain (KCTC13232BP), a culture of the strain, a lysate of the strain, or an extract of the strain.

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