Composition for improving sleep or maintaining deep sleep comprising lactobacillus reuteri LM1063 strain as active ingredient, and use thereof

Abstract

The composition disclosed by the invention not only can improve the sleep state or maintain deep sleep, but also can recover intestinal microorganism balance caused by sleep disorder, thereby being beneficial to health management of metabolic syndrome and the like.

Description

Technical Field

[0001] The present invention relates to a composition for improving sleep or maintaining deep sleep, comprising Lactobacillus reuteri LM1063 strain as an effective ingredient, and application thereof. Background Art

[0002] In recent years, various studies have shown that there is a two-way communication between the gut and the brain, that is, through an in vivo connection channel called the Microbiota-Gut-Brain Axis, the activities of intestinal microorganisms, the response of the autonomic nervous system connected to the intestine, and the regulation of brain function by metabolites or neurotransmitters produced by intestinal microorganisms. These studies have promoted the active exploration of using intestinal microorganisms to prevent, treat and improve depression, Alzheimer's disease, sleep disorders and other emotional diseases. For example, Korean Patent No. 10-2149404 discloses a method comprising a fermentation culture broth of Bacillus subtilis and vitamin B 6 A composition for preventing, improving or treating sleep disorders as an active ingredient, Japanese authorized patent No. 6127169 discloses a sleep improver containing lactic acid bacteria including Lactobacillus helveticus MIKI-020 strain, a fermentation product produced by the above lactic acid bacteria, and a bacterial cell crush product of the above lactic acid bacteria. These indicate that patent applications combining probiotics with sleep improvement have appeared both at home and abroad.

[0003] Representative substances of neurotransmitters produced by intestinal microorganisms include acetylcholine and γ-aminobutyric acid (GABA) produced by microorganisms of the genus Limosilactobacillus, serotonin and dopamine produced by Escherichia, and serotonin produced by Streptococcus and Enterococcus.

[0004] It has been reported in clinical studies (on people suffering from jet lag) and animal models (mouse models with forced interruption of 24-hour circadian rhythm) that intestinal microbial imbalance caused by lack of sleep can lead to increased inflammation, weight gain, increased blood sugar, etc., which in turn can cause diseases such as obesity, diabetes and metabolic syndrome.

[0005] Therefore, sleep health improvers made from intestinal microorganisms such as probiotics and lactic acid bacteria can not only improve sleep conditions, but also restore the balance of intestinal microorganisms caused by sleep disorders, thereby contributing to health management such as metabolic syndrome. With an eye on enabling health management, various sleep aids using probiotics have been developed.

[0006] The results of an analysis of a total of 14 clinical trials based on the effects of probiotics or heat-treated lactic acid bacteria on sleep improvement confirmed that continuous intake of probiotics or heat-treated lactic acid bacteria for more than 8 weeks can significantly improve the Pittsburgh Sleep Quality Index (PSQI).

[0007] A variety of neurotransmitter precursors produced by the intestinal microbiota can be converted into neurotransmitters through the BBB (Blood-Brain-Barrier), helping to improve brain function for sleep health and play 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 is no Food and Drug Administration-registered individual raw material for probiotics that improve sleep health in South Korea, and there is great potential for future commercialization. Summary of the invention

[0009] Technical issues

[0010] The problem to be solved by the present invention is to develop a probiotic that can prevent or treat sleep disorders, which are one of the diseases of modern people.

[0011] The above-mentioned problems are merely examples, and there may be other problems within the scope that can be understood by those skilled in the art.

[0012] Technical Solution

[0013] In order to solve the above problems, the present invention provides a pharmaceutical composition for improving sleep or maintaining deep sleep, comprising, as an active ingredient, one or more of Lactobacillus reuteri LM1063 strain (KCTC13232BP), a culture of the above strain, a crushed product of the above strain, and an extract of the above strain.

[0014] The present invention provides a health functional food, which contains, as an effective ingredient, one or more of Lactobacillus reuteri LM1063 strain (KCTC13232BP), a culture of the strain, a crushed product of the strain, and an extract of the strain.

[0015] The present invention provides a food raw material, comprising, as an effective ingredient, one or more of Lactobacillus reuteri LM1063 strain (KCTC13232BP), a culture of the strain, a crushed product of the strain, and an extract of the strain.

[0016] The above-mentioned food raw material may include a food composition, and is preferably a food raw material for health functional food, a functional raw material for health functional food, or a food composition for health functional food, but is not limited thereto.

[0017] The above solutions are only examples, and there may be other new technical solutions within the scope that can be understood by those skilled in the art.

[0018] Effects of the Invention

[0019] The composition of the present invention can not only improve the sleep state or maintain deep sleep, but also restore the intestinal microbial balance caused by sleep disorders, thereby contributing to health management such as metabolic syndrome. The strain of the present invention can not only be used as a healthy functional food, but also can be used as a raw material or auxiliary material of food according to the current development of probiotic research.

[0020] The above effects are merely examples, and there may be other effects within the scope that can be understood by those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a schematic diagram of the preparation of a sleep acceleration animal model using pentobarbital to induce sleep.

[0022] Figure 2 This is a schematic diagram of the animal model for inducing sleep using pentobarbital at a concentration below the sleep induction threshold.

[0023] Figure 3 is a schematic diagram showing melatonin production based on the living body's circadian rhythm.

[0024] Figure 4 The results of comparison of sleep latency in three groups of animal models induced with pentobarbital are shown.

[0025] Figure 5 The results of comparing the sleep time of three groups of animal models induced to sleep with pentobarbital are shown.

[0026] Figure 6 The figures show the results of comparison of sleep latency in three groups of animal models induced to sleep with pentobarbital below the sleep induction threshold.

[0027] Figure 7 The figures show the results of comparison of the sleep time of three groups of animal models induced to sleep with pentobarbital below the sleep induction threshold.

[0028] Figure 8 The figure shows the results of comparison of melatonin concentrations in the blood of three groups of animal models to which sleep was induced with pentobarbital.

[0029] Fig. 9 The graph shows the results of a comparison of blood GABA concentrations in three groups of animal models to which sleep was induced with pentobarbital. DETAILED DESCRIPTION

[0030] The embodiments of the present invention are described in detail below in conjunction with the accompanying drawings so that those skilled in the art can easily implement the present invention. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Moreover, in order to more clearly illustrate the present invention, parts that are not related to the description are omitted in the accompanying drawings, and similar reference numerals are given to similar parts throughout the specification.

[0031] Throughout the specification of the present invention, when a certain part is described as “including” a certain constituent element, other constituent elements are not excluded and other constituent elements may be included unless otherwise indicated.

[0032] Throughout the specification of the present invention, the term "combination thereof" included in the Markush form means a mixture or combination of one or more selected from the group consisting of the constituent elements represented by the Markush form, and means that one or more selected from the group consisting of the above constituent elements are included.

[0033] The Limosilactobacillus reuteri LM1063 strain based on the present invention was deposited in the Korean Collection for Type Cultures (KCTC) on April 4, 2017, with the deposit number KCTC13232BP.

[0034] Throughout the present invention, "pentobarbital" refers to (RS)-5-ethyl-5-(1-methylbutyl)-2,4,6(1H,3H,5H)-pyrimidinetrione, which is a substance used for short-term sedation, preoperative anesthesia with hypnosis or convulsion control in emergency situations. It is also used as an anesthetic in veterinary medicine.

[0035] Throughout the description of the present invention, "Diazepam" refers to 7-chloro-1-methyl-5-phenyl-3H-1,4-benzodiazepine-2-one, which is a benzodiazepine neuroleptic developed by Roche, Switzerland. It is usually used to treat various diseases including anxiety disorders, alcohol withdrawal syndrome, benzodiazepine withdrawal syndrome, convulsions, epileptic seizures, insomnia and restless leg syndrome, and is classified as a psychotropic drug due to the risk of addiction and abuse.

[0036] Throughout the description of the present invention, "×g" is a unit of G-force. G-force is a unit of gravity acceleration and is generally used to express the acceleration generated by centrifugal force or the like during centrifugation as a ratio. Specific embodiments

[0038] The following describes in detail the configuration examples and embodiments of the present invention in conjunction with the accompanying drawings. However, the present invention is not limited to these configuration examples, embodiments and drawings.

[0039] 1. Animal husbandry and feeding

[0040] Thirty four-week-old ICR male mice were selected as experimental animals and fed with Lab-chaw (Harlan, Madison, WI, USA) for one week. After one week of adaptive feeding, they were randomly divided into three groups according to their body weight: a control group (CON, sleep-induced group), a diazepam feeding group (DIZ), and a Limosilactobacillus reuteri LM1063 intake group (S). 10% kcal fat feed was added to the AIN-76 feed for three weeks. The composition according to the present invention was dissolved in distilled water and fed with 1x10 9 CFU / d, the normal control group and the stable feeding group were fed with distilled water once a day. The environment of the feeding room during the feeding period was maintained at a temperature of 22±3°C, a humidity of 55±5%, and a lighting time of 07:00-19:00 (12-hour cycle).

[0041]

Table 1

[0042] Design of an animal model for pentobarbital-induced improvement of sleep health

[0043]

[0044] 2. Pentobarbital-induced sleep test using sleep acceleration animal model

[0045] The pentobarbital-induced sleep test using rodents is widely known as a method for evaluating sleep-promoting effects on animals, which is simple and can obtain results in a short time.

[0046] As shown in Table 2 below, whether the test substance can further enhance the sleep effect induced by pentobarbital as an anesthetic was evaluated, and significant effects were obtained in two of the methods, and in the absence of a direct sleep effect, it was determined that the test substance has the function of improving sleep.

[0047]

Table 2

[0048]

[0049] (1) Sleep acceleration animal model using pentobarbital-induced sleep ( Figure 1 )

[0050] 1) Experimental groups were set up with 10 ICR mice per group and acclimatized to the laboratory environment for one week. During feeding, in order to eliminate factors that affect sleep, the experimental environment was maintained under the following conditions: ambient noise (below 40 dB), lighting (200-300 LUX 12 hours / day), constant temperature (22±3°C), humidity (55±5%), etc.

[0051] 2) The effective strains were divided into 1x10 9 The concentration of CFU / 100 μL was dissolved in distilled water and fed to mice daily by tube feeding for two weeks.

[0052] 3) After fasting for 24 hours immediately before the experiment, pentobarbital was injected intraperitoneally at a concentration of 45 mg / kg (hypnotic dose, sleep-inducing threshold concentration), the positive control group was injected with diazepam (2 mg / kg), and the negative control group was injected with distilled water.

[0053] 4) After pentobarbital treatment, the mice were transferred to independent spaces and their sleep latency and sleep duration were measured.

[0054] 5) Sleep latency was defined as the time from intraperitoneal injection of pentobarbital to loss of righting reflex for more than 1 minute, and sleep duration was defined as the time until righting reflex was restored.

[0055] (2) Sleep acceleration animal model using subthreshold concentrations of pentobarbital to induce sleep ( Figure 2 )

[0056] 1) Experimental groups were set up with 10 ICR mice per group and acclimatized to the laboratory environment for one week. During feeding, in order to eliminate factors that affect sleep, the experimental environment was maintained under the following conditions: ambient noise (below 40 dB), lighting (200-300 LUX 12 hours / day), constant temperature (22±3°C), humidity (55±5%), etc.

[0057] 2) The effective strains were divided into 1x10 9 The concentration of CFU / 100 μL was dissolved in distilled water and fed to mice daily by tube feeding for two weeks.

[0058] 3) After fasting for 24 hours before the experiment, pentobarbital was injected intraperitoneally at a concentration below the threshold, i.e., 30 mg / kg, with diazepam (2 mg / kg) injected into the positive control group and distilled water injected into the negative control group. At this time, in order to avoid the influence of the sleep induction threshold concentration experiment, the experiment was performed at a predetermined time interval.

[0059] 4) After pentobarbital treatment, the mice were transferred to independent spaces and their sleep latency and sleep duration were measured.

[0060] 5) Sleep latency was defined as the time from intraperitoneal injection of pentobarbital to loss of righting reflex for more than 1 minute, and sleep duration was defined as the time until righting reflex was restored.

[0061] 3. Analysis of melatonin in blood

[0062] It is known that changes in the composition of intestinal microorganisms when improving sleep health will lead to an increase in the concentration of melatonin in the human body. Therefore, in order to confirm the mechanism of improved sleep health, melatonin needs to be measured.

[0063] Blood separated from the experimental animals was centrifuged at 3000×g for 30 minutes to obtain serum, which was then analyzed for relative expression of melatonin in vivo using a mouse melatonin ELISA kit at room temperature.

[0064] The serum was placed in an ELISA plate for treatment and reacted at 37°C for 90 minutes. After the reaction, it was washed and then biotinylated antibody was added and reacted at 37°C for 60 minutes. After the reaction, it was washed again and treated with enzyme conjugate and reacted at 37°C for 30 minutes. After that, the reaction was terminated and the melatonin content in the blood was measured at a wavelength of 450nm ( Figure 3 ).

[0065] 4. Measurement of GABA in Blood

[0066] γ-Aminobutyric acid (GABA) is an inhibitory neurotransmitter acting on the central nervous system and has the function of regulating nerve excitation.

[0067] GABA inhibits neural activity by binding to receptors. It is released before the synapses of inhibitory neurons and binds to GABA receptors on the surface of neurons after the synapses. GABA receptors bound to GABA can inhibit the excitability of neurons. GABA's functions include preventing and relieving stress, tension, anxiety, and panic disorders, and can act as a substance that helps sleep activities stably. By measuring the GABA content in experimental animals, the effect of improving sleep activities can be confirmed.

[0068] The blood separated from the experimental animals was centrifuged at 3000×g for 30 minutes to obtain serum. The obtained serum was placed in the washed ELISA plate, and the serum and biotin-labeled antibody were treated together and reacted at 37°C for 45 minutes. After the reaction, the plate was washed, treated with HRP-Streptavidin Conjugate (SABC), and incubated at 37°C for 30 minutes. The reaction was then terminated and the GABA content in the blood was measured at a wavelength of 450nm.

[0069] 5. Statistical processing

[0070] All statistical processing of data (Tables 3 to 9) was analyzed using SPSS Statistics 20, and the results were expressed as mean ± standard error, and the significance of each mean was verified at a confidence level of 95% (p<0.05). The F value of each item was calculated by one-way ANOVA, and the significant difference of each interval was verified by Duncan's multiple range test. In each table or figure, the numerical values ​​with different superscripts or ordinary characters, such as a, b, c, ab, etc., indicate that the numerical values ​​are statistically significantly different from each other. For example, in Table 3, the sleep latency of the DIZ group and the S group both use a, indicating that there is no statistically significant difference between the two; while the CON group and the other two groups use the words b and a, indicating that there is a statistically significant difference in sleep latency. This principle also applies to Tables 3 to 9.

[0071] 6. Results

[0072] 1) Results of analysis of the effects of the composition of the present invention on sleep latency and sleep duration in a sleep acceleration animal model induced by pentobarbital

[0073] The composition of the present invention is 1x10 9 CFU / 100 μL concentration was dissolved and orally fed; the control group was distilled water, and diazepam was dissolved at a concentration of 2 mg / kg and orally fed. 45 minutes after the sample was fed, pentobarbital was intraperitoneally injected at a concentration of 45 mg / kg to analyze the sleep acceleration effect of the composition of the present invention.

[0074] The experimental animals were placed individually in transparent cages, and the time from the injection of pentobarbital to the onset of sleep was defined as sleep latency, and the time from the onset of sleep to the end of sleep was defined as sleep time, which were measured and recorded in seconds.

[0075] The sleep latency evaluation standard was set as the time from intraperitoneal injection of pentobarbital to loss of righting reflex for more than 1 minute, and the sleep time was set as the time until the righting reflex was restored again.

[0076] In the sleep experiment, if the sleep latency is reduced and the sleep time is increased compared to the control group, the composition of the present invention is considered to have a sleep promoting effect. In addition, if the sleep latency is reduced and the sleep time is increased compared to the drug control group, the sleep accelerating effect of the composition of the present invention can be considered to be very high.

[0077] For sleep latency, the sleep latency in the S group was significantly reduced by 25.0% compared with the CON group, and the S group also showed a decrease in sleep latency compared with the DIZ.

[0078] The above results show that the composition of the present invention has a significant effect on reducing sleep latency (Table 3, Figure 4 ).

[0079]

Table 3

[0080] Effect of ingestion of the composition of the present invention on sleep latency induced by pentobarbital (unit: minutes)

[0081]

[0082] Regarding sleep time, the DIZ group had significantly more sleep time than the CON group, and the S group also had significantly more sleep time than the CON group, and this effect was also very high when compared with the DIZ group. In addition, the S group had a significant increase in sleep time of 60.0% compared with the CON group, showing a significant sleep acceleration effect (Table 4, Figure 5 ).

[0083]

Table 4

[0084] Effect of ingestion of the composition of the present invention on sleep time induced by pentobarbital (unit: minutes)

[0085]

[0086] The above results show that in the sleep acceleration animal model fed with 45 mg / kg pentobarbital, the S group had a significant effect on the reduction of sleep latency and the increase of sleep time, and the composition of the present invention has the effect of improving sleep health.

[0087] 2) Analysis of the effects of the composition of the present invention on sleep latency and sleep duration in a sleep acceleration animal model using subthreshold concentrations of pentobarbital to induce sleep

[0088] The composition of the present invention is 1x10 9 CFU / 100 μL concentration was dissolved and then orally fed, the control group was distilled water, and diazepam was dissolved and then orally fed at a concentration of 2 mg / kg. 45 minutes after the sample was fed, a concentration below the threshold, i.e., 30 mg / kg, was intraperitoneally injected to analyze the sleep acceleration effect of the composition of the present invention.

[0089] The sleep latency of the CON group (16.8±3.2 minutes), which was fed with a subthreshold concentration of 30 mg / kg pentobarbital, increased by about 12 minutes (3.5 times) compared with the group (4.8±0.5 minutes) fed with 45 mg / kg pentobarbital. The sleep latency of the DIZ group (which was fed with diazepam) also increased from 3.2±0.2 minutes to 6.0±0.7 minutes, showing significant changes (Tables 3 and 5).

[0090] The sleep latency in the S group was significantly reduced compared with the CON group, and this effect was also very high when compared with the DIZ group (Table 5).

[0091] Compared with the CON group, the sleep latency of the S group was significantly reduced by 56.0%, showing a significant sleep acceleration effect (Table 5, Figure 6 ).

[0092]

Table 5

[0093] Effect of ingestion of the composition of the present invention on the sleep latency induced by pentobarbital at a concentration below the sleep induction threshold (unit: minutes)

[0094]

[0095] The following changes were confirmed: the sleep time of the CON group (22.1±4.4 minutes) fed with subthreshold concentration of pentobarbital (30 mg / kg) was reduced by about 35.4 minutes compared with that of the group (57.5±4.7 minutes) fed with 45 mg / kg of pentobarbital, and the sleep time of the DIZ group (fed with diazepam) was also reduced from 104.4±10.1 minutes to 82.4±7.7 minutes (Table 4, Table 6).

[0096] Compared with the CON group, the sleep time of the S group increased significantly by 154.8%, showing a significant sleep acceleration effect (Table 6, Figure 7 ).

[0097]

Table 6

[0098] Effect of ingestion of the composition of the present invention on sleep duration induced by pentobarbital at a concentration below the sleep induction threshold (unit: minutes)

[0099]

[0100] The above results show that the S group had an effect on reducing sleep latency and increasing sleep time in the sleep acceleration animal model fed with pentobarbital at a concentration below the threshold, i.e., 30 mg / kg. Therefore, the composition of the present invention has the effect of improving sleep health.

[0101] In addition, the sleep acceleration effect produced by ingestion of the composition of the present invention is more significant when 30 mg / kg of pentobarbital is administered compared to 45 mg / kg of pentobarbital, and thus it is considered to have higher sleep health improvement functionality.

[0102] 3) Results of analysis of the effect of ingestion of the composition of the present invention on the melatonin concentration in the blood

[0103] It is known that when sleep health improves, changes in the composition of intestinal microorganisms lead to an increase in melatonin concentration in the body, so in order to confirm the mechanism of improved sleep health, it is necessary to measure melatonin concentration.

[0104] Melatonin is involved in the regulation of the 24-hour circadian rhythm and changes physiological functions by regulating secretion. The physiological function of melatonin is a hormone that relaxes the body and induces sleep. When the concentration of melatonin in the blood increases, it can be considered to have an effect on accelerating sleep.

[0105] After taking the composition of the present invention, the concentration of melatonin in the blood was measured. The result showed that the melatonin content in the blood of the S group was significantly higher than that of the CON group by 49.8% (Table 7, Figure 8 ).

[0106]

Table 7

[0107] Effect of ingestion of the composition of the present invention on blood melatonin concentration

[0108]

[0109] The above results show that the composition of the present invention increases the concentration of melatonin in the blood, has the effects of reducing sleep latency and increasing sleep time.

[0110] 4) Results of analysis of the effect of ingestion of the composition of the present invention on blood GABA concentration

[0111] GABA inhibits nerve activity by binding to receptors. It is released before the synapse of inhibitory neurons and binds to GABA receptors on the surface of neurons after the synapse. GABA receptors bound to GABA can inhibit the excitability of neurons. When the secretion of GABA increases, it can act as a substance that stabilizes sleep-promoting activities.

[0112] The GABA concentration in the blood of the DIZ group was significantly increased compared with the CON group, and the GABA concentration in the blood of the S group was 25.5% higher than that of the CON group (Table 8, Fig. 9 ).

[0113] The above results show that the composition of the present invention increases the GABA concentration in the blood and has a significant effect on improving sleep.

[0114]

Table 8

[0115] Effect of ingestion of the composition of the present invention on blood GABA concentration

[0116]

Claims

1. A pharmaceutical composition for improving sleep or maintaining deep sleep, wherein: The active ingredient comprises one or more of Lactobacillus reuteri LM1063 strain (KCTC13232BP), a culture of the strain, a disrupted product of the strain, and an extract of the strain.

2. The composition according to claim 1, wherein The improving sleep or maintaining deep sleep includes reducing sleep latency or increasing sleep time.

3. The composition according to claim 1, wherein The composition is capable of increasing the formation of melatonin.

4. The composition according to claim 1, which is capable of increasing the formation of GABA (gamma-aminobutyric acid).

5. A health functional food, wherein: The active ingredient comprises one or more of Lactobacillus reuteri LM1063 strain (KCTC13232BP), a culture of the strain, a disrupted product of the strain, and an extract of the strain.

6. A food material, wherein: The active ingredient comprises one or more of Lactobacillus reuteri LM1063 strain (KCTC13232BP), a culture of the strain, a disrupted product of the strain, and an extract of the strain.

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