Composition for preventing or alleviating sleep disorders using combination regimen, comprising lactobacillus sp. strain and korean medicinal herbs

Abstract

The present invention relates to a composition which is for preventing or alleviating sleep disorders, and uses a combination regimen, the composition comprising Lactobacillus sp. strains and Korean medicinal herbs. According to one aspect of the present invention, the composition which is for preventing or alleviating sleep disorders and is co-administered with Lactobacillus sp. strains and Korean medicinal herbs can be effectively used for preventing or treating sleep disorder-related diseases such as alleviating defecation disorder symptoms caused by sleep disorders or preventing or alleviating depression caused by sleep disorders.

Description

Composition for preventing or improving sleep disorders using combination therapy containing a strain of the genus Lactobacillus and herbal medicine

[0001] The present invention relates to a composition for preventing or improving sleep disorders by administering a Lactobacillus strain and a herbal medicine in combination.

[0002] Sleep disorders are a common problem faced by modern people, affecting over 20% of the population at some point in their lives. These disorders are characterized by inability to achieve healthy sleep, difficulty maintaining daytime alertness despite adequate sleep, or difficulty falling asleep or staying awake due to disrupted sleep rhythms. Psychological issues can also play a significant role, resulting from excessive stress, anxiety, tension, and fear.

[0003] Sleep disorders can lead to a variety of personal and social problems, including learning disabilities, decreased productivity, traffic accidents, safety hazards, emotional disorders, social adjustment difficulties, marital dissatisfaction, and industrial accidents. Furthermore, if left untreated, sleep disorders can lead to constipation, bowel disorders like irritable bowel syndrome, depression, restless legs syndrome, myocardial infarction, and stroke.

[0004] Meanwhile, sleep disorders can also arise due to aging, and these sleep disorders, along with various physiological changes in the body, can lead to additional health problems such as bowel disorders and depression. Generally, as we age, the body's circadian rhythms and sleep patterns change, leading to decreased sleep quality. This increases the likelihood of various sleep disorders. Persistent sleep disorders can affect the autonomic nervous system, which regulates bowel function, leading to bowel problems. Furthermore, sleep deprivation and irregular sleep can lead to emotional instability, increasing the risk of depression.

[0005] To treat these sleep disorders, chemical hypnotics are used, which reversibly suppress the central nervous system to induce and maintain sleep. Conventional hypnotics have central depressant effects similar to anesthetics, producing sedation in small doses and sleep in moderate doses. However, larger doses can cause coma, paralysis, and respiratory depression. Barbiturates are known for their low safety profile, easily leading to tolerance and dependence. Discontinuation of long-term use can lead to sleep disturbances, such as nightmares.

[0006] Accordingly, for long-term users of sleeping pills and those with limited use, the need for alternatives is increasing, and the demand for compositions for preventing or improving sleep disorders with fewer side effects is also increasing.

[0007] One aspect is to provide a composition for preventing or improving sleep disorders, which comprises a first active substance including a Lactobacillus sp. strain or a culture thereof and a second active substance including a herbal medicine as active ingredients.

[0008] Another aspect is to provide a composition for preventing or improving sleep disorders, which comprises a first active ingredient including a Lactobacillus sp. strain or a culture thereof as an active ingredient, and a second active ingredient including a herbal medicine, which is administered in combination.

[0009] Another aspect provides a composition for preventing, improving or treating a sleep disorder-related disease, comprising a first active substance including a Lactobacillus sp. strain or a culture thereof and a second active substance including a herbal medicine as active ingredients.

[0010] Another aspect is to provide a composition for preventing, improving or treating a sleep disorder-related disease, which comprises a first active ingredient including a Lactobacillus sp. strain or a culture thereof as an active ingredient and a second active ingredient including a herbal medicine, which are administered in combination.

[0011] Another aspect is to provide a health functional food for preventing or improving bowel disorders or depression, which comprises a first active substance including a Lactobacillus sp. strain or a culture thereof and a second active substance including a herbal medicine as active ingredients.

[0012] Another aspect is to provide a health functional food for preventing or improving bowel disorders or depression, which comprises a first active ingredient including a Lactobacillus sp. strain or a culture thereof as an active ingredient, and a second active ingredient including a herbal medicine is administered in combination.

[0013] Another aspect provides a pharmaceutical composition for preventing or treating bowel disorders or depression, comprising a first active substance including a Lactobacillus sp. strain or a culture thereof and a second active substance including a herbal medicine as active ingredients.

[0014] Another aspect is to provide a pharmaceutical composition for preventing or treating bowel disorders or depression, which comprises a first active ingredient including a Lactobacillus sp. strain or a culture thereof as an active ingredient, and a second active ingredient including a herbal medicine, which is administered in combination.

[0015] Another aspect provides a method for preventing or improving sleep disorders, comprising administering to a subject in need thereof a composition comprising a first active ingredient comprising a Lactobacillus sp. strain or a culture thereof and a second active ingredient comprising a herbal medicine as active ingredients.

[0016] Another aspect provides a use of a composition comprising a first active ingredient including a Lactobacillus sp. strain or a culture thereof and a second active ingredient including a herbal medicine for preventing or improving sleep disorders.

[0017] Another aspect provides a use of a composition comprising a first active substance comprising a Lactobacillus sp. strain or a culture thereof for use in the manufacture of a medicine for preventing or improving sleep disorders, and a second active substance comprising a herbal medicine as active ingredients.

[0018] Another aspect provides a method for preventing or improving sleep disorders, comprising the steps of: administering to a subject in need thereof a first active substance comprising a Lactobacillus sp. strain or a culture thereof; and administering to a subject in need thereof a second active substance comprising a herbal medicine.

[0019] Another aspect provides a use of a composition comprising a first active ingredient including a Lactobacillus sp. strain or a culture thereof for preventing or improving sleep disorders, and a second active ingredient including a herbal medicine, which is administered in combination.

[0020] Another aspect provides a use of a composition comprising a first active ingredient including a Lactobacillus sp. strain or a culture thereof for use in the manufacture of a medicine for preventing or improving sleep disorders, and a second active ingredient including a herbal medicine, which is administered in combination.

[0021] Another aspect provides a use of a composition comprising a first active ingredient including a Lactobacillus sp. strain or a culture thereof for preventing or improving sleep disorders and a second active ingredient including a herbal medicine as active ingredients; or comprising the first active ingredient as an active ingredient and the second active ingredient being administered together.

[0022] Another aspect provides a use of a composition comprising a first active substance including a Lactobacillus sp. strain or a culture thereof for use in the manufacture of a medicine for preventing or improving sleep disorders and a second active substance including a herbal medicine as active ingredients; or comprising the first active substance as an active ingredient and the second active substance being administered together.

[0023] One aspect provides a composition for preventing or improving sleep disorders, or a pharmaceutical composition for preventing, improving or treating a disease related to sleep disorders, comprising a first active ingredient including a Lactobacillus sp. strain or a culture thereof and a second active ingredient including a herbal medicine as active ingredients; or comprising the first active ingredient and the second active ingredient as active ingredients, wherein the second active ingredient is administered in combination.

[0024] The term Lactobacillus in this specification refers to a gram-positive, rod-shaped microorganism of the genus Lactobacillus, which is widely distributed in nature, and the strain of the genus Lactobacillus is not limited in type as long as it is a strain of the genus Lactobacillus known in the art.

[0025] The above Lactobacillus has been renamed to Limosilactobacillus or Lactiplantibacillus, and the changed strain names can be used interchangeably in this specification. For example, Lactobacillus fermentum has been renamed to Limosilactobacillus fermentum, and Lactobacillus plantarum has been renamed to Lactiplantibacillus plantarum.

[0026] In this specification, Lactobacillus sp. means the old Lactobacillus sp. prior to the change of name to include Lactiplantibacillus plantarum and Limosilactobacillus fermentum.

[0027] In one specific example, the first active ingredient may be Lactobacillus fermentum and Lactobacillus plantarum.

[0028] In one specific example, the strain may be Lactobacillus fermentum GB102 strain belonging to the genus Lactobacillus sp. deposited under KCTC 14105BP, Lactobacillus fermentum GB103 strain belonging to the genus Lactobacillus sp. deposited under deposit number KCTC 14106BP, or Lactobacillus plantarum GB104 strain belonging to the genus Lactobacillus sp. deposited under KCTC 14107BP.

[0029] In one specific example, the first active ingredient may additionally include a Bifidobacterium animalissub sp. Lactis strain.

[0030] In one specific example, the first active ingredient may additionally include a Bifidobacterium animalissub sp. lactis strain and a Lactobacillus acidophilus strain.

[0031] In one specific example, the strain is freeze-dried and 10 3 10 inland 16 It may be included in an amount of CFU / g. Specifically, the strain may be, for example, 1 strain, 2 mixed strains, or 3 mixed strains, in an amount of 10 3 10 inland 16 CFU / g, 10 3 10 inland 15 CFU / g, 10 3 10 inland 14 CFU / g, 10 3 10 inland 13 CFU / g, 10 3 10 inland 12 CFU / g, 10 410 inland 16 CFU / g, 10 4 10 inland 15 CFU / g, 10 4 10 inland 14 CFU / g, 10 4 10 inland 13 CFU / g, 10 4 10 inland 12 CFU / g, 10 5 10 inland 16 CFU / g, 10 5 10 inland 15 CFU / g, 10 5 10 inland 14 CFU / g, 10 5 10 inland 13 CFU / g, 10 5 10 inland 12 CFU / g, 10 6 10 inland 13 CFU / g, 10 6 10 inland 12 CFU / g, 10 7 10 inland 13 CFU / g, 10 7 10 inland 12 CFU / g, 10 8 10 inland 13 CFU / g or 10 8 10 inland 12 It may include, but is not limited to, the amount of CFU / g.

[0032] For example, the composition according to one embodiment may comprise 0.001 wt% to 80 wt% of a Lactobacillus strain based on the total weight of the composition. In addition, the dosage of the Lactobacillus strain may be 0.01 mg to 10,000 mg, 0.1 mg to 1000 mg, 1 mg to 100 mg, 0.01 mg to 1000 mg, 0.01 mg to 100 mg, 0.01 mg to 10 mg, or 0.01 mg to 1 mg. The strain is included in the composition in a therapeutically effective amount or nutritionally effective concentration, for example, 1 strain, 2 mixed strains, or 3 mixed strains in an amount of 10 3 10 inland 16 CFU / g, 10 3 10 inland 15 CFU / g, 10 3 10 inland 14 CFU / g, 10 3 10 inland 13 CFU / g, 10 3 10 inland 12 CFU / g, 10 4 10 inland 16 CFU / g, 10 4 10 inland 15 CFU / g, 10 4 10 inland 14 CFU / g, 10 4 10 inland 13 CFU / g, 10 4 10 inland 12 CFU / g, 10 5 10 inland 16 CFU / g, 10 5 10 inland 15 CFU / g, 10 5 10 inland 14 CFU / g, 10 5 10 inland 13 CFU / g, 10 5 10 inland 12 CFU / g, 10 6 10 inland 13 CFU / g, 10 6 10 inland 12 CFU / g, 107 10 inland 13 CFU / g, 10 7 10 inland 12 CFU / g, 10 8 10 inland 13 CFU / g or 10 8 10 inland 12 It may be included in the composition as a content of CFU / g or as a culture of an equivalent number of live or dead cells.

[0033] The therapeutically effective amount refers to the amount of the pharmaceutical composition comprising the mixed strain and herbal medicine for the methods and uses of the present invention; or the mixed strain and herbal medicine for the methods and uses of the present invention; that will elicit a biological or medical response or desired therapeutic effect in a patient as desired by a researcher, physician, or other clinician. The therapeutically effective amount of the mixed strain and herbal medicine may vary depending on factors such as the disease state, age, sex, and weight of the subject, and the ability of the herbal medicine to elicit a desired response in the subject. A therapeutically effective amount is also the amount at which the therapeutically beneficial effects outweigh any toxic or detrimental effects.

[0034] In one specific example, the mixed strains of Lactobacillus may have sleep-inducing activity.

[0035] The term "sleep disorder" as used herein refers to a disrupted sleep pattern caused by a number of causes, including dysfunctional sleep mechanisms, abnormalities in physiological functions during sleep, abnormalities in the circadian rhythm, and sleep disorders induced by factors unrelated to the sleep process, and may be, for example, insomnia. Such insomnia may include middle-of-the-night insomnia, late-night insomnia, insomnia with prolonged wakefulness after sleep onset, sleep maintenance insomnia, and insomnia following nocturnal awakening.

[0036] The term “sleep disorder-related disease” used in this specification refers to diseases such as bowel disorders, constipation, irritable bowel syndrome, depression, restless leg syndrome, myocardial infarction, and stroke caused by physiological changes resulting from sleep disorders.

[0037] In one specific example, the second active ingredient may include a herbal medicine. For example, the second active ingredient may be selected from the group consisting of, but is not limited to, yam, job's tears, red ginseng, lingzhi, and combinations thereof.

[0038] In this specification, the term "herbal medicine" is defined in the Pharmaceutical Affairs Act as a raw medicinal material used to manufacture herbal medicine or herbal medicine preparations. Since the medicinal properties of herbal medicines can vary depending on the processing method, the processing method and manufacturing method are important. In the present invention, the herbal medicine may be, but is not limited to, yam, job's tears, red ginseng, or lingzhi. In this case, the herbal medicine may be used in powder or extract form.

[0039] The term "Dioscorea japonica" in this specification refers to a perennial vine of the Dioscorea family (Dioscoeaceae) in the order Dioscorea. Currently, there are approximately 650 species in 10 genera known, of which about 10 are used as food resources. It mainly grows in mountainous areas and is known to be distributed in Korea, Japan, and other regions.

[0040] The term "jobivar (Coix lachryma-jobivar. mayuen)" used herein refers to a plant belonging to the gramineae family, primarily cultivated in Southeast Asia. When used as a herbal medicine, it is called "Yiyiin" and has diuretic, analgesic, and tonic properties, making it useful for edema, bladder stones, rheumatism, and other conditions. Additionally, the raw leaves are used as a tea substitute, and the roots are used to treat jaundice and neuralgia.

[0041] The term "red ginseng" used herein refers to the steamed root of ginseng (Panax ginseng C. A. Meyer). It is known to have anti-aging, anti-fatigue, anti-stress effects, blood circulation, immune function, osteoporosis prevention, anemia treatment, male infertility treatment, hypertension and diabetes improvement, and cancer prevention. The red ginseng may be in liquid or powder form.

[0042] The term "lingzhi" or reishi mushroom in this specification refers to the elixir of youth (Ganoderma lucidum (Leyss. ex. Fr) Karst), also known as seoncho, gilsangbeoseot, and jeokji. Rich in minerals such as calcium and potassium, and containing a lot of unsaturated fatty acids, it is known to be effective in preventing adult diseases, removing phlegm, alleviating asthma, and aiding respiratory system diseases. It is also known to inhibit cancer growth, lower blood cholesterol levels, and lower blood pressure.

[0043] The herbal medicine of the present invention may be a mixture of one or more types.

[0044] Specifically, the herbal medicine may be a mixture of two types of herbal medicines. In one specific example, the herbal medicine may be a mixture of yam and job's tears. In one specific example, the herbal medicine may be a mixture of yam and red ginseng. In one specific example, the herbal medicine may be a mixture of yam and lingzhi. In one specific example, the herbal medicine may be a mixture of job's tears and red ginseng. In one specific example, the herbal medicine may be a mixture of job's tears and lingzhi. In one specific example, the herbal medicine may be a mixture of red ginseng and lingzhi.

[0045] The above herbal medicine may be a mixture of three kinds of herbal medicines. In one specific example, the herbal medicine may be a mixture of yam, job's tears, and red ginseng. In one specific example, the herbal medicine may be a mixture of yam, job's tears, and lingzhi. In one specific example, the herbal medicine may be a mixture of yam, red ginseng, and lingzhi. In one specific example, the herbal medicine may be a mixture of job's tears, red ginseng, and lingzhi.

[0046] The above herbal medicine may be a mixture of four kinds of herbal medicines. Preferably, the four kinds of herbal medicines of yam, job's tears, red ginseng, and lingzhi may be mixed in a predetermined ratio.

[0047] The above herbal medicine may contain substances other than the above yam, job's tears, red ginseng, and lingzhi.

[0048] The term "culture" in this specification may be used interchangeably with "culture supernatant", "conditioned medium" or "conditioned medium", and may mean the entire medium including the strain, its metabolites, extra nutrients, etc. obtained by culturing the strain for a certain period of time in a medium capable of supplying nutrients so that the strain of the genus Lactobacillus can grow and survive in vitro. In addition, the culture medium may mean a culture medium obtained by removing the cells from the cell culture medium obtained by culturing the strain. The medium may be selected from known liquid media or solid media, and may be, for example, but is not limited to, MRS liquid medium, GAM liquid medium, MRS agar medium, GAM agar medium, and BL agar medium.

[0049] In one specific example, the composition for preventing or improving sleep disorders may further comprise a third active substance including inulin or vitamins; or the third active substance may be additionally administered in combination.

[0050] In one specific example, the third active ingredient may be selected from the group consisting of, but is not limited to, inulin, vitamins, and combinations thereof.

[0051] The term "inulin" used herein refers to a polysaccharide found abundantly in root vegetables, a type of dietary fiber known as fructan. It is used as an energy storage medium in some plants. After ingestion, inulin is not broken down by digestive enzymes in the body, but rather is fermented by intestinal microorganisms, promoting bowel function before being excreted. It is known to suppress postprandial blood sugar levels, improve cholesterol levels, and alleviate constipation.

[0052] The term “vitamin” as used herein may be, but is not limited to, vitamin A, vitamin B (e.g., B1 or B2), vitamin C, vitamin E, vitamin K, or a combination thereof.

[0053] Vitamin B2, also known as riboflavin or lactoflavin, is a water-soluble vitamin. It is found in abundance in liver, egg yolks, spinach, milk, and other foods. Deficiency can lead to growth retardation, premature aging, meningitis, dermatitis, alopecia, sore throat, and gastrointestinal problems.

[0054] In one specific example, the composition may further comprise the first active ingredient being Lactobacillus fermentum and Lactobacillus plantarum, the second active ingredient being yam, Job's tears, red ginseng, and reishi, and the first active ingredient further comprising a Bifidobacterium animalissub sp. Lactis strain and a Lactobacillus acidophilus strain, and the third active ingredient further comprising inulin and vitamins.

[0055] In this specification, the term "including as an active ingredient" means that a Lactobacillus genus strain, a vesicle derived from the strain, a lysate of the strain, a culture medium, or an extract of the culture medium are added, and includes formulation in various forms by adding various components as auxiliary components for drug delivery and stabilization, etc.

[0056] In one specific example, the composition may be one in which the first active substance, the second active substance, and the third active substance are administered simultaneously, sequentially, or in reverse order.

[0057] The terms "combination administration," "combination therapy," "combined treatment," "combination administration," or "in combination" as used herein are used interchangeably and refer to any form of simultaneous or concurrent treatment using substances useful for treating at least two separate diseases. The components of the combination administration may be administered simultaneously, sequentially, in reverse order, or in any order. The components may be administered in different dosages, at different administration frequencies, or via different routes as appropriate.

[0058] As used herein, the term “administration” means introducing a given substance into an individual by an appropriate method.

[0059] The term "simultaneous administration" as used herein is not particularly limited and means that the components of the combination are administered substantially simultaneously, for example, as a mixture or in an immediately subsequent sequence.

[0060] The term "sequentially administered" as used herein is not particularly limited and means that the components of the combination are not administered simultaneously, but are administered one after the other or in clusters with a specific time interval between administrations. The time intervals may be the same or different between the individual administrations of the components of the combination, and may be selected, for example, from the range of 2 minutes to 96 hours, 1 day to 7 days, or 1 week, 2 weeks, or 3 weeks. Typically, the time interval between administrations may range from several minutes to several hours, for example, from 2 minutes to 72 hours, from 30 minutes to 24 hours, or from 1 to 12 hours. Additional examples include time intervals ranging from 24 to 96 hours, from 12 to 36 hours, from 8 to 24 hours, and from 6 to 12 hours.

[0061] In one specific example, the composition comprises a first oral formulation comprising the first active ingredient, a second oral formulation comprising the second active ingredient, and a third oral formulation comprising the third active ingredient, wherein the first, second, and third oral formulations may be administered orally.

[0062] In one embodiment, the oral formulation may be in the form of tablets, pills, capsules, lozenges, granules, powders, suspensions, sachets, or syrups.

[0063] The term "prevention" in this specification may mean any act of suppressing or delaying the onset of a disease state in an individual by administering a pharmaceutical composition according to one aspect.

[0064] The term "improvement" herein may mean any action that at least reduces a parameter associated with the condition being treated, for example, the severity of a symptom.

[0065] The term "treatment" in this specification may mean any action that improves or beneficially changes the symptoms of a disease state of an individual by administering a pharmaceutical composition according to one aspect.

[0066]

[0067] Another aspect provides a health functional food for preventing or improving sleep disorders, comprising a first active ingredient including a Lactobacillus sp. strain or a culture thereof and a second active ingredient including a herbal medicine as active ingredients; or comprising the first active ingredient and the second active ingredient being administered in combination.

[0068] Another aspect provides a health functional food for preventing or improving bowel disorders or depression, comprising a first active substance including a Lactobacillus sp. strain or a culture thereof and a second active substance including a herbal medicine as active ingredients; or comprising the first active substance as an active ingredient and co-administering the second active substance.

[0069] In one specific example, co-administration of the mixed strains of the Lactobacillus genus and / or herbal medicine can improve bowel disorders.

[0070] In one specific example, co-administration of mixed strains of Lactobacillus and / or herbal medicine can alleviate depression.

[0071] In this specification, the term “defecation disorder” may refer to diseases that occur in the lower part of the rectum, anus, and perianal tissues, such as hemorrhoids, anal fistula, anal fissure, proctitis, anal itching, rectal prolapse, or constipation. The “constipation” may refer to cases where the frequency of defecation is less than twice a week, the weight of the stool is less than 35 g per day, difficult defecation, hard stool, or a feeling of incomplete evacuation occurs in more than 25% of the defecation times, and two or more of the above symptoms persist for more than three months.

[0072] In this specification, the term “depression” refers to a mental illness that causes a decline in daily functioning by causing various cognitive, mental, or physical symptoms with a decrease in motivation and depression as the main symptoms, and makes daily life difficult due to a decline in overall functions such as thought processes, motivation, desire, behavior, and sleep, and is highly correlated with the prevalence and suicide rate.

[0073] In one specific example, the bowel disorder or depression may be caused by, but is not limited to, a sleep disorder.

[0074] In one specific example, the health functional food may further include a third active substance including inulin or a vitamin; or the third active substance may be additionally administered in combination.

[0075] In one specific example, the second active ingredient may be one or more selected from the group consisting of ginseng, yulmu, red ginseng, lingzhi, and combinations thereof, but is not limited thereto.

[0076] In one specific example, the first active substance, the second active substance, and the third active substance may be administered simultaneously, sequentially, or in reverse order.

[0077] In one specific example, the health functional food comprises a first oral formulation comprising the first active substance, a second oral formulation comprising the second active substance, and a third oral formulation comprising the third active substance, and the first, second, and third oral formulations may be administered orally.

[0078] The term "health functional food" as used herein refers to a food manufactured or processed for the purpose of health supplementation using a specific ingredient as a raw material or a specific ingredient contained in a food raw material through extraction, concentration, purification, mixing, etc., and refers to a food designed and processed so that the above-mentioned ingredient can sufficiently exert bioregulatory functions on the body, such as biological defense, biological rhythm regulation, disease prevention and recovery, etc. The above-mentioned health functional food can perform functions related to the prevention and improvement of metabolic diseases, etc.

[0079] There are no specific restrictions on the types of the above foods. Examples of foods to which the above extract can be added include formulations selected from the group consisting of powders, granules, tablets, capsules, pills, gels, jellies, suspensions, emulsions, syrups, tea bags, infused teas, gums, candies, and health drinks, and include all health foods in the conventional sense.

[0080] The above health functional food may include food additives that are food-related and acceptable, and may include an appropriate carrier commonly used in the manufacture of health functional foods.

[0081] As used herein, the term "food-acceptable" means that the compound exhibits the property of being non-toxic to cells or humans exposed to it.

[0082] The above health functional food may be formulated into one selected from the group consisting of tablets, pills, powders, granules, powders, capsules, and liquid formulations, further comprising one or more of a carrier, diluent, excipient, and additive. Foods to which compounds according to one aspect may be added include various foods, powders, granules, tablets, capsules, syrups, beverages, gum, tea, vitamin complexes, and health functional foods.

[0083] In addition to containing the above-mentioned effective ingredient, the above-mentioned health functional food may contain other ingredients as essential ingredients without special restrictions. For example, it may contain various flavoring agents or natural carbohydrates as additional ingredients, as in conventional beverages. Examples of the above-mentioned natural carbohydrates may include conventional sugars such as monosaccharides, such as glucose, fructose, etc.; disaccharides, such as maltose, sucrose, etc.; and polysaccharides, such as dextrin, cyclodextrin, etc.; and sugar alcohols, such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (thaumatin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) may be advantageously used. The proportion of the above-mentioned natural carbohydrates may be appropriately determined by a person skilled in the art.

[0084] In addition to the above, health functional foods according to the aspect may contain various nutrients, vitamins, minerals (electrolytes), flavorings such as synthetic flavorings and natural flavorings, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. These ingredients may be used independently or in combination, and the ratio of these additives may also be appropriately selected by those skilled in the art.

[0085] In the above health functional food, the active ingredient can be added directly to the food or used in combination with other foods or food ingredients, and can be used appropriately according to conventional methods. The amount of the active ingredient mixed can be appropriately determined depending on the intended use (prevention or improvement). Generally, when manufacturing a food or beverage, the health functional food can be added in an amount of about 15% by weight or less, more specifically about 10% by weight or less, based on the raw material. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount may be below the above range.

[0086]

[0087] Another aspect provides a food composition for preventing or improving sleep disorders, bowel disorders or depression, comprising a first active substance including a Lactobacillus sp. strain or a culture thereof and a second active substance including a herbal medicine as active ingredients; or comprising the first active substance as an active ingredient and co-administering the first active substance with the second active substance.

[0088] The above-mentioned food compositions for preventing or improving sleep disorders, bowel disorders, and depression include all forms of functional foods, nutritional supplements, health foods, and food additives, and the above-mentioned types of food compositions can be prepared in various forms according to conventional methods known in the art. For example, the compositions of the present disclosure may be considered food supplements. Food supplements, also known as dietary supplements or nutritional supplements, may be considered another specific pharmaceutical product. They are prepared for the purpose of supplementing the diet and are intended to provide nutrients or beneficial ingredients that may not be consumed or consumed in sufficient amounts in a normal diet. Most food supplements are considered foods, but sometimes they are considered drugs, natural health products, or nutraceutical products. In the sense of the present invention, food supplements include health functional foods. Dietary supplements are typically sold over the counter without a prescription. When taken in pill or capsule form, they contain the same excipients used in pharmaceuticals. However, dietary supplements can also take the form of foods fortified with certain nutrients (e.g., infant formula). Therefore, in certain embodiments, the composition of the present invention is a food supplement.

[0089] Furthermore, the composition of the present invention may be incorporated into various edible foods and foods for infants, such as dairy products. The term "edible product" as used herein broadly encompasses any product that can be ingested by an animal in any form (e.g., a product that can be perceived by the sense organs). The term "food product" is understood to refer to an edible product that provides nutritional support to the body. Food supplements and infant formulas are particularly interesting. The food preferably comprises a carrier material such as oatmeal gruel, lactic acid fermented foods, resistant starch, dietary fibers, carbohydrates, proteins, and glycosylated proteins. In certain embodiments, the bacterial cells of the present invention are homogenized with other ingredients, such as cereals or powdered milk, to form infant formula.

[0090]

[0091] Another aspect provides a feed composition for preventing or improving sleep disorders, bowel disorders or depression, comprising a first active substance including a Lactobacillus sp. strain or a culture thereof and a second active substance including a herbal medicine as active ingredients; or comprising the first active substance as an active ingredient and co-administering it with the second active substance.

[0092] At this time, the feed composition may further include one or more strains selected from the group consisting of Bifidobacterium animalis lactis strains and Lactobacillus acidophilus strains, or a culture thereof. In addition, the composition may additionally include inulin and vitamin B2.

[0093] The feed composition for preventing or improving the above sleep disorder, bowel disorder or depression can be manufactured by adding the strain or its culture and herbal medicine in an appropriate effective concentration range according to various feed manufacturing methods known in the art, and can be used as a feed additive composition for the purpose of preventing or improving diseases related to sleep disorder.

[0094] The above "feed" may mean any natural or artificial diet, meal, etc., or ingredients of the meal, which are suitable for or intended for an animal to eat, ingest, or digest. The type of feed is not particularly limited, and feed commonly used in the relevant technical field may be used. Non-limiting examples of the feed include plant feed such as grains, roots, fruits, food processing by-products, algae, fibers, pharmaceutical by-products, oils, starches, meal, or grain by-products; animal feed such as proteins, fat-free substances, oils, minerals, oils, single-cell proteins, zooplankton, or food.

[0095]

[0096] Another aspect provides a pharmaceutical composition for preventing or treating sleep disorders, comprising a first active ingredient including a Lactobacillus sp. strain or a culture thereof and a second active ingredient including a herbal medicine as active ingredients; or comprising the first active ingredient as an active ingredient and the second active ingredient being administered in combination.

[0097] Another aspect provides a pharmaceutical composition for preventing or treating bowel disorders or depression, comprising a first active ingredient including a Lactobacillus sp. strain or a culture thereof and a second active ingredient including a herbal medicine as active ingredients; or comprising the first active ingredient as an active ingredient and the second active ingredient being administered in combination.

[0098] The term “pharmaceutical composition” as used herein means a mixture containing one or more active ingredients and various ingredients that deliver or assist the same for a specific therapeutic, preventive or diagnostic purpose.

[0099] The pharmaceutical composition may additionally comprise a pharmaceutically acceptable diluent or carrier. The diluent may be lactose, corn starch, soybean oil, microcrystalline cellulose, or mannitol, or a combination thereof. The carrier may be an excipient, a disintegrant, a binder, a glidant, or a combination thereof. The excipient may be microcrystalline cellulose, lactose, low-substituted hydroxycellulose, or a combination thereof. The disintegrant may be calcium carboxymethylcellulose, sodium starch glycolate, calcium dihydrogen phosphate anhydrous, or a combination thereof. The binder may be polyvinylpyrrolidone, low-substituted hydroxypropyl cellulose, hydroxypropyl cellulose, or a combination thereof. The glidant may be magnesium stearate, silicon dioxide, talc, or a combination thereof.

[0100] When the above pharmaceutical composition is formulated, it can be prepared using diluents or excipients such as lubricants, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, fillers, bulking agents, binders, wetting agents, disintegrating agents, and surfactants that are commonly used. Solid preparations for oral administration may include tablets, pills, powders, granules, capsules, etc., and such solid preparations can be prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc., with the above composition. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, fragrances, and preservatives. Preparations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may include witepsol, macrogol, tween 61, cacao butter, laurin butter, and glycerogelatin. When manufacturing in the form of eye drops, known diluents or excipients may be used.

[0101] The pharmaceutical composition may contain carbohydrates such as glucose, sucrose or dextran, antioxidants such as ascorbic acid or glutathione, chelating agents, low-molecular-weight proteins or other stabilizers to increase stability or absorbability.

[0102] In one specific embodiment, the pharmaceutical composition may be formulated as an oral or parenteral dosage form. Oral dosage forms may include granules, powders, liquids, tablets, capsules, dry syrups, or combinations thereof. Parenteral administration may be administered topically, or by intraperitoneal injection, intrarectal injection, subcutaneous injection, intravenous injection, intramuscular injection, or intrathoracic injection.

[0103] In one embodiment, the route of administration of the pharmaceutical composition includes, but is not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal.

[0104] The above pharmaceutical composition is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment. The effective dosage level may be determined based on the type and severity of the patient's disease, the activity and sensitivity of the drug, the time of administration, the route of administration, and the excretion rate, the duration of treatment, concomitant medications, and other factors well known in the medical field.

[0105] In one aspect, the pharmaceutical composition may be administered once a day or in several divided doses. Specifically, the pharmaceutical composition may be administered in a cycle of 6 days of administration followed by 1 day of rest, 5 days of administration followed by 2 days of rest, or 4 days of administration followed by 3 days of rest, based on a 7-day schedule. More specifically, the pharmaceutical composition may be administered in a cycle of 5 days of administration followed by 2 days of rest.

[0106] In addition, the pharmaceutically effective amount and effective dosage of the pharmaceutical composition may vary depending on the method of formulation, administration method, administration time, and / or administration route of the pharmaceutical composition. In addition, the type and degree of the response to be achieved by administration of the pharmaceutical composition, the type, age, weight, general health condition, symptoms or degree of the disease, sex, diet, excretion, drugs used simultaneously or simultaneously in the subject, other components of the composition, etc., may vary depending on various factors, and similar factors well known in the pharmaceutical field. A person having ordinary skill in the art can easily determine and prescribe an effective dosage for the desired treatment. The pharmaceutical composition according to the present invention may be administered once a day or divided into several times. Therefore, the above dosage does not limit the scope of the present invention in any way. The dosage of the pharmaceutical composition may be 1 ug / kg / day to 1,000 mg / kg / day.

[0107] Another aspect provides a method for preventing or improving sleep disorders, comprising administering to a subject in need thereof a composition comprising a first active ingredient comprising a Lactobacillus sp. strain or a culture thereof and a second active ingredient comprising a herbal medicine as active ingredients.

[0108] Another aspect provides a use of a composition comprising a first active ingredient including a Lactobacillus sp. strain or a culture thereof and a second active ingredient including a herbal medicine for preventing or improving sleep disorders.

[0109] Another aspect provides a use of a composition comprising a first active ingredient comprising a Lactobacillus sp. strain or a culture thereof for use in the manufacture of a medicine for preventing or improving sleep disorders, and a second active ingredient comprising a herbal medicine as active ingredients.

[0110] Another aspect provides a method for preventing or improving sleep disorders, comprising the steps of: administering to a subject in need thereof a first active substance comprising a Lactobacillus sp. strain or a culture thereof; and administering to a subject in need thereof a second active substance comprising a herbal medicine.

[0111] Another aspect provides a use of a composition comprising a first active ingredient including a Lactobacillus sp. strain or a culture thereof for preventing or improving sleep disorders, and a second active ingredient including a herbal medicine, which is administered in combination.

[0112] Another aspect provides a use of a composition comprising a first active ingredient including a Lactobacillus sp. strain or a culture thereof for use in the manufacture of a medicine for preventing or improving sleep disorders, and a second active ingredient including a herbal medicine, which is administered in combination.

[0113] Another aspect provides a use of a composition comprising a first active ingredient including a Lactobacillus sp. strain or a culture thereof for preventing or improving sleep disorders and a second active ingredient including a herbal medicine as active ingredients; or comprising the first active ingredient as an active ingredient and the second active ingredient being administered together.

[0114] Another aspect provides a use of a composition comprising a first active substance including a Lactobacillus sp. strain or a culture thereof for use in the manufacture of a medicine for preventing or improving sleep disorders and a second active substance including a herbal medicine as active ingredients; or comprising the first active substance as an active ingredient and the second active substance being administered together.

[0115] Another aspect provides a method for preventing, improving or treating a sleep disorder-related disease, comprising administering to a subject in need thereof a composition comprising a first active ingredient comprising a Lactobacillus sp. strain or a culture thereof and a second active ingredient comprising a herbal medicine as active ingredients.

[0116] Another aspect provides a use of a composition comprising a first active ingredient including a Lactobacillus sp. strain or a culture thereof and a second active ingredient including a herbal medicine as active ingredients for preventing, improving or treating a sleep disorder-related disease.

[0117] Another aspect comprises the steps of administering to a subject in need thereof a first active substance comprising a Lactobacillus sp. strain or a culture thereof; and

[0118] A method for preventing, improving or treating a sleep disorder-related disease is provided, comprising the step of administering a second active substance including a herbal medicine to a subject in need thereof.

[0119] Another aspect provides a use of a composition comprising a first active ingredient including a Lactobacillus sp. strain or a culture thereof for preventing or improving sleep disorders, and a second active ingredient including a herbal medicine, which is administered in combination.

[0120] Another aspect provides a method for preventing or improving bowel disorders or depression, comprising administering to a subject in need thereof a composition comprising a first active ingredient comprising a Lactobacillus sp. strain or a culture thereof and a second active ingredient comprising a herbal medicine as active ingredients.

[0121] Another aspect provides a use of a composition comprising as active ingredients a first active substance comprising a Lactobacillus sp. strain or a culture thereof for preventing or improving bowel disorders or depression and a second active substance comprising a herbal medicine.

[0122] Another aspect provides a method for preventing or improving bowel disorders or depression, comprising the steps of: administering to a subject in need thereof a first active substance comprising a Lactobacillus sp. strain or a culture thereof; and administering to a subject in need thereof a second active substance comprising a herbal medicine.

[0123] Another aspect provides a use of a composition comprising a first active ingredient including a Lactobacillus sp. strain or a culture thereof for preventing or improving bowel disorders or depression, and a second active ingredient including a herbal medicine, which is administered in combination.

[0124] The terms and methods described for the above inventions apply equally to each invention.

[0125] Duplicate contents are omitted in consideration of the complexity of this specification, and terms not otherwise defined in this specification have meanings commonly used in the technical field to which the present invention belongs.

[0126] According to a composition for preventing or improving sleep disorders by co-administering a Lactobacillus strain and a herbal medicine according to the daily aspect, it can be usefully used for preventing or treating diseases related to sleep disorders, such as alleviating symptoms of bowel disorders induced by sleep disorders or reducing depression induced by sleep disorders.

[0127] Figure 1 shows changes in circadian rhythm gene expression according to combined administration of mixed strains and herbal medicines according to one specific example.

[0128] Figure 2 is a schematic diagram showing the process of recruiting research participants to participate in a human application test of combined administration of mixed strains and herbal medicines according to one specific example.

[0129] FIG. 3 is a graph showing the results of a human application test of combined administration of mixed strains and herbal medicines according to one specific example: FIG. 3a is a graph showing the results of measuring the change in the Insomnia Severity Index (ISI) of the mixed strain and herbal medicine composite preparation (Number Seven) and the placebo group according to one specific example, FIG. 3b is a graph showing the results of evaluating the change in the Gut Quotient (GQ) of the mixed strain and herbal medicine composite preparation (Number Seven) and the placebo group according to one specific example, and FIG. 3c is a graph showing the results of evaluating the change in the CES-D of the mixed strain and herbal medicine composite preparation (Number Seven) and the placebo group according to one specific example.

[0130] Hereinafter, preferred embodiments are presented to aid understanding of the present invention. However, the following embodiments are provided solely to facilitate a better understanding of the present invention and are not intended to limit the scope of the present invention. The embodiments are susceptible to various modifications, and thus the embodiments are not limited to the embodiments disclosed below and may be implemented in various forms.

[0131]

[0132] Example 1. Isolation and identification of Lactobacillus fermentum and Lactobacillus plantarum strains.

[0133] 1.1. Isolation of strains

[0134] The Lactobacillus fermentum strain and Lactobacillus plantarum strain of the present invention were isolated from vaginal samples of healthy women who visited a hospital for the purpose of a health checkup. Specifically, vaginal samples were collected with a cotton swab, inoculated onto Rogosa SL (MRS) plate medium, and cultured in an anaerobic chamber at 37°C for 48 hours. When bacterial colonies grew, single colonies were subcultured onto new MRS plate medium for pure isolation. After pure isolation, strain culture was performed using MRS medium.

[0135]

[0136] 1.2. Selection of strains with fat accumulation inhibition activity

[0137] In order to select a strain with fat accumulation inhibitory activity, the ability to inhibit pancreatic lipase activity and the ability to inhibit differentiation of 3T3-L1 preadipocytes into adipocytes were confirmed.

[0138] Specifically, the activity inhibition ability of pancreatic lipolytic enzyme was confirmed by diluting the strain 1-1 above to a concentration of 0.1 mg / mL, placing it on a plate with 0.167 mM p-nitrophenylpalmitate (PNP; Sigma, USA) solution, 0.061 M Tris-HCl buffer (pH 8.5), and 0.3 mg / mL lipase solution, reacting it at 25°C for 10 minutes, and measuring the absorbance at 405 nm.

[0139] In addition, the inhibition of 3T3-L1 preadipocyte differentiation into adipocytes was determined using Oil Red O (Sigma, USA), which specifically reacts with intracellular lipid droplets. After adipogenic differentiation, the medium was removed, washed twice with PBS, fixed with 10% formalin at 40°C for 1 hour, washed twice with 60% isopropanol, and stained with 0.5% Oil Red O solution for 30 minutes at room temperature. After staining, the staining solution was removed and washed twice with distilled water. After the distilled water completely dried, isopropyl alcohol was added, and the absorbance was measured at 520 nm.

[0140] Finally, the viability of 3T3-L1 cells was measured using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method for the strain obtained in the above 1-1. 3T3-L1 cells were cultured at 16×10 4 Cells / well were seeded into 96-well plates at a concentration of 10 cells / well, and the medium was removed after 24 hours of culture. Lactic acid bacteria samples diluted at various concentrations (100, 1,000 μg / mL) were added to 100 μL of fresh DMEM medium, and cultured for 24 hours. Afterwards, 20 μL of a 5 mg / mL MTT (Sigma, USA) solution was added, and the cells were cultured at 37°C for 4 hours. After culture, the supernatant was removed, 200 μL of dimethyl sulfoxide (DMSO) was added, and the absorbance was measured at 546 nm.

[0141] From the above results, the fat accumulation inhibitory effect of each strain could be confirmed, and among the prepared strains, Lactobacillus fermentum GB102 (hereinafter referred to as 'GB102'), Lactobacillus fermentum GB103 (hereinafter referred to as 'GB103'), and Lactobacillus plantarum GB104 (hereinafter referred to as 'GB104') strains with fat cell accumulation inhibitory effect and low cytotoxicity were finally selected. In addition, Lactobacillus fermentum was changed to Limosilactobacillus fermentum, and Lactobacillus plantarum was changed to Lactiplantibacillus plantarum. In the examples below, the changed strain names of the existing strains are described interchangeably.

[0142]

[0143] 1.3. Molecular biological identification of selected strains

[0144] The 16S rRNA gene sequences were used to identify the Lactobacillus fermentum GB102, GB103, and Lactobacillus plantarum GB104 strains selected above. The 16S rRNA gene sequences obtained through PCR using the 27F and 1492R primers targeting the 16S rRNA gene of bacteria were analyzed by Sanger sequencing, and the 16S rRNA sequences of Lactobacillus fermentum GB102, GB103, and Lactobacillus plantarum GB104 are represented by SEQ ID NOs: 1, 2, and 3, respectively.

[0145] In addition, a homology analysis was performed on this, and as a result, as shown in Table 1 below, strains GB102 and GB103 showed the highest sequence similarity to the type strain of Limosilactobacillus fermentum, followed by high sequence similarity to Limosilactobacillus gorilla. GB104 showed more than 98% sequence similarity with 18 species of Limosilactobacillus, including the type strain of Lactiplantibacillus plantarum, based on the 16S rRNA sequence, so species cannot be distinguished based on the 16S rRNA gene sequence. Accordingly, GB104 was confirmed to be a strain belonging to Lactiplantibacillus plantarum by comparing the average nucleotide identity (ANI) values ​​of the genome sequences after genome decoding. In conclusion, it was confirmed that the two strains above correspond to Limosilactobacillus fermentum, and one strain corresponds to Lactiplantibacillus plantarum.

[0146] 균주종 (Species)표준 균주명서열 유사도 (%)GB102Limosilactobacillus fermentumCECT 562(T)99.79Limosilactobacillus gorillaeKZ01(T)98.26GB103Limosilactobacillus fermentumCECT 562(T)99.93Limosilactobacillus gorillaeKZ01(T)98.13GB104Lactiplantibacillus pentosusDSM 20314(T)99.93Lactiplantibacillus argentoratensisDSM 16365(T)99.87Lactiplantibacillus plantarumATCC 14917(T)99.87Lactiplantibacillus paraplantarumDSM 10667(T)99.73Lactiplantibacillus paraplantarumDSM 10667(T)99.66Lactiplantibacillus herbarumTCF032-E4(T)98.92Lactiplantibacillus daoliensis116-1A(T)98.82Lactiplantibacillus pingfangensis382-1(T)98.81Lactiplantibacillus daowaiensis203-3(T)98.75Lactiplantibacillus nangangensis381-7(T)98.74Lactiplantibacillus plajomiNB53(T)98.72Lactiplantibacillus fabifermentansDSM 21115(T)98.72Lactiplantibacillus gariiFI11369(T)98.61Lactiplantibacillus xiangfangensisLMG 26013(T)98.59Lactiplantibacillus modestisalitoleransNB466(T)98.52Lactiplantibacillus mudanjiangensis11050(T)98.2Lactiplantibacillus songbeiensis398-2(T)98.19Lactiplantibacillus dongliensis218-3(T)98.12.

[0147]

[0148] Example 2. Genomic and comparative genomic analysis of Lactobacillus fermentum strains.

[0149] To investigate the genome-based species identification and characteristics of strains GB102 and GB103, next-generation sequencing technology (NGS) and bioinformatics were utilized to completely sequence the genomes of the strains and infer the functions of the genes contained in the genomes. In addition, the specificity of the strains was confirmed through comparative analysis with the completely sequenced genome of the same species. The strains were cultured in MRS broth at 37°C under anaerobic conditions for 4 hours, and genomic DNA was extracted from the cultures using the MG Genomic DNA purification kit (MGMED, Inc., Korea). Sequence analysis was performed using a PacBio RS II instrument to obtain long read data with an average length of 10 kb or more. To produce short read data with a sequence length of less than 500 bp with high accuracy to supplement the long read data with low accuracy, sequence analysis was performed using a NovaSeq 6000 instrument. Long read data were assembled into high-quality GB102 and GB103 genome drafts using the HGAP2 pipeline of the SMRT Analysis server. Potential SNP and InDel errors within the assembled genome draft sequences were corrected using long read and short sequence data. Coding sequences (CDSs) were predicted from the completed genome sequences using the Prodigal program, and rRNA and tRNAs were predicted using the RFAM tool. Functions of the predicted CDSs were predicted by homology searches (using the BLAST algorithm) against the publicly available UniProt database, GenBank nr database, Subsystem database, PFAM database, and COG database.Standard strain B1 28 of Lactobacillus fermentum species published in GenBank. T (= ATCC 14931 T ) and Lactobacillus gorillae standard strain KZ01 T , Lactobacillus gastricus standard strain DSM 16045 T The average nucleotide identity (ANI) of the genome sequences of strains GB102 and GB103 was calculated using the Jspecies program. The analysis results showed that both strains GB102 and GB103 showed ANI values ​​exceeding 95% compared to the reference strain of Lactobacillus fermentum, demonstrating their belonging to the species Lactobacillus fermentum (Table 2). Furthermore, the fact that strains GB102 and GB103 were not 100% identical to the reference strain indicates that they are novel species that have not been previously isolated and reported. Furthermore, the GB102 and GB103 strains were also confirmed to be different strains due to their large genomic distances. The present inventors named the GB102 and GB103 strains “Lactobacillus fermentum GB102” (Accession No.: KCTC 14105BP) and “Lactobacillus fermentum GB103” (Accession No.: KCTC 14106BP), and deposited them in the Korean collection for type cultures (KCTC) at the Korea Research Institute of Bioscience and Biotechnology on January 14, 2020, respectively.

[0150] StrainnameGenome size(bp)GC ratioANI valuesGB102GB103B1 28KZ01DSM16045GB1022,039,43251.88---98.4399.1479.3168.77GB1032,260,51351.2598.51---98.4279. 7269.23B1281,905,58752.3099.0998.51---79.4268.66KZ011,641,62148.1179.0579.3779.11---68.07DSM 160451,848,46141.6468.2268.3868.0268.18---

[0151]

[0152] Example 3. Genomic and comparative genomic analysis of Lactobacillus plantarum strains.

[0153] To investigate the genome-based species identification and characteristics of the GB104 strain, next-generation sequencing technology (NGS) and bioinformatics were utilized to completely sequence the genome of the strain and infer the functions of the genes contained in the genome. In addition, the specificity of the strain was confirmed through comparative analysis with the completely sequenced genome of the same species. This strain was cultured in MRS broth at 37℃ under anaerobic conditions for 4 hours, and genomic DNA was extracted from the culture using the MG Genomic DNA purification kit (MGMED, Inc., Korea). Sequence analysis was performed using the PacBio RS II instrument to obtain long read data with an average length of 10 kb or more. To produce short sequence data with a sequence length of less than 500 bp with high accuracy to supplement the long read data with low accuracy, sequence analysis was performed using the NovaSeq 6000 instrument. Long read data were assembled into a high-quality draft GB104 genome using the HGAP2 pipeline of the SMRT Analysis server. Potential SNP and InDel errors within the assembled draft genome sequence were corrected using long read data and short sequence data. CDSs were predicted from the completed genome sequence using the Prodigal program, and rRNA and tRNAs were predicted using the RFAM tool. Functions of the predicted CDSs were predicted by homology searches (using the BLAST algorithm) against the publicly available UniProt database, GenBank nr database, Subsystem database, PFAM database, and COG database. A summary of the GB104 genome is presented in Table 3.

[0154] FeaturesValues%Genome size (bp)3,247,930100.00DNA coding (bp)2,742,57584.44DNA G+C (bp)1,445,11744.49Total genes3,087100.00Protein coding genes2,99096.86Genes with function prediction2,51781.54RNA genes872.82rRNA genes160.52tRNA genes682.20Pseudo genes100.32

[0155]

[0156] Genomic analysis revealed that this strain possessed the plantaricin gene cluster, a group of bacteriocin biosynthetic genes associated with antimicrobial activity found in Lactobacillus plantarum. However, this did not align 100% with the standard and reference strains, indicating that this strain is a novel, previously unreported strain.

[0157] The present inventors named the GB104 strain as "Lactobacillus plantarum GB104" (accession number: KCTC 14107BP) and deposited it in the Korean collection for type cultures (KCTC) at the Korea Research Institute of Bioscience and Biotechnology on January 14, 2020.

[0158]

[0159] Example 4. Mixed strain and herbal medicine complex preparation

[0160] The mixed strain and herbal medicine complex preparation was used by adding inulin and vitamin B2 to the mixed strain and herbal medicine mixture consisting of Lactobacillus strains.

[0161] Specifically, the mixed strain consisting of the Lactobacillus genus strains was a mixture of three types of lactic acid bacteria, GB102, GB103, and GB104, and the herbal medicines used were Andong yam, Job's tears, red ginseng powder, and lingzhi.

[0162] The mixed strain of the three types of lactic acid bacteria, GB102, GB103 and GB104, was named Number Seven Lactic Acid Bacteria (#7 Lactic Acid Bacteria or No. 7 Lactic Acid Bacteria), and the mixed strain and herbal medicine complex preparation was named Number Seven (#7 or No. 7).

[0163]

[0164] Experimental Example 1. Confirmation of changes in circadian rhythm gene expression following combined administration of mixed strains and herbal medicines.

[0165] Aging can alter circadian rhythms, contributing to sleep disorders, bowel dysfunction, and depression. Aging weakens the expression of BMAL1 and CLOCK, as well as the regulatory mechanisms via REV-ERBα / β and RORα / γ proteins. Furthermore, decreased regulatory functions of PER and CRY proteins can lead to poor sleep quality and frequent awakenings. Furthermore, changes in the regulation of gene expression that bind to D-Box and E-Box can induce bowel disorders such as constipation and irritable bowel syndrome (IBS). These changes can impair the quality of life of older adults, increasing the risk of depression, cognitive decline, and frailty.

[0166] In order to confirm that the negative effects of circadian rhythm changes due to aging on physical and mental health can be improved by administering the mixed strain and herbal medicine complex preparation (Number Seven) of Example 4, the mixed strain and herbal medicine complex preparation (Number Seven) of Example 4 or PBS was orally administered daily to aged mice (16 months) for 8 months, and then tissue samples (intestine and muscle tissue) were collected from the aged experimental mice and the gene expression patterns were analyzed through RNA sequencing. As a control group, young mice aged 9 to 10 weeks were used, and the gene expression patterns of mice that were orally administered only probiotics (Limosilactobacillus fermentum, Lactiplantibacillus plantarum, Bifidobacterium animalis subsp., Lactobacillus acidophilus: Number Seven mixed strain) or prebiotics (inulin, fructooligosaccharide, hemp, brown rice, red ginseng, reishi mushroom) included in the mixed strain and herbal medicine complex preparation (Number Seven) for the same period were also analyzed.

[0167] The specific experimental process is as follows.

[0168]

[0169] 1.1 NGS performance

[0170] For RNA-Seq analysis of five tissues from five experimental groups of mice, which were repeated five times, tissue samples were collected from the experimental mice, immediately added an RNA preservative (RNAlater, USA), and stored at -80 degrees. Afterwards, each sample was thawed for RNA extraction, treated with DNase to degrade and remove DNA molecules, and the TruSeq Stranded mRNA Library Prep Kit was applied to selectively isolate and purify mRNA molecules from the obtained total RNA, and then a strand-specific RNA sequencing library was constructed. The production of the sequencing library was sequentially carried out through the processes of random fragmentation of extracted RNA, synthesis of cDNA fragments through reverse transcription, attachment of sequencing adapters to both ends of the cDNA fragments, PCR, and size selection to secure cDNA fragments of 200-400 bp in length. The final sequencing library produced was subjected to paired-end sequencing at a length of 100 bp x 2 using the NovaSeq6000 sequencer of the Illumina sequencing platform.

[0171]

[0172] 1.2 Bioinformatic Analysis

[0173] For the raw RNA-Seq sequence set of a total of 125 samples obtained through NGS, sequence preprocessing, reference genome mapping, and gene expression quantification processes were sequentially performed to obtain gene expression profiles for each sample. The reproducibility of gene expression profiles in repeated experiments and the relative similarity between experimental groups were evaluated through hierarchical clustering and principal coordinate analysis (PCoA). A statistical test was performed on the gene expression values ​​between comparative conditions to select significantly differentially expressed genes (DEGs). To more effectively understand the functional roles of the selected sets of differentially expressed genes, the functional categories to which the differentially expressed genes statistically significantly belonged were analyzed at the gene set level, including Gene Ontology and metabolic pathways. In addition, the differentially expressed gene sets were analyzed and reviewed together through direct functional category-level expression comparison. Sequence preprocessing was performed using the Trimmomatic program (Bolger, et al., 2014) to trim sequencing adapter sequences and low-quality base sequences, and reference genome mapping was performed using the HISAT2 program (Kim, et al., 2019) against the mouse reference genome sequence (Mus musculus reference genome, mm39). Gene expression quantification was performed using the StringTie program (Pertea, et al., 2015) was applied to obtain Fragments Per Kilobase Million (FPKM) and Transcripts Per Million (TPM) units, and differentially expressed genes were selected using the Ballgown program (Pertea, et al., 2016). Functional category analysis of differentially expressed genes was performed using the gProfiler program (Kolberg, et al., 2023), and direct functional category-level analysis of differentially expressed gene sets was performed using the Gene Set Enrichment Analysis (GSEA) program (Subramanian, et al., 2005).

[0174] The results of analyzing the gene expression pattern through the above process are shown in Figure 1.

[0175] Figure 1 shows changes in circadian rhythm gene expression according to combined administration of mixed strains and herbal medicines according to one specific example.

[0176] As shown in Fig. 1, when the mixed strain of the present invention and the herbal medicine complex preparation (Number Seven) were administered, it was confirmed that the expression pattern of genes related to circadian rhythm changes showed a pattern similar to that of young mice.

[0177] These results indicate that the mixed strain and herbal medicine complex preparation (Number Seven) of the present invention participates in the expression pattern of circadian-related genes and suppresses the weakening of circadian rhythm due to aging.

[0178]

[0179] Experimental Example 2. Human Application Test of Combined Administration of Mixed Strains and Herbal Medicines

[0180] In order to confirm the effects of taking the mixed strain and herbal medicine complex preparation (Number Seven) of Example 4 on improving sleep, bowel disorders, and depression, a human application test was conducted.

[0181]

[0182] 2.1 Selection of test subjects and final analysis subjects

[0183] We inquired about the eligibility of 65 adults aged 60 years or older to participate in the human application trial, and 60 subjects were recruited after excluding those who did not meet the selection criteria (2 subjects with underlying diseases and 3 subjects who refused to participate). Thirty subjects were randomly assigned to each group to take the mixed strain and herbal medicine complex preparation (Number Seven) and the placebo group and started taking the medication. Three subjects in the placebo group and six subjects in the mixed strain and herbal medicine complex preparation (Number Seven) group dropped out due to simple change of mind or antibiotic use. At the third visit (4 weeks of taking the medication), a questionnaire on medication adherence was administered, and each subject was asked to submit the remaining medication to evaluate medication compliance. Subjects with medication compliance of less than 80% were additionally excluded, and one case occurred in the placebo group and two cases occurred in the mixed strain and herbal medicine complex preparation (Number Seven) group. The final results were 26 cases in the placebo group and 22 cases in the mixed strain and herbal medicine complex preparation (Number Seven) group, and the effect of taking the mixed strain and herbal medicine complex preparation (Number Seven) was evaluated.

[0184] The gender ratio was assigned to be homogeneous between the two groups during random assignment, and other factors were analyzed without including other correction variables, assuming that they would be homogeneous between the two groups.

[0185] Specific details regarding the selection and analysis of test subjects are shown in Table 4 and Figure 2 below. Figure 2 illustrates the process of recruiting research participants to participate in a human application test of combined administration of mixed strains and herbal medicines according to one specific example.

[0186] Characteristics Total (n=48) Mixed strain and herbal medicine complex preparation (Number Seven) (n=22) Placebo (N=26) P *N(%)N(%)N(%)GenderMale14(29.2)5(22.7)9(34.6)0.171Female34(70.8)17(77.3)17(65.4)Age(years)60-6913(27.1)6(27.3)7(26.9)0.91870-7928(58.3)12(54.6)16(61.5)80+7(14.6)4(18.2)3(11.5)Highest level of educationElementary school18(37.5)6(27.3)12(46.2)0.367Middle school or high school20(41.7)11(50.0)9(34.6)University graduate Ideal 10(20.8) 5(22.7) 5(19.2) Living with a partner 32(66.7) 13(59.1) 19(73.1) 0.306 None 16(33.3) 9(40.9) 7(26.9) Monthly income (won) < 500,00014(29.2)6(27.3)8(30.8)0.374500,000~1,999,99917(35.4)10(45.5)7(26.9)≥2,000,00017(35.4)6(27.3)11(42.3)Smoking statusNon-smoker39(81.3)18(81.8)21(80.8)0.999Former smoker7(14.6)3(13.6)4(15.4)Current smoker2(4.2)1(4.6)1(3.9)Drinking statusYes27(56.3)10(45.5)17(65.4)0 .166No21(43.8)12(54.6)9(34.6)Physical activityYes19(39.6)8(36.4)11(42.3)0.675No29(60.4)14(63.6)15(57.7)No chronic disease18(37.5)7(31.8)11(42.3)0.455Yes30(62.5)15(68.2)15(57.7)High blood pressure24(50.0)14(63.6)10(38.4)Diabetes2(4.2)2(9.1)0(0)Hyperlipidemia17(35.4)10(45.5)7(26.9)

[0187] * Chi-square test was performed.

[0188]

[0189] 2.2 Research design

[0190] This study was conducted as a double-blind randomized controlled trial (RCT) divided into a mixed strain and herbal medicine complex preparation (Number Seven) group and a placebo group. The mixed strain and herbal medicine complex preparation (Number Seven) mainly consisted of probiotics (Limosilactobacillus fermentum, Lactiplantibacillus plantarum, Bifidobacterium animalis subsp., Lactobacillus acidophilus) and prebiotics (inulin, fructooligosaccharides, hemp, brown rice job's tears, red ginseng, and reishi mushroom) and was provided in three capsules. The mixed strain and herbal medicine complex preparation (Number Seven) contained at least 5×10 10 It was manufactured to contain a mixed strain of CFU (colony forming units). The group taking the mixed strain and herbal medicine complex preparation (Number Seven) took the three capsules daily for 4 weeks, while the group taking the placebo took three capsules containing mainly maltodextrin daily for 4 weeks. All participants were followed up for 4 weeks after stopping taking the mixed strain and herbal medicine complex preparation (Number Seven) or the placebo. Blood and stool samples were collected, physical ability was measured, and a questionnaire was completed at weeks 0, 2, 4, and 8. All measurement procedures were conducted at Seoul National University College of Medicine.

[0191]

[0192] 2.3 Survey

[0193] Demographic information, such as the participants' age, gender, education level, marital status, income, and occupational status, as well as behavioral characteristics such as smoking and drinking, were collected through questionnaires. Diagnosis and treatment history of chronic diseases, including hypertension, diabetes, and dyslipidemia, were also collected.

[0194]

[0195] Experimental Example 3. Confirmation of the effectiveness of combined administration of mixed strains and herbal medicines on improving sleep.

[0196] In order to confirm the effect of improving sleep disorders when co-administering a mixed strain of Lactobacillus strains and herbal medicine, the Insomnia Severity Index (ISI), which evaluates the sleep quality of participants recruited through the process of Experimental Example 2, was evaluated.

[0197] The Insomnia Severity Index (ISI) is a seven-item self-report questionnaire measuring sleep initiation, sleep maintenance, early morning awakening, sleep dissatisfaction, impact of sleep problems on daytime functioning, perceived sleep problems by others, and distress due to sleep problems. A higher ISI score indicates poorer sleep quality.

[0198] The Insomnia Severity Index (ISI) is a simple yet validated tool that is useful for measuring the severity of insomnia and evaluating the effectiveness of insomnia treatment. The reliability and validity of the Korean version of the ISI have been proven through a study that verified its validity.

[0199] The results of measuring the insomnia severity index of the group taking the mixed strain and herbal medicine complex preparation (Number Seven) and the group taking the placebo according to Example 2.2 are shown in Table 5 and Figure 3a.

[0200] MeasurePlaceboMixed strains and herbal medicine complex preparation (Number Seven)0 Weekly mean ± standard deviation4 Weekly mean ± standard deviationP-value0 Weekly mean ± standard deviation4 Weekly mean ± standard deviationp-valueISI6.1 ± 2.66.0 ± 3.80.9546.0 ± 2.34.4 ± 1.80.008

[0201] Figure 3a is a graph showing the results of measuring the change in the insomnia severity index (ISI) of a group taking a mixed strain and herbal medicine complex preparation (Number Seven) and a group taking a placebo according to one specific example.

[0202] As shown in Fig. 3a, in the group taking the mixed strain and herbal medicine complex preparation (Number Seven) according to one specific example, the ISI value tended to decrease throughout the taking period, and in the 4th week of taking it, the decrease in the ISI value was confirmed to be significantly greater than that in the placebo group. In addition, the decreasing trend in the ISI value was maintained even after taking the mixed strain and herbal medicine complex preparation (Number Seven) was discontinued (wash-out period).

[0203] This means that the mixed strain and herbal medicine complex preparation (Number Seven) according to one specific example is effective in improving sleep disorders.

[0204]

[0205] Experimental Example 4. Confirmation of the effectiveness of combined administration of mixed strains and herbal medicines for improving bowel disorders.

[0206] In order to confirm the effect of improving bowel disorders when co-administering a mixed strain of Lactobacillus strains and herbal medicine, the gut quotient (GQ) measurement scale of participants recruited through the process of Experimental Example 2 was evaluated.

[0207] The Gut Quotient (GQ) is a questionnaire-based tool developed to assess gut health in Koreans.

[0208] Specifically, the GQ is a validated questionnaire-based tool consisting of 17 items selected by clinicians and clinical experts based on the Rome criteria, the Irritable Bowel Syndrome Severity Scoring System (IBS-SSS), and the Irritable Bowel Syndrome Quality of Life Questionnaire (IBS-QOL). The GQ measures and scores discomfort with bowel movements, defecation, and bowel control, with higher GQ scores indicating better gut health.

[0209] The results of evaluating the gut quotient (GQ) of the group taking the mixed strain and herbal medicine complex preparation (Number Seven) and the group taking the placebo according to Example 2.2 are shown in Table 6 and Fig. 3b.

[0210] Measure Placebo Mean ± Standard Deviation Mixed strain and herbal medicine complex preparation (Number Seven) Mean ± Standard Deviation RM-ANOVA 0 wks4 wks0 wks4 wks Group Time Group X Time Gut Quotient 94.9 ± 5.8 92.0 ± 9.2 89.4 ± 12.7 93.8 ± 7.4 0.3 7 90.6 4 90.0 29*

[0211] (Mean ± SD,Two-way repeated measures ANOVA)

[0212] Figure 3b is a graph showing the results of evaluating the change in the gut quotient (GQ) measurement scale of the group taking the mixed strain and herbal medicine complex preparation (Number Seven) and the group taking the placebo according to one specific example.

[0213] As shown in Fig. 3b, in the group taking the mixed strain and herbal medicine complex preparation (Number Seven) according to one specific example, GQ tended to increase during the taking period, and in particular, after the second week of taking it, the increase in GQ in the group taking the mixed strain and herbal medicine complex preparation (Number Seven) was confirmed to be significantly greater than the decrease in GQ in the placebo group. In addition, the increasing trend in GQ was maintained even after taking the mixed strain and herbal medicine complex preparation (Number Seven) was discontinued (wash-out period).

[0214] This means that the mixed strain and herbal medicine complex preparation (Number Seven) according to one specific example is effective in improving bowel disorders.

[0215]

[0216] Experimental Example 5. Confirmation of the effectiveness of combined administration of mixed strains and herbal medicines for improving depression.

[0217] To confirm the effect of improving depression when co-administering a mixed strain of Lactobacillus strains and herbal medicine, the CES-D (Center for Epidemiological Studies-Depression scale) of participants recruited through the process of Experimental Example 2 was evaluated.

[0218] The Center for Epidemiological Studies-Depression scale (CES-D) is a self-report questionnaire developed to assess the severity of depressive symptoms. The CES-D is widely used in both clinical settings and research, particularly in epidemiological studies to identify risk factors for depression and assess psychological status (depression level).

[0219] Specifically, the Korean version of the Center for Epidemiological Studies Depression Scale (CES-D) was used. The CES-D consists of 20 depression-related items, each scored from 0 to 3. A higher total score indicates more severe depressive symptoms.

[0220] The results of evaluating the CES-D of the group taking the mixed strain and herbal medicine complex preparation (Number Seven) and the group taking the placebo according to Example 2.2 are shown in Table 7 and Fig. 3c.

[0221] MeasurePlaceboMixed strains and herbal medicine complex preparation (Number Seven)0 Weekly mean ± standard deviation4 Weekly mean ± standard deviationP-value0 Weekly mean ± standard deviation4 Weekly mean ± standard deviationP-valueCESD3.7 ± 5.73.3 ± 5.30.7406.1 ± 9.81.8 ± 2.30.053

[0222] (Mean ± SD, Paired t-test)

[0223] Figure 3c is a graph showing the results of evaluating the change in CES-D in a group taking a mixed strain and herbal medicine complex preparation (Number Seven) and a placebo group according to one specific example.

[0224] As shown in Fig. 3c, in the group taking the mixed strain and herbal medicine complex preparation (Number Seven) according to one specific example, CES-D tended to decrease during the taking period, and in particular, it was confirmed that the decrease in CES-D was significantly greater after the second week of taking the mixed strain and herbal medicine complex preparation (Number Seven). In addition, the decreasing trend in CES-D was maintained even after taking the mixed strain and herbal medicine complex preparation (Number Seven) was discontinued (wash-out period).

[0225] This means that the mixed strain and herbal medicine complex preparation (Number Seven) according to one specific example is effective in improving depression.

[0226]

[0227] Experimental Example 6. Statistical Analysis Process for Combined Administration of Mixed Strains and Herbal Medicines

[0228] The results of combined administration of mixed strains and herbal medicines were analyzed using parametric multiple comparison procedures, assuming data normality. A two-way repeated ANOVA (analysis of variance) was performed, and Bonferroni's multiple comparison test was used as a post-hoc test to analyze whether there were changes in evaluation indicators according to the progress of medication and whether there were differences between intervention factors. The statistics that simply compared the results before and after medication within the intervention factor between the group that did not take medication and the group that took medication for 4 weeks (0 week vs. 4 weeks) were analyzed using a paired t-test.

[0229]

[0230] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

[0231] [Accession number]

[0232] Name of depositor: Korea Research Institute of Bioscience and Biotechnology

[0233] Accession number: KTCT14105BP

[0234] Date of acceptance: 20200114

[0235]

[0236] Name of depositor: Korea Research Institute of Bioscience and Biotechnology

[0237] Accession number: KTCT14106BP

[0238] Date of acceptance: 20200114

[0239]

[0240] Name of depositor: Korea Research Institute of Bioscience and Biotechnology

[0241] Accession number: KTCT14107BP

[0242] Date of acceptance: 20200114

[0243]

Claims

1. Containing a first active substance including a Lactobacillus sp. strain or a culture thereof and a second active substance including a herbal medicine as active ingredients; or A composition for preventing or improving sleep disorders, comprising the first active ingredient as an effective ingredient and co-administering the second active ingredient.

2. In claim 1, a third active substance including inulin or vitamins is additionally included; or A composition for preventing or improving sleep disorders, wherein a third active ingredient is additionally administered in combination.

3. A composition for preventing or improving sleep disorders according to claim 1, wherein the first active ingredient is Lactobacillus fermentum and Lactobacillus plantarum.

4. A composition for preventing or improving sleep disorders according to claim 3, wherein the first active ingredient additionally contains a Bifidobacterium animalissub sp. lactis strain and a Lactobacillus acidophilus strain.

5. A composition for preventing or improving sleep disorders according to claim 1, wherein the second active ingredient is at least one selected from the group consisting of ginseng, yulmu, red ginseng, lingzhi, and combinations thereof.

6. A composition for preventing or improving sleep disorders according to claim 1 or 2, wherein the first active substance, the second active substance, and the third active substance are administered simultaneously, sequentially, or in reverse order.

7. A composition for preventing or improving sleep disorders, comprising a first oral preparation comprising the first active substance, a second oral preparation comprising the second active substance, and a third oral preparation comprising the third active substance according to claim 1 or 2, wherein the first, second, and third oral preparations are orally administered.

8. A composition according to claim 7, wherein the oral preparations are in the form of tablets, pills, capsules, lozenges, granules, powders, suspensions, sachets, or syrups.

9. In claim 1, the strain is freeze-dried and 10 3 10 inland 16 A composition comprising an amount of CFU / g.

10. Containing a first active substance including a Lactobacillus sp. strain or a culture thereof and a second active substance including a herbal medicine as active ingredients; or A health functional food for preventing or improving sleep disorders, comprising the first active ingredient as an effective ingredient and co-administering the second active ingredient.

11. A step of administering a first active substance containing a Lactobacillus sp. strain or a culture thereof to a subject in need thereof; and A method for preventing or improving sleep disorders, comprising the step of administering a second active substance containing a herbal medicine to a subject in need thereof.

12. Use of the composition of claim 1 for the manufacture of a medicament for preventing or improving sleep disorders.

13. Use of the composition of claim 1 for preventing or improving sleep disorders.

Images