Probiotics to prevent cognitive impairment

Abstract

The present invention relates to a bacterium of the species Lacticaseibacillus paracasei for use in the prevention or treatment of cognitive impairment in a subject in need thereof. The present invention also relates to a bacterium of the species Lacticaseibacillus paracasei for use in the prevention or treatment of cognitive impairment induced by sleep deprivation and / or sleep deprivation in a subject in need thereof.

Description

[Technical Field]

[0001] The present invention relates to the bacterium Lacticaseibacillus paracasei (formerly known as Lactobacillus paracasei) for use in the prevention or treatment of cognitive impairment in subjects requiring its use. The present invention also relates to the bacterium Lacticaseibacillus paracasei for use in the prevention or treatment of cognitive impairment induced by sleep deprivation and / or lack of sleep in subjects requiring its use. Furthermore, the present invention relates to compositions such as foods, nutritional supplements, and pharmaceutically acceptable preparations containing the bacterium Lacticaseibacillus paracasei, and to methods and uses of compositions containing the bacterium Lacticaseibacillus paracasei or the bacterium Lacticaseibacillus paracasei. [Background technology]

[0002] Sleep deprivation is a significant lifelong public health concern prevalent in today's 24-hour society, linked to a variety of harmful physical and mental impairments. The National Sleep Foundation recommends that adults (18-64 years) need 7-9 hours of sleep per day to maintain cognitive, emotional, and physical health, in addition to overall health and well-being (Hirshkowitz et al. 2015). In reality, it has been reported that in most developed countries, about one-third of adults are sleep-deprived (i.e., sleep less than 7 hours per day) (Owen and Veasey 2020), and repeated periods of sleep deprivation (SD) are linked to seven of the 15 leading causes of death in the United States, particularly those related to infections, cardiovascular complications, and metabolic disorders (Chattu et al. 2019). Furthermore, repeated sleep deprivation has serious consequences for work efficiency, public safety, and overall well-being (Magnavita and Garbarino 2017; Simon and Walker 2018), and is known to affect sleep disorders such as insomnia, mental illnesses such as mood disorders, depression, and anxiety, and addiction disorders (Geoffroy et al. 2020).

[0003] Numerous studies on sleep deprivation have revealed various aspects of sleep structure (Toth and Bhargava 2013), and the neurobehavioral effects of sleep deprivation on cognitive functions such as attention and working memory, as well as other functions such as long-term memory and decision-making, have been elucidated (Krause et al. 2017). Cognitive enhancement interventions such as caffeine and modafinil have been reported to reverse sleep deprivation-induced cognitive impairment in human and animal models (Colavito et al. 2013), but these types of interventions are accompanied by many side effects such as disorientation, addiction, and daytime fatigue. In other words, there is a need to research pharmacological interventions and nutritional supplements that are less side-effect-prone and more effective in counteracting cognitive deficits caused by sleep deprivation.

[0004] The gut-brain axis, which facilitates bidirectional communication between the gut microbiota and the central nervous system, presents an attractive target for developing novel therapeutic agents related to improving cognitive function and / or mitigating cognitive impairment. In this regard, various Lactobacillus strains, either alone or in combination, have been shown to improve cognitive function in various rodent models, for example (Stenman et al. 2020; Liang et al. 2015). In other studies, intervention with specific Bifidobacterium strains in healthy mice selectively improved object recognition memory, reduced errors in spatial memory tests, and induced better long-term learning in fear conditioning (Savignac et al. 2014). The safety and efficacy of Bifidobacterium breve A1 in preventing cognitive impairment in mouse models of Alzheimer's disease and in participants with mild cognitive impairment have been reported (Kobayashi et al. 2019; Kobayashi et al. 2017). Cognitive impairment after sleep deprivation in mice has also been reported, and the probiotic Lactobacillus plantarum MTCC 9510 bacterial strain has been demonstrated to be effective in mitigating such cognitive decline (Dhaliwal et al. 2018). Interestingly, high sleep quality in healthy older adults has been shown to be associated with improved performance on cognitive tasks and a higher proportion of specific phyla in the gut microbiota, suggesting a possible link between sleep quality, gut microbiota, and cognitive flexibility (Anderson et al. 2017). [Overview of the project] [Problems that the invention aims to solve]

[0005] Given the important connections between the gut microbiota, the gut-brain axis, sleep physiology, and cognition, directly supplementing with probiotics to regulate the composition of the gut microbiota or influencing the physiology of the mucosa may mitigate cognitive impairments induced by sleep deprivation. To test this hypothesis, we established a paradigm in which mice underwent repeated partial sleep deprivation in three types of cognitive behavioral tests, and investigated whether probiotic strains (Lactiplantibacillus plantarum Lp-115 (formerly known as Lactobacillus plantarum) (Lp-115), Lacticaseibacillus paracasei Lpc-37 (formerly known as Lactobacillus paracasei) (Lpc-37), Bifidobacterium animalis ssp. lactis 420 (B420)) and combinations thereof could prevent or treat, for example, cognitive impairment caused by sleep deprivation and / or sleep deprivation. [Means for solving the problem]

[0006] This invention is based on the research described herein, which has demonstrated that strains of Lacticaseibacillus paracasei, Lactiplantibacillus plantarum, Bifidobacterium animalis ssp. lactis, and combinations thereof, can remarkably prevent or treat cognitive impairment induced, for example, by sleep deprivation and / or lack of sleep.

[0007] Accordingly, in one embodiment, the present invention provides a bacterial strain of the species Lacticaseibacillus paracasei or a mixture thereof for use in the prevention or treatment of cognitive impairment in subjects requiring it.

[0008] In yet another embodiment, the present invention provides a composition comprising a strain of the species Lacticaseibacillus paracasei or a mixture thereof for use in the prevention or treatment of cognitive impairment in subjects requiring such treatment.

[0009] In a further embodiment, the present invention provides a method for preventing or treating cognitive impairment in a subject requiring such treatment, the method comprising administering to the subject a bacterial strain or a composition comprising a bacterial strain, the bacterial strain being a strain of the species Lacticaseibacillus paracasei or a mixture thereof. [Brief explanation of the drawing]

[0010] [Figure 1] Novel object recognition test (recognition memory). Effects of probiotic intervention on recognition memory impairment induced by sleep deprivation (SD). Cognitive function in the novel object recognition test when either a bacterial strain (Lp-115, Lpc-37, or B420) 1 × 10⁹ colony-forming units, a bacterial combination (Lp-115 + Lpc-37 + B420) 1.5 × 10⁹ colony-forming units, or a solvent was administered for 30 days before and 9 days during the behavioral test paradigm. Figure 1A shows the frequency (%) of object interaction in the same object trial on the training day (day 2), and Figure 1B shows the duration (%) of object interaction in the same object trial on the training day (day 2). Figure 1C shows the frequency (%) of object interaction in the novel object trial on the test day (day 3), and Figure 1D shows the duration (%) of object interaction in the novel object trial on the test day (day 3). Figure 1E shows the discrimination index (contact frequency) for new object trials on the third day of testing, and Figure 1F shows the discrimination index (contact time) for new object trials on the third day of testing. [Figure 2] Spontaneous alternation behavior test (spatial working memory) using a Y-maze. The effect of probiotic intervention on spatial working memory impairment induced by sleep deprivation (SD). Cognitive function in the spontaneous alternation behavior test using a Y-maze when any of the bacterial strains (Lp-115 or Lpc-37 or B420) at 1 × 109 colony forming units, the combination of bacteria (Lp-115 + Lpc-37 + B420) at 1.5 × 109 colony forming units, or the solvent was administered for 30 days before and 9 days during the behavioral test paradigm. Figure 2A shows the alternation behavior rate (%) on the training day (day 5), and Figure 2B shows the amount of spontaneous movement (number of arm entries) on the training day (day 5). Figure 2C shows the alternation behavior rate (%) on the test day (day 6), and Figure 2D shows the amount of spontaneous movement (number of arm entries) on the test day (day 6). [Figure 3] Step-through type passive avoidance task (context-dependent long-term memory). The effect of probiotic intervention on context-dependent long-term memory impairment induced by sleep deprivation (SD). Cognitive function in the step-through type passive avoidance task when any of the bacterial strains (Lp-115 or Lpc-37 or B420) at 1 × 109 colony forming units, the combination of bacteria (Lp-115 + Lpc-37 + B420) at 1.5 × 109 colony forming units, or the solvent was administered for 30 days before and 9 days during the behavioral test paradigm. Figure 3A shows the acquisition latency on the training day (day 8). Figure 3B shows the reaction latency on the test day (day 9). Figure 3C shows the escape latency on the test day (day 9). [Figure 4]Experimental design and treatment schedule in probiotic intervention. Three individual probiotic bacterial strains and combinations of these three strains were tested in mice subjected to sleep deprivation (SD), and compared with non-sleep-deprived solvent (No SD / Veh) group and sleep-deprived solvent (SD / Veh) group. A dosage containing either a single bacterial strain at 1×109 colony-forming units, a combination of bacteria at 1.5×109 colony-forming units, or a solvent was force-fed to the mice daily for 30 days before and 9 days during the behavioral test paradigm. To evaluate learning and memory functions, a behavioral test was performed after 5 hours of SD time. Behavioral tests including novel object recognition test (NOR; days 1 - 3), spontaneous alternation behavior test using a Y-maze (Y-maze; days 5 - 6), and step-through type passive avoidance task (STPA; days 8 - 9) were used, and days 4 and 7 were rest periods.

Mode for Carrying Out the Invention

[0011] The details of the present invention will be described below. Some of the detailed aspects are considered in separate sections. This is for ease of reference and is in no way limiting. Contextually, unless otherwise clearly indicated, all of the embodiments described below apply equally to all aspects of the present invention.

[0012] bacteria The bacteria used in aspects of the present invention are of the species Lacticaseibacillus paracasei. In one embodiment, the Lacticaseibacillus paracasei is strain Lpc-37, also known as Lbc81. This strain is commercially available from DuPont Nutrition Biosciences ApS. This strain of Lacticaseibacillus paracasei was also deposited on October 5, 2017, in accordance with the Budapest Treaty, by DuPont Nutrition Biosciences ApS at Langebrogade 1, DK-1411 Copenhagen K, Denmark, with the reference designation DGCC4981 at Leibniz-Institut Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) at Inhoffenstrasse 7B, 38124 Braunschweig, Germany, and is registered under registration number DSM 32661.

[0013] Other bacteria used in embodiments of the present invention are of the species Bifidobacterium animalis ssp. lactis. In particularly preferred embodiments, the bacteria used in the present invention is Bifidobacterium animalis ssp. lactis strain 420 (B420). This strain is commercially available from DuPont Nutrition Biosciences ApS. This strain of Bifidobacterium animalis ssp. lactis was also deposited on June 30, 2015, in accordance with the Budapest Treaty, by DuPont Nutrition Biosciences ApS at Langebrogade 1, DK-1411 Copenhagen K, Denmark, with the identification mark DGCC420 at Leibniz-Institut Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) at Inhoffenstrasse 7B, 38124 Braunschweig, Germany, and is registered under registration number DSM 32073.

[0014] Other bacteria used in embodiments of the present invention are of the species Lactiplantibacillus plantarum. In one embodiment, the Lactiplantibacillus plantarum strain is Lp-115, which is commercially available from DuPont Nutrition Biosciences ApS.

[0015] Preferably, the bacterial strains used in the present invention are generally recognized as safe (GRAS) strains, and preferably, have obtained GRAS certification. GRAS is a designation by the U.S. Food and Drug Administration (FDA) that means a chemical or substance added to food is recognized by experts as safe and is therefore exempt from the usual Federal Food, Drug, and Cosmetic Act (FFDCA) approval requirements for food additives.

[0016] In a first embodiment, the present invention provides a strain or mixture thereof of the species Lacticaseibacillus paracasei for use in the prevention or treatment of cognitive impairment in persons requiring it.

[0017] Cognitive impairment is often defined as a condition in which a person has difficulty remembering, learning new things, concentrating, or making decisions to such an extent that it affects their daily life. Cognitive impairment can range from mild to severe. In mild cases, a person may begin to notice changes in cognitive function, but they can still perform daily activities. In severe cases, the ability to understand the meaning or importance of things, and the ability to speak or write may be lost, making it impossible to live independently. Cognitive impairment is often caused by mental fatigue. Mental fatigue includes symptoms such as lack of attention, lack of focusing, forgetfulness, decreased productivity, difficulty concentrating, reduced mental clarity, problem paying attention, short attention span, and learning difficulties.

[0018] In another embodiment, the present invention provides a bacterial strain for use in the prevention or treatment of cognitive impairment, wherein the cognitive impairment is induced by sleep deprivation and / or lack of sleep. Sleep deprivation and / or lack of sleep may result from a sleep disorder, which may be or may be sleep apnea, narcolepsy, insomnia and / or parasomnia.

[0019] In one aspect, cognitive impairment is a disorder of learning and memory.

[0020] In another embodiment, cognitive impairment is impairment of recognition memory, spatial working memory, and / or context-dependent long-term memory.

[0021] Cognitive impairment is a contributing factor to neuropsychiatric conditions, mental illnesses, and / or neurodegenerative diseases. Neuropsychiatric conditions and / or mental illnesses include mood disorders, depression, anxiety, neurotic disorders, and addiction disorders. Neurodegenerative diseases include dementia, Parkinson's disease, Alzheimer's disease, vascular dementia, Lewy body dementia, Huntington's disease, progressive supranuclear palsy, frontotemporal dementia, Creutzfeldt-Jakob disease, and Wernicke-Korsakoff syndrome.

[0022] According to the present invention, Lacticaseibacillus paracasei can be used in combination with one or more other bacterial species that have the ability to provide positive health benefits to the administered host.

[0023] Lacticaseibacillus paracasei can be used in any form (e.g., live, dormant, inactivated, or dead) provided that the bacterium remains capable of exerting the effects described herein. Preferably, the Lacticaseibacillus paracasei used in the embodiments of the present invention is live.

[0024] Preferably, Lacticaseibacillus paracasei and other bacterial species (as used in embodiments of the present invention) are suitable for ingestion by humans and / or animals. Those skilled in the art will readily come up with specific strains of Lacticaseibacillus paracasei and other bacterial strains that are used in the food industry and / or agricultural industry and are generally considered suitable for ingestion by humans and / or animals.

[0025] Selectively, Lacticaseibacillus paracasei and other bacterial strains used in embodiments of the present invention are probiotic bacteria. The term “probiotic bacteria” is defined to encompass all non-pathogenic bacteria that, when administered to a host in appropriate amounts while alive, provide health benefits to that host. To be classified as “probiotics,” bacteria must pass through the upper digestive tract of the host alive. They are non-pathogenic and non-toxic and exert beneficial effects on health, on the one hand through ecological interactions with the commensal microbiota in the digestive tract, and on the other hand through their ability to favorably influence the host’s physiology and immune system. When administered to a host in sufficient numbers, probiotic bacteria have the ability to pass through the intestinal tract while maintaining viability and exerting their primary effects within the lumen and / or wall of the host’s digestive tract. Subsequently, probiotic bacteria transiently form part of the normal bacterial flora, and this colonization (or transient colonization) allows the probiotic bacteria to exert beneficial effects, such as suppressing potentially pathogenic microorganisms present in the microbiome or interacting with the host in the gut, including the immune system.

[0026] Therefore, in certain embodiments of the present invention, the bacterial strain for use in accordance with the present invention is a probiotic bacterial strain. In particular, the bacterial strain Lpc-37 of the Lacticaseibacillus paracasei species, registered with the DSMZ on October 5, 2017, under accession number DSM32661.

[0027] composition The term "composition" is used in a broad sense to mean the compositional form of something, i.e., its general composition. In aspects of the present invention, a composition may essentially consist of a single strain of the bacterial species Lacticaseibacillus paracasei.

[0028] Alternatively, the composition may contain the Lacticaseibacillus paracasei strain together with other components, such as other bacterial strains, biological and chemical components, active ingredients, metabolites, nutrients, fiber, prebiotics, etc. In certain embodiments of the present invention, other bacterial strains present in the composition are Lp-115 strain and / or B420 strain (B420 strain was registered with the DSMZ on June 30, 2015, under accession number DSM32073).

[0029] In one embodiment, the present invention provides a composition comprising a strain of the species Lacticaseibacillus paracasei or a mixture thereof for use in the prevention or treatment of cognitive impairment in subjects requiring such treatment.

[0030] According to the present invention, cognitive impairment is induced or potentially induced by sleep deprivation and / or sleep insufficiency. In certain embodiments of the present invention, cognitive impairment is impairment of learning and memory. In other specific embodiments of the present invention, cognitive impairment is impairment of recognition memory, spatial working memory and / or context-dependent long-term memory.

[0031] In certain embodiments, sleep deprivation and / or sleep insufficiency may result from a sleep disorder. In certain embodiments of the present invention, the sleep disorder is or may be sleep apnea, narcolepsy, insomnia and / or parasomnia.

[0032] In other embodiments of the present invention, cognitive impairment is a contributing factor to neuropsychiatric disorders, mental illnesses and / or neurodegenerative diseases. Neuropsychiatric disorders and / or mental illnesses are or may be mood disorders, depression, anxiety, neurotic disorders and addiction disorders.

[0033] In a further specific embodiment of the present invention, neurodegenerative diseases include dementia, Parkinson's disease, Alzheimer's disease, vascular dementia, Lewy body dementia, Huntington's disease, progressive supranuclear palsy, frontotemporal dementia, Creutzfeldt-Jakob disease, and Wernicke-Korsakoff syndrome.

[0034] In yet another aspect of the present invention, cognitive impairment is caused by mental fatigue. In a particular aspect, mental fatigue includes symptoms such as lack of attention, impaired focusing ability, forgetfulness, decreased productivity, lack of concentration, disorganization, inattention, distractibility, and learning difficulties.

[0035] In particular, the bacterial strain of the composition according to the present invention is a probiotic bacterial strain.

[0036] In other specific cases of the present invention, the strain of the composition is the Lpc-37 strain, which was registered with the DSMZ on October 5, 2017, under accession number DSM32661.

[0037] According to one aspect of the present invention, the composition is a spray-dried or freeze-dried composition.

[0038] According to another aspect of the present invention, the composition comprises a cryoprotectant.

[0039] In yet another aspect of the present invention, bacteria of the species Lacticaseibacillus paracasei are present in the composition in a quantity of 10 per dose. 6 ~10 12 (For example 10 8 ~10 12 ) Present in the form of colony-forming units (CFUs), and optionally, 10 per dose 10 It exists in the form of CFUs.

[0040] While it is not a requirement that the composition contain a carrier, diluent, or excipient, such carriers, diluents, or excipients may be added and used in a manner familiar to those skilled in the art. Examples of suitable excipients include, but are not limited to, microcrystalline cellulose, rice maltodextrin, silicon dioxide, and magnesium stearate. The compositions of the present invention may also contain cryoprotective components (e.g., glucose, sucrose, lactose, trehalose, sodium ascorbate, and / or other suitable cryoprotective agents).

[0041] The terms "composition" and "formulation" may be used synonymously.

[0042] The compositions used in embodiments of the present invention may take the form of solid formulations, liquid formulations, solution formulations, or suspension formulations. Examples of solid formulations include, but are not limited to, tablets, pills, capsules, granules, and powders, which may be wettable, spray-dried, or freeze-dried. The compositions may contain flavoring agents or coloring agents. The compositions may be formulated for immediate-release, delayed-release, controlled-release, sustained-release, pulsed-release, or controlled-release applications.

[0043] As an example, when the composition of the present invention is used in tablet form, the tablet may also contain one or more of the following: excipients, e.g., microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, calcium hydrogen phosphate, and glycine; disintegrants, e.g., starch (preferably corn starch, potato starch, or tapioca starch), sodium starch glycolate, croscarmellose sodium, and certain complex silicates); granulation binders, e.g., polyvinylpyrrolidone, hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose (HPC), sucrose, gelatin, and gum arabic; lubricants, e.g., magnesium stearate, stearic acid, glyceryl behenate, and talc.

[0044] Other acceptable carriers used in the preparation of the composition include water, salt solutions, alcohol, silicone, wax, petrolatum, vegetable oil, polyethylene glycol, propylene glycol, liposomes, sugars, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, oil perfumes, fatty acid monoglycerides and diglycerides, hydroxymethylcellulose, and polyvinylpyrrolidone.

[0045] In the case of aqueous suspensions and / or elixirs, the composition of the present invention can be combined with various sweeteners or flavoring agents, colorants or dyes, emulsifiers and / or suspending agents, diluents (e.g., water, propylene glycol, and glycerin), and combinations thereof.

[0046] Specific, non-limiting examples of compositions that can be used in embodiments of the present invention are described below for illustrative purposes. Such examples include, but are not limited to, foods, functional foods, nutritional supplements, pharmaceutical compositions, and drugs.

[0047] food The compositions of the present invention may take the form of food. In this specification, the term “food” is used in a broad sense and includes food and beverages for human consumption as well as food and beverages for animal consumption (i.e., animal feed). Preferably, the food is suitable for human consumption and is designed for human consumption.

[0048] Foods may be in the form of liquids, solids, or suspensions, depending on the method of use and / or application and / or administration.

[0049] When in food form, the composition may contain or be used in combination with one or more of the following: a nutritionally acceptable carrier, a nutritionally acceptable diluent, a nutritionally acceptable excipient, a nutritionally acceptable adjuvant, or a nutritionally effective ingredient.

[0050] As an example, the composition of the present invention can take one of the following forms: Fruit juice, whey protein beverages, health teas or herbal teas, cocoa drinks, dairy drinks, lactic acid bacteria drinks, yogurt and / or drinkable yogurt, cheese, ice cream, sherbet, desserts, confectionery, biscuits, cakes, cake mixes or cake fillings, snack foods, fruit fillings, cake or donut icing, instant filling creams for baking, cookie fillings, ready-to-use baking fillings, low-calorie fillings, adult nutritional drinks, acidified soy / fruit juice drinks, nutrition bars or health bars, powdered beverages, calcium-fortified soy milk or calcium-fortified coffee drinks.

[0051] Optionally, if the product is a food, the bacterium Lacticaseibacillus paracasei must remain active until the usual "best before" or "expiration date" on which the food is offered for sale by the retailer. Preferably, the shelf life should extend beyond such a date until the end of the usual freshness period on which spoilage of the food becomes apparent. The desired length of the period and the usual shelf life will vary depending on the ingredients, and those skilled in the art will recognize that the shelf life will vary depending on the type of ingredients, the size of the ingredients, the storage temperature, processing conditions, packaging materials and equipment.

[0052] food ingredients The composition of the present invention may take the form of food components and / or feed components.

[0053] As used herein, the terms “food ingredient” or “feed ingredient” include compositions that are functional foods or functional ingredients as nutritional supplements and / or health supplements for humans and animals, or compositions that may be added thereto.

[0054] This food ingredient may be in the form of a solution, suspension, or solid, depending on the method of use and / or application and / or administration.

[0055] functional food The composition of the present invention can take the form of a functional food.

[0056] As used herein, the term "functional food" refers to a food that not only provides nutritional benefits but also delivers additional beneficial effects to consumers.

[0057] Therefore, functional foods are familiar foods that incorporate components or ingredients (such as those described herein) that impart specific functional effects other than purely nutritional effects, such as medical or physiological benefits to the food.

[0058] While there is no legal definition of functional foods, most stakeholders in this field agree that functional foods are foods marketed as having specific health benefits beyond basic nutritional effects.

[0059] Some functional foods are nutraceuticals. As used herein, the term “nutraceutical” means a food that can not only provide nutritional effects and / or taste satisfaction, but also deliver therapeutic (or other beneficial) effects to consumers. Nutraceuticals transcend the traditional boundaries between food and medicine.

[0060] Nutritional supplements The compositions of the present invention may take the form of nutritional supplements or be used in combination with nutritional supplements, and in this specification, nutritional supplements are also referred to as food supplements.

[0061] As used herein, the term “dietary supplement” refers to a product intended for ingestion that contains “dietary components” intended to add (supplement) further nutritional value or health benefits to a diet. “Dietary components” may include, but are not limited to, one or any combination of the following substances: bacteria, probiotics (e.g., probiotic bacteria), vitamins, minerals, herbs or other plant substances, amino acids, dietary substances, concentrates, metabolites, components or extracts used in humans for the purpose of supplementing a diet by increasing total dietary intake.

[0062] Dietary supplements can be found in many forms, including tablets, capsules, soft gels, gel capsules, liquids, and powders. Some dietary supplements help ensure that essential nutrients are adequately obtained from the diet, while others may help reduce the risk of disease.

[0063] Pharmaceutical composition The compositions of the present invention can be used as pharmaceuticals or in the preparation thereof. The term "pharmaceutical" as used herein is used in a broad sense and includes pharmaceuticals for human use and pharmaceuticals for animal use (i.e., veterinary use). In preferred embodiments, the pharmaceuticals are for human use.

[0064] Pharmaceuticals may be intended for therapeutic purposes and may have curative, symptomatic, or preventive properties.

[0065] Pharmaceuticals may be in the form of compressed tablets, tablets, capsules, ointments, suppositories, or oral solutions.

[0066] When used as a pharmaceutical or in the preparation of a pharmaceutical, the compositions of the present invention may be used in combination with one or more of the following: a pharmaceutically acceptable carrier, a pharmaceutically acceptable diluent, a pharmaceutically acceptable excipient, a pharmaceutically acceptable adjuvant, or a pharmaceutically effective component.

[0067] Pharmaceuticals may be in liquid or solid form, depending on their use and / or mode of use and / or mode of administration.

[0068] Lacticaseibacillus paracasei used in the present invention may constitute a pharmaceutically active ingredient itself. In one embodiment, Lacticaseibacillus paracasei constitutes the sole active ingredient. Alternatively, Lacticaseibacillus paracasei may be at least one of many (i.e., two or more) pharmaceutically active ingredients.

[0069] medication The composition of the present invention can take the form of a pharmaceutical drug.

[0070] As used herein, the term "agent" encompasses agents utilized in both human and veterinary medicine for both humans and animals. Further, the term "agent" as used herein means any substance that produces a therapeutic, prophylactic, and / or beneficial effect. The term "agent" as used herein is not necessarily limited to substances that require marketing approval, and may also include substances that can be used in cosmetics, nutraceuticals, foods (such as feeds and beverages), probiotic cultures, and herbal medicines. Additionally, the term "agent" as used herein includes products designed to be incorporated into animal feeds, such as livestock feeds and / or pet foods.

[0071] Medical food The composition of the present invention can take the form of a medical food.

[0072] "Medical food" means a food that is formulated to be ingested or administered, either under the supervision of a physician or unsupervised, and for which specific dietary management or use under conditions is intended, based on recognized scientific principles, where distinctive nutritional requirements have been established by medical evaluation.

[0073] Dosage The composition of the present invention can contain from 10 6 ~10 12 colony-forming units (CFU) of the bacterial strain per dose or per gram of the composition, more specifically, from 10 8 ~10 12 CFU of the bacterial strain per dose or per gram of the composition. Optionally, the composition can contain about 10 10 CFU of the bacterial strain per dose or per gram of the composition.

[0074] The bacterial strain, such as Lacticaseibacillus paracasei (such as the Lpc-37 strain) and / or the Lp-115 strain and / or the B420 strain, can be from about 10 6 ~ about 10 12 CFU per dose, preferably from about 10 8 ~ about 1012 It can be administered in doses of CFU. The term "per dose" means that this number of bacteria is given to the subject per day or per single intake, preferably per day. For example, if the bacteria are administered in food, for example in yogurt, the yogurt contains about 10 6 ~about 10 12 It may contain a bacterial strain of CFU. Alternatively, this number of bacteria may be calculated as the total amount of bacterial strains ingested by the subject within any specific period, for example, every 24 hours, which is approximately 10 6 ~about 10 12 CFU, selectively selected bacteria 10 8 ~about 10 12 As long as it is a CFU, it can be divided into multiple doses, each consisting of a smaller amount of microbial additive.

[0075] According to the present invention, the effective amount of at least one bacterial strain is at least 10 6 CFU / dose, optionally, for approximately 10 bacteria 8 ~about 10 12 CFU / dose, e.g., about 10 bacteria 10 It may be CFU / dose.

[0076] In one embodiment, Lacticaseibacillus paracasei (e.g., strain Lpc-37 / DSM 32661) is used to form approximately 10 bacteria. 6 ~about 10 12 It can be administered at a dose of CFU / day, and optionally, approximately 10 bacteria 8 ~about 10 12 It can be administered at a dose of CFU / day. In other words, the effective dose in this embodiment is approximately 10 bacteria 6 ~about 10 12 CFU / day, selectively, approximately 10 bacteria 8 ~about 10 12 It could be CFU / day.

[0077] In a particular embodiment, a single bacterial strain is divided into 1 × 10⁶ 9 CFU, or multiple bacterial strains, 1.5 × 10 9CFU (for example, Lpc-37 5 × 10 8 CFU, Lp-115 5x10 8 CFU and B420 5×10 8 It was administered using CFU.

[0078] Action / Target / Medical Indications In one embodiment, the term “subject” as used herein means mammals, including, for example, livestock (e.g., cattle, horses, pigs, and sheep) and humans. In one embodiment, the subject is human. In one embodiment, the subject is female. In one embodiment, the subject is male. In other embodiments, the subject is a dog (e.g., a member of the genus Canis) or a cat (e.g., a member of the genus Felis or Panthera). In other embodiments, the subject is poultry, e.g., chickens, turkeys, ducks, and geese. In preferred embodiments, the bacterial strains and compositions are used for humans.

[0079] The bacterial strains and / or compositions of the present invention can be used to prevent or treat cognitive impairment in subjects requiring such treatment.

[0080] According to the U.S. Centers for Disease Control and Prevention (CDC), "cognitive impairment" is a condition in which a person has difficulty remembering, learning new things, concentrating, or making decisions to the extent that it affects their daily life. Cognitive impairment ranges from mild to severe. While the impairment is mild, a person may begin to notice changes in cognitive function, but they can still perform daily activities. As the impairment becomes more severe, the ability to understand the meaning or importance of things, and the ability to speak or write may be lost, making it impossible to live independently.

[0081] Cognitive impairment may be caused, for example, by sleep deprivation and / or lack of sleep. Sleep deprivation and / or lack of sleep may also be caused by sleep disorders such as sleep apnea, narcolepsy, insomnia and / or parasomnia.

[0082] Cognitive impairments include, for example, learning and memory impairments, recognition memory impairments, spatial working memory impairments, context-dependent long-term memory impairments, mood disorders, depression, anxiety, neurotic disorders, addiction disorders, dementia, Parkinson's disease, Alzheimer's disease, vascular dementia, Lewy body dementia, Huntington's disease, progressive supranuclear palsy, frontotemporal dementia, Creutzfeldt-Jakob disease, Wernicke-Korsakoff syndrome, attention deficit, impaired focusing ability, forgetfulness, decreased productivity, lack of concentration, disorganization, inattention, distractibility, and learning difficulties.

[0083] Prebiotics In one embodiment, the bacterial strains and compositions of the present invention may be further combined with or contain one or more types of fibers and / or prebiotics.

[0084] Prebiotics are defined as substrates that are selectively utilized by host microorganisms and provide health benefits. These are generally components that selectively stimulate the growth and / or activity of one or a limited number of bacterial species, thereby enhancing host health and having a beneficial effect on the host's health. Prebiotics can be applied orally, but they can also be applied to other sites where microorganisms form colonies. Typically, prebiotics are carbohydrates (such as oligosaccharides), but the definition does not exclude non-carbohydrates such as polyphenols, polyunsaturated fatty acids, or other components that can provide health benefits through selective utilization by a limited number of bacterial species. The most common form of prebiotics is those nutritionally classified as soluble dietary fiber. Many forms of dietary fiber exhibit a certain level of prebiotic effect to some extent.

[0085] In one embodiment, prebiotics are selectively fermented components that bring about specific changes in both the composition and / or activity of the gastrointestinal or skin microbiota, which are beneficial to the well-being and health of the host.

[0086] Preferably, the prebiotic can be used in an amount of 0.01 to 100 g / day, preferably 0.1 to 50 g / day, and more preferably 0.5 to 20 g / day, according to the present invention. In one embodiment, the prebiotic can be used in an amount of 1 to 10 g / day, preferably 2 to 9 g / day, and more preferably 3 to 8 g / day, according to the present invention. In another embodiment, the prebiotic can be used in an amount of 5 to 50 g / day, preferably 5 to 25 g / day, according to the present invention.

[0087] Examples of dietary sources of prebiotics include soybeans, inulin sources (such as Jerusalem artichoke, jicama, and chicory root), raw oats, unrefined wheat, unrefined barley, and yacon.

[0088] Suitable prebiotics include alginates, xanthan gum, pectin, locust bean gum (LBG), inulin, guar gum, galactooligosaccharides (GOS), fructooligosaccharides (FOS), polydextrose 10 (i.e., Litesse®), lactitol, L-arabinose, D-xylose, L-rhamnose, D-mannose, L-fucose, inositol, sorbitol, mannitol, xylitol, fructose, carrageenan, alginates, microcrystalline cellulose (MCC), betaine, lactosucrose, soy oligosaccharides, and isomaltulose (Palatinose). Examples include (TM), isomaltoligosaccharides, glucooligosaccharides, xylooligosaccharides, mannooligosaccharides, beta-glucans, cellobiose, raffinose, genthiobiose, melibiose, xylobiose, cyclodextrins, isomaltose, trehalose, stachyose, panose, pullulan, beruvascose, galactomannan, (human) milk oligosaccharides, and all forms of indigestible starch.

[0089] A combination of one or more bacterial strains according to the present invention and one or more fibers and / or prebiotics according to the present invention exhibits a synergistic effect (i.e., an effect exceeding the additive effect when the bacteria are used separately) in specific applications.

[0090] In one embodiment, the bacterial strain or mixture thereof according to the present invention is used in combination with one or more types of fiber and / or prebiotics.

[0091] Preferably, the prebiotics used are polydextrose, lactitol, inositol, L-arabinose, D-xylose, L-rhamnose, D-mannose, L-fucose, sorbitol, mannitol, xylitol, fructose, carrageenan, alginate, microcrystalline cellulose (MCC), milk oligosaccharides, or betaine.

[0092] In a further embodiment, the present invention relates to a composition, food, food ingredient, dietary supplement or pharmaceutically acceptable composition comprising a bacterial strain or mixture thereof according to the present invention and one or more types of fiber and / or prebiotics.

[0093] Method, Use, and Other Embodiments of the Invention In one embodiment, the present invention provides a method for preventing or treating cognitive impairment in a subject requiring such treatment, comprising administering to the subject a bacterial strain or a composition comprising a bacterial strain, wherein the bacterial strain is Lacticaseibacillus paracasei or a mixture thereof.

[0094] To avoid misunderstanding, the bacterial strains and any compositions described in this invention can be used in the methods and uses of this invention. For example, further embodiments, but not limited to these, include the following:

[0095] Embodiment 1. A bacterial strain or mixture thereof of the species Lacticaseibacillus paracasei for use in the prevention or treatment of cognitive impairment in persons requiring it.

[0096] Embodiment 2. A bacterial strain for use as described in Embodiment 1, wherein cognitive impairment is induced by sleep deprivation and / or lack of sleep.

[0097] Embodiment 3. The bacterial strain for use described in Embodiment 1, wherein cognitive impairment is a learning and memory impairment.

[0098] Embodiment 4. The bacterial strain for use described in Embodiment 1, wherein the cognitive impairment is an impairment of recognition memory, spatial working memory, and / or context-dependent long-term memory.

[0099] Embodiment 5. A bacterial strain for use as described in Embodiment 1, wherein cognitive impairment is a contributing factor to mental and neurological disorders, psychiatric disorders and / or neurodegenerative diseases.

[0100] Embodiment 6. A bacterial strain for use according to Embodiment 5, wherein the mental and neurological disorders and / or mental disorders are mood disorders, depression, anxiety, neurotic disorders and addiction disorders.

[0101] Embodiment 7. The neurodegenerative disease is dementia, Parkinson's disease, Alzheimer's disease, vascular dementia, Lewy body dementia, Huntington's disease, progressive supranuclear palsy, frontotemporal dementia, Creutzfeldt-Jakob disease, Wernicke-Korsakoff syndrome, and the bacterial strain for use described in Embodiment 5.

[0102] Embodiment 8. A bacterial strain for use as described in Embodiment 2, wherein sleep deprivation and / or sleep insufficiency result in a sleep disorder.

[0103] Embodiment 9. The bacterial strain for use described in Embodiment 8, wherein the sleep disorder is sleep apnea, narcolepsy, insomnia, and / or parasomnia.

[0104] Embodiment 10. A bacterial strain for use according to any one of Embodiments 1 to 9, wherein cognitive impairment is caused by mental fatigue.

[0105] Embodiment 11. A bacterial strain for use according to Embodiment 10, wherein mental fatigue includes symptoms such as attention deficit, impaired focusing ability, forgetfulness, decreased productivity, lack of concentration, disorganization, inattention, distractibility, and learning difficulties.

[0106] Embodiment 12. A bacterial strain of the species Lacticaseibacillus paracasei or a mixture thereof is a probiotic bacterial strain, the bacterial strain for use according to any one of Embodiments 1 to 11.

[0107] Embodiment 13. The bacterial strain for use according to any one of Embodiments 1 to 12 is strain Lpc-37 of the species Lacticaseibacillus paracasei, which was registered with the DSMZ on October 5, 2017, under accession number DSM32661.

[0108] Embodiment 14. A composition comprising a bacterial strain of the species Lacticaseibacillus paracasei, or a mixture thereof, for use in the prevention or treatment of cognitive impairment in a person requiring such treatment.

[0109] Embodiment 15. The composition for use described in Embodiment 14, wherein cognitive impairment is induced by sleep deprivation and / or lack of sleep.

[0110] Embodiment 16. The composition for use according to Embodiment 14, wherein the cognitive impairment is a learning and memory impairment.

[0111] Embodiment 17. The composition for use according to Embodiment 14, wherein the cognitive impairment is an impairment of recognition memory, spatial working memory, and / or context-dependent long-term memory.

[0112] Embodiment 18. The composition for use described in Embodiment 14, wherein cognitive impairment is a contributing factor to mental and neurological disorders, psychiatric disorders and / or neurodegenerative diseases.

[0113] Embodiment 19. The composition for use according to Embodiment 18, wherein the mental and neurological disorders and / or mental disorders are mood disorders, depression, anxiety, neurotic disorders and addiction disorders.

[0114] Embodiment 20. The composition for use according to Embodiment 19, wherein the neurodegenerative disease is dementia, Parkinson's disease, Alzheimer's disease, vascular dementia, Lewy body dementia, Huntington's disease, progressive supranuclear palsy, frontotemporal dementia, Creutzfeldt-Jakob disease, or Wernicke-Korsakoff syndrome.

[0115] Embodiment 21. The composition for use according to Embodiment 15, for sleep deprivation and / or sleep insufficiency resulting from a sleep disorder.

[0116] Embodiment 22. The composition for use according to Embodiment 21, wherein the sleep disorder is sleep apnea, narcolepsy, insomnia and / or parasomnia.

[0117] Embodiment 23. The composition for use according to Embodiments 14-22, wherein cognitive impairment is caused by mental fatigue.

[0118] Embodiment 24. The composition for use described in Embodiment 23, wherein mental fatigue includes symptoms such as lack of attention, impaired focusing ability, forgetfulness, decreased productivity, lack of concentration, inability to organize thoughts, distractibility, and learning difficulties.

[0119] Embodiment 25. A composition for use according to any one of Embodiments 14 to 24, wherein a bacterial strain of the species Lacticaseibacillus paracasei or a mixture thereof is a probiotic strain.

[0120] Embodiment 26. A composition for use according to any one of Embodiments 14 to 25, wherein the strain of the species Lacticaseibacillus paracasei is strain Lpc-37, which was registered with the DSMZ on October 5, 2017, under accession number DSM32661.

[0121] Embodiment 27. The composition for use according to any one of Embodiments 14 to 26, comprising further bacteria.

[0122] Embodiment 28. The composition for use according to Embodiment 27, wherein the further bacteria are strain Lp-115 and / or strain B420, registered with the DSMZ on June 30, 2015, under accession number DSM32073.

[0123] Embodiment 29. The composition according to any one of Embodiments 14 to 28, wherein the composition is a food, a food ingredient, a nutritional supplement, or a pharmaceutical composition.

[0124] Embodiment 30. A method for preventing or treating cognitive impairment in a subject requiring the use thereof, the method comprising administering to the subject a bacterial strain or a composition comprising a bacterial strain, wherein the bacterial strain is the species Lacticaseibacillus paracasei or a mixture thereof.

[0125] Embodiment 31. The method according to Embodiment 30, wherein the cognitive impairment is induced by sleep deprivation and / or lack of sleep.

[0126] Embodiment 32. The method according to Embodiment 30, wherein the cognitive impairment is a learning and memory impairment.

[0127] Embodiment 33. The method according to Embodiment 30, wherein the cognitive impairment is an impairment of recognition memory, spatial working memory, and / or context-dependent long-term memory.

[0128] Embodiment 34. The method according to Embodiment 30, wherein cognitive impairment is a contributing factor to mental and neurological disorders, psychiatric disorders and / or neurodegenerative diseases.

[0129] Embodiment 35. The method according to Embodiment 34, wherein the mental and neurological disorders and / or mental disorders are mood disorders, depression, anxiety, neurotic disorders and addiction disorders.

[0130] Embodiment 36. The method according to Embodiment 35, wherein the neurodegenerative disease is dementia, Parkinson's disease, Alzheimer's disease, vascular dementia, Lewy body dementia, Huntington's disease, progressive supranuclear palsy, frontotemporal dementia, Creutzfeldt-Jakob disease, or Wernicke-Korsakoff syndrome.

[0131] Embodiment 37. The method according to Embodiment 31, wherein the sleep deprivation and / or sleep insufficiency is caused by a sleep disorder.

[0132] Embodiment 38. The method according to Embodiment 37, wherein the sleep disorder is sleep apnea, narcolepsy, insomnia and / or parasomnia.

[0133] Embodiment 39. The method according to any one of Embodiments 30 to 38, wherein the cognitive impairment is caused by mental fatigue.

[0134] Embodiment 40. The method according to Embodiment 39, wherein mental fatigue includes symptoms such as attention deficit, impaired focusing ability, forgetfulness, decreased productivity, lack of concentration, disorganization, inattention, distractibility, and learning difficulties.

[0135] Embodiment 41. The method according to any one of Embodiments 30 to 40, wherein the bacterial strain or mixture thereof of the species Lacticaseibacillus paracasei is a probiotic bacterial strain.

[0136] Embodiment 42. The method according to any one of Embodiments 30 to 41, wherein the strain of the species Lacticaseibacillus paracasei is strain Lpc-37, which was registered with the DSMZ on October 5, 2017, under accession number DSM32661.

[0137] Embodiment 43. The method according to any one of Embodiments 30 to 41, wherein the composition comprises further bacteria.

[0138] Embodiment 44. The method according to Embodiment 43, wherein the further bacteria are strain Lp-115 and / or strain B420, which were registered with the DSMZ on June 30, 2015, under accession number DSM32073. [Examples]

[0139] The following examples are provided to illustrate and further explain specific embodiments and aspects of the present invention, and should not be construed as limiting its scope.

[0140] Theoretical basis of the exam Sleep deprivation is a serious public health epidemic in modern society, leading to impaired memory and other cognitive functions. Probiotics may offer a novel approach to improving cognitive function through the gut microbiota-gut-brain axis. In this study, cognitive impairment was induced in mice using partial sleep deprivation, and the effects of probiotics were evaluated. Lacticaseibacillus paracasei Lpc-37, Lactiplantibacillus plantarum Lp-115, Bifidobacterium animalis ssp. lactis 420, and combinations thereof were tested in partially sleep-deprived mice and compared with a non-sleep-deprivation solvent group and a sleep-deprivation solvent group (placebo group). Single bacterial strains were tested at a rate of 1 × 10⁶. 9 Colony-forming units (CFUs), multiple bacterial strains, 1.5 × 10⁻⁶ 9 Mice were orally administered daily with a dose containing either CFU or a solvent for 30 days prior to and 9 days during the behavioral testing paradigm. To evaluate learning and memory function after partial sleep deprivation, behavioral tests including novel object recognition (NOR), spontaneous alternation behavior in a Y-maze (Y-maze), and step-through passive avoidance (STPA) tasks were used.

[0141] Test design Overall test design and planning animal Five-week-old male Swiss mice weighing 30-35g were purchased from Janvier (Saint Berthevin, France). The mice were housed in groups in a temperature and humidity-controlled animal facility with a 12-hour light / dark cycle (lights off at 7pm). Six mice were housed per cage, and they were given free access to food (SAFE A04C, SAFE, Route de Saint Bris, 89290 AUGY, France) and water. All mice in the same cage received the same treatment. The experiment was conducted at the Amylgen animal facility (approval number A-34-169-002) in accordance with the recommendations of Directive 2010 / 63 / UE issued by the European Parliament and of the Council on September 22, 2010. The protocol was approved by the Languedoc Roussillon Ethics Review Board under CE2A-36. The overall health (hair and eyes) and activity / condition (spontaneous motility and posture) of the mice were regularly monitored visually on a daily basis. Body weight was measured and recorded three times a week.

[0142] Experimental design and probiotic intervention Mice were randomly assigned to one of six treatment groups (n=12 mice / group): a non-sleep-deprivation solvent group (no SD / Veh), a sleep-deprivation solvent group (SD / Veh), and four sleep-deprivation groups supplemented with either a single bacterial strain Lp-115 (SD / Lp-115), Lpc-37 (SD / Lpc-37), B420 (SD / B420), or multiple bacterial strains (Lpc-37+Lp-115+B420; SD / multiple strains).

[0143] The researchers involved in the treatment and care of the mice were blinded to the experimental treatment groups, and each treatment was assigned a number to maintain blindness. Mice in the probiotic treatment group were given a single bacterial strain at a rate of 1 × 10⁶. 9 Colony-forming units (CFUs) or multiple bacterial strains are measured in units of 1.5 × 10⁻⁶. 9 CFU (Lp-115 5 x 10 8 CFU, Lpc-37 5x10 8 CFU and B420 5×10 8One of the CFUs was administered orally. The lyophilized bacterial cultures were prepared by Danisco Cultures, a division of Danisco USA Inc. (DuPont Nutrition Biosciences; Madison, WI), and diluted daily with 100 μl of physiological saline (0.9% NaCl in double-de-ionized water) to ensure accurate dosage. Solvent mice were administered 100 μl of physiological saline orally per day. Bacterial intervention was continued daily for 30 days before and 9 days during the behavioral test paradigm, and until the final day of behavioral evaluation. On day 10, the day after the final day of the behavioral test, all mice (n=72) were anesthetized with 4% isoflurane and sacrificed by cervical dislocation. Figure 4 shows a schematic of the treatment schedule.

[0144] Behavioral testing paradigm Thirty days after probiotic or solvent intervention, all mice underwent behavioral testing paradigms to measure post-sleep deprivation cognitive function (learning and memory). A novel object recognition (NOR) test was used to measure short-term non-spatial recognition memory; a Y-maze spontaneous alternation behavior (Y-maze) test was used to measure spatial working memory for short-term tasks; and a step-through passive avoidance (STPA) task was used to measure context-dependent long-term memory.

[0145] Behavioral assessments were conducted for nine consecutive days: Day 1) NOR (Natural Occlusion); Day 2) NOR (Same Object Trial); Day 3) NOR (New Object Trial); Day 4) Rest; Day 5) Y-maze (training trial); Day 6) Y-shaped maze (test trial); 7th day) rest; Day 8) STPA (Training Trial) and Day 9) STPA (Trial Test).

[0146] On days 2, 5, and 8, immediately after the training trials for each behavioral test, the mice were deprived of sleep for 5 hours before the start of their active period (7 p.m.).

[0147] All behavioral tests were conducted in a specially equipped room within the animal housing facility. To minimize stress, the mice were allowed to acclimate to the laboratory environment by placing their cages in the laboratory for at least 30 minutes prior to each test. The same mice were evaluated in all behavioral tests. During each behavioral test, a researcher blinded to the designated group remained in the laboratory. In addition, an experimenter blinded to the experimental group measured all results.

[0148] Sleep deprivation procedure Prior to this procedure, mice were accustomed to the procedure and then partially deprived of sleep using a "gentle handling" technique by well-trained technicians. The "gentle handling" technique involves maintaining wakefulness by gently handling the mice, which were randomly assigned to the sleep-deprivation group, whenever behavioral signs of sleep (i.e., drowsiness or attempts to assume a sleep-inducing posture) were observed. Immediately after the training trials of each behavioral test on days 2, 5, and 8, the mice were deprived of sleep for 5 hours in their cages before the start of their active period (7 p.m.).

[0149] Endpoint measurement Novel Object Recognition (NOR) Test The NOR (Normative Object Reconciliation) test utilizes the innate tendency of mice to explore novel objects, measuring the time spent in contact with a novel object compared to the time spent in contact with a familiar object. This test consisted of a habituation period (day 1), a training period (day 2), and a test period (day 3).

[0150] Before evaluating NOR, all mice were acclimatized to the test arena, and anxiety-related behaviors, spontaneous motor activity, and stereotypic behaviors (e.g., standing up and grooming) were measured. The open field (OF) arena was a white plexiglass open arena (50cm x 50cm x 55cm) with infrared light-emitting diodes installed on the floor. Mice were placed in the center of the dimly lit (100 lux) OF arena and allowed to freely explore the arena for 10 minutes, during which time their spontaneous motor activity and behavior were recorded using an infrared detection camera connected to a computer. The time spent in the center of the arena and spontaneous motor activity, as well as overall spontaneous motor activity and stereotypic behavior, were analyzed using Ethovision® XT12 software (Noldus).

[0151] On the second day, 24 hours after the OF test, two identical objects were placed in the same arena, approximately 5 cm from each wall at the diagonal corners of the arena, and the mice were returned. The mice were allowed to explore the arena freely for 10 minutes, during which time their behavior was recorded using an infrared detection camera connected to a computer.

[0152] On the third day, 24 hours after the training period, the mice were returned to an arena containing one familiar object and one new object (the object that was in position 2 on the second day was replaced with a new object that differed in color, shape, and texture from the familiar object). The mice were allowed to explore the arena freely for 10 minutes, during which time their behavior was recorded using an infrared detection camera connected to a computer. The equipment and objects between the mice were cleaned with 50% vol / vol ethanol. At the end of each day, the mice were returned to their cages with their cagemates.

[0153] Cognitive function was analyzed by measuring the orientation index (PI) and discrimination index (DI). Object exploration was defined as the time a mouse's nose was within a 2 cm radius of an object and its head was facing the object. PI was calculated as the frequency or time spent in contact with the object at position 2 relative to the total frequency or time spent in contact with both objects (position 1 and position 2). The DI for new objects was calculated by dividing the difference between the time the mouse spent exploring new objects and the time it spent exploring familiar objects by the total exploration time for both objects. [Discrimination index DI = (Time spent exploring new objects - Time spent exploring familiar objects) / (Time spent exploring new objects + Time spent exploring familiar objects)]. The DI result could range from +1 (i.e., more time spent on new objects) to -1 (i.e., more time spent on familiar objects), with 0 representing equal time spent on new and familiar objects. Mice that had fewer than 10 contacts with objects on days 2 and 3 were excluded from evaluation.

[0154] Spontaneous alternating behavior test using a Y-maze The Y-maze test is based on the innate tendency of mice to alternately explore different arms of a maze. The test consisted of a training period (day 5) and an examination period (day 6).

[0155] The Y-shaped maze was constructed from gray polyvinyl chloride, with each arm measuring 40 cm long, 12 cm high, 3 cm wide at the base, and 10 cm wide at the top, connected in a central equilateral triangle. On days 5 and 6, one mouse was placed at the end of each arm of the maze and allowed to move freely within the maze for an 8-minute trial. Entry into each arm was visually observed and recorded by a trained technician. The equipment and objects between the mice were cleaned with 50% vol / vol water / ethanol. At the end of each day, the mice were returned to their cages with their cagemates. On day 5, the mice were deprived of sleep for 5 hours before the start of the activity phase immediately following the training trial (7 PM).

[0156] Alternating behavior was defined as continuous and complete entry (excluding the tail) into each of the three arms. The maximum number of alternating behaviors was determined by subtracting 2 from the total number of arm entries, and the alternating behavior rate was defined by the following formula: [(Number of alternating behaviors) / (Total number of entries - 2) × 100]. Mice that entered more than 10 arms on both days, or mice with an alternating behavior rate of less than 20% or more than 90%, were excluded from evaluation.

[0157] Step-Through Passive Avoidance (STPA) Challenge The STPA task is an index of context-dependent long-term memory used to assess memory formation. The test consisted of a training phase (day 8) and an examination phase (day 9).

[0158] The apparatus consisted of two separate rooms (15cm x 20cm x 15cm) connected by a retractable door: a bright room enclosed by illuminated white PVC walls and a dark room enclosed by black PVC walls. On day 8, one mouse was placed in each bright room. After 5 seconds, the retractable door was raised. Once the mouse entered the dark room, the door was closed, and an electric shock (0.3mA, 3 seconds) was delivered to the mouse's feet in a scrambling manner through a grid on the floor using a shock generator scrambler (Lafayette Instruments, Lafayette, USA). The time it took for each mouse to move from the bright room to the electric grid floor was used as an indicator of reaction latency, and the reaction latency until entry was recorded. On day 8, before the start of the activity phase immediately following the training trial (7pm), the mice were deprived of sleep for 5 hours.

[0159] On the ninth day, 24 hours after training, the time it took for the mice to return to the light room and re-enter the dark room was used as an indicator of learning. The door was lifted after 5 seconds. If the animal did not step into the dark room, the reaction latency cutoff time was set to 300 seconds. The reaction latency until retreating to the light room was also recorded. The equipment and objects between the mice were cleaned with 50% vol / vol water / ethanol. At the end of each day, the mice were returned to their cages with their cagemates.

[0160] statistical analysis All values ​​except passive avoidance latency are expressed as mean ± standard error (SEM). First, the normality of all data was determined using the Shapiro-Wilk test. If the data was normally distributed, a one-way ANOVA was performed, followed by Dunnett's multiple comparison test or a two-way ANOVA, followed by Sidak's multiple comparison test. Non-normally distributed data (i.e., passive avoidance latency) were analyzed using the non-parametric Kruskal-Wallis test, followed by Dunn's test for multiple comparisons. All data were analyzed using GraphPad Prism software v.6 (GraphPad Software Inc., La Jolla, CA, USA). A p-value less than 0.05 was considered statistically significant.

[0161] Results and Discussion Novel Object Recognition (NOR) Test (Recognition Memory; Figure 1) A non-spatial recognition (NOR) study was used to evaluate the effect of probiotics on short-term non-spatial recognition memory in sleep-deprived mice. The results are shown in Figure 1.

[0162] Prior to sleep deprivation on day 2 of the NOR trial, bacterial intervention did not significantly affect the frequency of contact between two similar objects (Figure 1A) or the duration of contact between objects (Figure 1B) compared to the solvent group.

[0163] Memory, i.e., the ability to recognize familiar objects by day 3 of the NOR study, was significantly impaired by sleep deprivation immediately after acquiring a familiar object (day 2 of the NOR study). The SD / Veh group had significantly higher contact frequency (p<0.05; Figure 1C) and contact duration (p<0.001; Figure 1D) with novel objects than the theoretical value of 50%, although the same results were not observed in the SD / Veh group. Mice in the SD / Lpc-37 group after Lpc-37 intervention had significantly higher contact frequency (p<0.05; Figure 1C) and contact duration (p<0.001; Figure 1D) with novel objects than the theoretical value of 50%, despite the sleep deprivation period. Similarly, SD / multiple bacterial strain treated mice showed increased contact frequency (p<0.01; Figure 1C) and contact duration (p<0.001; Figure 1D) with novel objects than the theoretical value of 50%. When Lp-115 or B420 was administered, the results were similar to those of the SD / Veh group, and sleep deprivation-induced recognition memory impairment could not be prevented (Figures 1C and 1D).

[0164] Since there was no significant difference in the DI (Depth Infection) related to object contact frequency between the SD / Veh group and the no SD / Veh group (Figure 1E), sleep deprivation did not appear to affect the DI related to object contact frequency. Furthermore, comparing the treatment group with SD / Veh (Figure 1E), bacterial intervention did not affect this DI index. In terms of DI related to contact time, the SD / Veh group showed a statistically significant difference compared to the no SD / Veh group (p<0.001; Figure 1F). The Lpc-37 group (p<0.001) and the multiple strain group (p<0.001) had significantly higher ability to identify novel and familiar objects compared to the SD / Veh group, even after partial sleep deprivation (Figure 1F), which is reflected in the DI related to contact time. Intervention with Lp-115 or B420 did not improve the ability to identify novel and familiar objects as represented by this index, similar to the SD / Veh group (Figure 1F). No significant differences were observed between the Lpc-37 group and the multiple strain group and the SD / Veh group (Figure 1F).

[0165] Statistical Analysis: One-way analysis of variance (F-score) was performed on all data, followed by Dunnett's multiple comparison test. Object contact frequency (%) and object contact time (%) were statistically analyzed by comparing them to the theoretical value of 50% (or DI "0"). Unless otherwise specified, n=12 in all groups. Data are expressed as mean ± SEM. $: p<0.05, $$: p<0.01 vs. the theoretical value of 50% (C); $$$: p<0.001 vs. the theoretical value of 50% (D); ***: p<0.001 vs. No SD / Veh group (F); ###: p<0.001 vs. SD / Veh group (1F).

[0166] Spontaneous alternating behavior test using a Y-maze (spatial working memory; Figure 2) To evaluate the effect of probiotics on spatial working memory in sleep-deprived mice, a spontaneous alternating behavior test using a Y-maze was employed. The results are shown in Figure 2.

[0167] On day 5, during the training trials of the spontaneous alternation behavior test using a Y-maze and before sleep deprivation, no significant difference was observed in spontaneous alternation behavior between the treatment group and the solvent group (Figure 2A). Furthermore, bacterial intervention did not affect spontaneous movement or the number of arm entries compared to the solvent group (Figure 2B). Sleep deprivation immediately after evaluating memory acquisition, i.e., spatial learning and spatial reference memory, significantly induced impairment in spatial working memory in the Y-maze on day 6 of the behavioral test paradigm. Spontaneous alternation behavior in mice in the SD / Veh group during the test trials was significantly reduced compared to the no SD / Veh group (p<0.001; Figure 2C). Sleep deprivation did not affect spontaneous movement or the number of arm entries, and no significant difference was observed between the solvent groups (Figure 2D). In the SD / Lpc-37 group of mice after Lpc-37 intervention, spontaneous alternation behavior was significantly increased compared to the SD / Veh group, despite the presence of sleep deprivation (p<0.001; Figure 2C). Furthermore, no significant difference was observed between the Lpc-37 and no SD / Veh groups (p>0.05; Figure 2C). After intervention with multiple bacterial strains, spontaneous alternation behavior was confirmed to recover to some extent compared to the SD / Veh group (p<0.05; Figure 2C). Intervention with Lp-115 or B420 did not increase spontaneous alternation behavior, similar to the SD / Veh group (Figure 2C). Compared to the solvent group, no significant effect of treatment was observed on spontaneous movement or the number of arm entry attempts (Figure 2D).

[0168] Statistical analysis: One-way analysis of variance (F-score) was performed on all data, followed by Dunnett's multiple comparison test. Unless otherwise specified, n=12 for all groups. Data are expressed as mean ± SEM. ***:p<0.001vs.No SD / Veh group (C);#:p<0.05, ###:p<0.001vs.SD / Veh group (2C).

[0169] Step-through passive avoidance (STPA) task (context-dependent long-term memory; Figure 3) The STPA task was used to evaluate the effects of probiotics on context-dependent long-term memory in sleep-deprived mice. The results are shown in Figure 3.

[0170] During the STPA task training trials on day 8 and before sleep deprivation, no significant difference in response latency was observed in any treatment group of mice compared to the solvent group (Figure 3A). Sleep deprivation immediately after memory acquisition, i.e., associating electric shock to the foot in a darkroom, induced significant context-dependent long-term memory collapse in the STPA task on day 9 of the behavioral test paradigm. The response latency in the no SD / Veh group during the test trials was similar to that of the SD / Veh group (Figure 3B), and bacterial intervention did not have a significant effect on response latency compared to the SD / Veh group (Figure 3B). Regardless of sleep deprivation, none of the mice attempted to enter the darkroom, and the maximum waiting time was set at 300 seconds, after which they were moved to the darkroom by hand. After entering the darkroom, the SD / Veh group had a significantly increased escape latency or was significantly slower to leave the darkroom compared to the no SD / Veh group (p<0.001; Figure 3C). The SD / Lpc-37 group of mice was the only treatment group that showed a significantly reduced escape latency time compared to the SD / Veh group (p<0.001; Figure 3C). Furthermore, the SD / Lpc-37 group was the only treatment group that did not show a significant difference in escape latency time compared to the no SD / Veh group, suggesting that the effects of sleep deprivation in the no SD / Veh group appear to have been normalized by the intervention of Lpc-37.

[0171] Statistical Analysis: All data were analyzed using Kruskal-Wallis nonparametric analysis of variance (H-value), followed by Dunn's test for multiple comparisons. Unless otherwise specified, n=12 for all groups. Data are expressed as mean ± SEM. *:p<0.05, ***:p<0.001vs.NoSD / Veh group;##:p<0.01vs.SD / Veh group (C).

[0172] Summary of the results From the results described above, we can draw the following conclusions. Partial sleep deprivation significantly affected cognitive function in sleep-deprived solvent mice (No SD / Veh and SD / Veh); Intervention with Lpc-37 significantly improved recognition memory impairment in NOR, spatial working memory impairment in Y-maze, and context-dependent long-term memory impairment in STPA, all induced by sleep deprivation. Multiple bacterial strains (Lp-115 + Lpc-37 + B420) significantly improved recognition memory impairment in the NOR (Natural Orientation Recognition) and spatial working memory impairment in the Y-maze, both induced by sleep deprivation.

[0173] All publications previously mentioned herein are incorporated herein by reference. Various modifications and variations of the methods and systems described herein, without departing from the scope and spirit of the present invention, will be apparent to those skilled in the art. While specific preferred embodiments of the present invention have been described, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. In fact, various modifications of the described methods for carrying out the present invention, which will be apparent to those skilled in the art in biochemistry and biotechnology or related fields, are intended to fall within the scope of the following claims.

Claims

1. A composition comprising the Lpc-37 bacterial strain of the Lacticaseibacillus paracasei species, registered with the DSMZ on October 5, 2017, under accession number DSM32661, for use in the prevention or treatment of sleep deprivation and / or sleep-deprived cognitive impairment in persons requiring such treatment, wherein the composition further comprises the Lp-115 strain of Lactiplantibacillus plantarum and the Bifidobacterium animalis ssp. lactis strain B420, registered with the DSMZ on June 30, 2015, under accession number DSM32073.

2. The composition for use according to claim 1, wherein the sleep deprivation and / or sleep insufficiency is caused by a sleep disorder.

3. The composition for use according to claim 2, wherein the sleep disorder is sleep apnea, narcolepsy, insomnia and / or parasomnia.

4. The composition according to any one of claims 1 to 3, wherein the composition is a food, a food ingredient, a nutritional supplement, or a pharmaceutical composition.

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