Lactococcus lactis strain and uses thereof

Abstract

The present application relates to a Lactococcus lactis strain and uses thereof.

Description

Lactococcus lactis strains and uses thereof

[0001] The present application relates to a novel Lactococcus lactis KBL1036 strain and its use.

[0002] Probiotics are microorganisms with antimicrobial and enzymatic activity that help maintain intestinal microbial balance, as well as their products. Probiotics are defined as single or complex strains of live bacteria administered to humans or animals in the form of dried cells or fermented products to improve intestinal flora. Probiotics must be able to thrive in the human intestine, be non-pathogenic and non-toxic, and survive until reaching the intestine. Furthermore, they must maintain viability and activity until they are incorporated into food and consumed, be sensitive to antibiotics used for infection prevention, and not harbor antibiotic-resistant plasmids. Furthermore, they must be resistant to acids, enzymes, and bile in the intestinal environment. Recently, probiotics have been reported to improve various health functions, including intestinal health, and are emerging as a key therapeutic agent that can replace existing compound-based treatments.

[0003] Meanwhile, in modern society, the prevalence of mental illnesses, including autism spectrum disorder, anxiety disorders, depression, and bipolar disorder, is increasing due to genetic factors and environmental factors such as rapidly increasing stress. Autism spectrum disorder, in particular, frequently develops in children, and if treatment is delayed, symptoms such as lack of social skills, repetitive behaviors, and emotional processing disorders can worsen, making it difficult for individuals to lead normal lives even into adulthood. Therefore, the need for early treatment for autism spectrum disorder is urgent. Autism spectrum disorder, anxiety disorders, depression, and bipolar disorder share many core symptoms.

[0004] Medications used to treat mental illnesses, including autism spectrum disorder, anxiety disorders, depression, and bipolar disorder, primarily work by modulating monoaminergic neurotransmitters. The most commonly used are those that inhibit the reuptake of serotonin, a type of monoaminergic neurotransmitter (e.g., SSRIs, SNRIs, and TCAs). However, approximately 40% of patients taking these medications discontinue treatment due to poor compliance due to side effects. Furthermore, 33% of patients remain non-responders, highlighting the need for the development of new, more effective treatments.

[0005] Meanwhile, it is known that the intestinal flora activates and suppresses immune cells in the intestines, thereby regulating the secretion of various immune cytokines and chemokines. Changes in the secretion of abnormal cytokines and chemokines by immune cells due to imbalance and reduced diversity in the intestinal flora are deeply related to the pathogenesis and development of mental illnesses including autism spectrum disorder, anxiety disorder, depression, and bipolar disorder, as they cause neuroinflammation, brain damage, and autoimmune reactions.

[0006] Against this backdrop, the inventors of the present invention conducted research on a probiotic strain that has the effect of increasing the secretion of serotonin in the body, increasing the differentiation ability of T helper 17 cells, thereby preventing, treating, and improving mental illness, and at the same time suppressing inflammatory immune responses, and verified that the strain according to an example of the present application exhibits an excellent effect in preventing and improving mental illness by increasing the secretion of serotonin in the intestine, increasing the differentiation ability of T helper 17 cells, and suppressing the increase in inflammatory immune responses.

[0007] An example of the present application relates to a strain of Lactococcus lactis KBL1036 having the accession number KCTC 14332BP.

[0008] Another example of the present application relates to a pharmaceutical composition for preventing or treating mental illness, comprising at least one selected from the group consisting of Lactococcus lactis KBL1036 strain having the deposit number KCTC 14332BP, a culture of the strain, a lysate of the strain, and an extract of the strain.

[0009] Another example of the present application relates to a food comprising at least one selected from the group consisting of Lactococcus lactis KBL1036 strain having the deposit number KCTC 14332BP, a culture of the strain, a lysate of the strain, and an extract of the strain.

[0010] Another example of the present application relates to probiotics comprising at least one selected from the group consisting of Lactococcus lactis KBL1036 strain having the deposit number KCTC 14332BP, a culture of the strain, a lysate of the strain, and an extract of the strain.

[0011] Another example of the present application relates to a quasi-drug comprising at least one selected from the group consisting of Lactococcus lactis KBL1036 strain having the deposit number KCTC 14332BP, a culture of the strain, a lysate of the strain, and an extract of the strain.

[0012] Another example of the present application relates to an animal feed comprising at least one selected from the group consisting of Lactococcus lactis KBL1036 strain having the deposit number KCTC 14332BP, a culture of the strain, a lysate of the strain, and an extract of the strain.

[0013]

[0014] Hereinafter, the present invention will be described in more detail.

[0015] Serotonin reuptake inhibitors, such as SSRIs, SNRIs, and TCAs, which are existing drugs used to treat mental illnesses including autism spectrum disorders, depression, bipolar disorder, and anxiety disorders, have limitations such as decreased drug compliance and non-responsiveness due to side effects in some patients taking the drugs. Therefore, in order to meet the technical needs of the art urging the development of effective treatments for mental illness, the inventors of the present invention considered that the health-promoting effects of probiotics are difficult to view as general characteristics of the genus and species, but rather correspond to the specific effects of specific strains, and completed the present invention by screening various strains to identify strains that exhibit excellent effects in improving mental illness. Accordingly, one example of the present application relates to the Lactococcus lactis KBL1036 strain having the deposit number KCTC 14332BP, which increases the intestinal secretion of serotonin, an effective substance for treating mental illness, and suppresses inflammatory immune responses.

[0016] The above strain may have one or more of the following characteristics (1) to (6):

[0017] (1) Having a 16S rRNA sequence of sequence number 1;

[0018] (2) Promoting the synthesis of serotonin, for example, promoting the expression of Tph-1 (tryptophan hydroxylase 1), which is a serotonin-producing enzyme, specifically, the expression of Tph-1 is 1.5 times or more, 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times or more, or 6.5 times or more compared to the untreated control group, the expression of Tph-1 is 1.5 times or more, or 2 times or more compared to the Lactococcus garvieae strain treatment group with accession number KCTC 3773, the expression of Tph-1 is 1.5 times or more, or 1.7 times or more compared to the Lactococcus piscium strain treatment group with accession number KCTC 3639, or the expression of Tph-1 is 1.5 times or more, or 1.8 times or more compared to the Lactococcus chunganggensis strain treatment group with accession number KCTC 13185,

[0019] (3) Promotes secretion of serotonin, specifically, serotonin secretion of 1.5 times or more, 2 times or more, 2.5 times or more, 3 times or more, 3.5 times or more, 4 times or more, 4.5 times or more, 5 times or more, 5.5 times or more, 6 times or more, or 6.5 times or more compared to the untreated control group, or serotonin secretion of 0.95 times or more, 1 times or more, 1.5 times or more, 2 times or more, 2.5 times or more, 3 times or more, or 3.5 times or more compared to the 2 mM concentration of tyramine treatment group.

[0020] (4) Promotes differentiation of naive T cells into T-helper 17 cells, specifically, promotes differentiation of naive T cells into T-helper 17 cells in the colon and / or meninges.

[0021] (5) Promotion of IL-17 secretion,

[0022] (6) Non-inducing inflammation, for example, non-inducing inflammatory cytokines, specifically non-inducing one or more inflammatory cytokines selected from the group consisting of IL-1β, IL-6, and TNF-α.

[0023] Even if the upper limit of the Tph-1 expression amount or the serotonin secretion amount is not specified, a person skilled in the art will be able to clearly implement a strain according to an example of the present application for the purpose of promoting the synthesis or secretion of serotonin, but for example, the upper limit of the Tph-1 expression amount or the serotonin secretion amount may be 10 times or less, 9 times or less, 8 times or less, 7 times or less, 6.5 times or less, 6 times or less, 5 times or less, 4 times or less, 3 times or less, or 2.5 times or less compared to the control group or the comparative treatment group, but is not limited thereto.

[0024] The above T helper 17 cells secrete the cytokine interleukin-17 (IL-17), and IL-17 has the effect of improving autism spectrum disorder and psychiatric disorder-like phenotypes. The immune system and neurological diseases including autism spectrum disorder and psychiatric disorders such as anxiety disorder, depression, and bipolar disorder are significantly related. T helper 17 cells are generally divided into pathogenic T helper 17 cells that also secrete the inflammatory cytokine interferon gamma (IFN-g) and homeostatic T helper 17 cells that play a role in protecting against diseases. Among these, homeostatic T helper 17 cells are promoted by the commensal microbiome in the gut. Therefore, it suggests the possibility of treating neurological diseases including autism spectrum disorder and psychiatric disorders through homeostatic T helper 17 cells promoted by the commensal microbiome in the gut.

[0025] The above-mentioned non-induction of inflammation or non-induction of inflammatory cytokines means that when the strain according to an example of the present application is treated, no significant level of inflammation or inflammatory cytokines is induced. For example, the above-mentioned non-induction of inflammation or non-induction of inflammatory cytokines may mean that the level of inflammatory cytokine expression is 30% or less, 20% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less compared to the group treated with LPS at a concentration of 1000 ng / mL. The above-mentioned inflammatory cytokine may be one or more selected from the group consisting of IL-1β, IL-6, and TNF-α.

[0026] Another example of the present application relates to a pharmaceutical composition for preventing or treating mental illness, comprising at least one selected from the group consisting of Lactococcus lactis KBL1036 strain having the deposit number KCTC 14332BP, a culture of the strain, a lysate of the strain, and an extract of the strain.

[0027] Another example of the present application relates to a composition for increasing or promoting serotonin levels, comprising at least one selected from the group consisting of Lactococcus lactis KBL1036 strain having the deposit number KCTC 14332BP, a culture of the strain, a lysate of the strain, and an extract of the strain. The serotonin level may be a serotonin level in the brain or intestine.

[0028] Serotonin is known as a neurotransmitter that plays a crucial role in the central nervous system and numerous biological functions, particularly influencing emotions, mood, sleep, appetite, and stress responses. Therefore, changes in serotonin levels are closely related to mental illness, and alterations in serotonin levels can trigger or worsen mental illness through other effects on the brain.

[0029] For example, it is known that patients with autism spectrum disorder have imbalanced serotonin levels. Serotonin plays a crucial role in regulating various aspects related to brain development and function, functions as a neurotransmitter in the central nervous system, and is related to emotional regulation, social interaction, and repetitive behaviors. Therefore, serotonin deficiency can cause neurodevelopmental disorders and lead to the development of autism spectrum disorder. Therefore, the autism spectrum disorder may be a serotonin-deficient autism spectrum disorder.

[0030] In this example, administering a strain according to an example of the present application to a mouse model of autism spectrum disorder significantly increased its sociability. Therefore, the strain according to an example of the present application may increase the sociability of individuals with autism spectrum disorder.

[0031] Additionally, it is generally known that patients with depression have low serotonin levels, which can make neurotransmission within the brain difficult and lead to symptoms of depression. Therefore, one of the treatments for depression is to alleviate depression by regulating serotonin levels.

[0032] Additionally, serotonin levels can change in conditions such as bipolar disorder or anxiety disorders, and decreased serotonin levels can increase stress responses and worsen psychological symptoms associated with anxiety.

[0033] Serotonin has also been implicated in mental illnesses related to drug and alcohol addiction, specifically drug addiction can disrupt the chemical balance in the brain, altering serotonin levels, which can lead to mental problems.

[0034] Therefore, it is widely known in the art that increasing the synthesis and / or secretion of serotonin in a subject with a mental illness can effectively prevent, improve, or treat the mental illness.

[0035] In the present invention, the strain or composition according to an example of the present application was treated to verify the effect on the synthesis and secretion of serotonin, and as a result, a significantly higher serotonin synthesis and / or secretion effect was observed compared to the untreated control group or comparison group, and thus the strain or composition according to an example of the present application is useful for the prevention, improvement, or treatment of mental illness.

[0036] The above mental illness may be a serotonin deficiency.

[0037] The above mental illness or the above serotonin deficiency may be at least one selected from the group consisting of autism spectrum disorder, depression, bipolar disorder, anxiety disorder, panic disorder, obsessive-compulsive disorder, memory impairment, delusions of grandeur, sleep disorder, eating disorder, cognitive disorder, hyperactivity, attention deficit, irritable bowel syndrome, schizophrenia, post-traumatic stress disorder (PTSD), neurosis, motor dysfunction, intellectual disability, drug addiction, and alcoholism.

[0038] The term "prevention" in this application refers to the inhibition or delay of the onset of a disease, disorder, or condition. Prevention may be considered complete if the onset of the disease, disorder, or condition is inhibited or delayed for a predetermined period of time.

[0039] The term “treatment” in this application means partially or completely alleviating, improving, palliating, inhibiting or delaying the symptoms of a specific disease, disorder and / or condition or condition, reducing the severity or reducing the occurrence of one or more symptoms or characteristics.

[0040] Another example of the present application relates to a food comprising at least one selected from the group consisting of Lactococcus lactis KBL1036 strain having the deposit number KCTC 14332BP, a culture of the strain, a lysate of the strain, and an extract of the strain. The food may be in the form of, for example, meat, bread, chocolate, candy, jelly, snacks, confectionery, kimchi, soy sauce, cheese, dairy products, powders, beverages, or vitamin complexes. The food may be a health functional food.

[0041] The above food may include, as active ingredients, ingredients commonly added during food manufacturing. The added ingredients include, for example, proteins, carbohydrates, fats, nutrients, seasonings, and flavoring agents. The carbohydrates include monosaccharides (e.g., glucose, fructose, etc.), disaccharides (e.g., maltose, sucrose, oligosaccharides, etc.), and polysaccharides (e.g., common sugars such as dextrin, cyclodextrin, etc., and sugar alcohols such as xylitol, sorbitol, erythritol, etc.). The flavoring agents include natural flavoring agents (thaumatin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.).

[0042] For example, when a food according to an example of the present application is manufactured as a drink, it may additionally include citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, fruit juice, jujube extract, or licorice extract.

[0043] Foods according to one example of the present application include processed forms of all natural ingredients, such as functional foods, nutritional supplements, health foods, and food additives. These types of foods can be manufactured in various forms using conventional methods known in the art. For example, as health foods, the strains described above can be manufactured into tea, juice, and drinks for consumption, or granulated, encapsulated, or powdered for consumption. In addition, the food may be in the form of beverages (including alcoholic beverages), fruits and processed foods thereof (e.g., canned fruits, bottled fruits, jams, marmalades, etc.), fish, meats and processed foods thereof (e.g., ham, sausages, corned beef, etc.), breads and noodles (e.g., udon, buckwheat noodles, ramen, spaghetti, macaroni, etc.), fruit juices, various drinks, cookies, taffy, dairy products (e.g., yogurt, fermented milk, butter, cheese, etc.), edible vegetable oils, margarine, vegetable proteins, retort foods, frozen foods, various seasonings (e.g., soybean paste, soy sauce, sauces, etc.). In addition, in order to use the strain according to an example of the present application, the culture of the strain, the pulverized product of the strain, and the extract of the strain, etc. in the form of a food additive, they may be manufactured in the form of a powder or a concentrate and used.

[0044] Another example of the present application relates to a probiotic comprising at least one selected from the group consisting of Lactococcus lactis KBL1036 strain having the deposit number KCTC 14332BP, a culture of the strain, a lysate of the strain, and an extract of the strain. The probiotic may be in the form of a powder, granules, tablets, or capsules.

[0045] Another example of the present application relates to a quasi-drug comprising at least one selected from the group consisting of Lactococcus lactis KBL1036 strain having the deposit number KCTC 14332BP, a culture of the strain, a lysate of the strain, and an extract of the strain. The quasi-drug composition may be in a form selected from the group consisting of a toothpaste, a mouthwash, an oral spray, an oral ointment, a mouthwash, a mouthwash, a bandage, and a pest repellent (e.g., mosquito, tick, etc.).

[0046] In the present application, the term “culture of a strain” means a product obtained by culturing a strain according to an example of the present application, and the culture may be a whole culture of the strain according to an example of the present application, a dilution thereof, a concentrate, a dried product, a lyophilized product, a lyophilized product, a fragment, and / or a fraction thereof, and the concentrate may be obtained by centrifuging or evaporating the culture, the dried product may be obtained by drying the culture using a dryer or the like, the lyophilized product may be obtained by lyophilizing the culture using a freeze dryer or the like, the fragment may be obtained by physically or ultrasonically treating the strain or culture, and the fraction may be obtained by subjecting the culture, the fragment, etc. to a method such as centrifugation, chromatography, etc. The culture may be in a solid phase (solid, for example, a dried product), a liquid phase (liquid), or a fluid phase, but is not limited thereto. In one example, the culture may refer to the entire medium including the cultured strain, its metabolites, and / or extra nutrients obtained by culturing the strain according to one example of the present application for a certain period of time. In one example, the culture may be one in which the strain according to one example of the present application is removed or not removed. In one example, the culture may refer to the remaining components excluding the strain (bacterial cells) in the culture obtained by culturing the strain according to one example of the present application in a medium. In one example, the culture may be a culture (or culture) obtained by removing the strain (bacterial cells) from the culture solution obtained by culturing the strain according to one example of the present application in a medium. The culture solution (or culture) obtained by removing the strain may be a cell-free culture solution (or culture) or a culture solution containing dead cells, and may be, for example, a filtrate (centrifuged supernatant) obtained by removing the strain by filtration or centrifugation, and / or a culture solution (or dried product of the culture solution) containing dead cells.Specifically, the culture may exhibit anti-inflammatory activity at a level equivalent to that exhibited by the strain according to an example of the present application, or activity for preventing, improving, or treating inflammatory diseases.

[0047] The term "strain fragment" in this application may refer to a product obtained by disrupting a strain according to an example of this application using chemical or physical force. Specifically, the fragment may exhibit anti-inflammatory activity equivalent to the activity exhibited by the strain according to an example of this application, or activity to prevent, improve, or treat inflammatory diseases.

[0048] The term “extract” in the present application may refer to a product obtained by extracting a strain according to an example of the present application, a culture of the strain, a lysate of the strain, or a mixture thereof, regardless of the extraction method, extraction solvent, extracted component, or form of the extract, and is a broad concept that includes all materials that can be obtained by processing or handling by another method after extraction. For example, the extract may be an extract of the strain according to an example of the present application, an extract of the culture of the strain, or an extract of the lysate of the strain. Specifically, the extract may exhibit anti-inflammatory activity, or activity for preventing, improving, or treating inflammatory diseases, at a level equivalent to the activity exhibited by the strain according to an example of the present application, the culture of the strain, or the lysate of the strain.

[0049] A composition, food, probiotics, over-the-counter drug, etc. according to an example of the present application may additionally include one or more types of effective ingredients exhibiting the same or similar function in addition to the above effective ingredients.

[0050] In addition, the composition, food, probiotics, quasi-drug, etc. according to an example of the present application can be manufactured in the form of a unit dose by formulating using a pharmaceutically acceptable carrier according to a method that can be clearly performed by a person having ordinary skill in the art to which the present invention pertains, or can be manufactured by placing it in a multi-dose container. The term “carrier” in the present application means a compound that facilitates the addition of a compound into a cell or tissue, and the term “pharmaceutically acceptable” means a composition that is physiologically acceptable and does not typically cause an allergic reaction such as gastrointestinal disorder or dizziness or a similar reaction when administered to a human.

[0051] The pharmaceutically acceptable carriers mentioned above are those commonly used in formulations, and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0052] In addition, compositions, foods, probiotics, over-the-counter drugs, etc. according to an example of the present application may additionally include additives such as fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers, and preservatives in addition to the above ingredients. In the present application, the content of the additives is not particularly limited and may be appropriately adjusted within the content range used in conventional formulations.

[0053] In addition, the composition, food, probiotics, over-the-counter drugs, etc. according to the present application may be formulated as oral preparations. Non-limiting examples of the oral preparations include tablets, troches, lozenges, aqueous suspensions, oily suspensions, prepared powders, granules, emulsions, hard capsules, soft capsules, syrups, or elixirs. In order to formulate the pharmaceutical composition or food composition according to the present application for oral administration, a binder such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose, or gelatin; an excipient such as dicalcium phosphate; a disintegrant such as corn starch or sweet potato starch; Magnesium stearate, calcium stearate, sodium stearyl fumarate, etc. can be used, and sweeteners, flavoring agents, syrups, etc. can also be used. Furthermore, in the case of capsules, in addition to the above-mentioned substances, a liquid carrier such as fatty oil can be additionally used.

[0054] The term "excipient" in this application refers to any substance, other than a therapeutic agent, that serves as a carrier or medium for the delivery of a therapeutic agent or is added to a pharmaceutical composition, thereby improving handling and storage characteristics or facilitating and facilitating the formation of a unit dosage of the composition.

[0055] The composition according to the present application, for example, a pharmaceutical composition, may be formulated and used in various forms, such as oral formulations such as liquids, suspensions, powders, granules, tablets, capsules, pills, extracts, emulsions, syrups, aerosols, and injections of sterile injectable solutions, according to conventional methods according to each intended use, and may be administered orally or through various routes, including intravenous, intraperitoneal, subcutaneous, rectal, and topical administration. The term 'oral administration' in the present application means that the active substance is administered to a substance prepared to be digested, i.e., to the gastrointestinal tract for absorption.

[0056] The preferred dosage of the composition or food according to the present application may vary depending on the patient's condition and weight, age, sex, health status, dietary constitution, nature of the preparation, degree of disease, administration time of the composition, administration method, administration period or interval, excretion rate, and drug form, and may be appropriately selected by a person skilled in the art.

[0057] The term “effective dosage” in this application means the amount of a composition of an active ingredient sufficient to treat a specific symptom. This may vary depending on the formulation method of the pharmaceutical composition or food composition, the administration method, the administration time, and / or the administration route, and may vary depending on various factors including the type and degree of the response to be achieved by administering the pharmaceutical composition or food composition, the type, age, weight, general health condition, symptoms or degree of the disease, sex, diet, excretion, drugs used simultaneously or at the same time in the subject, other components of the composition, and similar factors well known in the medical field. A person of ordinary skill in the art can easily determine and prescribe an effective dosage for the desired treatment.

[0058] The composition or food according to the present application may be administered once daily or divided into several doses. The composition may be administered as an individual treatment or in combination with another treatment, and may be administered sequentially or simultaneously with conventional treatments. Taking all of the above factors into consideration, the composition may be administered in an amount that achieves maximum effect with minimal side effects.

[0059] For example, the composition according to the present application may be administered in an amount of 0.001 to 10,000 mg, 0.001 to 5,000 mg, 0.001 to 1,000 mg, 0.001 to 500 mg, 0.001 to 300 mg, 0.001 to 100 mg, 0.001 to 50 mg, 0.001 to 30 mg, 0.001 to 10 mg, 0.001 to 5 mg, 0.001 to 1 mg, 0.001 to 0.5 mg, 0.001 to 0.1 mg, 0.001 to 0.05 mg, 0.001 to 0.01 mg, 0.01 to 10,000 mg, 0.01 to 5,000 mg, per kg of body weight. 0.01 to 1,000 mg, 0.01 to 500 mg, 0.01 to 300 mg, 0.01 to 100 mg, 0.01 to 50 mg, 0.01 to 30 mg, 0.01 to 10 mg, 0.01 to 5 mg, 0.01 to 1 mg, 0.01 to 0.5 mg, 0.01 to 0.1 mg, 0.01 to 0.05 mg, 0.1 to 10,000 mg, 0.1 to 5,000 mg, 0.1 to 1,000 mg, 0.1 to 500 mg, 0.1 to 300 mg, 0.1 to 200 mg, 0.1 to 100 mg, 0.1 to 50 mg, 0.1 to 30 mg, 0.1 to 10 mg, 0.1 to 5 mg, 0.1 to 1 mg, 0.1 to 0.5 mg, 1 to 10,000 mg, 1 to 5,000 mg, 1 to 1,000 mg, 1 to 500 mg, 1 to 300 mg, 1 to 200 mg, 1 to 100 mg, 1 to 50 mg, 1 to 10 mg, 1 to 5 mg, 10 to 10,000 mg, 10 to 5,000 mg, 10 to 1,000 mg, 10 to 500 mg, 10 to 300 mg, 10 to 200 mg, 10 to 100 mg, 10 to 50 mg, 10 to 40 mg, 10 to 30 mg, 10 to 20 mg, 100 to 10,000 mg, 100 to 5,000 mg, 100 The composition according to the present invention may be administered in a daily dosage of, but is not limited to, 1,000 mg, 100 to 500 mg, 100 to 300 mg, or 100 to 200 mg. For example, the daily dosage of the composition according to the present application may be 0.001 to 10 g / day, 0.001 to 5 g / day, 0.01 to 10 g / day, or 0.01 to 5 g / day on an oral administration basis. In addition, the total daily dosage may be divided and administered continuously or discontinuously as needed.

[0060] Another example of the present application relates to an animal feed comprising at least one selected from the group consisting of Lactococcus lactis KBL1036 strain having the deposit number KCTC 14332BP, a culture of the strain, a lysate of the strain, and an extract of the strain.

[0061] Various grains and soybean proteins, as well as peanuts, peas, sugar beets, pulp, grain by-products, animal viscera powder, and fish meal powder can be used as raw materials for feed, and these can be used without limitation in an unprocessed or processed form. An animal feed according to an example of the present application may be one in which at least one selected from the group consisting of a strain according to another example of the present application, a culture of the strain, a lysate of the strain, and an extract of the strain are added to the raw materials for the feed.

[0062] Another example of the present application relates to a method for preventing or treating mental illness, comprising administering to a subject in need thereof at least one selected from the group consisting of Lactococcus lactis KBL1036 strain having the deposit number KCTC 14332BP, a culture of the strain, a lysate of the strain, and an extract of the strain.

[0063] Another example of the present application relates to a method for increasing the serotonin level of a subject, comprising administering to a subject in need thereof at least one selected from the group consisting of Lactococcus lactis KBL1036 strain having the deposit number KCTC 14332BP, a culture of the strain, a lysate of the strain, and an extract of the strain. The serotonin level may be a serotonin level in the brain or intestine.

[0064] The strain and pharmaceutical composition according to one example of this application are non-toxic to the human body and thus safe, making them readily usable as therapeutic drugs. Furthermore, they exhibit excellent effects in improving mental illness without the risk of side effects associated with conventional psychiatric treatments, making them highly useful for industrial applications.

[0065] FIG. 1 is a drawing showing the effect of the KBL1036 strain according to an example of the present application on promoting the expression of Tph-1, a rate-limiting enzyme gene of the serotonin biosynthesis pathway.

[0066] Figure 2 shows the results of a concentration-dependent analysis of the effect of the KBL1036 strain according to an example of the present application on serotonin secretion of intestinal chromaffin cells.

[0067] Figures 3a to 3c are drawings verifying that the KBL1036 strain according to an example of the present application does not cause inflammation.

[0068] FIG. 4 is a drawing verifying the effect of the KBL1036 strain according to an example of the present application on increasing serotonin levels in the brain.

[0069] FIG. 5 is a drawing verifying the effect of the KBL1036 strain according to an example of the present application in treating autism spectrum disorder.

[0070] Figure 6 is a drawing verifying the effect of the KBL1036 strain according to an example of the present application on increasing Th17 cells.

[0071] Hereinafter, the present application will be described in more detail with reference to the following examples. However, these examples are merely intended to illustrate the present application, and the scope of the present application is not limited by these examples.

[0072]

[0073] Example 1. Identification of Lactococcus lactis strains

[0074] Fecal samples from a Korean woman in her 40s and a 1-year-old infant were provided, and the fecal samples were inoculated onto MRS medium supplemented with 0.5% L-cysteine. Subcultures were performed twice at 24-h intervals in an anaerobic environment at 37°C, and pure cultures were obtained by colony picking to isolate nine candidate strains. The candidate strains were identified as five strains of the genus Lactobacillus (hereinafter designated as KL1, KL2, KL3, KL4, and KL5), three strains of the genus Bifidobacterium (hereinafter designated as BF1, BF2, and BF3), and one strain of the genus Lactococcus (hereinafter designated as KBL1036).

[0075] Specifically, the strain isolated by colony picking after subculturing the above fecal sample was cultured in an anaerobic environment in a 37℃ CO2 incubator for 24 hours, and then centrifuged at 13,000 rpm at 4℃ to obtain only the strain. Then, 0.2 ul of the strain pellet was added to 25 ul of lysis buffer (pH 10; 2.5 M NaCl, 100 mM EDTA, 10 mM Trizma base, 1% Triton X-100) and heat treated at 95℃ for 10 minutes to obtain a template. After that, the V4 region of the 16S rRNA gene was amplified by polymerase chain reaction (PCR) using a G-Taq PCR kit to obtain a PCR product. The primers used were 27F and 1492R. The PCR product was purified using the Ultra Clean PCR clean-up Kit (Mobio Laboratories Inc.), and the strain was identified by genetic sequencing.

[0076] The 16S rRNA sequence of the KBL1036 strain is shown in Table 1. The sequence was compared with the NCBI Bacteria and Archaea: 16S ribosomal RNA project DB, and the isolated strain was confirmed to be Lactococcus lactis. The strain was deposited with the Korean Collection for Type Cultures, Korea Research Institute of Bioscience and Biotechnology, an international depository institution under the Budapest Treaty, and was assigned the accession number KCTC 14332BP.

[0077] 16S rRNA (5'->3') SEQ ID NO: GTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATTGTTAGTTGCCATCATTAAGTTGGGCACTCTAACGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTCGCGAGA CAGTGATGTTTAGCTAATCTCTTAAAACCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGGGAGTTGGGAGTACCCGAAGTAGGTTGCCTAACCGCAAGGAGGGCGCTCCTAAGGACGTTGACGTGATTCA1

[0078] In addition, as controls, Lactococcus garvieae strain with accession number KCTC 3773, Lactococcus piscium strain with accession number KCTC 3639, and Lactococcus chunganggensis strain with accession number KCTC 13185 were provided by the Korea Research Institute of Bioscience and Biotechnology and used.

[0079]

[0080] Example 2. Effect of increasing the expression level of serotonin synthase

[0081] TPH (tryptophan-hydroxlase) is an enzyme that produces 5-hydroxytryptophan (5-HTP), a precursor of serotonin. Serotonin production is activated by TPH, and among TPHs, TPH-1 is the enzyme responsible for most endogenous serotonin production. In order to verify the serotonin-inducing effect of the KBL1036 strain according to an example of the present application, the culture of the KBL1036 strain according to an example of the present application was treated to enterochromaffin cells that produce serotonin in the intestine, and the TPH-1 expression level was confirmed.

[0082] The strains prepared in Example 1 were each cultured in MRS medium supplemented with 0.05% cysteine, and subcultures were performed twice at 24-hour intervals. The culture supernatant containing the metabolites of each strain was obtained through centrifugation (13,000 × g, 5 min, 4°C), and then the pH was adjusted to 7.0. The supernatant was passed through a 0.22 μm pore size filter for sterilization and stored at 4°C until use.

[0083] The RIN14B (ATCC CRL-2059) cell line was cultured in RPMI 1640 medium supplemented with 10% FBS, penicillin (100 μg / mL), and streptomycin (100 μg / mL) at 37°C and 5% CO2, and passaged every 3 days. The RIN14B cell line was seeded in 24-well plates at a density of 4 × 10 5Cells were seeded per well and cultured for 24 hours. The medium was removed from the cultured RIN14B cell line and washed once with 500 μL of PBS per well. Then, 500 μL of the culture solution of the obtained strains KL1, KL2, KL3, Kl4, KL5, and KBL1036 was treated, or as a positive control, deoxycholate, which has been reported to induce serotonin synthesis, was diluted in MRS medium that had been filter-sterilized after adjusting the pH to 7.0, and the final concentration was 250 μM. After culturing for 1 hour under conditions of 37°C and 5% CO2, the cells and supernatant were collected.

[0084] To confirm the expression level of the TPH-1 gene, RNA was extracted from the cultured cells using the Easy-spin™ Total RNA Extraction Kit (Intron) according to the manufacturer's instructions. cDNA was synthesized from the extracted RNA using the High-Capacity RNA-to-cDNA™ Kit according to the manufacturer's instructions, and then qRT-PCR (quantitative real-time polymerase chain reaction) was performed using the Rotor-Gene Q (QIAGEN) equipment and the Rotorgene SYBR Green PCR kit. The Tph-1 expression level was calculated as the relative expression difference between the values ​​measured using the Tph-1 F and Tph-1 R primers of SEQ ID NOs: 2 and 3 and the values ​​measured using the GAPDH_F and GAPDH_F primers of SEQ ID NOs: 4 and 5 (delta-delta CT analysis), as shown in Table 2 below. The temperature conditions for qRT-PCR were 95°C for 5 minutes per cycle, 94°C for 15 seconds, 60°C for 30 seconds, and 72°C for 30 seconds, for a total of 50 cycles.

[0085] Primer base sequence SEQ ID NO:Tph-1 Forwardggctttgaggtcctctttcca2Tph-1 Reverseccccctttctgaggaatggtc3GAPDH ForwardAACTTTGGCATTGTGGAAGG4GAPDH ReverseGGATGCAGGGATGATGTTCT5

[0086] The Tph-1 expression promotion rate of each strain is shown in Table 3 and Fig. 1. As shown in Table 3 and Fig. 1, in the group treated with the Lactococcus lactis KBL1036 strain according to an example of the present application, the Tph-1 expression amount was about 2 to 5 times higher than that of the group treated with the Lactobacillus spp. strain, and the Tph-1 expression amount was about 4 to 6 times higher than that of the group treated with the Bifidobacterium spp. strain. In addition, compared to other Lactococcus strains belonging to the same genus as the strain according to an example of the present application, the Tph-1 expression amount was about 1.5 to 6 times higher. Therefore, the KBL1036 strain according to an example of the present application exhibited excellent serotonin synthesis induction ability.

[0087] GroupTph-1 relative expression levelMRS1.06Deoxycholate 250uM3.47KBL10366.72KCTC 37732.90KCTC 36393.79KCTC 131853.45KL11.13KL21.29KL32.70KL41.58KL53.00BF11.66BF21.10BF31.41

[0088] Example 3. Effect of inducing serotonin secretion in the intestine

[0089] In Example 2, the RIN14B cell line subcultured was seeded at 7.4 × 10 in a 96-well plate. 4Cells were seeded per well and cultured for 48 hours. After removing the medium from the RIN14B cell line, the cell line was washed twice with 300 μL of HBSS per well. Afterwards, the culture medium of Lactococcus lactis KBL1036 obtained in Example 2 was diluted in sterilized MRS medium to a final concentration of 0.5%, 1%, 2%, and 10%, and 200 μL was treated to the RIN14B cell line. As a positive control, tyramine, which has been reported to induce serotonin secretion, was diluted in filter-sterilized MRS medium to a final concentration of 2 mM, and 200 μL was treated. After culturing for 24 hours under conditions of 37°C and 5% CO2, the supernatant was collected.

[0090] To determine the amount of serotonin secretion, the supernatant of RIN14B cells cultured with MRS, Tyramine, or the culture medium of Lactococcus lactis KBL1036 strain was collected and filtered through a 0.22 μm pore size PVDF filter. Acetaminophen was added to the supernatant as an internal standard to a final concentration of 500 ng / mL. A mixture of 0.1% formic acid in distilled water (mobile phase A) and acetonitrile (mobile phase B) was used as the mobile phase solution for liquid chromatography, and an HSS T3 column was used as the column for separating serotonin. The separated analyte was detected using an Agilent 6546 qToF. The serotonin secretion amount of each group is shown in Table 4 and Figure 2 as a fold change value compared to the MSR control group.

[0091] As shown in Table 4 and Fig. 2, the group treated with the KBL1036 strain according to an example of the present application showed a serotonin secretion amount that was up to 6 times more than the MRS treated group, and showed a serotonin secretion amount that was up to 3.5 times more than the Tyramine treated group, which is a positive control group. In particular, when the KBL1036 strain according to an example of the present application was treated at increasing concentrations of 0.5%, 1%, 2%, and 10%, the serotonin secretion amount increased in a concentration-dependent manner. Therefore, the excellent serotonin secretion induction ability of the KBL1036 strain according to an example of the present application was verified. In Fig. 2, ** and **** indicate p<0.01 and p<0.0001, respectively, compared to the MRS treated group (control group) (student t-test).

[0092] Group Serotonin secretion amount MRS 1.00 Tyramine (2mM) 1.82 KBL 1036 0.5% 1.74 KBL 1036 1% 3.05 KBL 1036 2% 5.59 KBL 1036 10% 6.86

[0093] Example 4. Verification of inflammation induction

[0094] In order to verify whether the KBL1036 strain according to an example of the present application induces inflammation, the culture solution of the KBL1036 strain according to an example of the present application was treated to mononuclear cells, and the amount of inflammatory cytokine expression was confirmed.

[0095] The KBL1036, KL1, KL2, and KL3 strains prepared in Example 1 were cultured in MRS medium supplemented with 0.05% cysteine, and subcultured twice at 24-hour intervals. Each strain was obtained by centrifugation (13,000 × g, 5 min, 4°C), and the cell pellet was suspended in RPMI 1640 medium containing 10% FBS and used in the experiment.

[0096] THP-1 (ATCC TIB-202™) cell line was cultured in RPMI 1640 medium containing 10% FBS and gentamicin (100 μg / mL). Cultured cells were harvested and seeded at a density of 1 × 10 in RPMI 1640 medium containing 10% FBS and gentamicin (100 μg / mL). 5 After dissolving the cells at a concentration of 100 nM, PMA (phorbol-12-myristate-13-acetate) was treated to 100 nM. 200 μL of the culture medium containing the cells was dispensed into each well of a 96-well plate, and 2 × 10 4 After including the cells, they were cultured for 24 hours at 37°C and 5% CO2 conditions to differentiate them into mature macrophages. The culture medium was removed from the differentiated cells, and LPS was added as a control to one well at a concentration of 1 μg / mL, and 200 μL of each of the above strains was added to the remaining wells, so that 2 × 10 per well. 5 After including the cells, they were cultured under conditions of 37℃ and 5% CO2 (the cell ratio of THP-1 cells counted before differentiation and the strain was 1:10). The culture medium from each well was obtained 24 hours after culture and stored at -80℃ until used for analysis. The amounts of inflammatory cytokines IL-1β, IL-6, and TNF-α were measured in the culture medium of the strain-treated THP-1 cell line using the BD Cytometric Bead Array (CBA) human inflammation kit (Cat No. 551811) according to the manufacturer's method.

[0097] As shown in FIGS. 3A to 3C, the group treated with the KBL1036 strain according to an example of the present application to the THP-1 cell line had a significantly lower amount of inflammatory cytokines secreted than the group treated with LPS, and the amount of inflammatory cytokines secreted was also significantly lower compared to the groups treated with other Lactobacillus strains. Therefore, the KBL1036 strain according to an example of the present application was verified to have an excellent ability to induce serotonin synthesis without inducing inflammation. In FIGS. 3A to 3C, * indicates p<0.05 when compared to the LPS treatment group (student t-test).

[0098]

[0099] Example 5. Effect of increasing serotonin in the brain

[0100] According to an example of the present application, the original material of the KBL1036 strain was diluted in PBS containing 0.05% cysteine ​​(final concentration 2.5 X 10 10 CFU / ml) and vortexed for 30 minutes to prepare a sample for oral administration of the KBL1036 strain according to an example of the present application. The prepared sample was administered to 7-week-old C57BL / 6 mice at 200 μl per mouse (5x10 9CFU) were administered orally for 3 weeks. At 10 weeks of age, the mice were euthanized using CO2, the heads were separated, and foreign substances such as surrounding muscles and hair were removed, and the brain parenchyma was obtained. To measure serotonin in the brain parenchyma, 8 μl of ice-cold MeOH was added per 1 mg of tissue, and homogenization was performed (30 Hz, 5 min, 2 times) with a tissuelyser. Afterwards, the supernatant was obtained by centrifugation (15,000 g, 10 min, 4 °C), and filtered through a 0.22 μm pore size PVDF filter for use in the measurement. A mixture of 40:60 acetonitrile water (mobile phase A) containing 10 mM ammonium formate and 90:10 acetonitrile water (mobile phase B) containing 10 mM ammonium formate was used as the mobile phase solution for liquid chromatography, and a BEH Amide column was used as the column for separating serotonin. The separated analyte was detected using an Agilent 6546 qToF instrument.

[0101] As shown in Fig. 4, serotonin in the brain parenchyma was significantly increased by the KBL1036 strain according to an example of the present application. Therefore, the KBL1036 strain according to an example of the present application had an excellent ability to induce serotonin synthesis in the intestine, and at the same time, serotonin synthesized through the gut-brain axis had an effect of influencing the central nervous system. In Fig. 4, ** indicates p<0.01 compared to the vehicle-administered group (control group) (student t-test).

[0102]

[0103] Example 6. Effect of treatment on autism spectrum disorder

[0104] After administering the KBL1036 strain according to an example of the present application for three weeks to CNTNAP2 knockout mice, which are genetic autism spectrum disorder-like model mice, the sociality was evaluated using a three-chamber assay to verify the effect of increasing sociality. The mice used in this example are CNTNAP2 knockout (KO) mouse strains, which are single-gene modified mouse models known to exhibit autism spectrum disorder-like behavioral phenotypes.

[0105] Specifically, to eliminate segmented filamentous bacteria (SFB), which are known to induce Th17 differentiation in the intestine, CATNAP2 knockout mice were administered vancomycin (0.5 mg / mL) in their own water for 2 weeks, followed by a 1-week rest period. To prevent abnormalities in water intake in mice due to the bitter taste characteristic of antibiotics during antibiotic administration, Stevia, a sugar substitute, was administered concurrently at 2 g / L. After completing antibiotic administration, a three-chamber assay (pre-administration) was performed for 2 days.

[0106] The three-chamber assay is a representative behavioral experiment that can measure general sociality. It is a method to experimentally confirm the sociality of mice in the experimental group, which is in line with the habituation of mice to being isolated alone. The three-chamber assay is conducted over two days in two stages: habituation to the experimental chamber and a social run to meet new mice. On the first day, the adaptation stage was conducted by placing test mice in chambers consisting of three rooms in a row for 5 minutes. On the second day, the social run stage was conducted again after the previous day's adaptation stage. Then, a partition was used to place the mouse in the center chamber, and an inanimate object such as a rubber stopper (an inanimate target) and a new mouse (a social target) were placed in the center of each chamber using cups. After the partition was removed, the mice were measured to see which chamber they spent the most time in for 10 minutes. The sociability (%) of the mouse was calculated as “time spent on the novel mouse side / time spent on the novel mouse side + time spent on the inanimate object side) x 100.” Tracking of mouse movements was measured using ethovision software.

[0107] After completion of the pre-test, an oral administration sample of the KBL1036 strain according to an example of the present application prepared in Example 5 was administered to mice at a dose of 5x10 per mouse. 9 After oral administration of 200 μl of CFU for 3 weeks, a three-chamber assay (post-administration) was performed for 2 days. After completion of the post-test, colon and meninges tissues were obtained to confirm Th17 differentiation capacity.

[0108] As shown in Fig. 5, when the KBL1036 strain was administered to CNTNAP2 Knock Out (KO) mice, which are an ASD-like mouse model, it was confirmed that sociality significantly increased in the Post stage after administration compared to the Pre stage before administration compared to the Vehicle (0.05% L-cysteine ​​PBS) administration group. Therefore, the autism spectrum treatment effect of the KBL1036 strain according to an example of the present application was verified. In Fig. 5, ** indicates p<0.01 when comparing before (Pre) and after (Post) treatment with Vehicle and KBL1036 (2-way ANOVA).

[0109]

[0110] Example 7. Th17 cell increase effect

[0111] After administering the KBL1036 strain according to an example of the present application to CNTNAP2KO mice, which are genetic autism spectrum-like model mice, for 3 weeks, the immune cells of the colon and meninges tissues were analyzed using a flow cytometer, and the effect of significantly increasing T helper 17 cells in the immune cells of the colon and meninges tissues was verified.

[0112] Specifically, after euthanizing the CNTNAP2 Knock Out (KO) mouse using CO2, the colon tissue was isolated, and the fat and internal contents of the colon were removed on a tissue sufficiently soaked with HBSS, and then cut so that the internal tissue of the colon was visible. The colon tissue was washed in HBSS, wiped with a tissue sufficiently soaked with HBSS to remove mucus and foreign substances, and the colon tissue was placed in a 50 mL conical tube containing 15 mL of HBSS supplemented with EDTA (Ethylenediaminetetraacetic acid, 1 mM) and DTT (Dithiothreitol, 1 mM). The tissue was then reacted in a shaking incubator (250 rpm, 10 min, 37°C). After 15 min, the colon tissue was placed in a beaker containing 10 mL of RPMI 1640, shaken, and then dried with a dry tissue. Afterwards, the colon tissue was placed in a digestion solution (Digestion buffer) consisting of 5 mL of RPMI 1640 supplemented with Dnase I (50 μg / mL), Liberase (62.5 μg / mL), and FBS (2%), and the colon tissue was digested by reaction in a shaking incubator (250 rpm, 45 minutes, 37°C). The digested colon tissue was filtered using a new 50 mL conical tube equipped with a 100 μm cell strainer, and the colon cell pellet was obtained using a centrifuge (450 × g, 10 min, 4 °C). The pellet was resuspended in 4 mL of 40% percoll (in 2% FBS RPMI1640). 2.5 mL of 80% percoll (in HBSS) was added using glass pasteur pipettes, and centrifuged (860 × g, acceleration & deceleration 0, 20 min, 21 °C) to separate the immune cell layer by density difference.Afterwards, the immune cell layer between the separated 40% and 80% percoll was separated using a pipette, resuspended in 10 mL of HBSS, and centrifuged twice (450 × g, 10 min, 4°C) to isolate colonic immune cells.

[0113] In addition, after euthanizing the CNTNAP2 Knock Out (KO) mouse using CO2, the head was separated to remove the surrounding muscles, hair, and other foreign substances, as well as the lower jaw, to remove the brain tissue, and the edge of the skull was scraped to obtain the meninges tissue. Afterwards, the meninges tissue was placed in a digestion solution (Digestion buffer) consisting of 1.5 mL RPMI supplemented with DNase I (15 μg / mL), Liberase (15 μg / mL), and FBS (2%), and the meninges tissue was digested by reaction in a shaking incubator (250 rpm, 45 min, 37°C). The digested meninges tissue was filtered using a 5 mL strainer tube and centrifuged (450 × g, 10 min, 4°C) to obtain a meninges cell pellet. Afterwards, 2 mL of HBSS was added and centrifuged (450 × g, 10 min, 4°C) to obtain meningeal immune cells.

[0114] The separated colon and meningeal immune cell pellets were resuspended with 200 μL of T cell culture medium (10% FBS, Gentamicin sulfate 50 μg / mL, 100×GlutaMAX 2x, 2-mercaptoethanol 55 μM, 100×Penicillin / Streptomycin 1x in RPMI1640), and 100 μL was dispensed into a round-bottom 96-well plate. Then, 100 μL of T cell stimulation culture medium (PMA 100 ng / mL, Ionomycin 2 μM, Brefeldin 2 μg / mL in T cell culture medium) was dispensed into a round-bottom 96-well plate, pipetted, and cultured in a cell incubator (5% CO2, 37°C) for 3 hours. After incubation, centrifugation (450 × g, 2 min, 4 ° C) was performed, the supernatant was removed, and 200 μL of HBSS was added per well and centrifugation (450 × g, 2 min, 4 ° C) was performed twice. After that, Live Dead fluorescence was diluted in a ratio of 1:500 in HBSS, 50 μL was added per well, and mixed well. After 15 minutes of reaction at room temperature under light-blocking conditions, 200 μL of HBSS was added per well and centrifugation (450 × g, 2 min, 4 ° C) was performed twice. After that, surface marker antibodies were diluted in HBSS at a ratio of 1:500, 1:400, and 1:200, and then 50 μL was added to each well, pipetted, and reacted for 30 minutes at 4°C in a dark environment. Then, 200 μL of HBSS was added to each well, and centrifuged (450 × g, 2 minutes, 4°C) to wash twice. 150 μL of fixation / permeabilization buffer was added to each well, and reacted for 20 minutes at room temperature or 12-16 hours in a 4°C refrigerator, and then washed twice with 200 μL of permeabilization wash buffer per well.Afterwards, intracellular antibody was diluted 1:200 in the permeabilization wash buffer, 50 μL per well was added, and after pipetting, the cells were incubated for 1 hour at 4°C in a dark environment, and washed three times with 200 μL per well of the permeabilization wash buffer. Afterwards, the cells were resuspended in 200 μL of FACS buffer (EDTA 2 mM, 100x Penicillin / Streptomycin 1x, BSA 0.5% in HBSS), transferred to a 1.5 mL FACS tube, and analyzed for colonic and meningeal immune cells using a flow cytometer.

[0115] As shown in FIGS. 6a and 6b, when the KBL1036 strain according to an example of the present application was administered to CNTNAP2Knock Out (KO) mice, which are ASD-like mouse models, it was confirmed that Th17 cells significantly increased in the colon and meninges compared to the vehicle (0.05% L-cysteine ​​PBS) administration group. Therefore, it was verified that the KBL1036 strain according to an example of the present application has an effect of increasing T helper 17 cells in the colon and meninges immune cells. In FIG. 6a, * means p<0.05 when compared to the vehicle administration group, and in FIG. 6b, ** means p<0.01 when compared to the vehicle administration group (student t-test).

[0116] [Accession number]

[0117] Name of depositor: Korea Research Institute of Bioscience and Biotechnology, Biological Resource Center

[0118] Accession number: KCTC14332BP

[0119] Date of acceptance: 20201014

[0120]

Claims

1. Lactococcus lactis KBL1036 strain with accession number KCTC 14332BP.

2. A strain according to claim 1, wherein the strain has a 16S rRNA sequence of sequence number 1.

3. A strain according to claim 1, wherein the strain promotes the synthesis and / or secretion of serotonin.

4. A strain according to claim 1, wherein the strain promotes the expression of Tph-1 (tryptophan hydroxylase 1).

5. In the first paragraph, the strain promotes differentiation of naive T cells into T-helper 17 cells.

6. In the first paragraph, the strain is a strain that promotes secretion of IL-17.

7. A strain according to claim 1, wherein the strain does not cause inflammation.

8. A strain according to claim 1, wherein the strain does not induce inflammatory cytokines.

9. A strain according to claim 8, wherein the inflammatory cytokine is at least one selected from the group consisting of IL-1β, IL-6, and TNF-α.

10. A pharmaceutical composition for preventing or treating mental illness, comprising at least one selected from the group consisting of Lactococcus lactis KBL1036 strain having the deposit number KCTC 14332BP, a culture of the strain, a lysate of the strain, and an extract of the strain.

11. A pharmaceutical composition according to claim 10, wherein the mental illness is serotonin deficiency.

12. A pharmaceutical composition according to claim 10, wherein the mental illness is at least one selected from the group consisting of autism spectrum disorder, depression, bipolar disorder, anxiety disorder, panic disorder, obsessive-compulsive disorder, memory impairment, delusions of grandeur, sleep disorders, eating disorders, cognitive disorders, hyperactivity, attention deficit, irritable bowel syndrome, schizophrenia, post-traumatic stress disorder (PTSD), neurosis, motor dysfunction, intellectual disability, drug addiction, and alcoholism.

13. A food comprising at least one selected from the group consisting of Lactococcus lactis KBL1036 strain having the deposit number KCTC 14332BP, a culture of the strain, a lysate of the strain, and an extract of the strain.

14. Probiotics comprising at least one selected from the group consisting of Lactococcus lactis KBL1036 strain having the deposit number KCTC 14332BP, a culture of the strain, a lysate of the strain, and an extract of the strain.

15. A non-medical product comprising at least one selected from the group consisting of Lactococcus lactis KBL1036 strain having the deposit number KCTC 14332BP, a culture of the strain, a lysate of the strain, and an extract of the strain.

16. Animal feed comprising at least one selected from the group consisting of Lactococcus lactis KBL1036 strain having the deposit number KCTC 14332BP, a culture of the strain, a lysate of the strain, and an extract of the strain.

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