Composition for preventing, improving, or treating depression comprising lactobacillus buchneri 200793 strain

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating depression comprising a Lactobacillus buchneri 200793 strain, and specifically, to a composition for preventing, improving, or treating depression comprising the strain or a culture of the strain, a food composition for alleviating tension, and an animal feed additive composition for preventing or improving depression. When the composition according to the present invention is administered to a subject, sucrose preference is increased, and the expression level of TPH-1 or SERT in small intestine and large intestine tissue is increased, thereby being highly effective in alleviating depression, and thus the composition can be effectively employed for alleviating and improving various symptoms of depression.

Description

Composition for preventing, improving or treating depression comprising Lactobacillus buchneri 200793 strain

[0001] The present invention relates to a pharmaceutical composition for preventing or treating depression, comprising Lactobacillus buchneri 200793 strain (KCCM 12593P) or a culture of the strain as an active ingredient.

[0002] Probiotics, when consumed in appropriate amounts, are beneficial to the health of the host. The known effects of probiotics include antioxidant, anti-inflammatory, cholesterol-lowering, and anti-diabetic properties, and these effects are reported to vary depending on the strain.

[0003] Lactobacillus is a Gram-positive bacterium belonging to the genus Lactobacillus, a subgroup of the Lactobacillus family, comprised of oxygen-tolerant anaerobic bacteria. Lactobacillus is widely used in food and probiotic products and is known to have beneficial effects on the digestive system, including improving digestive health and strengthening the immune system.

[0004] Lactobacillus buchneri is a gram-positive, non-spore-forming, anaerobic, rod-shaped prokaryotic bacterium of the Lactobacillus family. It is a heterofermentative bacterium that produces lactic acid and acetic acid during fermentation.

[0005] The gut environment has been reported to influence the brain, transmitting bidirectional signals. Furthermore, it is known to influence brain health, including anxiety, depression, and dementia, through neural, hormonal, and immune regulation.

[0006] According to Non-Patent Documents 1 and 2, there are reports suggesting that probiotics can help improve mental health and alleviate stress. However, these are still preliminary hypotheses, and research demonstrating their impact on improving depression is lacking. Furthermore, while Patent Document 1 discloses the protective effect of Lactobacillus buchneri 200793 against neuronal cell death, no research has yet been conducted on its effect on depression.

[0007] Accordingly, the inventors of the present invention completed the present invention by injecting the Lactobacillus buchneri 200793 strain into mice and conducting an experiment related to depression, and confirming that the strain can prevent, improve, or treat depression.

[0008] [Prior Art Literature]

[0009] [Patent Document]

[0010] Republic of Korea Patent No. 10-2221579 (February 23, 2021)

[0011] [Non-patent literature]

[0012] Ma L et al, Stress / strain characterization in electronic packaging by micro-Raman spectroscopy: A review. Microelectronics Reliability. 118: 114045 (2021)

[0013] Takada M et al, Probiotic Lactobacillus casei strain Shirota relieves stress-related symptoms by modulating the gut-brain interaction in human and animal models. Neurogastroenterol Motil. 28: 1027-1036 (2016)

[0014] The present invention aims to provide a novel use of Lactobacillus buchneri 200793 strain (KCCM 12593P).

[0015] To achieve the above purpose, the present invention provides a pharmaceutical composition for preventing or treating depression, which comprises Lactobacillus buchneri 200793 strain (KCCM 12593P) or a culture of the strain as an active ingredient.

[0016] The present invention also provides a health functional food composition for preventing or improving depression, comprising Lactobacillus buchneri 200793 strain (KCCM 12593P) or a culture of the strain as an active ingredient.

[0017] The present invention also provides a health functional food composition for stress relief comprising Lactobacillus buchneri 200793 strain (KCCM 12593P) or a culture of the strain as an active ingredient.

[0018] The present invention also provides an animal feed additive composition for preventing or improving depression, comprising Lactobacillus buchneri 200793 strain (KCCM 12593P) or a culture of the strain as an active ingredient.

[0019] The Lactobacillus buchneri 200793 strain (KCCM 12593P) according to the present invention increases sucrose preference when administered to a subject and enhances mRNA expression of TPH-1 or SERT in small and large intestine tissues, and thus can be utilized as a composition for preventing, improving, or treating various diseases related to depression.

[0020] Figure 1 illustrates the experimental design for inducing depression through restraint stress.

[0021] Figure 2 is a graph showing the results of measuring body weight changes according to the intake of selected strains in a restraint stress depression model.

[0022] Figure 3 is a bar graph showing the results of a sucrose preference test according to the intake of a selected strain in a restraint stress depression model.

[0023] Figure 4 is a bar graph showing the results of the tail suspension test according to the intake of the selected strain in the restraint stress depression model.

[0024] Figure 5 is a bar graph showing the results of open field tests according to the intake of selected strains in a restraint stress depression model.

[0025] Figure 6 is a bar graph showing the results of the elevated plus maze test according to the intake of the selected strain in the restraint stress depression model.

[0026] Figure 7 is a bar graph showing the results of serum corticosterone content according to the intake of selected strains in a restraint stress depression model.

[0027] Figure 8 is a bar graph showing the results of SERT mRNA expression in small and large intestine tissues according to the intake of selected strains in a restraint stress depression model.

[0028] Figure 9 is a bar graph showing the results of TPH-1 mRNA expression in small and large intestine tissues according to the intake of selected strains in a restraint stress depression model.

[0029] Figure 10 shows a bar graph showing the results of SERT and HTR2A mRNA expression levels in the prefrontal cortex tissue according to the intake of selected strains in a restraint stress depression model.

[0030] Hereinafter, the present invention will be described in detail with examples to aid understanding. However, the following examples are merely illustrative of the content of the present invention and are not intended to limit the scope of the present invention. The examples of the present invention are provided to more fully explain the present invention to those of average skill in the art.

[0031] The term "depression" in the present invention refers to a disease that causes a variety of cognitive and psychosomatic symptoms, with a decrease in motivation and feelings of depression as the main symptoms, resulting in a decline in daily functioning. The causes are very diverse, including chemical, biological, psychological, sociological, pharmacological, and pathological causes. One of them is a chemical imbalance of neurotransmitters such as serotonin, melatonin, dopamine, or norepinephrine. Chronic stress, medications such as antihypertensives, anti-anxiety drugs, narcotics, and central nervous system stimulants, or diseases such as diabetes, pancreatic cancer, and endocrine diseases can also cause depression.

[0032] In the present invention, the term "prevention" may include without limitation any act that can block, suppress or delay symptoms caused by sepsis by using the composition of the present invention.

[0033] In the present invention, the term "treatment" may include, without limitation, any act that can improve or benefit symptoms caused by sepsis by using the composition of the present invention.

[0034] In the present invention, the term "improvement" may include without limitation any act in which symptoms caused by sepsis are improved or beneficially changed by using the composition of the present invention.

[0035] In the present invention, the term "culture" may include the culture itself obtained by culturing a strain, a concentrate or lyophilized product of the culture supernatant obtained by removing the strain therefrom.

[0036] The above culture medium refers to a result obtained by culturing the strain of the present invention. In a broad sense, the culture medium may be a whole culture of the strain, a culture supernatant, a lysate, a dried product, or a fraction thereof. At this time, the culture supernatant may be obtained by centrifuging the culture of the strain, the lysate may be obtained by physically or ultrasonically treating the strain, and the fraction may be obtained by centrifuging the culture, culture supernatant, lysate, etc., or by a method such as chromatography.

[0037] In the present invention, when a part is said to "include" a certain component, this does not mean that other components are excluded, but rather that other components may be further included, unless specifically stated otherwise.

[0038] The present invention relates to a pharmaceutical composition for preventing or treating depression, comprising Lactobacillus buchneri 200793 strain (KCCM 12593P) or a culture thereof as an active ingredient.

[0039] The above strain (KCCM 12593P) is a subspecies of Lactobacillus buchneri and was deposited with the Korea Microbiological Conservation Center on September 30, 2019, and was assigned the accession number KCCM 12593. Lactobacillus buchneri is a type of lactic acid bacteria known to aid in the digestive process and promote the growth of beneficial bacteria in the intestines.

[0040] The above Lactobacillus buchneri 200793 strain can be cultured in a known liquid medium or solid medium, and preferably, can be cultured in MRS, GAM, TSB or NB medium, but is not limited thereto.

[0041] The composition may contain live or dead cells of the Lactobacillus buchneri 200793 strain, preferably, but not limited to, live cells.

[0042] The above probiotics refer to bacteria that can survive and reproduce independently.

[0043] The above-mentioned dead cells refer to live cells cultured under certain conditions, and the growth of the cells is no longer possible through methods such as collection, heat drying, pressurization, and drug treatment.

[0044] The composition may include, but is not limited to, proteins and metabolites of the strain, concentrates, dried products, fragments and extracts of the culture.

[0045] The above composition comprises 10 Lactobacillus buchneri 200793 strains. 8 10 inland 11 Contains a concentration of CFU / ml, preferably 10 10 It may be included at a concentration of CFU / ml, but is not limited thereto.

[0046] The composition may increase sucrose preference when administered to a subject, and may increase mRNA expression levels of TPH-1 or SERT in small and large intestine tissues, but is not limited thereto.

[0047] The above-mentioned subject means any animal, including a human, that has developed or may develop depression, preferably a mammal, including a human, but is not limited thereto.

[0048] The aforementioned sucrose preference can be measured using the Sucrose Preference Test (SPT). This test is used to study and assess depression-related characteristics in animal models of depression, and sucrose preference tends to decrease in these animals.

[0049] The above TPH-1 (tryptophan hydroxylase-1) is mainly expressed in the intestines and sensory nerve cells, and is an enzyme that synthesizes tryptophan into the neurotransmitter serotonin, so it can be used as an indicator of the level of serotonin expression in the brain.

[0050] Serotonin, also known as 5-hydroxytryptamine (5-HT), is a monoamine neurotransmitter derived from tryptophan. A serotonin deficiency can lead to depression.

[0051] The above-mentioned SERT (serotonin transporter) is a protein that plays a crucial role in the serotonin signaling process. Serotonin is responsible for various neurotransmission functions in the central nervous system, some of which involve binding to receptors and transmitting signals. The serotonin transporter is located in the cell membrane and regulates serotonin levels and signaling.

[0052] Chromogranin A (ChgA) is a protein expressed primarily in the nervous and endocrine systems, and is one of the hormones secreted when under stress.

[0053] 5-HT receptors (5HTRs; 5-HT receptors) are proteins that serotonin binds to and transmits signals, and are classified into subtypes from 5-HT1 to 5-HT7.

[0054] The pharmaceutical composition is administered in a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment. The effective dosage level may be determined based on factors including the type and severity of the patient's disease, the activity and sensitivity of the drug, the time of administration, the route of administration and excretion rate, the duration of treatment, concurrently used drugs, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or in multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that achieves the maximum effect with the minimum amount without causing side effects, and this can be easily determined by those skilled in the art.

[0055] The pharmaceutical composition according to the present invention can be formulated and used in the form of oral preparations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injection solutions, according to conventional methods, and may include appropriate carriers, excipients, or diluents conventionally used in the manufacture of pharmaceutical compositions for formulation.

[0056] The carrier or excipient or diluent may include various compounds or mixtures including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

[0057] When formulating, it can be manufactured using diluents or excipients such as fillers, weighting agents, binders, wetting agents, disintegrants, and surfactants that are commonly used.

[0058] Solid preparations for oral administration can be prepared by mixing the strain (KCCM 12593P) with at least one excipient, such as starch, calcium bonate, sucrose or lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc can also be used.

[0059] Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, fragrances, and preservatives.

[0060] Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solutions and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, Tween 61, cocoa butter, laurin, and glycerol gelatin.

[0061] The preferred dosage of the pharmaceutical composition according to the present invention varies depending on the patient's condition, body weight, severity of disease, drug form, route of administration, and period of administration, but can be appropriately selected by those skilled in the art. Administration may be administered once a day or divided into several doses. However, the scope of the present invention is not limited by the above dosage.

[0062] The pharmaceutical composition according to the present invention can be administered to mammals such as rats, mice, livestock, and humans via various routes. All modes of administration can be administered, for example, orally, rectally, or by intravenous, intramuscular, subcutaneous, intrauterine, or intracerebroventricular injection.

[0063] The present invention also relates to a health functional food composition for preventing or improving depression, comprising Lactobacillus buchneri 200793 strain (KCCM 12593P) or a culture thereof as an active ingredient.

[0064] The composition may contain live or dead cells of the Lactobacillus buchneri 200793 strain, preferably, but not limited to, live cells.

[0065] The above composition comprises 10 Lactobacillus buchneri 200793 strains. 8 10 inland 11 Contains a concentration of CFU / ml, preferably 10 10 It may be included at a concentration of CFU / ml, but is not limited thereto.

[0066] The present invention also relates to a health functional food composition for stress relief comprising Lactobacillus buchneri 200793 strain (KCCM 12593P) or a culture thereof as an active ingredient.

[0067] In the present invention, "tension relief" refers to relieving an individual's physical, mental, or emotional tension in a specific situation or condition. This tension may be related to stress, anxiety, discomfort, depression, or pressure in daily life. Furthermore, tension relief can have the effect of reducing depression and promoting mental stability.

[0068] The composition may contain live or dead cells of the Lactobacillus buchneri 200793 strain, preferably, but not limited to, live cells.

[0069] The above composition comprises 10 Lactobacillus buchneri 200793 strains. 8 10 inland 11 Contains a concentration of CFU / ml, preferably 10 10 It may be included at a concentration of CFU / ml, but is not limited thereto.

[0070] The above health functional food may include all foods in the conventional sense, and may be used interchangeably with terms known in the art, such as functional food.

[0071] There is no limitation on the types of foods in which the composition of the present invention can be used. Furthermore, the composition of the present invention can be prepared by mixing other appropriate auxiliary ingredients and known additives that can be included in foods, as selected by those skilled in the art. Examples of foods to which the composition can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes. In addition, foods applicable to the present invention may include all foods, such as special nutritional foods (e.g., formulated milk, infant and toddler foods, etc.), processed meat products, fish products, tofu, starch, noodles (e.g., ramen, noodles, etc.), health supplements, seasoned foods (e.g., soy sauce, soybean paste, red pepper paste, mixed sauce, etc.), sauces, confectionery (e.g., snacks), processed dairy products (e.g., fermented milk, cheese, etc.), other processed foods, kimchi, pickled foods (various kimchi, pickled vegetables, etc.), beverages (e.g., fruit and vegetable beverages, soy milk, fermented beverages, etc.), natural seasonings (e.g., ramen soup, etc.).

[0072] In addition, the food composition may further include a physiologically acceptable carrier, and the type of the carrier is not particularly limited, and any carrier commonly used in the art may be used. In addition, the food composition may include additional ingredients commonly used in food compositions to improve odor, taste, sight, etc. For example, the food composition may include vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, pantothenic acid, etc. In addition, the food composition may include minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), chromium (Cr), etc.; and amino acids such as lysine, tryptophan, cysteine, etc. In addition, the food composition may include food additives such as preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), sterilizers (bleaching powder and high-purity bleaching powder, sodium hypochlorite, etc.), antioxidants (butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), etc.), colorants (tar colorants, etc.), color developers (sodium nitrite, sodium nitrite, etc.), bleaching agents (sodium sulfite), seasonings (MSG, monosodium glutamate, etc.), sweeteners (dulcin, cyclamate, saccharin, sodium, etc.), flavorings (vanillin, lactones, etc.), leavening agents (alum, D-potassium hydrogen tartrate, etc.), reinforcing agents, emulsifiers, thickeners (glucose fillers), film-forming agents, gum-forming agents, foam suppressants, solvents, and improvers. The above additives can be selected according to the type of food and used in an appropriate amount.

[0073] When the health functional food composition of the present invention is used in the form of a beverage, it may contain various sweeteners, flavoring agents, or natural carbohydrates as additional ingredients, just like conventional beverages. In addition to the above, the health functional food composition of the present invention may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, and the like. In addition, it may contain fruit pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks.

[0074] The present invention also relates to an animal feed additive composition for preventing or improving depression, comprising Lactobacillus buchneri 200793 strain (KCCM 12593P) or a culture thereof as an active ingredient.

[0075] The composition may contain live or dead cells of the Lactobacillus buchneri 200793 strain, preferably, but not limited to, live cells.

[0076] The above composition comprises 10 Lactobacillus buchneri 200793 strains. 8 10 inland 11 Contains a concentration of CFU / ml, preferably 10 10 It may be included at a concentration of CFU / ml, but is not limited thereto.

[0077] The above feed additive composition can be added to feed, and "feed" can mean any natural or artificial diet, meal, etc., or an ingredient of the meal, which is intended for or suitable for an animal to eat, ingest, or digest. The type of the feed is not particularly limited, and feed commonly used in the relevant technical field can be used. Non-limiting examples of the feed include plant feed such as grains, roots, fruits, food processing by-products, algae, fibers, pharmaceutical by-products, oils, starches, meal, or grain by-products; and animal feed such as proteins, inorganic substances, oils, minerals, oils, single-cell proteins, zooplankton, or food.

[0078] The above feed additive composition may be in the form of a dry or liquid formulation, and may further include other non-pathogenic microorganisms in addition to the above strain.

[0079] When the above feed additive composition is used as an animal feed additive, 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 feed raw materials, and these can be used without limitation in an unprocessed or processed form.

[0080] Hereinafter, preferred examples are presented to aid in understanding the present invention. However, the following examples are provided solely to facilitate a better understanding of the present invention, and the scope of the present invention is not limited by the following examples.

[0081] [Example 1]

[0082] Strains and animals used in the experiment

[0083] 1-1. Strains used

[0084] In an embodiment of the present invention, Lactobacillus buchneri strain (KU200793) was cultured in MRS medium at 30°C to 40°C for 10 to 50 hours. 9 It was used as a CFU live cell.

[0085] 1-2. Experimental animals

[0086] To establish an animal model of restraint stress-induced depression for the screening of novel probiotics, we conducted a study using C57BL / 6 mice (WIKIM IACU 202140). Four-week-old, 14-17 g mice were purchased from Z-Bio Co., Ltd. (Gwangju, Korea). Experiments were conducted after a one-week acclimatization period. Fluoxetine (Sigma, MO, USA), the positive control used in the experiment, was suspended in phosphate-buffered saline (PBS) and administered at a dose of 20 mg / kg.

[0087] The experimental animals were divided into control group (PBS administration, n=10), restraint group (restraint + PBS administration, n=8), fluoxetine group (restraint + fluoxetine, n=8), and Lactobacillus buchneri KU200793 group (10 9 CFU / 200 μL, n=8) and administered orally. Fluoxetine (Sigma, MO, USA), the positive control group used in the experiment, was suspended in PBS and set at a concentration of 20 mg / kg.

[0088] Lactic acid bacteria and PBS were administered for 7 weeks, and restraint stress and fluoxetine were administered 5 days a week for 4–7 weeks until sacrifice (Fig. 1). Restraint stress was induced using a 50 mL conical tube, and depression was induced by restraining the mice for 4 hours per day (Fig. 1).

[0089] 1-3. Check your weight changes

[0090] Depressed mice induced by restraint stress were weighed weekly after being placed in the cage. Lactic acid bacteria and PBS were administered orally daily for 4 weeks, and depression was induced by stress through daily restraint starting in the 4th week. Changes in body weight were observed from the 5th week, one week after restraint.

[0091] 1-4. Sucrose Preference Test (SPT)

[0092] SPT eliminated learning about the location of sucrose and water by providing 1% (w / v) sucrose and water in the same tube, respectively, for 72 h, while rotating the locations at irregular intervals. After 72 h, lipopolysaccharide (LPS) and phosphate-buffered saline (PBS) were administered intraperitoneally in proportion to body weight, and mice were separated into cages, one per cage, after fasting for 24 h. 1% (w / v) sucrose and water were provided for 4 h, with the tubes rotated every 2 h. The tubes were weighed before and after provision, and the weight was entered into Equation 1 below to measure sucrose preference.

[0093] [Formula 1]

[0094] Sucrose preference (%) = Sucrose intake / (Water intake + Sucrose intake)

[0095] 1-5. Tail Suspension Test (TST)

[0096] The mice were moved to their cages one hour prior to the behavioral analysis test to acclimate them to the laboratory's lighting and environment. A climbstopper was attached to the mouse's tail, and the tail was taped 20-25 cm from the nose to the floor. Immobility was recorded for 6 minutes, and the total immobility time was calculated for 360 seconds.

[0097] 1-6. Open Field Test (OFT)

[0098] The open field used was a closable box measuring 45 cm (l) X 45 cm (w) X 40 cm (h) (TSE Systems LTD, Germany), and the noise level inside the open field was maintained at 10 dB before the experiment. Mice were placed in the open field and filmed for 10 minutes to measure the time in the center, the time in the periphery, and the time spent looking around with both paws raised.

[0099] 1-7. Elevated plus maze test (EPM)

[0100] The EPM test is a behavioral analysis method for anxiety and depression symptoms. It measures the time and distance spent in the open and close arms, and can be used as an indicator to confirm the induction of depression.

[0101] The EPM used in the analysis was a cross-shaped (+) maze made of black acrylic and installed 50 cm above the floor. Two of the four passages (25 cm long and 5 cm wide) facing each other were open, and the other two passages of equal size had walls 16 cm high. The central platform was 5 cm wide and 5 cm high, and 6 minutes of filming was conducted from the ceiling in the center of the maze for analysis.

[0102] 1-8. Serum corticosterone content analysis

[0103] For analysis of serum corticosterone, mice were sacrificed, blood was collected, and serum was separated by centrifugation. The amount of corticosterone in the serum was measured using an ELISA assay (MyBioSource, Inc. CA, USA).

[0104] 1-9. Confirmation of mRNA expression levels of depression-related genes in intestinal tissue

[0105] After sacrificing the mice, the small and large intestines were separated and the mRNA expression levels of tryptophan hydroxylase-1 (TPH-1), serotonin transporter (SERT), chromogranin A (ChgA), and 5-HT receptors (5HTRs) were investigated.

[0106] The tissue was transferred to a pre-filled bead tube (Lysing Matrix E, MP Biomedicals, CA, USA) and 1 mL of TRIzol reagent (Invitrogen, MA, USA) per 50-100 mg of tissue was added. The mixture was beaten twice for 40 seconds using a bead beater (BEADBLASTER 24, Benchmark Scientific, NJ, USA). The mixture was left to stand at room temperature for 5 minutes, and 0.2 mL of chloroform per 1 mL of TRIzol was added and centrifuged at 4°C, 12,000 rpm, for 15 minutes. 0.5 mL of isopropanol was added to the supernatant and centrifuged at 4°C, 12,000 rpm, for 10 minutes. The supernatant was removed, and 1 mL of 75% (v / v) ethanol was added and centrifuged for 5 minutes. After removing the supernatant and drying for 30 minutes to 1 hour, the RNA was dissolved in 0.2-0.3 mL of nuclease-free water (Noble Bio, Gyeonggi, Korea) and used.

[0107] RNA concentration was measured using Nanodrop 2000 (Thermo Fisher Scientific, USA), and cDNA was synthesized using a cDNA synthesis kit (TOPscriptTM, Daejeon, Korea) using 1 ng of RNA. The synthesized cDNA was analyzed by real-time quantitative PCR (Bio-Rad CFX-96, CA, USA) using TOPreal qPCR 2Х premix (Enzynomics, Korea). Primer sequences are shown in [Table 1].

[0108]

[0109] 1-10. Confirmation of mRNA expression levels of depression-related genes in brain tissue

[0110] Brain-Derived Neurotrophic Factor (BDNF) is one of the nerve growth promoting factors and plays an important role in the maintenance and development of the nervous system, such as inducing growth and differentiation during nerve development. BDNF is related to the pathophysiological mechanism of depression. Brain tissue was obtained, and the expression level was analyzed using the BDNF primer sequences F-CAC TTT TGA GCA CGT CAT CGA A and R-CAC CCG GGA AGT GTA CAA GTC (SEQ ID NO: 15) for mRNA expression.

[0111] [Example 2]

[0112] Weight changes in a model of depression induced by restraint stress

[0113] Depressed mice induced by restraint stress were weighed weekly after introduction. Lactic acid bacteria and PBS were administered orally daily for 4 weeks, and from the 4th week onward, depression was induced by stress through daily restraint.

[0114] Changes in body weight were observed from week 5, one week after the restraint (Fig. 2). The control group showed the greatest weight change, followed by the positive control group, fluoxetine. At week 5, there were significant differences between the control group and the other restraint stress groups and the probiotic-administered group, excluding the fluoxetine group.

[0115] [Example 3]

[0116] Sucrose Preference Test (SPT)

[0117] Through a sucrose preference test, it was confirmed that depression was induced by a decrease in sucrose preference due to restraint stress, and the sucrose preference of selected lactic acid bacteria was confirmed to have an effect of alleviating depression.

[0118] The group that received restraint stress and PBS (Restraint) showed a significantly decreased preference for sucrose compared to the control group and the fluoxetine group (p<0.05), and the group that received oral administration of Lactobacillus buchneri strain (KU200793) showed a significantly higher preference for sucrose (p<0.01) (Fig. 3).

[0119] Through these results, it was confirmed that depression was induced by restraint stress in the sucrose preference test, and the depression alleviation effect was confirmed in the group administered the selected lactic acid bacteria, Lactobacillus buchneri strain (KU200793).

[0120] [Example 4]

[0121] Tail Suspension Test (TST)

[0122] In the TST test, the immobility time of animals was measured to confirm the induction of depression due to restraint stress and the depression-relieving effect of selected lactic acid bacteria.

[0123] In the TST test, the group that received restraint stress and PBS (Restraint) had a significantly higher immobility time than the control group and fluoxetine group (p<0.05), and there was no significant difference in immobility time between the control group and the group administered the selected lactic acid bacteria (Fig. 4).

[0124] The TST results showed that depression was induced by restraint stress, and that there was no difference in immobility time between the control group and the positive control group, fluoxetine administration group, confirming that the selected probiotics were effective in alleviating depression.

[0125] [Example 5]

[0126] Open Field Test (OFT)

[0127] We measured the time spent in the center and the time spent in the periphery of the OFT to determine whether depression was induced by restraint stress, and confirmed the depression-alleviating effect of selected lactic acid bacteria.

[0128] The control group had the highest central dwell time, while the other groups subjected to restraint stress showed lower central dwell times compared to the control group. Compared to the control group, the group administered Lactobacillus buchneri strain (KU200793) showed no significant difference, and the periphery dwell time did not show any significant difference among the groups (Fig. 5).

[0129] The control group, which spent a lot of time in the center, showed active behavior, unlike the group that received restraint stress. When Lactobacillus buchnery was administered to the group that received restraint stress, it was determined that Lactobacillus buchnery, a selected lactic acid bacteria, was effective in alleviating depression, as it was found that the group spent a lot of time in the center.

[0130] [Example 6]

[0131] Elevated plus maze test (EPM)

[0132] Through the EPM test, the time spent in the open arm and the time spent in the close arm were measured to confirm whether depression was induced by restraint stress, and the depression-relieving effect of the selected lactic acid bacteria was confirmed through the retention time.

[0133] The time spent in the open arm was significantly shorter in the group induced with depression through restraint stress compared to the control group. However, the group administered the selected lactic acid bacteria Lactobacillus buchneri showed no significant difference in the time spent in the open arm compared to the control group. The time spent in the closed arm was significantly longer in the restraint group compared to the control group (Fig. 6).

[0134] Through these results, it was confirmed that depression was induced by restraint stress in the EPM test, and in the group administered with the selected lactic acid bacteria, Lactobacillus buchneri, there was no significant difference in the time spent in the open arm and the close arm, suggesting that it had an effect of alleviating depression.

[0135] [Example 7]

[0136] Biochemical analysis of animal models of depression induced by restraint stress

[0137] After the behavioral analysis of the depression-induced model through restraint stress was completed, the corticosterone content in the serum and the mRNA expression levels of TPH-1 and SERT in the small and large intestines were investigated, and the mRNA expression levels of BDNF, 5-HT transporter 2A, and SERT in the prefrontal cortex of the brain tissue were investigated.

[0138] [Example 8]

[0139] Serum corticosterone content analysis

[0140] To assess stress responses and depression induced by restraint stress, changes in serum corticosterone levels were measured. Physical stress, such as anger, increases blood corticosterone levels, and increased serum corticosterone levels are associated with activation of the HPA (Hypothalamus-Pituitary-Adrenal) axis in response to repeated stress.

[0141] The serum corticosterone levels of mice induced with depression by restraint stress are shown in Figure 7. Lower corticosterone levels were observed in the control and fluoxetine groups, suggesting that the mice experienced less stress than the restraint group. Compared to the control group, the restraint group showed significantly higher serum corticosterone levels. However, the group administered Lactobacillus buchneri showed no significant difference from the control group.

[0142] These results have been reported in many studies, and it has been reported that the longer the time of exposure to restraint stress, the higher the corticosterone content, and that increased corticosterone content is accompanied by decreased feed intake due to restraint stress.

[0143] Through the above results, it was confirmed that depression was induced by restraint stress, and the serum corticosterone content in the group administered Lactobacillus buchneri was not significantly different from the control group and fluoxetine group, confirming the effect of alleviating depression and stress.

[0144] [Example 9]

[0145] Confirmation of mRNA expression levels of depression-related genes in intestinal tissue

[0146] mRNA was isolated from the small and large intestine tissues of a depression model induced by restraint stress, and the expression levels of the SERT gene, which transports serotonin to enterocytes, and the TPH-1 gene, which is an indicator of serotonin production, were confirmed.

[0147] Comparison of SERT mRNA expression levels in small intestinal tissues revealed that the Lactobacillus buchneri group showed a significant difference from the restraint group, which had the lowest expression levels. SERT expression levels in large intestinal tissues were similar across all groups, and no significant results were observed (Fig. 8).

[0148] In the small intestine tissue, the expression level of TPH-1 mRNA was significantly higher in the Lactobacillus buchneri group compared to the restraint group, similar to the results of the SERT expression level (Fig. 9). In the large intestine tissue, the expression level of TPH-1 was measured to be the highest in the control group, and the expression level of the Lactobacillus buchneri group showed no significant difference compared to the control group, and the expression level of the Lactobacillus buchneri group was significantly higher compared to the restraint group.

[0149] The selected strain, Lactobacillus buchneri, was found to have elevated mRNA expression levels of SERT and TPH-1 in the intestinal tissue of mice induced with depression. These results suggest that Lactobacillus buchneri is effective in alleviating depression by influencing the mechanisms of serotonin synthesis and uptake.

[0150] [Example 10]

[0151] Determination of mRNA expression levels of depression-related genes in brain tissue

[0152] Brain-derived neurotrophic factor (BDNF) is a neurotrophic factor that plays a crucial role in the maintenance and development of the nervous system, inducing growth and differentiation during neural development. BDNF has been linked to the pathophysiological mechanisms of depression. Among these, the prefrontal cortex, a crucial part of the brain responsible for regulating thought and behavior, is a brain region frequently analyzed in depression-related research.

[0153] In the prefrontal cortex, the mRNA expression level of BDNF was highest in the control group, while the groups subjected to restraint stress showed a tendency toward decreased BDNF expression. However, no significant difference was found compared to the control group.

[0154] As a result of measuring the mRNA expression levels of SERT and HTR2A in the prefrontal cortex tissue (Fig. 10), the expression level of SERT was highest in the fluoxetine group. This result was similar to the result of SERT mRNA expression level in the intestinal tissue, but no significant difference was observed. HTR2A, one of the serotonin receptors, is a serotonin receptor in brain tissue and is a receptor frequently analyzed in depression research. The mRNA expression level of HTR2A was similar to the expression pattern of SERT, and the expression levels in the control group and fluoxetine group were higher than in the other groups, but no significant difference was observed.

[0155]

[0156] The present invention is an invention carried out through the following tasks.

[0157] [National Research and Development Project Supporting This Invention]

[0158] [Project ID]1545027078

[0159] [Assignment Number]321035053SB010

[0160] [Ministry Name] Ministry of Agriculture, Food and Rural Affairs

[0161] [Name of project management (specialized) organization] National Institute of Agricultural and Food Technology Planning and Evaluation

[0162] [Research Project Name] Development of High Value-Added Food Technology

[0163] [Research Project Name] Microbiome-targeted probiotic discovery and materialization technology

[0164] development

[0165] [Contribution rate] 1 / 1

[0166] [Name of the project performing organization] Konkuk University Industry-Academic Cooperation Foundation

[0167] Research Period: April 1, 2021 - December 31, 2025

Claims

1. A pharmaceutical composition for preventing or treating depression, comprising Lactobacillus buchneri 200793 strain (KCCM 12593P) or a culture thereof as an active ingredient.

2. A pharmaceutical composition according to claim 1, wherein the composition comprises a live cell of the Lactobacillus buchneri 200793 strain.

3. In the first paragraph, the composition comprises 10 Lactobacillus buchneri 200793 strains. 8 10 inland 11 A pharmaceutical composition comprising a concentration of CFU / ml.

4. A pharmaceutical composition according to claim 1, wherein the composition increases sucrose preference.

5. A pharmaceutical composition according to claim 1, wherein the composition increases the mRNA expression level of TPH-1 or SERT in small and large intestine tissues.

6. A health functional food composition for preventing or improving depression, comprising Lactobacillus buchneri 200793 strain (KCCM 12593P) or a culture thereof as an active ingredient.

7. A health functional food composition according to claim 6, wherein the composition comprises live cells of the Lactobacillus buchneri 200793 strain.

8. In the 6th paragraph, the composition comprises 10 Lactobacillus buchneri 200793 strains. 8 10 inland 11 A health functional food composition comprising a concentration of CFU / ml.

9. A health functional food composition for stress relief containing Lactobacillus buchneri 200793 strain (KCCM 12593P) or a culture thereof as an active ingredient.

10. A health functional food composition according to claim 9, wherein the composition comprises live cells of the Lactobacillus buchneri 200793 strain.

11. In the 9th paragraph, the composition comprises 10 Lactobacillus buchneri 200793 strains. 8 10 inland 11 A health functional food composition comprising a concentration of CFU / ml.

12. An animal feed additive composition for preventing or improving depression, comprising Lactobacillus buchneri 200793 strain (KCCM 12593P) or a culture thereof as an active ingredient.

13. An animal feed additive composition according to claim 12, wherein the composition comprises a live cell of the strain.

14. In the 12th paragraph, the composition comprises 10 Lactobacillus buchneri 200793 strains. 8 10 inland 11 An animal feed additive composition comprising a content of CFU.

Images