JP7595342B2 - Anti-stress composition - Google Patents

Description

NPMD NITE P-03300

Article 30, Paragraph 2 of the Patent Act, Japan Society for Bioscience, Biotechnology, and Agricultural Chemical Sciences, 2020 (Reiwa 2) Annual Meeting Abstracts (online), cover, table of contents, abstract, supporting documents, conference cancellation notice, published on March 5, 2020

The present invention relates to a composition for improving or suppressing changes in the physical condition caused by stress.

Psychological and social stress, which can be considered a modern disease, are believed to cause various diseases and symptoms in the body. In particular, these stresses can lead to disorders of the central nervous system and brain function.

Stress-related diseases and symptoms include many, including depression, mania, loss of appetite, loss of motivation, psychosomatic disorders, anxiety neurosis (panic disorder), adjustment disorder, mood disorder, PTSD (post-traumatic stress disorder), ASD (acute stress disorder), eating disorders, drinking disorders, alcoholism, drug addiction, inflammation, insomnia, Alzheimer's disease, cognitive impairment, memory disorders, speech disorders, stroke, cerebral infarction, Parkinson's disease, irritable bowel syndrome, gastric ulcers, asthma, atopic dermatitis, hives, alopecia, high blood pressure, lifestyle-related diseases, headaches, and rheumatism.

For example, mental and neurological problems caused by stress can lead to unstable food intake, hyperactivity, and increased energy consumption, resulting in weight loss. In particular, when depression appears in children and adolescents, not only does it lead to weight loss, but it can also lead to failure to achieve the expected weight gain. Non-Patent Document 1 describes how depression patients have experienced increased insomnia and fatigue, as well as a marked decrease in weight and appetite.

In addition, a condition in which stress or tension causes thirst, excessive fluid intake, and polyuria is known as psychogenic polydipsia. Clinical findings have also been obtained for severe cases of psychogenic polydipsia, in which the amount of fluid consumed reaches 15 liters per day.

On the other hand, one of the factors that causes stress-induced illnesses such as depression and dementia is inflammatory reactions in brain tissues. Inflammatory cytokines are involved in inflammatory reactions. Examples of inflammatory cytokines include interleukin (IL)-6, IL-1, and TNF-α. Of these, it has been reported that IL-6 is associated with stress-related illnesses such as depression and anxiety (see Non-Patent Document 2).

Psychiatry Journal, Volume 2015, Article ID 575931 Neurobiology of Stress 4 (2016) 15-22

As mentioned above, stress can cause external abnormalities in the body and behavior, as well as internal neurological and psychological abnormalities.

If there were an active ingredient that could improve both the external and internal abnormalities caused by stress, it would be possible to improve the mental and physical abnormalities caused by stress. However, little is known about such active ingredients to date.

The problem that the present invention aims to solve is to provide a composition containing an active ingredient that can improve both external and internal abnormalities caused by stress.

The present inventors have conducted intensive research to solve the above problems, and through trial and error to confirm the improving effect of various physiologically active ingredients on mental and physical abnormalities caused by stress, have surprisingly found that Bifidobacterium bifidum has the effect of improving external abnormalities caused by stress, such as suppressing a decrease in food intake, mitigating the suppression of weight gain, and suppressing an increase in water intake, as well as the effect of improving internal abnormalities caused by stress, such as suppressing an increase in inflammatory cytokines. Based on this knowledge, the present inventors have finally succeeded in creating an anti-stress composition containing Bifidobacterium bifidum and its components as active ingredients, which can solve the problems of the present invention. The present invention has been completed based on the knowledge and successful examples first discovered by the present inventors.

Thus, according to the present invention, there are provided compositions and methods according to the following aspects:
[1] An anti-stress composition comprising at least one active ingredient selected from the group consisting of Bifidobacterium bifidum cells, cell components, cultures, and processed products thereof.
[2] An antidepressant composition or anti-anxiety composition comprising at least one active ingredient selected from the group consisting of Bifidobacterium bifidum cells, cell components, and cultures, as well as processed products thereof.
[3] A composition for improving or suppressing at least one disease and/or symptom selected from the group consisting of weight loss, excessive drinking, eating disorders and inflammation, comprising at least one active ingredient selected from the group consisting of Bifidobacterium bifidum cells, cell components, and cultures, and processed products thereof.
[4] The composition described in [3], wherein the disease or symptom is a disease or symptom caused by stress.
[5] A composition for suppressing interleukin-6 production or a composition for suppressing TNF-α production, comprising at least one active ingredient selected from the group consisting of Bifidobacterium bifidum cells, cell components, and cultures, as well as processed products thereof.
[6] The composition according to any one of [1] to [5], wherein the Bifidobacterium bifidum is the Bifidobacterium bifidum TMC3115 strain having the accession number NITE P-03300.

According to the present invention, it is expected that the active ingredient will improve or suppress mental and physical abnormalities caused by stress in an individual organism through its effect of improving diseases and symptoms caused by stress, thereby achieving a healthy state of both mind and body.

FIG. 1A is a copy of the accession certificate for accession number NITE P-03300 (Bifidobacterium bifidum strain TMC3115). FIG. 1B is a copy of the certificate of viability for accession number NITE P-03300 (Bifidobacterium bifidum strain TMC3115). FIG. 2 is a schematic diagram showing an overview of the sCSDS model together with the test plan. FIG. 3 is a schematic diagram of a cage in an open field used in a social behavior test. FIG. 4 shows the results of measuring the amount of weight gain, as described in the Examples below. FIG. 5 shows the results of measuring the amount of water consumed, as described in the Examples below. FIG. 6 shows the results of measuring IL-6, as described in the Examples below.

Each aspect of the present invention will be described in detail below, but the present invention can take various forms as long as it achieves its objectives.

Unless otherwise specified, the terms used in this specification are used in the sense commonly used by those skilled in the art of food, pharmaceuticals, cosmetics, etc., and should not be construed as having an unduly restrictive meaning. Furthermore, the speculations and theories made in this specification are based on the inventors' knowledge and experience to date, and therefore the present invention is not limited solely to such speculations and theories.

The term "composition" is not particularly limited to the meaning in which it is usually used, and is, for example, a combination of two or more components.
The term "and/or" means any one or any or all combinations of two or more of the associated listed items.
"Comprising" means that elements other than the elements explicitly stated as being included can be added (same meaning as "comprising at least"), but also encompasses "consisting of" and "consisting essentially of." That is, "comprising" can mean including the elements explicitly stated and any one or more elements, consisting of the elements explicitly stated, or consisting essentially of the elements explicitly stated. Elements include limitations such as ingredients, steps, conditions, parameters, etc.

"Anti-stress effect" refers to improving or suppressing at least one abnormality selected from the group consisting of external and internal abnormalities caused by exposure to stress. Note that "improving or suppressing" an abnormal state includes making an abnormal state into a healthy state, bringing an abnormal state closer to a healthy state, maintaining a healthy state, and mitigating or preventing a healthy state from changing into an abnormal state.
"Antidepressant effect" and "anti-anxiety effect" refer to improving or suppressing depressive symptoms and anxiety symptoms, respectively. Depressive symptoms include those described in the Japanese Society of Mood Disorders Treatment Guideline "II. Depression (DSM-5)/Major Depressive Disorder 2016". Depressive symptoms also include decreased motivation and mood disorders.
Regarding "weight loss", "polydipsia", "eating disorder" and "inflammation", weight loss includes weight loss and failure to gain weight as expected; polydipsia includes drinking a lot of water and urinating a lot; eating disorder includes eating a little; inflammation includes inflammation confirmed by cytological and histological findings and an increase in the amount of inflammatory cytokines. These may not be caused by stress, but are preferably caused by stress.
The "interleukin-6 production inhibitory effect" includes reducing the amount of interleukin (IL)-6, inhibiting an increase in the production of IL-6, and maintaining the amount of IL-6. Whether or not a certain active ingredient has an interleukin-6 production inhibitory effect can be evaluated by testing and measuring IL-6 according to the method described in the Examples below.

The anti-stress composition of one embodiment of the present invention contains at least one active ingredient selected from the group consisting of Bifidobacterium bifidum cells, cell components, cultures, and processed products thereof. The anti-stress composition of one embodiment of the present invention exhibits an anti-stress effect by containing the active ingredient.

The anti-stress effect of the anti-stress composition of one embodiment of the present invention can be confirmed by evaluating the improvement or suppression of at least one type of abnormality caused by stress selected from the group consisting of reducing the effect of stress on body weight, suppressing an increase in water intake, suppressing a decrease in food intake, and suppressing an increase in IL-6 and TNF-α when the anti-stress composition of one embodiment of the present invention is applied to B6 mice for at least three days, as described in the Examples below, compared to when the anti-stress composition of one embodiment of the present invention is not applied.

Bifidobacterium bifidum may be any microorganism classified as Bifidobacterium bifidum. There is no particular limitation on the method of obtaining Bifidobacterium bifidum, and examples of such methods include a method of using commercially available or deposited Bifidobacterium bifidum, and a method of isolating Bifidobacterium bifidum from natural or commercially available dairy products. Deposited Bifidobacterium bifidum includes Bifidobacterium bifidum TMC3115 strain (deposit number: NITE P-03300). TMC3115 strain can be obtained from the National Institute of Technology and Evaluation, Patent Microorganism Deposit Center (NPMD) (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818). As Bifidobacterium bifidum, the TMC3115 strain is preferred because it has an excellent anti-stress effect.

Bifidobacterium bifidum can be used as a single strain or as a combination of two or more strains.

The active ingredient in the anti-stress composition of one embodiment of the present invention may be Bifidobacterium bifidum itself, i.e., in addition to the cells of Bifidobacterium bifidum, a cell component of Bifidobacterium bifidum and a culture of Bifidobacterium bifidum. The active ingredient in the anti-stress composition of one embodiment of the present invention may also be a processed cell, cell component and/or culture of Bifidobacterium bifidum.

Examples of the Bifidobacterium bifidum cells include cells obtained by removing the medium from a culture of Bifidobacterium bifidum using a commonly known solid-liquid separation method such as centrifugation. Examples of the cell components include purified or non-purified components present within the cells of Bifidobacterium bifidum or secreted outside the cells. Examples of the culture of Bifidobacterium bifidum include the culture liquid itself obtained by culturing Bifidobacterium bifidum.

The bacterial cells, bacterial components and cultures of Bifidobacterium bifidum can be used alone or in combination of two or more. The method for obtaining the bacterial cells, bacterial components and cultures of Bifidobacterium bifidum is not particularly limited, but an example of a method for obtaining the bacterial cells of Bifidobacterium bifidum is to inoculate the cells into a medium suitable for the growth of Bifidobacterium bifidum, preferably a modified FG medium, and anaerobic culture at a temperature suitable for the growth of Bifidobacterium bifidum, preferably about 37°C, for several hours to several tens of hours, preferably 18 hours to 30 hours, to obtain a culture of Bifidobacterium bifidum, and then collect the bacterial cells using a conventional bacterial cell collection means such as centrifugation. For example, extracellular components of Bifidobacterium bifidum can be obtained from the culture (culture solution) after the cells are harvested, and intracellular components can be obtained by disrupting the harvested cells, for example by homogenizing them.

Examples of processed products of Bifidobacterium bifidum cells, cell components, and cultures include dried products of Bifidobacterium bifidum cells, and from the viewpoint of obtaining a stable anti-stress effect, a freeze-dried product of Bifidobacterium bifidum is preferable. The freeze-drying conditions may be those of ordinary microorganisms that can obtain a freeze-dried powder of the microorganism.

Since Bifidobacterium bifidum has been consumed and its safety has been confirmed, the anti-stress composition of one embodiment of the present invention is highly practical and can be used for various purposes in oral or parenteral forms, either as is or after processing. Parenteral applications include, but are not limited to, injection and infusion by administration intradermally, subcutaneously, intravenously, intramuscularly, etc.; transdermally; and inhalation through mucous membranes such as the nose and pharynx.

The individual organism to which the anti-stress composition of one embodiment of the present invention is applied is not particularly limited, and examples thereof include animals, particularly mammals, and examples of mammals include humans, dogs, cats, cows, horses, pigs, sheep, etc., and among these, humans are preferred. The individual organism may be a healthy individual, but is preferably an individual that is currently under stress, an individual that is easily susceptible to stress, or an individual at risk of developing abnormalities due to stress.

The anti-stress composition of one embodiment of the present invention may contain other ingredients depending on the application, so long as it has an anti-stress effect. Thus, the anti-stress composition of one embodiment of the present invention may contain various other ingredients in addition to the active ingredient, so long as the object of the present invention can be achieved. The content of the other ingredients may be appropriately determined by a person skilled in the art, so long as it does not interfere with the solution of the problem of the present invention.

The other ingredients are not particularly limited, but for example, when the anti-stress composition of one embodiment of the present invention is used as a food or beverage composition, ingredients used in foods and seasonings can be mentioned, and specifically, dairy products such as fermented milk such as yogurt and drink yogurt, milk drinks, ice cream, and foods that use milk as a main ingredient. In addition, examples of solid ingredients include salt, sugars (sugar, glucose, fructose, starch syrup, isomerized liquid sugar, etc.), grain ingredients (bread crumbs, wheat flour, oatmeal, etc.), spices (ginger, chili pepper, pepper, basil, oregano, ginger, mixed spices, etc.), thickeners (thickening polysaccharides such as carrageenan, starch, processed starch, gums, etc.), processed meat ingredients (chicken powder, meat powder, fish powder, etc.), chemical seasonings (monosodium glutamate, etc.), and the like. , glycine, sodium inosinate, sodium guanylate, etc.), flavors, miso, curry powder, etc.; liquid components include water, alcohol, sweet components (mirin, liquid sugar, starch syrup, etc.), sour components (vinegar, citrus fruits such as apple, yuzu, lemon, etc.), oil and fat components (sesame oil, olive oil, salad oil, soybean oil, chili oil, butter, beef tallow, lard, etc.), alcoholic beverage components (wine, sake, etc.), fruit juice (apple juice, etc.). In this case, the other components may be extracts such as seafood extract, seaweed extract, meat extract, vegetable extract, yeast extract, protein hydrolysate, etc.; broth obtained by extracting fish, seaweed, mushrooms, etc. with a solvent such as hot water or ethanol; food ingredients such as vegetables, spicy vegetables, mushrooms, fruits, meat, seafood, seaweed, etc.

In addition, when the anti-stress composition of one embodiment of the present invention is used as a cosmetic composition or a pharmaceutical composition, the other components may include components that are typically used in cosmetics and pharmaceuticals, such as bases such as water, oily components, moisturizers, cooling agents, preservatives, chelating agents, pH adjusters, antioxidants, UV absorbers, UV scattering agents, thickeners, whitening agents, emulsifiers, vitamins, various other medicinal ingredients, powders, fragrances, coloring materials, excipients, binders, lubricants, etc.

The anti-stress composition of one embodiment of the present invention is not particularly limited as long as it is in a commonly used form, and may be in any form, such as a solid, liquid, gel, suspension, cream, sheet, stick, powder, granules, granules, tablet, rod, plate, block, paste, capsule, or caplet.

The content of the active ingredient is not particularly limited as long as it is an amount that exhibits an anti-stress effect, but is, for example, 1.0×10 ⁴ CFU/g to 1.0×10 ¹² CFU/g, preferably 1.0×10 ⁵ CFU/g to 1.0×10 ¹⁰ CFU/g, more preferably 1.0×10 ⁶ CFU/g to 1.0×10 ⁹ CFU/g; or 0.01% to 99.9% by mass, preferably 0.05% to 99% by mass, more preferably 0.1% to 90% by mass, relative to the total amount of the composition.

The dosage and administration method of the anti-stress composition of one embodiment of the present invention, such as the amount used at one time, the amount used per day, the period of use, and the interval between uses, are not particularly limited and can be set appropriately depending on the mode of use, the condition of the individual organism to which it is applied, etc.

The amount used at one time is, for example, preferably 0.01 mg to 10,000 mg, more preferably 0.1 mg to 1,000 mg, and even more preferably 1 mg to 500 mg of the active ingredient. The daily amount may be set appropriately according to the symptoms and physical build of the individual organism, but is preferably, for example, 100 mg/60 kg body weight/day to 10 g/60 kg body weight/day.

The interval of use may be, for example, once, twice, three times, or several times a day, continuously applied for a certain period of time, i.e., one day or more, preferably two days or more, three days or more, four days or more, five days or more, one week or more, two weeks or more, one month or more, six months or more, or one year or more. It is preferable to apply the anti-stress composition of one embodiment of the present invention every day, but the anti-stress composition of one embodiment of the present invention does not have to be applied every day as long as it is applied continuously during the period.

The anti-stress composition according to one embodiment of the present invention can be used alone or as an additive to food and beverages, cosmetics, medicines, etc. Specific embodiments of the present invention include so-called health foods and beverages, such as foods and beverages for specified health uses, foods and beverages with functional claims, foods and beverages with nutrient functions, foods and beverages with health functions, foods and beverages for special uses, foods and beverages with nutritional supplements, foods and beverages with health supplements, supplements, and beauty foods and beverages; foods and beverages for specified persons, such as foods and beverages for infants, foods and beverages for pregnant women, and foods and beverages for the elderly; cosmetics; medicines; quasi-drugs; animal feed; and raw materials for producing these products.

The anti-stress composition according to one embodiment of the present invention can be packed in a container and sealed to form a packaged composition. The container is not particularly limited, but examples include packaging containers such as pouches, sachets, bottles, cans, and jars made of metals such as aluminum, plastics such as PET and PTP, single-layer or laminated films, and glass. It is preferable that the packaged composition is one that can be distributed and sold commercially by itself independently after dispensing, filling, and/or individually packaging the manufactured anti-stress composition.

Bifidobacterium bifidum can improve or suppress various external and internal physical and mental abnormalities caused by stress. It is well known that stress can cause depression and anxiety symptoms. Therefore, another aspect of the present invention is an antidepressant composition and an anti-anxiety composition containing at least one active ingredient selected from the group consisting of Bifidobacterium bifidum cells, cell components, and cultures, as well as processed products thereof.

It has been known that exposure to stress, and the manifestation of depressive and anxiety symptoms due to stress, can lead to the development of diseases and symptoms associated with brain dysfunction, such as cognitive impairment, memory impairment, Alzheimer's disease, speech disorders, stroke, and cerebral infarction. Thus, the composition of one embodiment of the present invention has anti-stress, anti-depressant, and anti-anxiety effects and can be used to improve or suppress diseases and symptoms associated with brain dysfunction.

As described in the Examples below, stressed organisms exhibit abnormalities such as suppression of expected weight gain, increased water intake, decreased food intake, and increased levels of inflammatory cytokines such as IL-6 and TNF-α. In contrast, Bifidobacterium bifidum can improve or suppress these abnormalities. Another aspect of the present invention is a composition for improving or suppressing at least one disease and symptom selected from the group consisting of weight loss, excessive drinking, eating disorders, and inflammation, comprising at least one active ingredient selected from the group consisting of Bifidobacterium bifidum cells, cell components, and cultures, and processed products thereof. These diseases and symptoms are not particularly limited in terms of their relationship to stress, but are preferably diseases and symptoms caused by stress.

Bifidobacterium bifidum can also suppress the production of IL-6 and TNF-α, which are types of inflammatory cytokines, and is particularly effective in suppressing IL-6 production. Therefore, another aspect of the present invention is a composition for suppressing interleukin-6 production and a composition for suppressing TNF-α production, which contain at least one active ingredient selected from the group consisting of Bifidobacterium bifidum cells, cell components, and cultures, as well as processed products thereof.

The method for producing the composition of one embodiment of the present invention is not particularly limited, but may include, for example, a method including mixing the active ingredient with other ingredients and molding the resulting mixture into a desired shape.

Another aspect of the present invention is a method for improving or suppressing at least one abnormality selected from the group consisting of external abnormalities and internal abnormalities caused by exposure to stress, a method for improving or suppressing depressive symptoms and anxiety symptoms, a method for improving or suppressing at least one disease and symptom selected from the group consisting of weight loss, excessive drinking, eating disorders and inflammation, and a method for suppressing the production of interleukin-6 and TNF-α, by applying an active ingredient or a composition containing the active ingredient to an individual organism.

The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples, and the present invention can take various forms as long as the object of the present invention can be solved.

1. Test material (1-1) Bacterial strain Bifidobacterium bifidum TMC3115 strain (hereinafter simply referred to as TMC3115) was used, which was stored in the microorganism collection of Takanashi Dairy Co., Ltd., the applicant of the present application. TMC3115 has the identification mark of the microorganism "TMC3115" and the deposit number "NITE P-03300" at the Patent Microorganism Deposit Center of the National Institute of Technology and Evaluation (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818) on the deposit date of October 6, 2020. A copy of the receipt is shown in FIG. 1A and the certificate of viability is shown in FIG. 1B.

TMC3115 was cultured anaerobically at 37°C for 18 hours using modified FG medium (glucose 3% by mass, peptone 3% by mass, yeast extract 0.5% by mass, sodium acetate 0.5% by mass, diammonium hydrogen phosphate 0.2% by mass, dipotassium hydrogen phosphate 0.2% by mass, magnesium sulfate 0.05% by mass, polysorbate 80 0.1% by mass, water balance). The culture solution after the culture was centrifuged (8,000g, 8 minutes, 4°C) to recover the bacterial cells, which were then washed twice with sterilized water. The washed bacterial cells were freeze-dried and stored at -80°C until use. The viable cell count of the freeze-dried bacterial cell powder was 1 x 10 ¹⁰ CFU/g.

(1-2) Mice Seven-week-old male C57BL/6JJcl (B6) mice were purchased from CLEA Japan, and retired male ICR mice were purchased from Nippon SLC.

Mice were individually housed in cages with wooden bedding and housed in an environment with free access to water and food in a breeding room controlled by a 12-hour light/dark cycle.

B6 mice were fed AIN-93G feed (powder type; manufactured by Oriental Yeast Co., Ltd.) (hereafter referred to as standard feed) or a test feed (4.2 x 10 ⁸ CFU/g) containing 2.4 g of AIN-93G mixed with 10 mg of TMC3115 freeze-dried cell powder in a Rodencafe. The feed was changed every day.

ICR mice were fed MF feed (pellet type; Oriental Yeast Co., Ltd.) in a regular feeding bowl.

The body weight, food intake, and water intake of the B6 mice were measured daily. After purchase, the mice were allowed to acclimate to the breeding environment for one week.

2. Test method (2-1) sCSDS model Based on the body weight at the end of the acclimation, B6 mice were divided into three groups: a non-stressed control group (non-stressed; n=15), a stressed control group (stressed; n=24), and a TMC3115 group (n=25). Throughout the entire test period (10-day pre-administration period (Day 0-9), 10-day stressed period (Day 10-19), 1-day behavioral test (Day 20), and the end of the test (Day 21)), both control groups were fed with standard feed, which was AIN-93G, and the TMC3115 group was fed with test feed, which was a mixture of AIN-93G and TMC3115 freeze-dried cell powder.

Stress loading on mice was performed using the subchronic and mild social defeat stress (sCSDS) model (Goto et al., 2014; Goto T, Kubota Y, Tanaka Y, Iio W, Moriya N, Toyoda A, "Subchronic and mild social defeat stress accelerates food intake and body weight gain with polydipsia-like features in mice.", Behav Brain Res 270:339-348 (2014)). Figure 2 shows a schematic overview of the sCSDS model along with the test plan. In summary, the sCSDS model is a test model in which a B6 mouse is placed as an intruder in the cage of a previously selected highly aggressive resident (ICR mouse), and the resident ICR mouse is made to directly attack the B6 mouse for a certain period of time.

The stress period for the B6 mice in the stressed control group and the TMC3115 group was 10 days, and the duration of the direct attack was 5 minutes on the first day, and was reduced by 30 seconds each day thereafter. After the direct attack, the ICR mice and the B6 mice were separated by a transparent acrylic partition with small holes for the remainder of the day. This meant that the B6 mice were not directly attacked by the ICR mice, but were exposed to sensory stimuli such as the appearance and smell of the attacked ICR mice. The ICR mice providing stimuli to each B6 mouse were changed every day, and the B6 mice in each test group were exposed to direct attacks and indirect stimuli from different ICR mice each day for 10 days, thereby loading stress on the B6 mice.

The B6 mice in the non-stress control group were housed in the same cages, separated by an acrylic panel similar to that described above, with one mouse on each side. Therefore, the B6 mice in the non-stress control group were not exposed to direct attacks or indirect stimuli from the ICR mice.

(2-2) Social interaction test
The social behavior test was carried out on the day after the stress load by the sCSDS model for 10 days was completed (Day 20). A plastic cage (100 mm long, 100 mm wide, 130 mm high) covered with wire mesh was installed near the center of one wall of an open field (a lidless box with a length of 400 mm, a width of 400 mm, and a height of 300 mm). The area of about 7 cm around the cage was defined as the social zone. In addition, the two corners of the wall opposite to the wall where the cage was installed were defined as corner zones. Figure 3 shows a schematic diagram of the relationship between the cage, the social zone, and the corner zone in the open field.

First, without placing an ICR mouse in the cage, the B6 mice from each test group were released from the corner zone into the open field and allowed to explore freely for 2 minutes and 30 seconds. After exploring, the mice were retrieved and the open field was cleaned with disinfectant ethanol to remove odors and create a clean space.

Next, an ICR mouse was placed in a cage, and the same B6 mouse was released again from the corner zone into the open field to explore. The sociality score was calculated based on the time spent in the social zone when an ICR mouse was present, relative to the time spent in the social zone when an ICR mouse was not present. When the time spent in the social zone when an ICR mouse was absent was equal to the time spent in the social zone when an ICR mouse was present, the sociality score was calculated as 100. Individuals with a sociality score of less than 100% (sociality score less than 100) were classified as stress-sensitive individuals. On the other hand, individuals with a sociality score of 100 or more were classified as stress-resistant individuals ("resilient").

(2-3) Measurement of serum inflammatory cytokine concentrations To collect blood from the abdominal vena cava, B6 mice were dissected on the day after the social behavior test (Day 21). Specifically, B6 mice were fasted for 3 hours before dissection, anesthetized with 3% isoflurane, and then subjected to abdominal incision to collect blood from the abdominal vena cava.

The collected blood was kept at 37°C for 20 minutes, then left to cool at 4°C for 2 hours. After cooling, the blood was centrifuged (1,000g, 15 minutes, 4°C) to separate and collect serum. The serum was stored at -80°C until analysis.

The concentrations of interleukin 6 (IL-6) and tumor necrosis factor (TNF-α) in serum were measured using the corresponding ELISA kits (#BMS607HS for IL-6, Invitrogen; #BMS603HS for TNF-α, Invitrogen) according to the kit manual.

(2-4) Statistical Analysis Body weight gain (change in body weight from the start of the study), food intake, and water intake were analyzed by repeated analysis of two-way variance. Comparisons among the three groups were performed using the Tukey-HSD test, with P<0.05 considered statistically significant.

3. Test results and evaluation The measurement results of weight gain are shown in Figure 4. As shown in Figure 4, the weight gain was significantly lower in the stressed control group compared to the non-stressed control group and the TMC3115 group during the stress period. However, the decrease in weight gain was suppressed in the TMC3115 group, and there was no significant difference from the non-stressed control group. This shows that TMC3115 has the effect of reducing the effect of stress on body weight.

The results of measuring water intake are shown in Figure 5. As shown in Figure 5, the amount of water intake increased significantly in the stressed control group compared to the non-stressed control group during the stress period. However, the increase in water intake was inhibited in the TMC3115 group. This demonstrated that TMC3115 has the effect of inhibiting an increase in water intake due to stress.

Regarding food intake, the average food intake during the stress period was 3.13g for the non-stressed control group, 3.04g for the stressed control group, and 3.11g for the TMC3115 group. Statistical analysis showed that the stressed control group had a significantly lower intake than the non-stressed control group. However, there was no significant difference between the TMC3115 group and the non-stressed control group. This shows that TMC3115 has the effect of suppressing the decrease in food intake due to stress.

The results of the sociality score measurements are shown in Table 1. As shown in Table 1, the stressed control group had a higher proportion of stress-sensitive individuals than the non-stressed control group. However, in the TMC3115 group, the proportion of stress-sensitive individuals was as low as in the non-stressed control group. This demonstrated that TMC3115 has the effect of improving sociality scores and enhancing stress resistance.

The results of measuring IL-6 are shown in Figure 6. As shown in Figure 6, IL-6 increased significantly in the stressed control group compared to the non-stressed control group and the TMC3115 group. However, the increase in IL-6 was inhibited in the TMC3115 group, and there was no significant difference from the non-stressed control group. This shows that TMC3115 has the effect of inhibiting the increase in IL-6 caused by stress.

The results of measuring TNF-α are shown in Table 2. As shown in Table 2, TNF-α increased in the stressed control group and the TMC3115 group compared to the non-stressed control group. However, the increase in TNF-α was slightly suppressed in the TMC3115 group compared to the stressed control group. This shows that TMC3115 has the effect of suppressing the increase in TNF-α.

The composition of one embodiment of the present invention contains an active ingredient that can be administered either orally or parenterally, and is useful for individual organisms in which anti-stress activity, antidepressant activity, anti-anxiety activity, anti-anxiety activity, and anti-mood disorder activity are desired, and further for individual organisms in which improvement of central nervous function and brain function is desired, through the effects of reducing the impact of stress on body weight, suppressing an increase in water intake, suppressing a decrease in food intake, and suppressing an increase in inflammatory cytokines, and can be used as foods and beverages, medicines, quasi-drugs, cosmetics, and the like that contribute to the health and welfare of such individual organisms.

Claims (5)

An anti-stress composition comprising an active ingredient of freeze-dried powder of bacteria consisting only of Bifidobacterium bifidum TMC3115 strain , which has a deposit number of NITE P-03300 . An antidepressant composition or an anti-anxiety composition comprising an active ingredient of a freeze-dried powder of bacteria consisting only of Bifidobacterium bifidum TMC3115 strain , which has a deposit number of NITE P-03300 . A composition for improving or suppressing at least one disease and/ or symptom selected from the group consisting of weight loss, excessive drinking, eating disorders and inflammation, comprising an active ingredient of a freeze-dried powder of bacterial cells consisting solely of Bifidobacterium bifidum TMC3115 strain, which has an accession number of NITE P-03300. The composition according to claim 3, wherein the disease or condition is a disease or condition caused by stress. A composition for suppressing interleukin-6 production or a composition for suppressing TNF-α production, comprising as an active ingredient a freeze-dried powder of bacterial cells consisting solely of Bifidobacterium bifidum TMC3115 strain, which has a deposit number of NITE P-03300 .