JP7536504B2 - Agent for improving depressive symptoms and composition containing same - Google Patents

Description

NPMD NITE BP-72

Applicable under Article 30, Paragraph 2 of the Patent Act [Publication date] May 29, 2019 [Website address] http://pharmacology.pupu.jp/135kinki/index.html, http://pharmacology.pupu.jp/135kinki/pdf/pdf/135kinki_abstract_b.pdf [Disclosed content of the invention] "Effect of Lactobacillus gasseri OLL2809 on depression-like behavior in mice" was disclosed in the "PDF version of Abstracts of the 135th Kinki Branch Meeting of the Japanese Pharmacological Society" published on the website at the address listed above.

The present invention relates to a depressive symptom improving agent used to improve depressive symptoms, and a pharmaceutical composition, a quasi-drug composition, or a food and drink composition containing the same. The present invention also relates to a composition for increasing Akkermancia bacteria present in the intestine.

Depression is a mood disorder (depressive disorder) in which emotions are suppressed for some reason, and the person loses the energy to live a social life and has difficulty adapting to society. Depression has a wide variety of symptoms, including mental symptoms such as depressed mood, sadness, loss or decline in motivation, interest, and joy, self-blame and guilt, loss or decline in self-esteem, inhibition of thought, restlessness, decreased concentration, and suicidal ideation (repeated thoughts of death or suicide), as well as physical symptoms such as loss of appetite (weight loss), insomnia (difficulty falling asleep, difficulty sleeping soundly, early morning awakening), fatigue, easy fatigue, decreased libido, headache, heavy head, dizziness, and diarrhea and constipation. The number of people with depression or those considered to be at risk of depression is steadily increasing, and in recent years it has been reported that one in 15 people may suffer from depression at least once in their lifetime (Non-Patent Document 1).

One method of treating depression is pharmacotherapy using antidepressants. Tricyclic or tetracyclic antidepressants are commonly used as antidepressants, but they are known to cause strong side effects such as low blood pressure, drowsiness, dry mouth, constipation, forgetfulness, gastrointestinal disorders, and headaches. For this reason, selective serotonin reuptake inhibitors (SSRIs) and serotonin-noradrenaline reuptake inhibitors (SNRIs), which have fewer side effects, are often used in recent years. However, these drugs are known to cause strong nausea when first started taking them, and to cause headaches, dizziness, and general fatigue if suddenly stopped taking them or forgotten to take them. For this reason, there is a demand for treatment methods that are gentle on the body and have few or no side effects.

Incidentally, Lactobacillus gasseri, a type of Lactobacillus lactic acid bacteria, has long been known to have an effect of reducing psychological stress such as anxiety and tension (stress-reducing effect) (Patent Document 1). In addition, a fermented product of Lactobacillus pentosus, another type of Lactobacillus lactic acid bacteria, is also known to have an anti-stress effect (Patent Document 2). Stress has a variety of causes, including mental tension, mental instability during menopause, and anxiety and tension about the future, and excessive stress can lead to undesirable physical and mental symptoms such as irritability, social anxiety disorder, mental fatigue, and sleep disorders.

When the body is subjected to such stress, serotonin, a type of monoamine in the brain, is released, and the amount of serotonin in the brain decreases. This state continues for a certain period of time even after the stress is released, and the brain becomes deficient in serotonin, causing various symptoms such as lethargy, laziness, depressed mood, sleep disorders, chronic fatigue, loss of appetite, migraines, and hypersensitivity (Patent Document 3). These depressive symptoms observed after stress loading also support the theory that depression is related to a decrease in serotonin function in the brain (Non-Patent Document 2). The above-mentioned Patent Document 3 describes that Lactobacillus casei, a lactic acid bacterium of the Lactobacillus genus, has the effect of improving serotonin deficiency after stress is released. However, it is not clear whether Lactobacillus gasseri, which is known to have a stress-reducing effect, has an effect on depressive symptoms that occur after stress loading.

International Publication No. 2014/132982 JP 2010-155790 A International Publication No. 2017/047777

Yusuke Sakamoto, Takumi Ogawa, Mami Ogawa, Yumi Matsuo, Naoya Hashikawa, Narumi Hashikawa. Effects of 15 days of stress on behavior and hippocampal neurons in ICR mice. Yakugaku Zasshi. 2015, Vol. 135, No. 1, p. 151-158. Soichi Nomura; Special Edition Japanese Clinical Psychiatric Syndrome I 38:236-239, 2003 Collad M.C. et al, Intestinal integrity and Akkermansia muciniphila, a mucin-degrading member of the intestinal microbiota present in infants, adults, and the elderly. Appl. Environ. Microbiol. 73, 7767-7770 (2007). Everard A, et al. Cross-talk between Akkermansia muciniphila and intestinal epithelium controls diet-induced obesity. Proc. Natl. Acad. Sci. USA. 110: 9066-9071 (2013).

As described above, the object of the present invention is to provide an agent for improving depressive symptoms that has no or few side effects and is gentle on the body. More preferably, the object of the present invention is to provide an agent for improving depressive symptoms that can be suitably used to prepare food and drink for improving depressive symptoms that can be continuously ingested, and a pharmaceutical composition, a quasi-drug composition, and a food and drink composition for improving depressive symptoms that contain the agent for improving depressive symptoms.
Another objective of the present invention is to provide a composition for increasing Akkermancia bacteria present in the intestine.

The inventors of the present invention have conducted intensive research to solve the above problems, and have discovered that oral ingestion of Lactobacillus gasseri has the effect of improving depressive symptoms in addition to the aforementioned stress-reducing effect, and that it also acts on the intestinal flora to increase the bacteria belonging to Akkermancia, a type of beneficial bacteria present in the intestine. After further investigation, the inventors have completed the present invention.

The present invention has the following embodiments.
(I) Depression symptom improving agent
(I-1) An agent for improving depressive symptoms, comprising as an active ingredient Lactobacillus gasseri cells, a processed product thereof, or a fermented product containing them, the depressive symptoms including those occurring after being relieved from stress (hereinafter the same).
(I-2) The agent for improving depressive symptoms according to (I-1), wherein the Lactobacillus gasseri is Lactobacillus gasseri OLL2809 strain deposited under the accession number NITE BP-72.

(II) Composition for Improving Depressive Symptoms (II-1) A composition for improving depressive symptoms, comprising an effective amount of the agent for improving depressive symptoms described in (I-1) or (I-2).
(II-2) The composition according to (II-1), which is a pharmaceutical composition, a quasi-drug composition, or a food and drink composition.

(III) Method for using Lactobacillus gasseri cells, etc. (III-1) A method for using Lactobacillus gasseri cells or a processed product thereof, or a fermented product containing these, for imparting an effect of improving depressive symptoms to a pharmaceutical composition, a quasi-drug composition, or a food and beverage composition, the method comprising the step of preparing a pharmaceutical composition, a quasi-drug composition, or a food and beverage composition by blending Lactobacillus gasseri cells, a processed product thereof, or a fermented product containing these.
(III-2) The method of use according to (III-1), wherein the Lactobacillus gasseri is Lactobacillus gasseri OLL2809 strain deposited under the accession number NITE BP-72.

(IV) Use of Lactobacillus gasseri cells, etc. (IV-1) Use of Lactobacillus gasseri cells or a processed product thereof, or a fermented product containing these, for producing an agent for improving depressive symptoms, or a pharmaceutical composition, a quasi-drug composition, or a food and beverage composition containing the same.
(IV-2) The use according to (IV-1), wherein the Lactobacillus gasseri is the Lactobacillus gasseri OLL2809 strain deposited under the accession number NITE BP-72.

(V) Method for improving depressive symptoms (V-1) A method for improving depressive symptoms in a human or non-human mammal having depressive symptoms, comprising the step of administering to the human or non-human mammal a composition containing as an active ingredient Lactobacillus gasseri cells or a processed product thereof, or a fermented product containing these.
(V-2) The method according to (V-1), wherein the Lactobacillus gasseri is Lactobacillus gasseri OLL2809 strain deposited under the accession number NITE BP-72.

(VI) Composition for increasing intestinal Akkermancia bacteria (VI-1) A composition for increasing intestinal Akkermancia bacteria, comprising Lactobacillus gasseri cells or a processed product thereof, or a fermented product containing these as an active ingredient.
(VI-2) The composition for increasing intestinal Akkermancia bacteria according to (VI-1), wherein the Akkermancia genus is Akkermancia muciniphila.
(VI-3) A composition for increasing intestinal Akkermancia bacteria according to (VI-1) or (VI-2), wherein the Lactobacillus gasseri is Lactobacillus gasseri OLL2809 strain deposited under the accession number NITE BP-72.
(VI-4) Use of Lactobacillus gasseri cells or a processed product thereof, or a fermented product containing these, for producing a composition for increasing intestinal Akkermancia bacteria.
(VI-5) A method for increasing Akkermancia bacteria in the intestines of a human or non-human mammal, comprising the step of administering to a human or non-human mammal a composition containing Lactobacillus gasseri cells, a processed product thereof, or a fermented product containing these as an active ingredient. This method can also be said to be a method for changing the intestinal flora of a human or non-human mammal so that Akkermancia bacteria are increased.

The depressive symptom improving agent of the present invention can be added during the production of a pharmaceutical composition, a quasi-drug composition, or a food and beverage composition to impart a depressive symptom improving effect to the pharmaceutical composition, a quasi-drug composition, or a food and beverage composition. In other words, the depressive symptom improving agent of the present invention can be produced and provided as a pharmaceutical composition, a quasi-drug composition, or a food and beverage composition that exerts a depressive symptom improving effect. By using the composition for improving depressive symptoms of the present invention (pharmaceutical composition, quasi-drug composition, or food and beverage composition) thus produced, it is possible to improve and treat depressive symptoms in humans or non-human mammals.

The figures show the results of the (1) forced swimming test carried out in Experimental Example 1. In the figure, "*" indicates a significant difference of p<0.05 compared to the non-stressed group (control group), and "#" indicates a significant difference of p<0.05 compared to the stressed group (vehicle group) (t-test). 1 shows the results of the (2) sucrose preference test conducted in Experimental Example 1. In the figure, "*" indicates a significant difference of p<0.05 compared to the non-stressed group (control group) (t-test). 1 shows the results of the forced swimming test carried out in Experimental Example 2. In the figure, "*" indicates a significant difference of p<0.05 compared to the non-stressed group (control group). This shows the results of an analysis of the number of bacteria (Akkermancia muciniphila) in feces performed in Experimental Example 2. In the figure, "*" indicates a significant difference of p<0.05 compared to the non-stressed group (control group), and "#" indicates a significant difference of p<0.05 compared to the stressed group (vehicle group) (t-test).

(I) Agent for Improving Depressive Symptoms The agent for improving depressive symptoms that is the subject of the present invention is characterized in that it contains Lactobacillus gasseri cells as lactic acid bacteria as an active ingredient.

As long as it exerts the effect of the present invention (effect of improving depressive symptoms), any Lactobacillus gasseri may be used. Preferably, it is Lactobacillus gasseri OLL2809 (hereinafter, simply referred to as "OLL2809 strain") isolated from human adult feces. The strain has been deposited under the identification mark "Lactobacillus gasseri OLL2809" on February 1, 2005 (original deposit date) at the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) under the deposit number "NITE P-72", and on January 18, 2006, it was transferred from the original deposit to an international deposit based on the Budapest Treaty and deposited under the deposit number "NITE BP-72".

Lactobacillus gasseri, especially the OLL2809 strain, is a gram-positive rod-shaped bacterium, and its colonies on Lactobacilli MRS Agar (Difco) are round, pale yellow, and flat. Its physiological characteristics include homolactic fermentation, growth at 45°C, and fermentation of glucose, mannose, fructose, galactose, sucrose, cellobiose, lactose, and trehalose.

The medium for culturing Lactobacillus gasseri, particularly the OLL2809 strain, is not particularly limited and may be any medium normally used for culturing lactic acid bacteria. In other words, any medium that contains a suitable amount of a nitrogen source, inorganic substances and other nutrients in addition to a main carbon source may be used. As carbon sources, lactose, glucose, sucrose, fructose, starch hydrolysate, blackstrap molasses, etc. may be used depending on the assimilation ability of the bacteria. As nitrogen sources, organic nitrogen-containing substances such as casein hydrolysate, whey protein hydrolysate, and soy protein hydrolysate may be used. In addition, meat extract, fish meat extract, yeast extract, etc. may be used as a growth promoter, if necessary.

Cultivation is preferably carried out under anaerobic conditions, but may be carried out under microaerobic conditions using liquid static culture, which is commonly used. For anaerobic cultivation, known techniques such as cultivation under a carbon dioxide gas layer can be applied, but other methods may also be used. The cultivation temperature is generally preferably around 30 to 40°C, but other temperature conditions may be used as long as the bacteria can grow at that temperature. The medium during cultivation is preferably maintained at a pH range of 6 to 7, but other pH conditions may be used as long as the bacteria can grow at that pH. Cultivation may also be carried out under batch cultivation conditions. The cultivation time is usually preferably 10 to 24 hours, but other cultivation times may be used as long as the bacteria can grow.

As long as the effect of the present invention is achieved, the Lactobacillus gasseri cells may be live or dead. Live cells are preferred. As long as the effect of the present invention is achieved, the cells may be wet or dry. The depressive symptom improving agent of the present invention contains Lactobacillus gasseri cells and, as long as the effect of the present invention is achieved, is not limited to those containing the cells in an isolated and purified state, and may have a processed product thereof, or may have a fermented product (fermented product) using the cells or the processed product thereof. Here, the "processed product" is not limited, but may include, for example, a culture of Lactobacillus gasseri and various processed products thereof (for example, fractionated products such as culture supernatant and culture pellets; heat-treated products; sterilized products; crushed products; concentrated/diluted products; dried products such as spray drying, freeze drying, and vacuum drying; and products obtained by combining various processes). Furthermore, examples of "fermented products" include, but are not limited to, products obtained by fermenting edible animal ingredients (e.g., milk, seafood, meat, or processed products thereof) or edible plant ingredients (soybeans, vegetables, or processed products thereof) using Lactobacillus gasseri cells.

In addition, in order to enhance palatability, in the preparation of the fermented product, in addition to the active ingredient Lactobacillus gasseri, preferably the OLL2809 strain, other microorganisms can be used in combination for purposes other than the effect of the present invention, such as enhancing palatability. Examples of such microorganisms include, but are not limited to, Lactobacillus bacteria other than Lactobacillus gasseri, Bifidobacterium bacteria, Streptococcus bacteria, Lactococcus bacteria, Enterococcus bacteria, Bacillus bacteria, yeasts belonging to the Saccharomyces or Candida genera, etc. However, it is preferable that the depressive symptom improving agent of the present invention does not contain lactic acid bacteria or yeasts other than Lactobacillus gasseri, and contains only Lactobacillus gasseri, preferably only the OLL2809 strain, as the bacterial cells, processed products thereof, and fermented products thereof.

The depressive symptom improving agent of the present invention is used to impart a depressive symptom improving effect to a pharmaceutical composition, a quasi-drug composition, or a food and drink composition (including a feed composition; the same applies below) for humans or non-human mammals. In particular, it is used to manufacture a pharmaceutical, a quasi-drug, or a food and drink composition used to improve depressive symptoms in humans or non-human mammals. Here, non-human mammals include non-human primates, rodents (e.g., mice, rats, rabbits, guinea pigs, etc.), dogs, cats, etc. These non-human mammals include laboratory animals in which symptoms mimicking depressive symptoms have been artificially developed, such as the social defeat stress mouse described below. Thus, the depressive symptom improving agent of the present invention can be used as a raw material for manufacturing a pharmaceutical composition, a quasi-drug composition, or a food and drink composition, particularly a composition for improving depressive symptoms described below. The amount of the depressive symptom improving agent of the present invention in a pharmaceutical composition, a quasi-drug composition, or a food and drink composition varies depending on the form (dosage form) of the composition, the subject (human or non-human mammal, sex, etc.) and its symptoms, etc., and is not particularly limited, but can be appropriately selected and set within the range of 0.001 to 100% by mass. Therefore, the amount of Lactobacillus gasseri cells contained in the depressive symptom improving agent of the present invention can be set so that the finally prepared pharmaceutical composition, quasi-drug composition, or food and drink composition exhibits the effects of the present invention, and is preferably 1 x ¹⁰⁶ cells/g or more, although it is not limited.

The form of the depressive symptom improving agent of the present invention is not particularly limited, and may be in any form that can be added to and blended in the production of a pharmaceutical composition, a quasi-drug composition, or a food and drink composition. It may be a culture or processed product of Lactobacillus gasseri cells, or it may be prepared into various formulation forms (e.g., tablets, pills, powders, granules, capsules, suspensions, etc.) by blending any carrier or additive therewith.

The "depressive symptoms" targeted by the present invention are depressive symptoms in humans or non-human mammals that are mimicked in a social defeat stress model mouse (Kudryavtseva N.N., et al., Pharmacol. Biochem. Behav., 38, 315-320 (1991)), which is an animal model of depression, and are mood disorders or mood disorders that include any of the following symptoms: social avoidance (social withdrawal), lethargy (reduced energy), anhedonia, loss of interest, reduced concentration, decreased or increased appetite, abnormal sleep rhythm, increased fatigue or tiredness, etc. Preferably, lethargy or anhedonia. Various subtypes and symptoms of each type of depression, as well as methods for diagnosing them, are described in the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM IV), which can be referred to. Depressive symptoms include depressive symptoms that occur after release from stress load.

The social defeat stress model is created by placing a test animal (intruder animal) in the same cage as an aggressive animal (resident animal) and subjecting the test animal to social stress such as mental tension, anxiety, and fear. In particular, animals sensitive to stress, such as mice and rats, show social avoidance, lethargy (reduced vitality), and/or anhedonia even after being released from the stress load, and show behavioral changes similar to human depression in behavioral tests. Examples of behavioral tests include the forced swim test (e.g., Porsolt et al., Nature 266,730 (1977); and Petit-Demouliere et al., Psychopharmacology, 177, 245 (2005)), and the sucrose preference test (e.g., Kurre Nielson, et al., Behavioural Brain Research 107, 21-33, (2000)), as shown in the experimental examples described below. These tests are widely used to screen antidepressants.

[Forced swimming test]
Social defeat stress model animals are placed in a cylindrical container filled with water to a depth where the tail cannot reach the bottom, and swimming time and immobility time are measured during the test period. In animals in a depressed state, immobility time is prolonged, and administration of antidepressants reduces immobility time. This test allows us to measure the aforementioned depressive symptoms, particularly lethargy (reduced energy).
[Sucrose preference test]
A bottle containing sucrose water and a bottle containing drinking water are given to a social defeat stress model animal at the same time, and the percentage of animals that drink the sucrose water is calculated as sucrose preference. Normal animals that are not depressed prefer to drink the sucrose water, but the percentage of animals that drink the sucrose water decreases in depressed animals, and the sucrose preference is restored when an antidepressant is administered. This test can be used to measure the aforementioned depressive symptoms, particularly anhedonia.

The term "improvement" in the present invention includes the temporary, intermittent or sustained alleviation, mitigation, reduction or elimination of depressive symptoms occurring in humans or non-human mammals. It preferably includes recovery to a normal state or a state close to the normal state free of depressive symptoms. For this reason, pharmaceutical compositions or quasi-drug compositions prepared using the depressive symptom improving agent of the present invention can be used to treat depressive symptoms in patients with depression. Furthermore, food and drink compositions (including feed compositions) prepared using the depressive symptom improving agent of the present invention can be used to alleviate, mitigate, reduce or eliminate the symptoms in humans or non-human mammals having depressive symptoms.

(II) Composition for improving depressive symptoms The composition for improving depressive symptoms (hereinafter also referred to as "the composition for improving depressive symptoms") of the present invention contains the above-mentioned agent for improving depressive symptoms (hereinafter also referred to as "the agent for improving depressive symptoms") of the present invention, and can be prepared using the agent for improving depressive symptoms. That is, the composition for improving depressive symptoms contains Lactobacillus gasseri cells or a processed product thereof, or a fermented product containing these as an active ingredient. The composition for improving depressive symptoms includes pharmaceutical compositions, quasi-drug compositions, and food and drink compositions. In addition, the food and drink compositions include feed compositions as food and drink when the target is a non-human mammal, unless otherwise specified.

The ratio of the depressive symptom improving agent contained in the composition for improving depressive symptoms of the present invention is not particularly limited, as long as the composition for improving depressive symptoms contains the effective amount of Lactobacillus gasseri cells that exhibits the effect of the present invention.Specifically, although it varies depending on the use, form, and symptoms of the subject of the composition for improving depressive symptoms, when the composition for improving depressive symptoms is a pharmaceutical composition or a quasi-drug composition, it can be set and adjusted to contain 1 x ¹⁰⁶ or more, preferably 1 x ¹⁰⁷ or more, more preferably 1 x ¹⁰⁸ or more, and particularly preferably 1 x ¹⁰⁹ or more Lactobacillus gasseri cells per day of intake. Furthermore, in the case of a food or beverage composition, the settings can be adjusted so that the Lactobacillus gasseri cells are contained in a daily intake of 1 x 10 ^or more for humans, preferably 1 x ^{10 or} more, more preferably 1 x 10 ^or more, and particularly preferably 1 x 10 ^or more for non-human mammals, respectively.

The composition for improving depressive symptoms can be prepared in the conventional form of each product as a drug, quasi-drug, or food or beverage, depending on the application and method of administration. Food or beverage products are preferred.

When the present composition for improving depressive symptoms is prepared as a pharmaceutical or quasi-drug, examples of the form include tablets, pills, capsules, granules, powders, powders, liquids, syrups, suspensions, emulsions, suppositories, injections, etc. Oral administration is preferable. These various preparations can be formulated according to conventional methods using known auxiliary agents that can be normally used in the fields of pharmaceuticals and quasi-drugs, such as excipients, binders, disintegrants, lubricants, dissolving agents, suspending agents, coating agents, fillers, bulking agents, moisturizing agents, surfactants, and pH adjusters, in addition to the main agent, the present composition for improving depressive symptoms. In addition, various additives such as preservatives, antioxidants, colorants, flavorings, flavorings, sweeteners, and odorants can also be blended. The manufacturing method can be performed by a method well known in the art. The pharmaceutical composition or quasi-drug composition of the present invention prepared using the present composition for improving depressive symptoms can be administered to a patient with depression, and can be used for the treatment of the patient with depression, such as improving the depressive symptoms of the patient with depression. For patients with depression, a pharmaceutical composition or quasi-drug composition containing an effective amount of Lactobacillus gasseri bacteria described above can be administered once a day or in divided doses about two to three times a day.

When the composition for improving depressive symptoms is prepared as a food or drink, the form of the food or drink can be the various formulation forms described above, as well as the general form of processed food or drink. Examples of processed food or drink include, without limitation, dairy processed foods such as fermented milk, lactic acid bacteria drinks, dairy drinks, yogurt, cheese, and powdered milk (skim milk powder, modified milk powder, milk powder for lactating women, fat milk powder, etc.); beverages (non-alcoholic beverages, alcoholic beverages); vegetable processed foods; fruit processed foods; oil and fat processed foods; seafood processed foods; meat processed foods; luxury foods; seasonings; confectionery (Western confectionery, Japanese confectionery, chocolate, frozen desserts, ice cream, fresh confectionery, baked confectionery, candy, gummy, jelly, tablet confectionery, etc.); frozen foods; retort foods; canned foods; bottled foods; instant foods; liquid foods, etc. These foods and drinks include, for example, specific health foods and functional foods that claim to reduce, alleviate, or lower depressive symptoms, as well as functional foods such as nutritional composition-labeled foods, foods for the sick, and foods for special uses.

The manufacturing method of these foods and beverages can be performed by a method well known in the art. For example, in the case of yogurt, yogurt can be manufactured through a process of preparing a starter using Lactobacillus gasseri (preferably OLL2809 strain), a process of adding the starter to pretreated milk and culturing it, a cooling process, a flavoring process, a filling process, etc. Cheese can be manufactured through a process of adding Lactobacillus gasseri (preferably OLL2809 strain) as a starter to milk that has been pretreated such as pasteurized and then lactic acid fermenting it, a process of adding rennet to produce cheese curds, a curd cutting process, a whey discharge process, a salting process, a maturation process, etc. Alternatively, in the manufacture of the various dairy products, other lactic acid bacteria may be used as a starter, and Lactobacillus gasseri (preferably OLL2809 strain) or a processed product thereof may be added during the manufacturing process. The food and beverage composition of the present invention prepared in this manner can be ingested by a human or non-human mammal with depressive symptoms to alleviate, reduce, decrease or eliminate the depressive symptoms. Humans or non-human mammals can be administered the food and drink composition containing the above-mentioned effective amount of Lactobacillus gasseri cells once a day or in divided doses about two to three times a day.

Regarding the composition for improving depressive symptoms, the definitions of depressive symptoms, improvement, Lactobacillus gasseri, OLL2809 strain, bacterial cells, processed products, fermented products, etc., and the preparation methods thereof are as explained in section (I) above, and the descriptions therein also apply to this section.

(III) Composition for increasing intestinal Akkermancia bacteria As shown in Experimental Example 2, the Lactobacillus gasseri bacteria or its processed product, or the fermented product containing them, when orally ingested, act on the intestinal flora to promote the proliferation of Akkermancia bacteria present in the intestine, and exert the effect of increasing Akkermancia bacteria in the intestine (see Experimental Example 2). Therefore, the Lactobacillus gasseri bacteria or its processed product, or the fermented product containing them can be suitably used as a composition for increasing intestinal Akkermancia bacteria.

The Lactobacillus gasseri cells or processed products thereof, or fermented products containing these, used herein, are as described in (I) above, and the description can be used herein as well. The composition for increasing intestinal Akkermancia bacteria that is the subject of the present invention is a composition to be taken orally, and is preferably a food or drink composition (including a feed composition). The form (including dosage form), the amount of Lactobacillus gasseri cells to be blended in the composition, and the number of times of administration (ingestion), etc., can be described in (I) and (II) above.

The bacteria belonging to Akkermancia are not particularly limited as long as they are intestinal bacteria present in the intestines of humans or non-human mammals. Akkermancia muciniphila is preferred. A. muciniphila is a gram-positive bacterium capable of assimilating mucin. It has been reported that this bacterium has an anti-inflammatory effect and blood glucose level improving effect via the effect of improving the intestinal mucosal barrier (Non-Patent Document 4), and it is an intestinal bacterium that has attracted attention, including the development of it as a pharmaceutical product. Therefore, the composition for increasing intestinal Akkermancia bacteria of the present invention can be orally ingested to improve the intestinal flora, and is expected to have an effect of improving the intestinal mucosal barrier, or various health functional effects mediated by this.

As mentioned above, in this specification, the terms "comprise" and "contain" include the meanings of "consist of" and "consist essentially of."

The present invention will be explained below using experimental examples to aid in understanding the configuration and effects of the present invention. However, the present invention is not limited in any way by these experimental examples. Unless otherwise specified, the following experiments were carried out at room temperature (23±5°C) and atmospheric pressure. In addition, the animals used in the following experiments, as well as the experiments and care of the animals, were approved by the Okayama University of Science Animal Experiment Management Committee and were conducted under conditions that conform to the guidelines set forth by the university, which comply with domestic and international laws and policies.

Experimental Example 1 Improvement of depressive symptoms
1. Preparation of Lactobacillus gasseri OLL2809
Lactobacilli gasseri OLL2809 was stimulated and cultured twice in Lactobacilli MRS Broth (Difco) (37°C, 18 hours). The stimulated culture was inoculated into the same medium at 1% and cultured at 37°C for 18 hours. After harvesting, the cells were washed once with sterile distilled water.

2. Creation of a social defeat stress model animal Eight-week-old male C57BL/6J mice (average body weight 23.3±0.47g) were divided into two groups: a non-stressed group (control group) (non-STRESS: n=13 mice) and a stressed group (STRESS: n=18 mice). For the stressed group (stressed mice), a male ICR mouse (resident mouse) aged about 30 weeks, which is larger and more aggressive than the mice, was placed in the same cage for 10 minutes per day for 4 weeks (Day 1 to Day 29) to expose the mice to fear and defeat stress. Specifically, one resident mouse was placed in one cage, and one stressed mouse was placed in the cage and allowed to face each other for 10 minutes. The cage was then separated by a transparent partition with holes and the mice were allowed to stay there for 24 hours. The next day, the stressed mouse was placed in a cage with another resident mouse and exposed to the same stress. Both the non-stressed group (control group) and the stressed group were housed under controlled environmental conditions (23±2°C, 60% relative humidity, 12-hour light/dark cycle (lights on at 7am, lights off at 7pm), food and water available ad libitum).

3. Behavioral test A forced swimming test was performed on the day after the last day of stress loading (Day 30). The stressed groups were then divided into two groups, a normal diet group (vehicle group) and a Lactobacillus gasseri OLL2809-ingested group (OLL2809 group), and were reared for 2 weeks (Day 30 to Day 44) under the same controlled environmental conditions as above. The L. gasseri OLL2809-ingested group (OLL2809 group) was given one ball-shaped ball made by adding 3.5 g of powdered AIN-93M (manufactured by CLEA Japan, Inc.) to 1.75 mL of water and L. gasseri OLL2809 (2 x 10 ⁹ cfu) per day. On the other hand, the vehicle group was given one ball-shaped ball made by adding 3.5 g of powdered AIN-93M to 1.75 mL of water per day. After two weeks of rearing, a forced swimming test was performed as a behavioral test on Day 45. In addition, a sucrose preference test was conducted for four days from Day 45 to Day 48.

(1) Forced swimming test: 800 mL of water adjusted to 28°C in a thermostatic bath was placed in a 1 L glass cylindrical beaker (height 15 cm, diameter 11.5 cm), and mice (non-stressed group [control group] (n=9), stressed group [vehicle group (n=9), OLL2809 group (n=9)]) were immersed in the water for 6 minutes and forced to swim. Behavior was observed for 5 minutes from 1 minute after immersion, and the time the mice remained floating on the water surface without struggling (including the time when the mice made slight movements to get their head out of the water) was recorded as immobility time (seconds).

(2) Sucrose Preference Test Two water bottles, one containing water and the other containing a 2% by mass sucrose solution, were prepared and mice (control group (n=4), vehicle group (n=9), OLL2809 group (n=9)) were allowed to freely consume water for two days from Day 45 to Day 46 (habituation learning). On the second day, the two water bottles were switched and the mice were allowed to freely consume water and sucrose solution for two days (Day 47 to Day 48), and the amount of water consumed by each of the water bottles was measured every four hours. The value of "(sucrose solution intake/total amount of water consumed) x 100" (%) was calculated from the average values of the total amount of water consumed and the amount of sucrose solution consumed over four hours for two days, and this was used to evaluate sucrose preference.

4. Test Results The results of the forced swimming test are shown in Figure 1, and the results of the sucrose preference test are shown in Figure 2.
As shown in Figure 1, in the forced swimming test, the immobility time (seconds) was significantly increased in the stressed group (stressed group [vehicle group]) compared with the non-stressed group (non-stressed group [control group]). In contrast, the immobility time was significantly decreased by ingestion of L. gasseri OLL2809 (OLL2809 group). Although we are not bound by the reason (cause), it is considered that the immobility time was significantly increased in the stressed group due to the occurrence of depressive symptoms (lethargy, decreased vitality, easy fatigue), but the depressive symptoms were improved (alleviated, mitigated, decreased) by ingestion of L. gasseri OLL2809.

As shown in Figure 2, in the sucrose preference test, the amount of sucrose intake was significantly decreased by stress (vehicle group), whereas the amount of sucrose intake was significantly increased by feeding L. gasseri OLL2809 (OLL2809 group) to the same level as the non-stressed group [control group]. Although we are not bound by the reason (cause), it is considered that the stressed group developed depressive symptoms (decrease/loss of interest and pleasure) and no longer preferred to consume sucrose, but the depressive symptoms were improved (alleviated/mitigated/reduced) by feeding L. gasseri OLL2809.
From the above results, it is recognized that L. gasseri OLL2809 has the effect of improving depressive symptoms.

Experimental Example 2: Effect on gut flora Stress load changes gut flora through nervous system, endocrine system or immune system, while gut flora also affects host stress response through these signal transduction pathways.So, the effect of stress on gut flora, especially the effect of Lactobacillus gasseri administration on gut flora, was evaluated by preparing social defeat model mice in the same manner as in Experimental Example 1, and performing forced swimming test.

1. Test Method A social defeat model mouse (stressed group) was prepared in the same manner as described in Experimental Example 1. Specifically, 8-week-old male C57BL/6J mice were divided into a non-stressed group (control group, n=7) and a stressed group (OLL2809 group [n=5], vehicle group [n=5]), and among them, the stressed group (stressed mice) was subjected to social defeat stress every day for 4 weeks, and then, for 2 weeks, the OLL2809 group was fed a diet containing L. gasseri OLL2809 (2 x ¹⁰⁹ cfu) added to rice balls, and the vehicle group was fed a diet containing only water added to rice balls.
After feeding each diet every day for 2 weeks, feces were collected from each group of mice before the forced swimming test. 20 mg of feces was weighed into EasyBeads (AMR). Feces were crushed using FastPrep (MP-Biomedicals), and DNA was extracted using the RSC PureFood GMO and Authentication Kit (Promega) and an automated nucleic acid extractor (Maxwell, Promega). The extracted DNA was used to quantify the number of Akkermancia muciniphila bacteria by real-time PCR.

Real-time PCR was performed with reference to the method of Collado et al. (Non-Patent Document 3). The PCR reaction solution consisted of 10 μL of PowerUP SYBR Green Master Mix (Applied Biosystems), 4 μL of diluted solution of extracted DNA, 0.18 μL each of forward and reverse primers adjusted to 100 pmol/μL, and 5.64 μL of ultrapure water, totaling 20 μL. The PCR reaction conditions were as follows: the reaction solution was heated at 50°C for 2 minutes and 95°C for 2 minutes, followed by 40 cycles of denaturation at 95°C for 15 seconds and annealing/extension reaction at 60°C for 1 minute.
PCR reactions were carried out using a QuantStudio 3 real-time PCR system (Applied biosystems).
The forward and reverse primers used had the following sequences:
AM1: 5'CAG CAC GTG AAG GTG GGG AC (SEQ ID NO: 1);
AM2: 5'CCT TGC GGT TGG CTT CTT CAG AT (sequence number 2).

Akkermancia muciniphila ATCC BAA-835 obtained from ATCC (American Type Culture Collection) was used to create a standard curve. The strain was cultured according to a prescribed method, and DNA was extracted from 10 ¹¹ cfu of the prepared cells in the same manner as above, and a standard curve was created in the range of 10 ⁵ to 10 ⁹ cfu. The ΔΔCt method was used for analysis.

2. Test results (1) Forced swimming test The results of the forced swimming test are shown in Figure 3. As in Experimental Example 1, the immobility time (seconds) was significantly increased in the vehicle group compared to the control group. On the other hand, because the number of subjects was small, the OLL2809 group did not show a significant decrease compared to the vehicle group, but did not show a significant increase compared to the control group. As in Experimental Example 1, it was observed that oral intake of Lactobacillus gasseri can improve (reduce, alleviate, or reduce) depressive symptoms.

(2) A. muciniphila count in feces Figure 4 shows the results of the analysis of A. muciniphila count in feces. As shown in Figure 4, the fecal A. muciniphila count did not change in the vehicle group compared to the control group. On the other hand, a significant increase was observed in the OLL2809 group compared to the vehicle and control groups. A. muciniphila is an intestinal bacterium that has been reported to have anti-inflammatory effects and blood glucose improvement effects via the intestinal mucosal barrier improvement effect, so oral intake of OLL2809 can be expected to improve the intestinal mucosal barrier and various health function effects mediated by it.

SEQ ID NOs: 1 and 2 show the base sequences of the forward and reverse primers for PCR used to quantify the number of Akkermancia muciniphila bacteria.

Claims (3)

A depressive symptom improving agent comprising, as an active ingredient, Lactobacillus gasseri cells, a processed product thereof, or a fermented product containing the same,
A depressive symptom improving agent, wherein the Lactobacillus gasseri is Lactobacillus gasseri OLL2809 strain deposited under accession number NITE BP-72. 2. A pharmaceutical composition, a quasi-drug composition, or a food and drink composition for improving depressive symptoms, comprising an effective amount of the agent for improving depressive symptoms according to claim 1 . A method for using Lactobacillus gasseri cells or a processed product thereof, or a fermented product containing the same, for imparting a depressive symptom improving effect to a pharmaceutical composition, a quasi-drug composition, or a food and drink composition, the method comprising the step of preparing a pharmaceutical composition, a quasi-drug composition, or a food and drink composition by blending Lactobacillus gasseri cells or a processed product thereof ,
The method for use, wherein the Lactobacillus gasseri is Lactobacillus gasseri OLL2809 strain deposited under accession number NITE BP-72.

Images