TW202243684A - Antidepressants, antiaging agents and antiobesity agents to determine novel properties of Lactobacillus kosoi10H strain of lactic acid bacteria - Google Patents

Abstract

An object of the present invention is to determine novel properties of Lactobacillus kosoi10H strain of lactic acid bacteria, and to provide new uses based on the novel properties. A solution of the present invention is to provide an antidepressant, antiaging agent or antiobesity agent containing bacterial cells of Lactobacillus kosoi (Lactobacillus kosoi) 10H strain deposited under the registration number NITE BP-02811 as an active ingredient.

Description

Antidepressants, antiaging agents and antiobesity agents

The present invention relates to an antidepressant, antiaging agent and antiobesity agent containing bacterium of Lactobacillus kosoi10H strain of lactic acid bacteria.

In the past, it has been known that lactic acid bacteria or fermentation products thereof have various physiological functions. For example, Patent Document 1 discloses that plant-derived lactic acid bacteria belonging to the genus Lactobacillus or the like have an action of activating intestinal immunity and an action of promoting IgA antibody production of Peyer's patch cells. Moreover, there is the following report in Patent Document 2: the active substance produced by germ lactic acid bacteria (Lactobacillus plantarum) contributes to the increase of Cisd2 gene expression (gene expression), the reduction of mitochondrial damage (mitochondrial damage), and the delay of neurodegeneration ( neurodegeneration) and sarcopenia (sarcopenia) and other aging states to promote longevity. Furthermore, Patent Document 3 discloses that a composition containing a specific strain of lactic acid bacteria isolated from kimchi has a preventive or therapeutic effect on aging and dementia.

The applicant found that the Lactobacillus kosoi10H strain isolated from the vegetable brown sugar fermentation liquid is a good fructophilic lactic acid bacterium (fructophilic lactic acid bacteria) with a novel genome structure (genome structure) different from other lactic acid bacteria, and has excellent immunity. Activation (refer to Patent Document 4).

[Patent Document 1] Japanese Patent Laid-Open No. 2007-308419 [Patent Document 2] Japanese Patent Laid-Open No. 2020-59694 [Patent Document 3] Japanese Patent No. 6009080 [Patent Document 4] Japanese Patent Laid-Open No. 2020-92704

The active substance described in Patent Document 2 is considered to be produced by a specific strain of Lactobacillus germinus GKM3, and is based on the effect of increasing the expression of longevity genes. In Patent Document 3, it is described that a large number of lactic acid bacteria strains isolated from kimchi have been examined, and only specific strains such as Lactobacillus pentosus (Lactobacillus pentosus) variant plantarum C29 have antioxidant and anti-aging properties. and the prevention or treatment of dementia. On the other hand, the Lactobacillus kosoi10H strain recorded in Patent Document 4 has relatively small genosomes for other lactic acid bacteria, and has low resource conversion ability of glucose (glucose), and uses fructose (fructose) as an appropriate carbon source ( Carbon source) is characterized by capitalization. Although it was revealed that this strain promotes the production of IgA in the intestinal mucosa and has an excellent immunostimulatory effect, other effects are not clear.

The object of the present invention is to clarify the novel properties of the Lactobacillus kosoi10H strain of such lactic acid bacteria, and to provide new uses based on the novel properties.

In order to solve the above-mentioned problems, the inventors of the present invention found that a composition containing bacterial cells of the Lactobacillus kosoi10H strain has an antidepressant effect as a result of diligently examining the new usefulness of the Lactobacillus kosoi10H strain in an experimental system using an animal model. , anti-aging effect and anti-obesity effect and completed the present invention. That is, the present invention includes the following embodiments.

(1) An antidepressant comprising, as an active ingredient, a lactic acid bacterium kosoi (Lactobacillus kosoi) 10H strain deposited under deposit number NITE BP-02811. (2) The antidepressant as in (1), wherein the bacteria are dead bacteria. (3) The antidepressant according to (1) or (2), which is in the form of a drug, food or drink, or an active ingredient composition mixed with a drug or a food or drink. (4) An anti-aging agent containing as an active ingredient the bacterial cells of Lactobacillus kosoi (Lactobacillus kosoi) 10H strain deposited under the registration number NITE BP-02811. (5) The anti-aging agent as in (4), wherein the bacteria are dead bacteria. (6) The anti-aging agent of (4) or (5), which is in the form of a pharmaceutical, food or drink, or an active ingredient composition blended with a medicine or food or drink. (7) The antiaging agent according to (6), wherein the aforementioned food and beverages are food and beverages for beauty or health promotion. (8) An anti-obesity agent comprising, as an active ingredient, cells of Lactobacillus kosoi (Lactobacillus kosoi) 10H strain deposited under the registration number NITE BP-02811. (9) The use of a lactic acid bacterium kosoi (Lactobacillus kosoi) 10H strain, which is used to improve depressive symptoms in humans or non-human mammals, and to manufacture anti-aging or anti-obesity medicines , food and beverages, or active ingredient compositions compounded with pharmaceuticals and food and beverages.

According to the present invention, antidepressants, antiaging agents, antiobesity agents, beauty food and beverages, and health improvement food and beverages containing the lactic acid bacteria kosoi (Lactobacillus kosoi) 10H strain can be provided as novel applications.

Next, various embodiments of the present invention will be described with reference to the drawings. In addition, each embodiment described below does not limit the invention related to the scope of claims, and all elements and combinations thereof described in each embodiment are not necessarily essential to the solution means of the present invention.

(I), the lactic acid bacteria of active ingredient The active ingredient in one embodiment of the present invention is a specific strain of lactic acid bacteria isolated from a vegetable brown sugar fermentation liquid and a variant thereof. It is preferably a bacterium belonging to the genus Lactobacillus obtained from a culture solution under the trade name [Georina (registered trademark) enzyme]), more preferably Lactobacillus kosoi (Lactobacillus kosoi) 10H strain (registration number NITE BP-02811) or a variant thereof. [Variant strain] refers to a specific strain that does not mutate within the range that a person skilled in the art does not impart major changes in its properties by a method known to those skilled in the art, or if it is equivalent Those who are familiar with the technology can confirm it.

In addition, the lactic acid bacterium kosoi (Lactobacillus kosoi) 10H strain was deposited on November 7, 2018 (the original deposit date) in Japan's independent administrative agency product evaluation technology base institution, Patent Microorganism Deposit Center (292-0818 Japan Room 122, 2-5-8 Kamizu Kamiso, Kisarazu City, Chiba Prefecture, Japan). The registration number is NITE BP-02811 (hereinafter referred to as the strain [10H]). Furthermore, the taxonomic properties of the 10H strain are described in papers published by the present inventors (Chiou T-Y et al, Antonie van Leeuwenhoek (2018), 111:1149-1156) and Patent Document 4, all of which are incorporated by reference constituted by this manual.

(characteristic of lactic acid bacteria kosoi10H strain) This strain can be considered to be a bacterium evolved from a known lactic acid bacteria (Filannino et al. "Fructose-rich niches traced the evolution of lactic acid bacteria toward fructophilic species”Critical Reviews in Microbiology, Vol. 45, No. 1, 2019, pp.65-81). FLAB inhabits environments rich in fructose, such as the digestive tract of insects that feed on flowers, fruits, fermented foods, or fructose as a staple food. It is said that FLAB is a heterofermentative lactic acid bacterium that prefers to use fructose instead of glucose as a carbon source, and promotes growth in the presence of glucose by adding an electron acceptor substrate such as oxygen. The 10H strain has a relatively small genome size and low GC content for other FLABs and lactic acid bacteria (see Figure 3 of Filannino et al.). Therefore, reclassifying the genus of Lactobacillus and classifying this strain as a new genus of Apilactobacillus has also been proposed (refer to Zheng et al.International Journal of Systematic and Evolutionary Microbiology, Vol. 70, No. 4, 2020, pp. 1-77). Therefore, the active ingredient of the present invention has a bacterial cell component different from conventional lactic acid bacteria, and there is a possibility of exhibiting a new action by the bacterial cell component.

(II), antidepressants In this specification, the term [depression] is broadly defined as a depressed mood, a marked loss of interest or joy as the main symptom, and further includes sleep disturbance or thinking, loss of concentration, suicidal ideation or suicide attempt, etc. Mood or psychiatric disorders, regardless of the physiological or pharmacological background to the onset. Furthermore, the term [depression] also includes in particular anxiety states. Therefore, the term [antidepressants] refers to preventive or therapeutic agents widely used in the treatment of these disorders.

The antidepressant of this embodiment means a composition effective for improving the depression state of the subject, and contains bacteria of Lactobacillus kosoi (Lactobacillus kosoi) 10H strain (registration number NITE BP-02811) as an active ingredient. The term "improvement" used in this specification includes temporary, intermittent or continuous alleviation, alleviation, reduction or disappearance of the melancholic state that occurs in humans or non-human mammals. Preferably a normal state or near normal state comprising a return to a depressive state. Therefore, the antidepressant of the present embodiment includes pharmaceuticals, food and beverages, or forms of active ingredient compositions mixed with pharmaceuticals and food and beverages, etc., as will be described in detail below. Furthermore, health food is also preferable among medicines and foods, and can be used to alleviate, relieve, decrease or disappear the symptoms of depression in humans or non-human mammals.

(III), anti-aging agent and anti-obesity agent The term "anti-aging" refers to those broadly defined to alleviate, improve, suppress, prevent and treat skin diseases such as spots, wrinkles and dullness, myocardial infarction, cerebral infarction, osteoporosis ( Osteoporosis), Alzheimer's disease, Rheumatoid arthritis, diabetic angiopathy, cataract, etc. Preferably, it means that it has the effect of preventing or improving skin aging. Specifically, it refers to the prevention or improvement of external factors (ultraviolet rays, drying of the air, etc.) or aging of the skin's moisturizing function or elasticity, loss of skin tension and radiance, roughness, wrinkles, dullness, etc. symptom. The term "anti-aging agent" refers to agents used for such anti-aging. Furthermore, since the above-mentioned aging symptoms are also related to the reduction of metabolic function or weight gain (obesity) accompanied by excessive intake of energy, it is also considered useful to suppress obesity in order to exert an anti-aging effect. Therefore, one embodiment of the present invention provides an anti-obesity agent containing the bacterial cells of Lactobacillus kosoi (Lactobacillus kosoi) 10H strain as an active ingredient.

(IV), form of agent The antidepressant, antiaging agent, or antiobesity agent of this embodiment can be used in the form of pharmaceuticals, food or beverages, or active ingredient compositions blended with pharmaceuticals or food or beverages. In the case of using the active ingredient composition, the lactic acid bacteria cells of the active ingredient are cultivated according to the conventional method of lactic acid bacteria culture, and not only those isolated from the obtained culture by means of collecting bacteria such as centrifugation can be used as they are, but also can be used. : The culture, fermentation liquid (including culture supernatant), crude or refined products of the culture, freeze-dried products of the above, or the cytoplasm or cell wall division of the bacteria treated with enzymes or physical means.

In addition, the bacterial cells are not limited to live bacterial cells, but may be those sterilized by a usual general heat sterilization operation. Lactobacillus kosoi (Lactobacillus kosoi) 10H strain is expected to have a firm cell surface structure because it grows under severe growth conditions of high sugar concentration (high osmotic pressure). The heat treatment is preferably at 65-85°C for 1 minute or more, such as about 10 minutes, 30 minutes or 60 minutes, and more preferably at 70-75°C for 1 minute or more, such as about 5 minutes, 10 minutes or 30 minutes. Even heat-treated bacteria can not only expect anti-depressant, anti-aging and anti-obesity effects, but also live bacteria may cause morphological changes during distribution or display after product manufacturing, so no further Morphologically altered heat-sterilized dead bacteria can be suitably used.

Furthermore, the active ingredient composition of the present embodiment can also contain an appropriate amount of nutritional components suitable for maintenance, growth, etc. of the 10H strain as needed. Specific examples of the nutrient components include: fructose, glucose, sorbose (sorbose), ribose (ribose), lyxose (lyxose), xylose (xylose), arabic Carbon sources such as arabinose, lactulose, sucrose, etc.; nitrogen sources such as yeast extract, peptone, etc.; vitamins, minerals, trace metal elements, and other nutrients ingredients etc. Examples of vitamins include vitamin B, vitamin D, vitamin C, vitamin E, and vitamin K. As trace metal elements, zinc, selenium, etc. can be illustrated, for example. Examples of other nutritional components include lactooligosaccharides, soybean oligosaccharides, lactulose, lactitol, fructooligosaccharides, and galactooligosaccharides. ) and other various oligosaccharides (oligosaccharide). The blending amount of these oligosaccharides is not particularly limited, but it is usually preferably selected from an amount range of about 1 to 30% by weight in the composition of the present embodiment.

The compounding amount of the 10H strain in the active ingredient composition of the present embodiment can generally be appropriately selected so that the number of bacteria is about 10 ⁸ to 10 ¹³ (not necessarily the number of viable bacteria) in 100 g of the composition. The number of viable bacteria can be determined by the limiting dilution-culture method using MRS liquid medium containing 10% fructose. Since the number of viable bacteria is related to turbidity, if the correlation between the number of viable bacteria and turbidity is obtained in advance, instead of measuring the number of viable bacteria, the number of viable bacteria can be calculated by measuring the turbidity. The active ingredient composition of this embodiment is suitably blended with an appropriate edible carrier (food material) or a pharmaceutically acceptable carrier, and it is preferably prepared into a form such as a food or drink or a pharmaceutical to be described later. The form of pharmaceuticals and food and beverages containing the active ingredient composition will be specifically described below.

(Pharmaceuticals) In the case of pharmaceuticals, the antidepressant, antiaging agent, or antiobesity agent of this embodiment is prepared in the form of a general pharmaceutical composition using an appropriate formulation carrier that is pharmaceutically acceptable together with the 10H strain. practical. As the preparation carrier, diluents such as fillers, extenders, binders, moisturizers, disintegrating agents, surfactants, lubricants, etc. known in this field are generally exemplified. or excipient. These are appropriately selected and used according to the dosage unit form of the preparation to be obtained.

Various forms can be selected as the dosage unit form of the pharmaceutical composition, but preferred ones include oral administration preparations and external administration preparations. Typical preparations for oral administration include tablets, pills, powders, solutions, suspensions, emulsions, fine granules, capsules and the like.

When forming into the form of tablets, as the above-mentioned preparation carrier, for example, lactose, white sugar, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin (kaolin), crystalline cellulose, silicic acid, potassium phosphate, etc. can be used. Excipients: water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethyl cellulose, hydroxypropyl cellulose, Binders such as methylcellulose and polyvinylpyrrolidone; carboxymethylcellulose sodium, carboxymethylcellulose calcium, low-substituted hydroxypropyl cellulose ( Disintegrants such as hydroxypropyl cellulose with low degree of substitution), dry starch, sodium alginate, agar powder, laminaran powder, sodium bicarbonate, calcium carbonate, etc. ; Surfactants such as polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, monoglyceride stearate, etc.; white sugar, stearin ), cocoa butter (cocoa butter), disintegration inhibitors such as hydrogenated oil (hydrogenated oil), absorption accelerators such as quaternary ammonium base (ammonium base), sodium lauryl sulfate, etc.; moisturizers such as glycerin (glycerin) and starch ;Adsorbent of starch, lactose, kaolin, bentonite, colloidal silicic acid, etc.; Lubricant of refined talc, stearate, boric acid powder, polyethylene glycol, etc. agent etc. Furthermore, the lozenge can be a lozenge coated with a usual coating as needed, such as a sugar-coated lozenge, a gelatin-coated lozenge, an enteric-coated lozenge, a film-coated lozenge, or a double or multi-layer lozenge.

When forming into a pill form, as a preparation carrier, for example, excipients such as glucose, lactose, starch, cocoa butter, hardened vegetable oil, kaolin, and talc; gum arabic powder, tragacanth, etc. can be used. Gum (tragacanth) powder, binders for gelatin, ethanol, etc.; disintegrating agents for kelp sugar, agar, etc.

In addition, coloring agents, preservatives (preservatives), fragrances, flavoring agents, sweeteners (edulcorants), and other pharmaceuticals can also be contained in the pharmaceutical composition as needed.

The administration method of the above-mentioned pharmaceutical composition is not particularly limited, and is determined according to various preparation forms, age, sex and other conditions of the patient, degree of disease, and the like. In addition, although the dose is appropriately selected according to the usage, age, sex and other conditions of the patient, the degree of the disease, etc., it is usually about 0.5 to 100 mg per 1 kg of the body weight of the above-mentioned active ingredient composition. , the preparation can be divided into 1 to 4 times a day and administered to humans.

(drinks) In the case of the antidepressant, antiaging agent, or antiobesity agent of the present embodiment, for example, fermented milk, lactic acid bacteria beverage, fermented vegetable beverage, fermented fruit beverage, fermented soybean milk beverage, etc. are exemplified. [Fermented milk] refers to milk or dairy products fermented with lactic acid bacteria, bifidus or yeast to make a paste or liquid. Therefore, the fermented milk includes a beverage form and also includes a yogurt form. Furthermore, [lactic acid bacteria beverage] refers to a beverage that uses the following as the main ingredient and dilutes it in water: milk or dairy products are fermented with lactic acid bacteria, bifidus or yeast to make a paste or liquid.

Examples of other forms of food and drink include fermented foods such as pickled vegetables, miso, fermented tea, and bread; foods for infants such as weaning foods, milk powder, and infant foods; foaming preparations, chewing gum, and soft candies Cakes, puddings, etc.; noodles; nutritional supplements such as capsules, granules, powders, lozenges, etc.; dairy products other than the aforementioned fermented milk and lactic acid bacteria beverages, etc. In particular, processed foods that require a heating process are preferable because they contain active ingredients that maintain their functionality even when heated. As a particularly preferred form, processed foods that require heating and conditioning in terms of hygiene management, such as nursing foods, are mentioned. The food and drink of this embodiment can provide an antidepressant, an antiaging agent, or an antiobesity agent which is stable even by heating at 75° C., which is effective for preventing food poisoning.

The term "drinks" in this specification refers to all forms (including beverages, for example) that are exclusively orally used for eating and drinking. Even if it is a form such as a lozenge, as long as it is exclusively used for eating and drinking, it is included in this specification. food and drink. For example, based on the concept of improving depression or anti-aging, etc., health food, health supplement food, food for the sick, nutritional supplement food or health functional food (food for specific health use) stipulated by the Japanese Ministry of Health, Labor and Welfare, etc. , nutritional functional food) are also included in the food and beverages in this specification.

As a preferable embodiment, food and drink for cosmetics are mentioned. The term "cosmetic use" refers to the purpose of suppressing the progress of aging of the subject and maintaining or improving the beautiful appearance, which also includes improving the beautiful appearance of skin and hair, preventing obesity, and the like. In particular, in the examples described later, aging indicators such as the appearance of hair growth, the luster of the hair, and the condition of the skin around the eyes of the aging mice that orally ingested the active ingredient of the present embodiment are improved, so it can be expected Improvement of the appearance of beauty obtained by suppressing the aging of the subject, that is, use as a food and drink for beauty. Furthermore, the cosmetic food and drink of this embodiment can also be used as an anti-obesity agent for oral administration, a skin (skin) anti-aging (prevention) agent, a skin (skin) elasticity improving agent, an anti-wrinkle agent, and a skin texture improving agent. , Use as a hairdressing improver, laxative improver, body condition improver, and physique improver.

The above-mentioned food and beverages for beauty can be used as food and beverages for health improvement from a different point of view because they can adjust the physical condition of the subject and improve their physique through the anti-aging effect and anti-obesity effect. In this specification, "health improvement" refers to a "healthy" state that not only means not getting sick or weak, but also enhancing the physical, mental, and social well-being.

Furthermore, the food and beverages for beauty and health improvement may contain physiologically active substances, (food) raw materials, (food) additives, etc. as appropriate, in addition to the above-mentioned active ingredients. As additives/raw materials, for example, they are used by adding, mixing, impregnating and other methods in the manufacture, processing and storage of food, such as excipients, extenders, thickeners, Binders, lubricants, stabilizers, colorants, emulsifiers, solubilizers, food colorings, fragrances, seasonings, spices, sweeteners, preservatives, and are commonly used as spices.

Content of the said active ingredient composition in the food-drinks according to this embodiment is not specifically limited, It can determine suitably. From the viewpoint of achieving the improvement of depression and the anti-aging effect, the total mass of each food and drink is, for example, preferably 0.001% by mass or more, more preferably 0.01% by mass or more, and even more preferably 0.1% by mass or more. On the other hand, the upper limit of the content of the active ingredient composition in food and beverages is not particularly limited. For example, it is also possible to ingest freeze-dried bacteria as the active ingredient composition as it is in the examples described later, but usually it can be combined with a diet. The shape of the product is adjusted appropriately.

(V), the preparation method of the active ingredient composition with anti-depressant, anti-aging or anti-obesity effect In another aspect of the present invention, a method for producing an active ingredient composition having antidepressant, antiaging or antiobesity effects is provided. Fig. 1 is a flowchart showing the manufacturing process of the active ingredient composition. The method comprises: the seed culture program (S01) of preparing the seed culture solution from the preserved strain of the lactic acid bacteria kosoi10H strain; the main culture program (S02) of using the obtained seed culture solution to cultivate the culture medium for bacterium production; The bacteria cleaning procedure (S03) for recovering and cleaning the bacteria from the culture medium; the heat treatment procedure (S04) for heat-treating the cleaned bacteria; the recovery procedure (S05) for recovering the heat-treated bacteria.

The seed culture program (S01) is to plant the preserved strain of lactic acid bacteria kosoi10H strain such as freeze-dried bacteria or frozen preserved bacteria and use the medium suitable for the growth of this bacterial strain, such as the MRS medium that contains D-fructose, etc. It is obtained by culturing at C for about 16-96 hours. The seed culture solution is inoculated into the main culture solution, and the inoculation amount is preferably in the range of 1-10% (v/v).

The main culture step (S02) is not particularly limited as long as the seed bacteria obtained in the above-mentioned step (S01) can be grown, and a method generally used for culturing lactic acid bacteria can be appropriately modified and used as needed. For example, the culture temperature is preferably 20-40°C, more preferably 25-35°C. Furthermore, the cultivation may be carried out under either aerobic conditions or anaerobic conditions, but it is better to carry out under anaerobic conditions, for example, while passing an anaerobic gas such as carbonic acid gas (carbonic acid gas). To cultivate. Furthermore, culture under microaerobic conditions such as liquid static culture can also be used.

The culture medium is not particularly limited, and a medium generally used for the culture of lactic acid bacteria can be appropriately modified and used as needed. That is, as a carbon source, for example, fructose, glucose, sorbose, ribose, lyxose, xylose, arabinose, lactulose, sucrose, starch hydrolyzate (starch hydrolysate), molasses ( molasses) and other sugars. As the nitrogen source, for example, ammonia, ammonium sulfate, ammonium chloride, ammonium nitrate and other ammonium salts and nitrates can be used. Furthermore, as inorganic salts, for example, sodium chloride, potassium chloride, potassium sulfate, magnesium sulfate, calcium chloride, calcium nitrate, manganese chloride, ferrous sulfate and the like can be used. In addition, organic ingredients such as egg whites, soybean flour, defatted soybean meal, meat extract, and yeast extract can also be used. Furthermore, as the prepared medium, for example, MRS medium can be suitably used.

As one of the embodiments, it is preferable to inoculate and culture lactic acid bacteria kosoi10H strain in a medium containing high sugar concentration and D-fructose. In this embodiment, [high sugar concentration] means culturing with a medium with increased sugar concentration, for example, by adding fructose, glucose, sorbose, ribose, lyxose, xylose, arabinose, lactulose, Sugar solutions such as sucrose are used for adjustment. The high-sugar-concentration medium refers to a medium having a higher osmotic pressure than a normal medium. In the high sugar concentration medium, for example, the final concentration of added sugars is at least 10% by mass, preferably 10 to 40% by mass, and can grow even at a sugar concentration of 40% by mass or more. Furthermore, the carbon source of the 10H strain is a medium containing 5 to 20% by mass, preferably 5 to 15% by mass, of D-fructose. D-fructose may be added not only as a monosaccharide, but also as a medium component added to sucrose, oligosaccharides, or polysaccharides (including vegetables, mushrooms, etc.) and decomposed during cultivation to be supplied. Therefore, as a high-sugar-concentration medium, it is preferable to contain saccharides derived from natural products, such as sucrose, brown sugar (muscovado), or brown sugar, whose constituents are D-fructose. By culturing in a medium containing D-fructose at such a high sugar concentration, even in the presence of other lactic acid bacteria strains, the 10H strain can be grown as a dominant strain.

The bacterial cell cleaning process (S03) is a process of separating bacterial cells from the above-mentioned cultivated culture solution by centrifugation or filtration, and cleaning the bacterial cells with physiological saline or the like. The number of times of washing can be appropriately selected according to the medium components used in the main culture process (S02) and the purpose of use of the bacteria. In addition, depending on the situation, the aseptic body cleaning procedure (S03) may be used to directly heat-treat the culture solution obtained above.

The heat treatment process (S04) is a process of sterilizing the bacterial cells obtained in the above bacterial cell cleaning process (S03) by heating. The heating temperature can be 65-100°C, and the heating time can be suitably selected from about 1 minute to 120 minutes. The preferred heat treatment is 65-85°C for more than 1 minute, such as about 10 minutes, 30 minutes or 60 minutes, more preferably 70-75°C for more than 1 minute, such as about 5 minutes, 10 minutes or 30 minutes.

Next, in the recovery process (S05), the bacterial cells are recovered by filtering or centrifuging the treatment liquid after the heat treatment. For example, the treated solution can be recovered by centrifuging at 5000 rpm, 4°C, for 20 minutes and collecting bacteria. The bacterial cells may be frozen and stored as they are, and the bacterial cells may also be recovered as dry matter by freeze-drying, spray drying, vacuum drying, drum drying, or the like. The bacterial cells thus obtained can be used as active ingredients of the composition of the present invention. Furthermore, ground or crushed products subjected to treatment such as grinding or crushing may be used for the obtained bacterial cells.

Next, examples are given to illustrate the present invention in more detail, but the present invention is by no means limited by these examples. In addition, in the following examples, the unit % of the numerical value which shows the addition amount of each component means mass %.

[Example] (Example 1) Production of freeze-dried cells of lactic acid bacteria kosoi The preservation strain of lactic acid bacteria kosoi10H (registration number NITE BP-02811) was inoculated to 500 mL of MRS medium (Difco Laboratories company) adding 10% by mass of fructose, and left standing at 30°C for 48 hours Cultivate (seed culture). This culture solution was added to a medium (5% inoculation) in which 10 mass % of fructose was added to 10 L of MRS medium, and the main culture was performed by standing still at 30° C. for 48 hours. Then, the culture solution was centrifuged at 5000 rpm for 20 minutes, and the supernatant was removed. The obtained bacterial cells were suspended in 10 L of sterilized physiological saline phosphate buffer solution, and then centrifuged at 5000 rpm for 20 minutes to obtain washed bacterial cells. Repeat this operation twice. Further, 10 L of sterilized physiological saline phosphate buffer solution was added to the obtained bacterial cells to suspend, and then heat-treated at 70° C. for 30 minutes. Next, centrifugation was performed at 5000 rpm for 20 minutes to obtain heat-treated bacterial cells. Furthermore, the operations of suspension and centrifugation with 10 L of sterilized distilled water were repeated three times. The finally obtained precipitate was freeze-dried to obtain 14.3 g of dried bacterial cells. The freeze-drying device uses EYELA U-2200, and operates for 72 hours under a vacuum condition of 2 to 3 Pa and a trap temperature of -80°C.

(Example 2) Evaluation of antidepressant effect by animal model of depression Corticosterone (CORT: corticosterone), a rodent stress hormone similar to human cortisol, has been shown to be effective in humans with major depressive disorder and those exposed to chronic stress. ) worsened in rodents. Therefore, corticosterone-induced depression-like behavior is widely used as a chronic model of stress-induced depression in animals. By chronically administering CORT, the effects of stress on anxiety-related approach-avoidance-flee behavior in animals can be mimicked.

2-1. Mice Male C57BL/6 mice were obtained from the National Laboratory Animal Center (Taiwan). All mice were housed in 365×207×140mm cages with 5 mice each, and were kept at 23±2°C under a 12-hour light-dark cycle starting from 7:30 am. The state of health of the animals was observed daily. No major adverse events were observed. Water and feed were freely available. During the administration period, the amount of drinking water for 24 hours was measured once a week, and the body weight was measured twice a week. The rate of change in body weight of the mice was calculated. All animal experiments and procedures were approved by the Institutional Animal Care and Use Committee (ITRI-IACUC-2019-083M) of the Taiwan Industrial Technology Research Institute.

2-2. Administration method Eight-week-old male C57BL/6 mice were divided into 4 groups (n = 10 for each group). (1), sham operation group (sham group), (2) carrier group (vehicle group) (solvent control group), (3) LK600 group (administration of lactic acid bacteria kosoi prepared in embodiment 1 with 600mg/kg/day) Freeze-dried cells), (4) fluoxetine group (fluoxetine hydrochloride administered at 15 mg/kg/day, product number 1279804, manufactured by Sigma-Aldrich) ). The mice in the sham operation group were subcutaneously injected with normal saline 3.25mL/kg/day and administered for 21 days (the 0-20th day), and the mice in other groups were subcutaneously injected with CORT (corticosterone, product number C2505, Sigma-Aldrich) 40mg/kg/day (3.25mL/kg/day) and induced depression. For the CORT system, 2% DMSO (Product No. D2650, manufactured by Sigma-Aldrich) was added to sesame oil (Product No. S3547, manufactured by Sigma-Aldrich) and dissolved. Carrier (solvent), LK (the aqueous solution in which the freeze-dried thalline suspension of the lactic acid bacteria kosoi prepared in Example 1 was suspended) and fluoxetine (aqueous solution) (5mL/kg/day) were orally administered every day. 21 days (days 0 to 20). Behavioral tests were performed between days 21 and 25, and mice were sacrificed on day 28.

2-3. Statistical analysis The data were analyzed using one-way layout ANOVA (analysis of variance), and Dunnett's post hoc analysis was performed. All statistical analyzes were performed using EZR (R version 4.0.3, R Commander 2.7-1, reference: Bone Marrow Transplantation 2013: 48, 452-458.). A p-value less than 0.05 was considered statistically significant.

2-4. Elevated cross test The elevated plus maze (EPM: elevated plus maze) test is based on the fact that animals hate high open spaces and tend to want to stop in closed spaces. In the EPM test, anxiety is manifested by spending more time in the closed arm. In the EPM test, a lifting cross (+) device with a height of 50 cm was used, with two open arms and two closed arms. Each arm has a length of 30 cm and a width of 5 cm. On both arms closed there are 15 cm tall walls surrounding the arms. Mice did not receive acclimation to the maze prior to the experiment. To start the behavioral analysis, each mouse was placed in the central area (5 x 5 cm) of the maze and allowed to explore for 5 minutes. Time spent on each arm was analyzed as a ratio of time spent on the open arm to the time spent on the closed arm. The maze was then cleaned with 70% ethanol between tests so that the odor would not be a hindrance to the behavior of the next mouse.

2-5. Results The results are shown in Fig. 2 and Fig. 3 . For the sham group, a tendency towards reduction in the total travel distance ( FIG. 2 ) and the ratio of open portion retention ( FIG. 3 ) was recognized in the vehicle group. However, the LK600 group (600 mg/kg/day freeze-dried bacterium administration group of lactic acid bacteria kosoi) significantly increased the total moving distance (Fig. 2, Dunnett, p<0.05) and the retention ratio of the open part (Fig. 3, Dunnett, p<0.01). This effect was equal to or exceeded that of the 15 mg/kd/day administration group of fluoxetine used as an antidepressant.

(Example 3) Evaluation of anti-aging effect through aging animal model Aging-promoted mice (SAM) were produced by selective inbreeding of the AKR/J system. This mouse exhibits premature aging phenomena such as senile amyloidosis, senile osteoporosis, cataract, decreased immune response, and learning/memory impairment. Senescence-accelerated mouse prone-8 (SAMP8: Senescence-accelerated mouse prone-8) is a particularly widely used model for dementia research because brain atrophy begins early, accompanied by lethargy/memory impairment and mood disorders (T. Takeda, et al., A new murine model of accelerated senescence. Mech Aging Dev. 17(1981) 183-194). The lifespan of SAMP8 was about half that of SAM/resistant 1 (SAMR1) of the reference strain. It is known that the aging-promoting mouse SAMP8 further accelerates aging by feeding a high fat diet (high fat diet), and becomes a pathological model of Alzheimer dementia (Alzheimer dementia) while developing type II diabetes ( Mehta et al., Experimental Induction of Type 2 Diabetes in Aging-Accelerated Mice Triggered Alzheimer-Like Pathology and Memory Deficits. Journal of Alzheimer's Disease 39:145-162, 2014).

3-1. Mice Male SAMP8/TaHsd (aging-promoting mice) were obtained from Envigo RMS B.V. (Netherlands). Keep SAMP8 mice solitary at 6 weeks of age according to the supplier's housing policy. Mice were 4–7 weeks old on arrival. SAMP8 mice aged 6–7 weeks became solitary before shipment and were assigned to batch 1. SAMP8 mice aged 4–5 weeks became solitary at 5–6 weeks (after quarantine) and were assigned to batch 1. Batch 2. All mice were housed individually in 365 × 207 × 140 mm cages (without lids) at a temperature of 23 ± 20°C under a 12-h light-dark cycle starting at 7:30 am. Batch 2 mice started the experiment two weeks later than Batch 1 mice and were matched for age. For acclimatization, all mice were given a control diet (LFD, D12450Ji, Research Diets) from 17 days before the start of the test. Mice with a subset of SAMP8 were switched to a high-fat diet (HFD, D12492i, Research Diets) at 10–11 weeks of age. The HFD and LFD lines were fed 3 times a week. The health status of the animals was monitored daily. Water and feed were freely available. Daily consumption of feed was determined weekly. Water consumption is determined by measuring 24-hour drinking water every 2 to 3 weeks. Body weight was determined twice a week during the test period. The rate of change in body weight of the mice was calculated. All animal experiments and procedures were approved by the Institutional Animal Care and Use Committee (ITRI-IACUC-2019-069V1) of the Taiwan Industrial Technology Research Institute.

3-2. Administration method Male SAMP8 mice aged 10-11 weeks were divided into 3 groups (n = 9 in each group, consisting of two parties of batch 1 and batch 2, and 4-5 mice/group in batch 2). There were 3 groups as follows: (1), LFD (low fat diet) group, (2), vehicle (high fat diet) group, and (3), LK600 (high fat diet) group. In the carrier (high-fat diet) group and the LK600 (high-fat diet) group (administering the freeze-dried thalline body of the lactic acid bacteria kosoi prepared in Example 1 with 600mg/kg/day) from the 0th day of the start of the test, the They freely ingested a high-fat diet, and they freely ingested a low-fat diet in the LFD group. Furthermore, 1500 mg/kg/day of dextrin (dextrin) was administered to the LFD (low-fat diet) group and the vehicle (high-fat diet) group, and 600 mg/kg/day of freeze-dried cells of lactic acid bacteria kosoi were administered to the LK600 (high-fat diet) group. kg/day, and at the same time administer dextrin 900 mg/kg/day. As an administration (administration) method, 5 mL/kg/day was orally administered every day for 122 days (0 to 121 days from the start of the test) using a probe (sonde). From the 122nd day to the 132nd day, the test was continued without administration of the sample, and the mice were sacrificed on the 133rd day.

Statistical analysis was carried out by analysis software EZR (R version 4.0.3, R commander 2.7-1). Figure 4 and Figure 6 are analysis of variance (analysis of variance) (Repeated measures ANOVA) for repeated measures. The paired t test (unpaired t test) and Figures 7 to 10 were analyzed using ANOVA with a one-way configuration, and Dunnett's post hoc analysis was performed. *p<0.05, **p<0.01, ***p<0.001.

3-3. Determination of weight gain rate Body weight is often used as an indicator of the health status of mice. Compared with the LFD (low-fat diet) group, the weight gain rate of the vehicle (high-fat diet) group (represented as HFD Vehicle (vehicle) in FIG. 4 ) ingested a high-fat diet was significantly higher (Repeated measures ANOVA, P< 0.001) (Figure 4). In the group administered together with LK600 and a high-fat diet (represented as HFD LK600 in FIG. 4 ), although higher than the LFD (low-fat diet) group, Significantly lower (Repeated measures ANOVA, P<0.05) (Figure 4). The body weight of the mice on day 133 from the start of the test was measured, and the results of comparing the groups are shown in FIG. 5 . As shown in Figure 5, by unpaired t-test, a significant difference (p<0.05) was identified between the group administered together with LK600 and the high-fat diet and the vehicle (high-fat diet) group ).

3-4. Determination of aging index Aging Evaluation Score In order to evaluate the degree of aging in SAMP8 mice, an aging evaluation score was used, which is a total of 11 items representing changes in behavior and appearance from 0 to 4 (see Table 1). Fig. 6 shows the change over time of the average value (total aging index) of the 11-item aging degree evaluation scores of aging-promoting mice administered with the active ingredient composition of the present invention and the like.

[Table 1]

The average score of each administration group gradually increased from the age of 9 weeks to the age of 25 weeks. There was no significant difference until 15 weeks of age, but there was a significant difference after 22 weeks of age (Fig. 6) (Repeated measures ANOVA; LK600 vs. vehicle: P<0.01, LFD vs. vehicle: P.<0.001). The sum of the mean values of the aging degree evaluation points of each group at the age of 22 weeks is shown in FIG. 7 . As shown in Figure 7, the Vehicle (High Fat Diet) group showed significantly higher scores compared to the LFD (Low Fat Diet) group (Dunnett, P<0.001). On the other hand, the LK600 (high-fat diet) group significantly suppressed this increase compared with the vehicle (low-fat diet) group (Dunnett, P<0.01). The change in score is mainly centered on the categories of luster and denseness, which are indicators of the appearance of hair. Therefore, the scores of the glossiness of fur (Glossiness of fur) of 22-week-old hair in each administration group are shown in FIG. 8 . As shown in Figure 8, the Vehicle (High Fat Diet) group showed significantly higher scores compared to the LFD (Low Fat Diet) group (Dunnett, P<0.001). On the other hand, the LK600 (high-fat diet) group significantly suppressed this increase compared with the vehicle (low-fat diet) group (Dunnett, P<0.01).

3-5. Tissue recovery When the mice were sacrificed, the serum and brain tissue of the mice were preserved. Mice were deeply anesthetized by inhalation of 2.5% isoflurane using an induction box and mask, and anesthesia was confirmed by toe pinch reflex. The chest was opened, and 0.35 mL of whole blood was collected from the right ventricle with a syringe (25G needle) as serum. The intracardiac perfusion system uses a peristaltic pump to infuse 15-20 mL (10 mL/min) of ice-cold artificial cerebrospinal fluid (ACSF) from the left ventricle just after cutting the right atrium. ACSF consisted of 124mM NaCl, 2.5mM KCl, 1.2mM NaH ₂ PO ₄ , 24mM NaHCO ₃ , 5mM HEPES, 12.5mM Glucose, 2mM MgSO ₄ .7H ₂ O, 2mM CaCl ₂ .2H ₂ O. The pH of ACSF was adjusted to 7.4 after supplying oxygen using carbogen (95% O ₂ /5% CO ₂ ) at room temperature. ACSF was stored at 4°C or on ice and reoxygenated with carbonated oxygen for 20 minutes before use. After intracardiac perfusion, the brain was quickly recovered and separated into two hemispheres. The left hemisphere was snap frozen in liquid nitrogen and stored at -80°C. After coagulating whole blood samples at room temperature for 2 hours, centrifuge at 10,000 × g for 10 minutes. Serum was recovered and stored at -80°C.

3-6. Determination of amyloid-β in the brain with 800 μL of T-PER ^™ Tissue Protein Extraction Reagent (78510, Thermo Fisher Scientific) added with protease inhibitor (protease inhibitor) cocktail (K266-100, BioVision) Brain homogenization of frozen (-80°C) left hemisphere. Then, the homogenate was centrifuged at 10000 xg for 5 minutes. The supernatant was recovered and stored at -80°C. The total concentration of amyloid-beta _1-42 in the hemispheres was determined using an ELISA kit (Mouse amyloid beta peptide 1-42 ELISA Kit, CSB-E10787m, CUSABIO) according to the manufacturer's instructions.

It is known that the aging-promoting model mouse SAMP8 becomes a model of Alzheimer's dementia caused by increased oxidative stress caused by high expression of amyloid-β (Morley et al., 2020 The senescence accelerated mouse (SAMP8) as a model for oxidative stress. and Alzheimer's disease. Biochimica et Biophysica Acta 1822:650-656, 2012). The results of evaluating the effect on amyloid-β in the LK600 (high-fat diet) group using brains of SAMP8 mice are shown in FIG. 9 . As shown in Figure 9, the level of amyloid-β in the brain homogenate supernatant was compared with the LFD group, in the vehicle (high-fat diet) group significantly (Dunnett, p<0.001) increased, but in the LK600 (high Fat diet) group, compared with the vehicle (high fat diet) group, the increase of amyloid-β was significantly suppressed (Dunnett, p<0.01).

3-7. Determination of inflammatory factor (inflammatory factor) interleukin 1β (interleukin 1β) (IL-1β) in serum It is known that the production of inflammatory cytokines (inflammatory cytokines) such as IL-1 is increased in the elderly (Isobe, Perspectives in Geriatric Medicine-Aging and Immunity-Journal of the Japanese Geriatrics Society, 48: 205-210, 2011 and Licastro, et al., Innate immunity and inflammation in ageing: a key for understanding age-related diseases. Immunity & Aging 2:8, 2005). ELISA was performed to investigate whether the IL-1β level in SAMP8 mice was changed by the administration of LK600. The serum samples of each group were pooled, and the results of detecting IL-1β using Abcam’s High Sensitivity IL-1β ELISA Kit (abl97742) are shown in Figure 10. As shown in Figure 10, the IL-1β level was significantly increased in the carrier (high-fat diet) group compared with the LFD (low-fat diet) group (Dunnett, p<0.001), but in the LK600 (high-fat diet) group , compared with the vehicle (high-fat diet) group, the increase of IL-1β was significantly suppressed (Dunnett, p<0.001).

(prescription example 1 lozenge) The following ingredients are mixed according to the usual method to make lozenges. In addition, the freeze-dried cells of the lactic acid bacteria kosoi obtained in Example 1 were used for the active ingredient composition. composition Active ingredient composition (Example 1) 150mg Cellulose 80mg Starch 20mg Sucrose fatty acid esters 2mg The above-mentioned ingredients are mixed and made into tablets to obtain lozenges.

(Prescription example 2 capsules) The following ingredients are mixed according to the usual method to obtain a soft capsule. In addition, the freeze-dried cells of the lactic acid bacteria kosoi obtained in Example 1 were used for the active ingredient composition. composition Active ingredient composition (Example 1) 100mg Beeswax 10mg Grape seed oil 110mg The above ingredients are mixed and filled into a gelatin- and glycerin-mixed capsule base to obtain soft capsules.

(Prescription example 3 drinking water for beauty) The following ingredients were mixed according to the usual method, and the whole amount was prepared with 100g of drinking water for beauty. In addition, the freeze-dried cells of the lactic acid bacteria kosoi obtained in Example 1 were used for the active ingredient composition. composition Active ingredient composition (Example 1) 500mg Coenzyme Q10 500mg Vitamin B2 5mg Vitamin B6 5mg Nicotinamide 10mg Vitamin E 100mg L-ascorbic acid (L-ascorbic acid) 100mg Brown sugar glucose liquid sugar 10g Citric acid 1g Spices 100mg Color 100mg Water 97.5g

The application of the composition containing lactic acid bacteria kosoi was confirmed to exhibit antidepressant and antiaging effects (including suppression of obesity) in the evaluation of drug administration to depression model mice and high-fat diet administration to aging promotion model mice . Therefore, the antidepressant, antiaging agent, and antiobesity agent of the present invention have the possibility of being utilized as pharmaceuticals, food and beverages, especially food and beverages for beauty and health improvement.

Fig. 1 is a flowchart showing the manufacturing process of the active ingredient composition of the present invention. Fig. 2 is a graph showing the results of an elevated cross test using an animal model of depression with respect to the active ingredient composition of the present invention. The total distance after the depression-induced mice administered with each sample moved in the elevated plus maze shown on the horizontal axis is shown on the vertical axis. Fig. 3 is a graph showing the results of an elevated cross test using an animal model of depression with respect to the active ingredient composition and the like of the present invention. The ratio of the time that the depression-induced mice administered with each sample stayed in the open part of the elevated plus maze shown on the horizontal axis is shown on the vertical axis. Fig. 4 is a graph showing the results of measuring the rate of body weight gain in senescence accelerated mice with respect to the active ingredient composition of the present invention and the like. Fig. 5 is a graph showing the results of body weight gain on the 133rd day after the start of the test in aging-promoting mice administered the active ingredient composition of the present invention and the like. Fig. 6 is a graph showing changes over time in the average value of 11 items of aging degree evaluation scores of aging-promoting mice administered with the active ingredient composition of the present invention and the like. Fig. 7 is a graph showing the average values of 11-item aging degree evaluation scores in aging-promoting mice administered with the active ingredient composition of the present invention, etc., at the age of 22 weeks. Fig. 8 is a graph showing gloss scores of hair of 22-week-old aging-promoting mice administered with the active ingredient composition of the present invention and the like. Fig. 9 is a graph showing the concentration of amyloid β in the brain homogenate supernatant of 22-week-old mice administered with the active ingredient composition of the present invention and the like. Fig. 10 is a graph showing the amount of IL-1β in the brain homogenate supernatant of 22-week-old aging-promoting mice administered with the active ingredient composition of the present invention and the like.

Claims (8)

An antidepressant containing, as an active ingredient, bacterial cells of Lactobacillus kosoi (Lactobacillus kosoi) 10H strain deposited under deposit number NITE BP-02811. The antidepressant as claimed in item 1, wherein the bacterium is a dead bacterium. The antidepressant according to Claim 1 or Claim 2, which is in the form of pharmaceuticals, food or beverages, or active ingredient compositions mixed with pharmaceuticals or food or beverages. An anti-aging agent containing as an active ingredient the bacterial cells of Lactobacillus kosoi (Lactobacillus kosoi) 10H strain deposited under the deposit number NITE BP-02811. The anti-aging agent as claimed in item 4, wherein the bacterium is a dead bacterium. The anti-aging agent according to Claim 4 or Claim 5, which is in the form of pharmaceuticals, food and beverages, feeds, or active ingredient compositions compounded with pharmaceuticals, food and beverages, and feeds. The anti-aging agent according to claim 6, wherein the food and beverage is a food and drink for beauty or health improvement. An anti-obesity agent comprising, as an active ingredient, bacterial cells of Lactobacillus kosoi (Lactobacillus kosoi) 10H strain deposited under the deposit number NITE BP-02811.

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