Novel lactic acid bacteria and their use

Abstract

To provide novel lactic acid bacteria derived from the Awa-bancha, which is a kind of green tea traditionally produced in the Tokushima Prefecture, and a composition including the same.SOLUTION: Lactic acid bacteria according to the present invention are Lactiplantibacillus pentosus AWA1922 strain (accession number NBRC115326), Lactiplantibacillus pentosus AWA1955 strain (accession number NBRC115327), Levilactobacillus brevis AWA1978 strain (accession number NBRC115323), Levilactobacillus brevis AWA1984 strain (accession number NBRC115324), or Levilactobacillus brevis AWA1985 strain (accession number NBRC115325).SELECTED DRAWING: None

Description

[Technical Field]

[0001] This article relates to novel lactic acid bacteria and their uses. [Background technology]

[0002] Lactic acid bacteria, a general term for bacteria that produce lactic acid, have been deeply involved in people's lives since ancient times. For example, lactic acid fermentation significantly influences the flavor of traditional fermented foods such as pickles, yogurt, miso paste, and soy sauce. The lactic acid produced by lactic acid bacteria not only influences the flavor of fermented foods, but also lowers the pH and suppresses the growth of unwanted harmful bacteria. Various strains of lactic acid bacteria have been reported to have various functions. For example, lactic acid bacteria that are resistant to digestive juices can reach the intestine alive when ingested orally. The lactic acid they produce is believed to suppress harmful bacteria (bad bacteria) that can adversely affect the body, improving the intestinal environment and promoting intestinal regulation. Furthermore, lactic acid bacteria that remain in the intestine are believed to exhibit greater intestinal regulation. Some strains have also been reported to produce gamma-aminobutyric acid, which has blood pressure-lowering and relaxing effects.

[0003] Awa Bancha, a fermented food made by lactic acid bacteria, has long been produced in Tokushima Prefecture. Awa Bancha is classified as a post-fermented tea produced by microbial fermentation, and post-fermented tea is produced in only a few regions around the world. Awa Bancha tea leaves are harvested in midsummer. In the production of Awa Bancha, the harvested tea leaves are typically boiled in a kettle to inactivate the enzymes contained in the tea leaves, rolled, and then packed into barrels. A weight is placed on top and the tea is subjected to anaerobic fermentation for approximately two to four weeks. After fermentation, the tea leaves are dried in the sun for one to two days, completing the Awa Bancha. Anaerobic fermentation changes the components of the tea leaves, resulting in a unique sour flavor. Lactic acid bacteria are the dominant bacteria in the anaerobic fermentation of Awa Bancha. These lactic acid bacteria grow in an environment rich in antibacterial components such as catechins, potentially giving the tea unique properties. Lactic acid bacteria isolated from Awa Bancha tea have been reported to date, including pyrogallol-producing lactic acid bacteria (Patent Document 1) and lactic acid bacteria useful for infection prevention (Patent Document 2), but no lactic acid bacteria with other functions are known. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Laid-Open No. 2016-167984 [Patent Document 2] Japanese Patent Application Laid-Open No. 2010-227043 Summary of the Invention [Problem to be solved by the invention]

[0005] The object of the present invention is to provide a new lactic acid bacterium derived from Awa Bancha tea and a composition utilizing the same. [Means for solving the problem]

[0006] The present inventors have focused on a traditional fermented tea from Tokushima Prefecture and conducted extensive research in light of the above-mentioned problems, and have succeeded in isolating novel lactic acid bacteria from Awa Bancha tea that have at least one of the following properties: digestive juice resistance, intestinal epithelial cell adhesion, γ-aminobutyric acid production, and tea leaf extract resistance. The present invention was completed as a result of further research based on this finding, and includes, for example, the following aspects. Item 1. A lactic acid bacterium selected from the group consisting of Lactiplantibacillus pentosus strain AWA1922 (accession number NBRC115326), Lactiplantibacillus pentosus strain AWA1955 (accession number NBRC115327), Levilactobacillus brevis strain AWA1978 (accession number NBRC115323), Levilactobacillus brevis strain AWA1984 (accession number NBRC115324), and Levilactobacillus brevis strain AWA1985 (accession number NBRC115325). Item 2. A composition containing at least one lactic acid bacterium selected from the group consisting of Lactiplantibacillus pentosus strain AWA1922 (accession number NBRC115326), Lactiplantibacillus pentosus strain AWA1955 (accession number NBRC115327), Levilactobacillus brevis strain AWA1978 (accession number NBRC115323), Levilactobacillus brevis strain AWA1984 (accession number NBRC115324), and Levilactobacillus brevis strain AWA1985 (accession number NBRC115325). Item 3. The composition according to Item 2, wherein the composition is an oral composition. Item 4. The composition according to Item 2 or 3, wherein the composition is at least one selected from the group consisting of fermented tea, yogurt, pickles, and supplements. [Effects of the Invention]

[0007] According to the present disclosure, novel lactic acid bacteria can be provided. In particular, according to the present disclosure, novel lactic acid bacteria can be provided that have at least one of the following effects: digestive fluid resistance, intestinal epithelial cell adhesion, γ-aminobutyric acid production, and tea leaf extract resistance. Furthermore, because the lactic acid bacteria have at least one of the useful effects of digestive fluid resistance, intestinal epithelial cell adhesion, γ-aminobutyric acid production, and tea leaf extract resistance, they can be preferably used in various compositions, particularly oral compositions. [Brief explanation of the drawings]

[0008] [Figure 1] FIG. 1 shows the results of a digestive fluid resistance test (artificial gastric fluid pH 3.0) of lactic acid bacteria. [Figure 2] FIG. 2 shows the results of a digestive fluid resistance test (artificial gastric fluid pH 2.0) of lactic acid bacteria. [Figure 3]FIG. 3 shows the results of an adhesion test of lactic acid bacteria to intestinal epithelial cells, Caco-2. [Figure 4] FIG. 4 shows the results of a test of γ-aminobutyric acid productivity of lactic acid bacteria. [Figure 5] FIG. 5 shows the results of a growth test of lactic acid bacteria in tea leaf extract. DETAILED DESCRIPTION OF THE INVENTION

[0009] lactic acid bacteria The lactic acid bacteria disclosed herein are novel lactic acid bacteria belonging to the genus Lactiplantibacillus pentosus or Levilactobacillus brevis, which were isolated by the present inventor from Awabancha tea produced in Tokushima Prefecture, and are the following five strains named by the present inventor as follows: LactipranchiBacillus pentosus strain AWA1922, Lactipranchi Bacillus pentosus strain AWA1955, Lactobacillus brevis strain AWA1978, Lactobacillus brevis strain AWA1984, Lactobacillus brevis strain AWA1985.

[0010] More specifically, the isolation was carried out by adding phosphate-buffered saline to Awabancha tea leaves, shaking the mixture at room temperature, spreading the supernatant onto an MRS agar plate containing 2% agar, and culturing the resulting lactic acid bacteria colonies. The resulting strains were then genetically analyzed to isolate and identify them. The genetic analysis involved multiplex PCR (Polymerase Chain Reaction) for the recA gene or based on homology to the 16S rRNA gene. The isolation and identification were described in more detail in the Examples below.

[0011] Both Lactipranchibacillus pentosus and Leviractobacillus brevis were previously classified in the genus Lactobacillus and were called Lactobacillus pentosus and Lactobacillus brevis, respectively. However, in recent years (2020), lactic acid bacteria have been reclassified, and the genus and species names mentioned above follow this reclassification.

[0012] The lactic acid bacteria disclosed herein have been deposited at the Biotechnology Center of the National Institute of Technology and Evaluation (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) (October 29, 2021). Specifically, the Lactipranchibacillus pentosus AWA1922 strain has been deposited under accession number NBRC115326, the Lactipranchibacillus pentosus AWA1955 strain under accession number NBRC115327, the Leviractobacillus brevis AWA1978 strain under accession number NBRC115323, the Leviractobacillus brevis AWA1984 strain under accession number NBRC115324, and the Leviractobacillus brevis AWA1985 strain under accession number NBRC115325. These lactic acid bacteria can be obtained from the depository institution following the prescribed procedures. These lactic acid bacteria have the following characteristics:

[0013] Colony color: Milky white (MRS agar plate) Colony shape: Mainly circular to oval (MRS agar plate) Fungal morphology: rod-shaped Motility: None Spore formation: None Growth temperature: 25-37°C (good growth) Sugar assimilation: shown in Table 1 in the Examples below.

[0014] The strains shown in Table 1 are Lactiplantibacillus pentosus strains AWA1922 and AWA1955, and the known lactic acid bacterium Lactiplantibacillus pentosus NBRC106467, which is classified into the same species as these strains. TStrain (according to the old classification, Lactobacillus pentosus NBRC106467 T ), Levilactobacillus brevis AWA1978, AWA1984, and AWA1985 strains, and the known lactic acid bacteria Levilactobacillus brevis NBRC107147, which is classified into the same species as these strains. T Strain (according to the old classification, Lactobacillus brevis NBRC107147 T )

[0015] Lactipranchibacillus pentosus strains AWA1922 and AWA1955 were identified under the ID number NBRC106467. T The AWA1922 strain also has the following characteristics that differentiate it from the other strains: When cultured in MRS liquid medium at 35°C for 24 hours, the AWA1922 strain aggregates when the test tube is shaken. When cultured in MRS liquid medium at 35°C for 24 hours, the AWA1955 strain produces a highly viscous substance. This is presumably because these two strains produce exopolysaccharides, with AWA1922 producing capsular polysaccharides bound to the cell outer membrane and AWA1955 producing free slime polysaccharides. NBRC106467 T The strain does not undergo such aggregation, nor does it produce highly viscous material.

[0016] Furthermore, Lactipranchibacillus pentosus strains AWA1922 and AWA1955, and Reviractobacillus brevis strains AWA1978, AWA1984, and AWA1985 have at least one characteristic selected from the group consisting of the following (1) to (4): (1) digestive juice resistance, (2) intestinal epithelial cell adhesion, (3) gamma-aminobutyric acid production, and (4) tea leaf extract resistance.

[0017] Regarding the above (1) digestive juice resistance, as will be shown in the Examples below, the Lactipranchibacillus pentosus AWA1922 and AWA1955 strains are similar to the known lactic acid bacterium Lactipranchibacillus pentosus NBRC106467, which is classified into the same species as these strains. TThe AWA1922 strain is generally equivalent to or better than the NBRC106467 strain (used as the standard strain in the Examples described later), and survives even when in contact with gastric juice and proliferates even when in contact with intestinal juice. T The resistance to digestive fluids can be confirmed and evaluated in detail according to the experimental examples described below.

[0018] Furthermore, as shown in the Examples below, the Reviractobacillus brevis AWA1978, AWA1984, and AWA1985 strains survive even when they come into contact with gastric juice and grow even when they come into contact with intestinal juice. Thus, these strains also have resistance to digestive juice. Furthermore, as shown in the Examples below, these strains have a higher resistance to digestive juice than the known lactic acid bacterium Reviractobacillus brevis NBRC107147. T The resistance to digestive fluids is higher than that of the strain (used as a standard strain in the Examples described later). Confirmation and evaluation of the resistance to digestive fluids can also be carried out in detail according to the Experimental Examples described later.

[0019] Furthermore, the AWA1922, AWA1978, AWA1984, and AWA1985 strains all survive and / or grow even when in contact with digestive fluids, and are particularly resistant to digestive fluids. For these reasons, these lactic acid bacteria can be said to be useful, for example, in that they easily reach the stomach and intestines alive.

[0020] Regarding (2) intestinal epithelial cell adhesion, Lactipranchibacillus pentosus strains AWA1922 and AWA1955 have high adhesion to intestinal epithelial cells, as shown in the Examples below. This suggests that AWA1922 and AWA1955 strains can remain in the digestive tract for a certain period of time, making them useful for, for example, improving the intestinal environment, ameliorating allergies, preventing infection by pathogenic bacteria, ameliorating colitis, improving lipid metabolism, and alleviating the worsening of allergies and other conditions. Confirmation and evaluation of this adhesion can be carried out in detail according to the Experimental Examples below.

[0021] Regarding the (3) γ-aminobutyric acid productivity, the Leviractobacillus brevis AWA1978, AWA1984, and AWA1985 strains were found to be as effective as Leviractobacillus brevis NBRC107147, as shown in the Examples below. T The AWA1978, AWA1984, and AWA1985 strains produce more γ-aminobutyric acid than the AWA1978 strain. γ-Aminobutyric acid, also known as GABA, is known to have various useful effects. For this reason, the AWA1978, AWA1984, and AWA1985 strains are useful in that they produce more γ-aminobutyric acid. Furthermore, these strains are also useful as lactic acid bacteria used for the purpose of obtaining useful effects based on γ-aminobutyric acid (e.g., improvement of high blood pressure, stress relief, improvement of sleep quality, reduction of fatigue, maintenance of skin elasticity, and improvement of cognitive function). The confirmation and evaluation of the productivity can be carried out in detail according to the experimental examples described below.

[0022] Regarding (4) resistance to tea leaf extract, all five strains of the present disclosure are resistant to tea leaf extract, as shown in the Examples below. T In addition, the Lactobacillus brevis AWA1978, AWA1984, and AWA1985 strains were more resistant to tea leaf extract than the strain NBRC107147, as shown in the Examples below. T The five strains of the present disclosure, particularly the AWA1922, AWA1978, AWA1984, and AWA1985 strains, are useful in that they have resistance to tea leaf extracts. Therefore, these strains of the present disclosure can be preferably used in, for example, tea products (including fermented tea) and products containing such antibacterial components. Confirmation and evaluation of this resistance can be carried out in detail according to the experimental examples described below.

[0023] Thus, the present disclosure provides novel lactic acid bacteria. These lactic acid bacteria are useful, for example, in that they possess at least one characteristic selected from the group consisting of (1) to (4) above, and are useful in that they can be used to obtain beneficial effects based on these characteristics. Furthermore, the lactic acid bacteria of the present disclosure can be used in a variety of compositions, and are preferably used, for example, to obtain compositions with these beneficial effects. Thus, the lactic acid bacteria of the present disclosure are also useful as probiotic bacteria. Examples of such compositions include the following:

[0024] composition The composition of the present disclosure can be produced by combining the lactic acid bacteria of the present disclosure, i.e., at least one lactic acid bacterium selected from the group consisting of AWA1922 strain, AWA1955 strain, AWA1978 strain, AWA1984 strain, and AWA1985 strain, with optional components depending on the form and mode of use, etc., and may be produced by culturing (including fermentation) etc. as necessary. Examples of the optional components include edible components, pharmaceutically acceptable components, cosmetically acceptable components, and medium components.

[0025] The composition is preferably used orally, regardless of whether it is orally or parenterally.The form of the composition is also not limited, and it may be solid, semi-solid, or liquid.For example, when the composition of the present disclosure is solid, it may be mixed with water or the like before use.

[0026] The manner of use of the composition is not limited either and can be appropriately set depending on the purpose. The composition can be used as a food composition (including beverages, health functional foods (foods for specified health uses, foods with nutrient functions, foods with functional claims, etc.), supplements, foods for the sick, etc.), pharmaceutical compositions, quasi-drug compositions, cosmetic compositions, feed compositions, or as an additive to food compositions, pharmaceutical compositions, quasi-drug compositions, cosmetic compositions, feed, etc. The composition also includes compositions used as a fermentation starter that can be used in producing fermented products.

[0027] The lactic acid bacteria may be, for example, cultured by mixing the lactic acid bacteria with a medium under known lactic acid bacteria culture conditions, and then harvested and separated from the resulting culture by means of centrifugation or the like, and used as is. Alternatively, the culture (including fermented product, supernatant, etc.), its crude product, purified product, or processed product thereof (dried product (lyophilized product, spray dried product, heat dried product, etc.), crushed product, etc.) may also be used as desired. These can be carried out according to conventionally known procedures for culturing lactic acid bacteria, etc. These may be used alone or in combination of two or more types.

[0028] The lactic acid bacteria of the present disclosure may be cultured in any medium as long as the lactic acid bacteria can grow and / or proliferate, and may be cultured in accordance with conventionally known general lactic acid bacteria culture procedures, for example, using a medium for culturing lactic acid bacteria or modifying the medium as appropriate, and the culture method is not limited to this. An example of a method that does not limit the present disclosure is a method using MRS medium, which is commonly used for culturing lactic acid bacteria, under conditions that allow the lactic acid bacteria of the present disclosure to grow.

[0029] Furthermore, without limiting the present disclosure, for example, the medium may contain, as necessary, any components such as glutamic acid or its salts (e.g., sodium salt), which are substrates for the production of γ-aminobutyric acid, components capable of producing glutamic acid or its salts, and components capable of promoting the growth (including proliferation) of lactic acid bacteria (e.g., carbon sources such as glucose, starch, sucrose, lactose, dextrin, sorbitol, fructose, etc.; nitrogen sources such as yeast extract, peptone, etc.; vitamins, minerals, fatty acids, trace metal elements, etc.). These may be contained alone or in combination of two or more.

[0030] Although not limiting the present disclosure, the culture temperature is, for example, 5 to 45°C, preferably 25 to 37°C, from the viewpoint of good bacterial growth. The pH of the medium (at the start of culture) is also not limited, but is, for example, 4 to 8 at 35°C, preferably 6 to 7. The culture time may be determined taking into consideration the culture conditions, culture scale, target bacterial amount, etc., and is, for example, 24 to 48 hours. The culture may be either aerobic or anaerobic, with anaerobic culture being preferred.

[0031] The lactic acid bacteria of the present disclosure may be cultured using a known medium or the like. Alternatively, the lactic acid bacteria may be cultured using ingredients for the composition (composition ingredients), such as food ingredients or foods, instead of or together with the medium. Examples of food ingredients and foods include, but are not limited to, tea leaves, vegetables (Chinese cabbage, radish, carrot, cucumber, eggplant, cabbage, etc.), milk (yogurt, cow's milk, skim milk, skim milk powder, etc.), grains (rice, barley, wheat, etc.), seafood (sweetfish, mackerel, herring, crucian carp, salmon, char, etc.), livestock products, and seasonings. These ingredients may be used alone or in combination of two or more. When using such composition ingredients, as described above, the culture conditions are not limited as long as the lactic acid bacteria can grow and / or proliferate. Furthermore, the composition ingredients may be subjected to pretreatment such as washing, crushing, squeezing, heating, or mixing, as necessary.

[0032] The composition of the present disclosure may be, for example, the culture obtained in this manner (including fermented products, crude products, purified products, and processed products thereof) itself, or may be a product produced by further mixing the culture with any of the edible components, pharmaceutically acceptable components, cosmetically acceptable components, medium components, etc., and may be a product further cultured (fermented) as needed. As mentioned above, without limiting the present disclosure, the composition of the present disclosure may be used as a fermentation starter for fermentation using the lactic acid bacteria of the present disclosure. Furthermore, without limiting the present disclosure, as mentioned above, the Leviractobacillus brevis AWA1978 strain, AWA1984 strain, and AWA1985 strain are Leviractobacillus brevis NBRC107147.T Since the composition of the present disclosure can produce more γ-aminobutyric acid than the strain, the composition of the present disclosure may be used, for example, as a composition for enhancing γ-aminobutyric acid production.

[0033] In the composition of the present disclosure, the lactic acid bacteria may be in a live state or a dead state, which may be appropriately determined depending on the intended form, mode of use, action, etc.

[0034] Although not limiting the present disclosure, it is generally known that ingesting lactic acid bacteria as live bacteria in dairy products such as yogurt is useful for maintaining health by regulating the intestines and improving the intestinal flora. Furthermore, as described above, the lactic acid bacteria of the present disclosure have at least one of the properties of digestive juice resistance and intestinal epithelial cell adhesion, and are expected to have effects such as regulating the intestines and improving the intestinal flora. From this perspective, a preferred example is when the lactic acid bacteria of the present disclosure are contained in the composition as live bacteria.

[0035] In this way, the composition of the present disclosure can be produced, and as mentioned above, the form of the composition can be any of solid, semi-solid, and liquid.For example, when the composition of the present disclosure is a food composition, it can be in the form that is generally ingested as food.In addition, although not limiting the present disclosure, an example of an embodiment of the food composition is a supplement, and its dosage form can be exemplified as tablets, capsules (including microcapsules), pills, powder, granules, lozenges, paste, gel, liquid (including syrup, emulsion, suspension) and the like, and can also be coated with sugar coating or the like.

[0036] Furthermore, without limiting the present disclosure, for example, when the composition of the present disclosure is a pharmaceutical composition, a quasi-drug composition, or a cosmetic composition, there are no limitations on the dosage form as long as it is an acceptable dosage form for these, and examples of dosage forms include oral compositions such as tablets, capsules, pills, powders, granules, lozenges, pastes, gels, and liquids (including syrups, milks, and suspensions), as well as parenteral compositions such as ointments, creams, lotions, gels, liquids, aerosols, sprays, patches (cataplasms, plasters, transdermal absorbents, and transmucosal absorbents), injections (e.g., intraperitoneal injections), and suppositories. Furthermore, without limiting the present disclosure, there are no limitations on the dosage form for, for example, a feed composition, as long as it is an acceptable dosage form for a feed composition.

[0037] For this reason, optional components such as the edible components, pharmaceutically acceptable components, and cosmetically acceptable components also include carriers (water, ethanol, propylene glycol, glycerin, dextrin, cyclodextrin, etc.), excipients, binders, lubricants, coating agents, disintegrants, disintegration aids, diluents, stabilizers, preservatives, dispersants, humectants, solubilizers, solubilizers, isotonicity adjusters, pH adjusters, colorants, sweeteners, flavorings, flavorings, gelling agents, thickeners, emulsifiers, acidulants, amino acids, vitamins, minerals, and other nutritional components, as well as components commonly used in foods (including the food raw materials and foods). Furthermore, as mentioned above, the medium components are not limited as long as they can culture the lactic acid bacteria of the present disclosure, and the above-mentioned explanation for the culture method applies. These components may be used alone or in combination of two or more, and the amounts thereof may be determined appropriately depending on the form and manner of use.

[0038] Without being limiting the present disclosure, specific examples of oral compositions include tea (including tea leaves, fermented tea leaves, tea drinks, etc.), dairy products such as yogurt, cheese, and milk drinks, pickles such as kimchi, bran pickles, and sauerkraut, confectioneries such as natto, chocolate, candy, and biscuits, bread, noodles, (processed) seafood foods (such as fermented sushi), (processed) livestock foods (such as ham), oils and fats, seasonings, beverages such as amazake, soft drinks, carbonated drinks, beauty drinks, nutritional drinks, fruit drinks, and vegetable drinks, beverages such as concentrated beverage concentrates and powders for adjustment, and fermented and non-fermented foods such as supplements.

[0039] Although not limiting the present disclosure, for example, tea is a preferred example of an oral composition, given that the lactic acid bacteria have high resistance to tea leaf extracts (tea leaf extract components). Furthermore, for example, given that the lactic acid bacteria have resistance to digestive juices, adherence to intestinal epithelial cells, and / or γ-aminobutyric acid production, the present disclosure can easily provide tea with even better intestinal regulating effects or tea with a high γ-aminobutyric acid content. As described above, such tea can be produced by, for example, mixing the lactic acid bacteria of the present disclosure with the raw materials for the tea (such as tea leaves or tea leaf extracts) according to conventional tea production procedures (including fermented tea).

[0040] Furthermore, while not limiting the present disclosure, examples of embodiments of the composition of the present disclosure include yogurt, kimchi, bran pickles, supplements, etc. Yogurt can be produced, for example, by mixing raw materials (foodstuffs) such as dairy ingredients with the lactic acid bacteria of the present disclosure according to conventionally known yogurt production procedures. Kimchi can be produced, for example, by mixing any raw materials, such as vegetables such as Chinese cabbage and seasonings, with the lactic acid bacteria of the present disclosure according to conventionally known kimchi production procedures. Bran pickles can be produced, for example, by mixing any raw materials, such as vegetables such as radish, Chinese cabbage, carrot, cucumber, and eggplant, rice bran, salt, and sake lees, with the lactic acid bacteria of the present disclosure according to conventionally known bran pickle production procedures. Furthermore, while not limiting the present disclosure, the composition of the present disclosure can be produced in the form of a tablet or the like by mixing, for example, a dried product of the lactic acid bacteria of the present disclosure with ingredients such as starch, lactose, and cellulose according to conventionally known supplement production procedures.

[0041] In the present disclosure, the subject (subject animal) of the composition is not limited, and examples thereof include humans, non-human mammals, etc. Examples of non-human mammals include mice, rats, guinea pigs, rabbits, dogs, cats, monkeys, pigs, cows, horses, etc., preferably mice, rats, guinea pigs, rabbits, dogs, monkeys, etc.

[0042] The amount of the composition of the present disclosure to be applied to a subject (subject animal) is not particularly limited, and may be appropriately determined depending on the physique, age, symptoms, application form, purpose of use, etc. of the subject (subject animal). For example, when the composition of the present disclosure is an oral composition, the amount of the composition to be applied to a human (body weight 60 kg) is 10 4 ~10 11 cells, preferably 10 8 ~10 11 When the composition of the present disclosure is other than an oral composition, the amount of the lactic acid bacteria of the present disclosure in the composition may be appropriately determined with reference to the above range.

[0043] According to the present disclosure, compositions containing the lactic acid bacteria can be easily provided. Furthermore, since the lactic acid bacteria of the present disclosure have at least one of the properties (1) to (4) above, compositions intended to obtain the beneficial effects resulting from these properties can be easily provided.

[0044] Examples of useful effects resulting from resistance to digestive juices and adhesion to intestinal epithelial cells include, but are not limited to, intestinal regulation, improvement of intestinal flora, improvement of bowel movements (e.g., regulating stomach condition), allergy relief, prevention of infection by pathogenic bacteria, improvement of colitis, and improvement of lipid metabolism. Examples of useful effects resulting from the ability to produce γ-aminobutyric acid include those conventionally known as effects based on γ-aminobutyric acid, such as improvement of high blood pressure, stress relief, improvement of sleep quality, reduction of fatigue, maintenance of skin elasticity, and improvement of cognitive function. Furthermore, as described above, the resistance to tea leaf extracts allows the lactic acid bacteria of the present disclosure to be preferably applied to tea products, and in particular, to the production of fermented tea to which the above-described useful effects have been imparted. Thus, the present disclosure makes it possible to easily provide compositions intended to maintain or improve health.

[0045] As mentioned above, the lactic acid bacteria of the present disclosure can also be used as a starter by mixing them with raw material compositions, and producing fermented foods using the lactic acid bacteria of the present disclosure can impart beneficial effects attributable to the lactic acid bacteria while suppressing the growth of unwanted harmful bacteria (contaminants) by lowering the pH in the fermented food. Furthermore, the use of the lactic acid bacteria of the present disclosure facilitates management of the production process (including the fermentation process, etc.) and also leads to improved stability of the quality of the final product. [Example]

[0046] The present disclosure will be described in more detail below with reference to examples, but the present disclosure is not limited thereto. Test Example 1 Awa Bancha tea leaves (wet weight after fermentation before drying) were fermented according to the standard Awa Bancha manufacturing procedure. 1 g of Awa Bancha tea leaves was added to 10 mL of phosphate-buffered saline (PBS) (composition: 6.5 g sodium chloride, 5.15 g disodium phosphate dodecahydrate, 0.3 g monopotassium phosphate per liter, pH 7.2 ± 0.2) and shaken at room temperature (25°C) for 1 minute. The supernatant was smeared onto an MRS agar plate (Becton, Dickinson and Company) containing 2% agar and cultured at 35°C for 48 hours in an anaerobic jar. Bacterial DNA was extracted from the resulting colonies and identified based on the results of multiplex PCR for the recA gene and 16S rRNA homology.

[0047] In this identification, the Lactiplantibacillus plantarum group (L. plantarum, L. pentosus, and L. paraplantarum) had approximately 99.8% homology in their 16S rRNA genes, making it difficult to distinguish between them based on 16S rRNA gene homology. Therefore, multiplex PCR was performed on the recA gene, and the PCR amplified products were subjected to 2% agarose gel electrophoresis. Bands were identified at 318 bp for L. plantarum, 218 bp for L. pentosus, and 107 bp for L. paraplantarum, confirming the identification of the L. plantarum group. Based on the bands obtained, two isolates belonging to L. pentosus were designated strains AWA1922 and AWA1955, respectively. On the other hand, no bands were observed in species outside this group, so based on the homology of the 16S rRNA gene, three isolated strains belonging to Levilactobacillus brevis were named AWA1978, AWA1984, and AWA1985, respectively.

[0048] These strains were deposited at the National Institute of Technology and Evaluation Biotechnology Center (2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan, October 29, 2021). The AWA1922 strain is deposited under accession number NBRC115326, the AWA1955 strain under accession number NBRC115327, the AWA1978 strain under accession number NBRC115323, the AWA1984 strain under accession number NBRC115324, and the AWA1985 strain under accession number NBRC115325.

[0049] The sugar assimilation ability of the five lactic acid bacteria strains was evaluated according to the usual evaluation procedures known in the art. T Strain (Lactobacillus pentosus NBRC106467 T ), Lactobacillus brevis NBRC107147 T Strain (Lactobacillus brevis NBRC107147 T ) were used as standard strains for Lactipranchibacillus pentosus and Lactobacillus brevis, respectively. The evaluation results are shown in Table 1.

[0050] [Table 1]

[0051] As shown in Table 1, all of these lactic acid bacteria had different sugar assimilation abilities from the standard strain.

[0052] Test Example 2 Using the lactic acid bacteria (5 strains) obtained in Test Example 1 and the above-mentioned standard strains (2 strains), tests were conducted on (1) digestive fluid resistance, (2) intestinal epithelial cell adhesion, (3) γ-aminobutyric acid production, and (4) tea leaf extract resistance as follows.

[0053] (1) Digestive fluid resistance (1A) Test Procedure The artificial gastric juice was prepared using MRS liquid medium (Becton, Dickinson and Company) containing 0.04% pepsin and adjusted to pH 3.0 or 2.0 with hydrochloric acid. The pH was measured at 25°C using a pH meter LAQUA (Model F-74, Horiba, Ltd.). The artificial intestinal juice was prepared using MRS liquid medium containing 0.2% bile powder, 0.01% trypsin, and 0.01% pancreatin.

[0054] 100 μL (10 9 cfu / mL) was inoculated into 10 mL of MRS liquid medium and statically cultured at 35°C for 24 hours to obtain a lactic acid bacteria culture solution. 1 mL of the thus obtained lactic acid bacteria culture solution was added to 9 mL of the artificial gastric juice (pH 3.0 or pH 2.0) and allowed to come into contact at 35°C for 3 hours. The resulting solution was then smeared on an MRS agar plate and cultured in an anaerobic jar at 35°C for 24 hours, and the number of colonies that formed was counted (artificial gastric juice treatment: 3 hours). Additionally, the treatment time with artificial gastric juice was set to 0, and the lactic acid bacteria culture solution obtained as described above was mixed with the artificial gastric juice and immediately smeared on an MRS agar plate, and the colonies were counted in the same manner as above (artificial gastric juice treatment: 0).

[0055] Furthermore, 100 μL of the lactic acid bacteria solution that had been contacted with artificial gastric fluid for 3 hours as described above was mixed with 10 mL of the artificial intestinal fluid, and the mixture was cultured at 35° C. for 24 hours. The resulting colonies were counted in the same manner as described above and cultured on an MRS agar plate (artificial intestinal fluid treatment: 24 hours). Furthermore, the treatment time with artificial intestinal fluid was set to 0, and the lactic acid bacteria solution that had been contacted with artificial gastric fluid for 3 hours as described above and the artificial intestinal fluid were immediately mixed and smeared on an MRS agar plate. The mixture was cultured in the same manner as described above and the resulting colonies were counted (artificial intestinal fluid treatment: 0).

[0056] (1B) Results The results are shown in Figures 1 and 2. Figure 1 shows the results using artificial gastric juice adjusted to pH 3.0, and Figure 2 shows the results using artificial gastric juice adjusted to pH 2.0.

[0057] As shown in Figures 1 and 2, Lactipranchibacillus pentosus strains AWA1922 and AWA1955 are the same strains as the standard strain NBRC106467. T The AWA1922 strain was found to have a higher resistance to digestive juice than the AWA1955 strain. As shown in Figures 1 and 2, the AWA1978, AWA1984, and AWA1985 strains were found to have a higher resistance to digestive juice than the standard strain NBRC107147. T Higher resistance to digestive fluids was observed than in the AWA1922, AWA1978, AWA1984, and AWA1985 strains. In particular, significantly higher resistance to digestive fluids was observed in the AWA1922, AWA1978, AWA1984, and AWA1985 strains. This indicates that these strains have excellent resistance to digestive fluids, and that the AWA1922, AWA1978, AWA1984, and AWA1985 strains in particular have extremely high resistance to digestive fluids.

[0058] (2) Adhesion to intestinal epithelial cells (Caco-2) (2A) Test Procedure Lactic acid bacteria cultured in MRS liquid medium at 35°C for 24 hours were collected by centrifugation, replaced with phosphate-buffered saline, and the bacterial count was measured using a bacteria counter (product name: Bacteria Counter, manufactured by Sunlead Glass Co., Ltd.). The lactic acid bacteria suspension obtained by replacing with phosphate-buffered saline was then transferred to cell culture medium (MEM (Minimum Essential Medium), manufactured by Thermo Fisher Scientific) containing 20% ​​fetal bovine serum and non-essential amino acids (8.9 mg / L L-alanine, 15 mg / L L-asparagine·H2O, 13.3 mg / L L-aspartic acid, 14.7 mg / L L-glutamic acid, 7.5 mg / L glycine, 11.5 mg / L L-proline, and 10.5 mg / L L-serine) at 1 × 10 7Separately, human colon cancer-derived Caco-2 cells were cultured in MEM containing 20% ​​fetal bovine serum and non-essential amino acids (8.9 mg / L L-alanine, 15 mg / L L-asparagine·H2O, 13.3 mg / L L-aspartic acid, 14.7 mg / L L-glutamic acid, 7.5 mg / L glycine, 11.5 mg / L L-proline, 10.5 mg / L L-serine) at a concentration of 2 × 10 5 The cells were planted in a 6-well plate at a concentration of 1000 cells / mL and cultured at 37°C in 5% CO2 for 48 hours. After culture, the culture supernatant was replaced with lactic acid bacteria suspension medium (2 mL) and incubated at 35°C for 2 hours. The lactic acid bacteria suspension medium was then removed and the cells were washed three times with phosphate-buffered saline. After that, sterilized ultrapure water was added and the cells were suspended, then smeared on an MRS agar plate and cultured in an anaerobic jar at 35°C for 48 hours, after which the number of colonies was counted.

[0059] (2B) Results The results are shown in Figure 3. As shown in Figure 3, the Lactipranchibacillus pentosus AWA1922 and AWA1955 strains were significantly higher than the standard strain NBRC106467. T The AWA1922 and AWA1955 strains adhered to the intestinal epithelial cell Caco-2 more effectively than the AWA1922 strains. This indicates that the AWA1922 and AWA1955 strains can remain in the digestive tract for a certain period of time, making them particularly useful for regulating the intestinal environment.

[0060] (3) γ-aminobutyric acid production (3A) Test Procedure 100 μL of a lactic acid bacteria culture solution cultured in MRS liquid medium at 35° C. for 24 hours was added to 10 mL of MRS liquid medium containing 5% sodium glutamate, and the mixture was cultured at 35° C. for 48 hours. The resulting culture solution was centrifuged, and 100 μL of the supernatant was mixed with 900 μL of 10% sulfosalicylic acid and then diluted with lithium citrate buffer P-21 (manufactured by JEOL Ltd.). The amount of γ-aminobutyric acid was measured using a fully automated amino acid analyzer JLC500 / V2 (manufactured by JEOL Ltd.).

[0061] (3B) Results The results are shown in Figure 4. As shown in Figure 4, the Lactobacillus brevis AWA1978, AWA1984, and AWA1985 strains were significantly different from the standard strain NBRC107147. T It was found that Lactiprunci Bacillus pentosus strains AWA1922, AWA1955, and their standard strain NBRC106467 produced more γ-aminobutyric acid than the Lactiprunci Bacillus pentosus strains AWA1922, AWA1955, and their standard strain NBRC106467. T In both cases, no production of γ-aminobutyric acid was observed in the test.

[0062] (4) Growth in tea leaf extract (4A) Test Procedure Boiling ultrapure water was added to commercially available, standard green tea leaves to a tea leaf concentration of 0.02 g / mL, and glucose was added to a concentration of 1%. The mixture was then allowed to stand for 1 minute. The supernatant of the resulting solution was used as a tea leaf extract containing 1% glucose. 100 μL of a lactic acid bacteria culture solution cultured in MRS liquid medium at 35°C for 24 hours was added to 10 mL of the resulting tea leaf extract. After 24 hours of incubation at 35°C, the mixture was smeared on an MRS agar plate and the number of colonies was counted. The relative growth rate of lactic acid bacteria in contact with the tea leaf extract was calculated using the number of colonies when the lactic acid bacteria culture solution cultured in MRS liquid medium (not in contact with the tea leaf extract) was smeared on an MRS agar plate as 100%.

[0063] (4B) Results The results are shown in Figure 5. Although tea leaf extract is known to have antibacterial activity, as shown in Figure 5, all strains were found to be resistant to the components of green tea leaf extract, and in particular, Lactipranchibacillus pentosus AWA1922, Reviractobacillus brevis AWA1978, AWA1984, and AWA1985 strains were found to be highly resistant to the tea leaf extract compared to the respective standard strains. [Accession number]

[0064] NBRC115323 NBRC115324 NBRC115325 NBRC115326 NBRC115327

Claims

1. The lactic acid bacterium is Lactiplantibacillus pentosus strain AWA1922 (accession number NBRC115326), Lactiplantibacillus pentosus strain AWA1955 (accession number NBRC115327), Levilactobacillus brevis strain AWA1978 (accession number NBRC115323), Levilactobacillus brevis strain AWA1984 (accession number NBRC115324), or Levilactobacillus brevis strain AWA1985 (accession number NBRC115325).

2. A composition containing at least one lactic acid bacterium selected from the group consisting of Lactiplantibacillus pentosus AWA1922 strain (accession number NBRC115326), Lactiplantibacillus pentosus AWA1955 strain (accession number NBRC115327), Levilactobacillus brevis AWA1978 strain (accession number NBRC115323), Levilactobacillus brevis AWA1984 strain (accession number NBRC115324), and Levilactobacillus brevis AWA1985 strain (accession number NBRC115325).

3. The composition of claim 2 , wherein the composition is an oral composition.

4. The composition according to claim 2 or 3, wherein the composition is at least one selected from the group consisting of fermented tea, yogurt, pickles, and supplements.

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