JP7677635B2 - Antiallergic agents, intestinal immunity enhancers, agents for improving the intestinal adhesion of lactic acid bacteria - Google Patents

Description

The present invention relates to an antiallergic agent, an intestinal immunity enhancer, and an agent for improving the intestinal adhesion of lactic acid bacteria.

Several types of T cells act as the command center for the human immune system. There are two types of T cells: Th1 cells, which mainly stimulate the production of IgA antibodies and activate cellular immunity, and Th2 cells, which stimulate the production of IgE antibodies that cause type I allergies and activate humoral immunity. These Th1 and Th2 cells suppress each other, and allergic symptoms are more likely to occur when Th2 cells are dominant over Th1 cells.

It is said that 70% of the body's immune cells are concentrated in the intestinal tract, where they play a major role in the body's immune system. In recent years, it has become clear that lactic acid bacteria play an important role in intestinal immunity. For example, it is known that the bacterial components of lactic acid bacteria promote cytokine secretion from macrophages and dendritic cells in the intestinal tract, thereby activating the activity of Th1 cells and promoting the production of IgA antibodies. This is thought to suppress the activity of Th2 cells, inhibiting the production of IgE antibodies, and improving allergies.

The adhesion of lactic acid bacteria (and their bacterial components) to the intestinal tract is thought to be extremely important for enhancing this intestinal immune stimulating effect and the associated anti-allergic effect. If oral agents that improve the intestinal adhesiveness of lactic acid bacteria can be used to promote intestinal adhesion, it is expected that allergies can be prevented or improved. Such oral agents must be highly safe and inexpensive to obtain, but their development has not progressed sufficiently and the diverse needs of patients and consumers have not been fully met.

On the other hand, green juice is a product that uses processed plant stems and/or leaves, such as dried powder or juice powder, and is used as a health food that allows easy intake of vegetable ingredients. The plant stems and/or leaves used as ingredients for green juice are known as inexpensive food ingredients that can be safely ingested, and various effects have been investigated so far (e.g., Patent Document 1), but there has been no study of the anti-allergic effect focusing on the role of lactic acid bacteria in intestinal immunity.

JP 2003-267880 A

Therefore, the object of the present invention is to provide a new agent for improving the intestinal adhesion of lactic acid bacteria, an agent for enhancing intestinal immunity, and an antiallergic agent.

The present inventors have conducted extensive research into substances that can increase the intestinal adhesiveness of lactic acid bacteria. As a result, they have discovered that the intestinal adhesiveness of lactic acid bacteria can be increased by using the stems and/or leaves of plants that are used as green juice ingredients, which are inexpensive and safe to obtain.

The present invention is based on the above findings and provides an anti-allergy agent (excluding anti-allergy agents that suppress interleukin 4 production) that contains the stems and/or leaves of plants that are green juice ingredients.

The present invention also provides an intestinal immunity enhancer and an intestinal adhesiveness improver for lactic acid bacteria that contain the stems and/or leaves of plants that are green juice ingredients.

The present invention provides an antiallergic agent, an intestinal immunity enhancer, and an agent for improving the intestinal adhesion of lactic acid bacteria that can be safely ingested and is available at low cost and has excellent effects of improving the intestinal adhesion of lactic acid bacteria.

FIG. 1 is a graph showing the results of evaluating the intestinal adhesive action of the agents of the Examples and Comparative Examples.

The present invention will be described below based on its preferred embodiments. The agent of the present invention contains the stems and/or leaves (hereinafter simply referred to as "stems and leaves") of a plant that is a raw material for green juice. Unless otherwise specified, the following description applies to the anti-allergy agent, intestinal immunity enhancer, and agent for improving the intestinal adhesion of lactic acid bacteria of the present invention. The stems and leaves may be only the stems or only the leaves, or a mixture thereof, but are preferably only the leaves or a mixture of stems and leaves. Furthermore, the agent of the present invention may contain flowers, roots, etc. in addition to the stems and leaves as the raw material for green juice, or may not contain any parts other than the stems and leaves.

Preferred plants that are the raw materials for green juice include (1) barley, (2) sweet potato, (3) kale, (4) mulberry, and (5) Peony root. These are explained below.

(1) Barley (scientific name: Hordeum vulgare) includes two-rowed barley, six-rowed barley, naked barley, etc., and these may be used alone or in combination of two or more. Barley stalks and leaves are preferably harvested before maturity, that is, from the time when tillers start to the time when heading starts. Specifically, although it varies depending on the variety, it is generally preferable to harvest stalks and leaves from barley that is 10 cm or more tall, preferably about 10 to 90 cm, particularly preferably about 20 to 80 cm, and especially about 30 to 70 cm tall, but this is not limited thereto. Barley stalks and leaves used in the present invention are not particularly limited as long as they are normally available, and those obtained by the processing method described below or commercially available products can be used.

(2) Examples of sweet potatoes (scientific name: Ipomoea batatas) include Suio, Joy White, Koganesengan, Shiroyutaka, Satsuma Starch, and Ayamurasaki, and these can be used alone or in combination of two or more. Among sweet potatoes, Suio, which has a high polyphenol content, is preferred. Suio has the characteristic that young sweet potato stems and leaves regenerate from the tip of the same stem even after the stems and leaves have been harvested once, so it is also suitable for use in terms of productivity. As sweet potato stems and leaves, it is preferable to use stems and/or leaves that are exposed above the ground during sweet potato cultivation. Sweet potato stems and leaves that have grown 5 cm or more, more preferably 10 cm or more, and even more preferably 20 cm or more out of the ground are preferred. In addition, the length from the position where the sweet potato stem is exposed above the ground to the tip of the sweet potato stem and leaves is preferably 300 cm or less, more preferably 200 cm or less, and even more preferably 150 cm or less. As sweet potato stalks and leaves, the tip portion of the sweet potato stalks and leaves ("young sweet potato stalks and leaves") is preferred, and young sweet potato stalks and leaves that retain a yellowish green color compared to other stalks and leaves are even more preferred. As young sweet potato stalks and leaves, stalks and leaves within 100 cm from the tip of the sweet potato stalks and leaves are preferably used. Young sweet potato stalks and leaves are easy to process because the plant body itself is soft. Furthermore, when dried and powdered, young sweet potato stalks and leaves have a good texture and can be easily used in various foods. The sweet potato stalks and leaves used in the present invention are not particularly limited as long as they are commonly available, and those obtained by the processing method described below or commercially available products can be used.

(3) Kale is Brassica oleracea L. (a crop of the genus Brassica). Specific examples of kale include kitchen kale, tree kale, bush kale, marrow kale, collard, green-leaf kale, drinking leaf, Portuguese kale, Chinese kale, thousand-headed kale, and Jersey kale, and these can be used alone or in combination of two or more. The kale stems and leaves used in the present invention are not particularly limited as long as they are normally available, and those obtained by the processing method described below or commercially available products can be used.

(4) Mulberry is a general term for the genus Morus (scientific name: Morus) of the family Moraceae. Examples of mulberry include Japanese mulberry, Japanese mulberry, log mulberry, striped mulberry, Korean mulberry, and hybrids and hybrids thereof. These may be used alone or in combination of two or more. The mulberry stems and leaves used in the present invention are not particularly limited as long as they are normally available, and those obtained by the processing method described below or commercially available products may be used.

(5) Peucedanum japonicum (scientific name: Peucedanum japonicum) is a perennial plant of the Apiaceae family, Peucedanum genus. It is also known as Chou-mei-sou, and grows wild mainly from southern Kyushu to Okinawa. The stems and leaves of Peucedanum japonicum used in the present invention are not particularly limited as long as they are normally available, and may be obtained by the processing method described below or commercially available products.

In the present invention, various processed products obtained from the stems and leaves of plants that are the green juice ingredients can be used. Examples of such processed products include dried powder (sometimes called pulverized powder) obtained by drying and pulverizing the stems and leaves, shredded stems and leaves and dried powder thereof, juice obtained by squeezing the stems and leaves and dried powder thereof, and extract obtained by extracting the stems and leaves with water, an organic solvent or a mixture thereof and dried powder thereof. These may be products obtained through a process of fermenting the stems and leaves (fermented products) or products obtained without a fermentation process (non-fermented products).

For example, a conventional method can be used to dry and powder the stems and leaves (to produce a pulverized powder). As such a method, a combination of drying and pulverization can be used for the stems and leaves. Either the drying or pulverization can be performed first, but it is preferable to perform the drying first. This method can be combined with one or more treatments selected from blanching, sterilization, etc., as necessary, to dry and powder the stems and leaves. The pulverization can be performed once or twice or more times, but it is preferable to combine a coarse pulverization process with a fine pulverization process to pulverize the stems and leaves more finely.

Blanching is a process for keeping the green color derived from the stems and leaves vivid, and examples of the method of blanching include hot water treatment and steaming. The heating temperature in the blanching process is preferably higher than 80°C, more preferably 85°C or higher, and even more preferably 90°C or higher. The heating temperature is preferably 100°C or lower. The heating time in the heat treatment is preferably 10 minutes or less, more preferably 5 minutes or less, even more preferably 3 minutes or less, and particularly preferably 10 seconds to 1 minute. In addition, when hot water treatment is performed as the blanching process, it is preferable to dissolve carbonates such as magnesium carbonate or bicarbonates such as sodium bicarbonate in the hot water, since this makes the green color of the stems and leaves more vivid. In this case, the pH of the hot water is preferably 5.4 or higher, more preferably 5.6 to 8.4, and even more preferably 5.6 to 8.0. In addition, as the steaming process, an intermittent steaming process in which the process of steaming the stems and leaves with water vapor and the process of cooling are repeated under normal pressure or pressure is preferable. In the intermittent steaming process, steaming with water vapor is preferably performed for 20 to 40 seconds, more preferably 30 seconds. Cooling after the steaming process is preferably performed immediately, and the method is not particularly limited, but may be immersion in cold water, refrigeration, cooling with cold air, evaporative cooling with hot air, or evaporative cooling combining hot air and cold air. Of these, evaporative cooling combining hot air and cold air is preferred. Such cooling is performed so that the product temperature of the stems and leaves is preferably 60°C or less, more preferably 50°C or less, and most preferably 40°C or less. In order to produce a powder of stems and leaves rich in nutrients such as vitamins, minerals, and chlorophyll, it is preferable to repeat the intermittent steaming process 2 to 5 times.

Sterilization is usually a process of physically and chemically killing microbial cells using temperature, pressure, electromagnetic waves, chemicals, etc. Among these methods, heat sterilization is a preferred process because it can improve the flavor of the stems and leaves. The temperature of the heat treatment is preferably 110°C or higher, and specific equipment that can be used includes a high-pressure sterilizer, a heat sterilizer, and a pressurized steam sterilizer. For example, in the case of heat treatment by pressurized steam sterilization, the coarsely crushed sweet potato stems and leaves are preferably heat sterilized for 2 to ¹⁰ seconds with saturated steam at 110°C to 200°C under a pressure of ^0.5 kg/cm2 to 10 kg/cm2. After these sterilization processes, the moisture contained during heating with saturated steam can be further dried as necessary.

In addition, the drying process is preferably such that the moisture content of the stems and leaves after drying is 10% or less, particularly 5% or less, since a dried powder of stems and leaves with a good flavor and bright color can be obtained. This drying process can be performed by any method known to those skilled in the art, such as hot air drying, high-pressure steam drying, electromagnetic wave drying, freeze drying, etc. Drying by heating can be performed at a temperature and for a time period at which the stems and leaves do not discolor due to heating. In terms of obtaining a dried powder of stems and leaves with a good flavor and bright color, it is preferable to perform the drying process at 60°C to 150°C, preferably 70°C to 100°C. When drying the stems and leaves as they are, it is preferable to perform drying in two stages. The two-stage drying can be performed, for example, using a hot air dryer. In the two-stage drying, first, the stems and leaves are primarily dried at a temperature of 60°C to 80°C until the moisture content is 25% by mass or less. Then, the stems and leaves that have been primarily dried are secondary dried at a temperature higher than that of the primary drying, preferably 70°C to 90°C, until the moisture content is 5% by mass or less.

The crushing process may be performed by any method commonly used by those skilled in the art, using a crusher, mill, blender, stone mill, etc. The crushed stems and leaves may be sieved as necessary.

When blanching is performed in addition to drying and grinding, it is preferable that the blanching is performed before the drying. Also, when sterilization is performed in addition to drying and grinding, it is preferable that the sterilization is performed after the drying, or before or after the grinding.

Specific methods for drying and powdering include, for example, cutting the stems and leaves, blanching them, then drying them so that the moisture content is 10% by mass or less, preferably 5% by mass or less, and then pulverizing them (see JP 2004-000210 A). Another example is cutting the stems and leaves, blanching them, rolling them, drying them, and pulverizing them (see JP 2002-065204 A). Another example is drying the stems and leaves, coarsely pulverizing them, heating them at 110°C or higher, and further finely pulverizing them (see JP 2003-033151 A). It is preferable that the particle size of the powder is such that 50% by mass or more, especially 70% by mass or more, passes through a 200 mesh.

The method of shredding the stems and leaves can be any method that is commonly used by those skilled in the art when shredding plants, such as slicing, crushing, or chopping. One example of shredding is slurrying. Slurrying is performed by putting the stems and leaves through a mixer, juicer, blender, mass colloider, or the like to make the stems and leaves into a mushy gruel (a suspension of liquid and solids). By slurrying in this way, the stems and leaves are shredded so that 80% or more by mass of the shredded pieces preferably have an average diameter of 1 mm or less, more preferably 0.5 mm or less, even more preferably 0.1 mm or less, and most preferably 0.05 mm or less, and the shredded pieces become fluid.

Methods for squeezing the stems and leaves include squeezing the stems and leaves or shredded material thereof, or centrifuging or filtering the shredded material. A representative example is a method in which the juice is extracted by mechanical crushing means such as a mixer or juicer, and if necessary, the crude solid content is removed by means of sieving, filtration, or the like to obtain a squeezed juice, and specific examples of such methods are described in JP-A-08-245408 and JP-A-09-047252.
In addition, the method for obtaining an extract of stems and leaves includes a method in which an extraction solvent commonly used by those skilled in the art, such as ethanol, water, or aqueous ethanol, is added to the stems and leaves or shredded material thereof, and extraction is performed by heating as necessary. The extract may be concentrated as necessary.

Among processed stems and leaves, it is particularly preferable to use crushed stems and leaves (crushed powder, etc.) or squeezed juice (squeezed juice powder, etc.) because this makes the agent of the present invention more vivid in color and has a better flavor, and because it is rich in dietary fiber. Commercially available processed products can be used. For example, in the case of barley, commercially available crushed barley stems and leaves powder (product name "Barley Young Leaf Powder", etc.) or squeezed juice powder (product name "Barley Young Leaf Extract", etc.) can be used, and in the case of sweet potato, commercially available crushed sweet potato stems and leaves powder (product name "Suio", etc.) can be used.

In terms of enhancing the intestinal adhesion activity of lactic acid bacteria, it is even more preferable that the plant that is the raw material for green juice contains two or more of the above-mentioned (1) barley, (2) sweet potato, (3) kale, (4) mulberry, and (5) Peony Root. It is particularly preferable that the plant that is the raw material for green juice contains (1) barley and at least one selected from (2) sweet potato, (3) kale, (4) mulberry, and (5) Peony Root. It is more preferable that the plant contains (1) barley and two or more selected from (2) sweet potato, (3) kale, (4) mulberry, and (5) Peony Root. When the agent of the present invention contains (1) and two or more selected from (2) to (5), it is particularly preferable that the plant contains (1) barley, (3) kale, and (5) Peony Root. Most preferably, it contains all of (1) barley, (2) sweet potato, (3) kale, (4) mulberry, and (5) Peony root.

The total amount of stems and leaves of plants that are green juice ingredients in the agent of the present invention is preferably 0.01% to 90% by mass, more preferably 0.05% to 80% by mass, and particularly preferably 0.1% to 70% by mass, in terms of dry mass in the agent of the present invention. The total amount referred to here is the content of only one plant that is a green juice ingredient when the agent of the present invention contains only one plant. When the agent of the present invention contains the stems and leaves of any of the plants (1) to (5) above, the preferred range of the total amount of stems and leaves of plants (1) to (5) in the agent of the present invention is the same as the total amount of stems and leaves of the plants that are green juice ingredients.

In particular, in order to enhance the intestinal adhesion effect of lactic acid bacteria by the agent of the present invention, when the agent contains the stems and leaves of any of the plants (1) to (5), the stems and leaves of each of the plants (1) to (5) are preferably contained in the agent of the present invention in an amount of 0.01% to 80% by mass, more preferably 0.05% to 70% by mass, and particularly preferably 0.1% to 50% by mass, on a dry mass basis.

When the agent of the present invention contains the above-mentioned (1) and one or more of (2) to (5), the total dry mass of (2) to (5) is preferably 0.1 parts by mass to 1,000 parts by mass, more preferably 0.5 parts by mass to 800 parts by mass, and particularly preferably 1 part by mass to 500 parts by mass, per 100 parts by mass of dry mass of (1).

The agent of the present invention may consist essentially of only (1) to (5) as plant-derived components, or may contain other plant-derived components. For example, the plant-derived components other than (1) to (5) in the agent of the present invention preferably account for 70% by mass or less, and more preferably 50% by mass or less, of the agent of the present invention.

The agent of the present invention may not contain lactic acid bacteria, but it is preferable to contain lactic acid bacteria in order to further enhance the intestinal adhesion effect of lactic acid bacteria.Lactic acid bacteria may be live or dead.When it is live, it is preferable from the viewpoint of improving the intestinal environment, but it may be dead.This is based on the fact that not only live bacteria but also dead bacteria are considered to stimulate intestinal immunity.The lactic acid bacteria used in the present invention are not particularly limited as long as they produce lactic acid as a metabolic product, and include those that have been orally ingested in animals such as humans, for example, those of the genus Bifidobacterium, Lactobacillus, Enterococcus, Leuconostoc, Pediococcus, Staphylococcus, Tetragenococcus, and Bacillus.
Examples of the genus Bifidobacterium include Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Bifidobacterium adolescentis, and Bifidobacterium mongoliense.
As the Lactbacillus genus, Lactbacillus brevis, Lactbacillus gasseri, Lactobacillus acidophilus, Lactobacillus buchneri, Lactobacillus bulgaricus, Lactobacillus delburvecki, Lactobacillus casei, Lactobacillus crispatus, Lactobacillus curvatus, Lactobacillus halivaticus, Lactobacillus pentosus, Lactobacillus plantarum, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus salivarius, Lactobacillus sporogenes, Lactobacillus sakei, Lactobacillus fructivorans, Lactobacillus hilgardii, Lactobacillus reuteri, Lactobacillus fermentum.
Examples of Enterococcus include Enterococcus faecalis (sometimes referred to as Streptococcus faecalis), Enterococcus faesium (sometimes referred to as Streptococcus faesium), Streptococcus thermophilus, and Lactococcus lactis (sometimes referred to as Streptococcus lactis).
The genus Leuconostoc includes Leuconostoc mesenteroides and Leuconostoc oenos.
Examples of the genus Pediococcus include Pediococcus acidilactici and Pediococcus pentosaceus.
The genus Staphylococcus includes Staphylococcus carnosus and Staphylococcus xylosus.
The genus Tetragenococcus includes Tetragenococcus halophilus. The genus Bacillus includes Bacillus coagulans and Bacillus mesentericus.
Among these, Bacillus coagulans, Enterococcus faecalis, Bifidobacterium bifidum, Enterococcus faesium, and Lactobacillus acidophilus are preferable. These may be used alone or in combination of two or more.

When the agent of the present invention contains lactic acid bacteria, the content of the lactic acid bacteria is not particularly limited, but for example, the number of bacterial cells in the agent of the present invention is preferably 1 x ¹⁰ or more, preferably 1 x ¹⁰ or more and 1 x ^{10 or} less, and more preferably 1 x ¹⁰ or more and 1 x ¹⁰ or less, from the viewpoints of ease of formulation and enhancing the effect of the lactic acid bacteria adhering to the intestinal tract.

When the agent of the present invention contains lactic acid bacteria, the lactic acid bacteria and the stems and leaves of the plant may be contained in the same agent or in separate agents. When contained in separate agents, the agent of the present invention is a multi-drug type of two or more agents. When contained in the same agent, the agent of the present invention may be a single-drug type or a multi-drug type, but a single-drug type is preferable in terms of ease of intake, etc. The dosage form of the agent of the present invention, regardless of whether it is a single-drug type or a multi-drug type, may be a solid, liquid, paste, gel, etc. For example, solid forms include powder, granules, tablets, rods, plates, blocks, solid forms, capsules such as hard capsules and soft capsules, caplets, tablets, chewable forms, sticks, etc. Examples of liquid forms include fluid forms and syrup forms. These can be formulated into various dosage forms by known formulation methods by adding pharma-ceutically acceptable base materials or carriers.

For example, preferred dosage forms of the agent of the present invention containing lactic acid bacteria include a single dosage form that contains lactic acid bacteria and the stems and leaves of the plant and is in any of the following forms: powder, fine granules, granules, tablets, capsules such as soft capsules and hard capsules, or liquid; and a two-dosage form that has a first agent that contains the stems and leaves of the plant and is in any of the following forms: powder, fine granules, granules, tablets, capsules such as soft capsules and hard capsules, or liquid; and a second agent that contains lactic acid bacteria and is in any of the following forms: powder, fine granules, granules, tablets, capsules such as soft capsules and hard capsules, or liquid. In the present invention, the intestinal adhesiveness of lactic acid bacteria and bifidobacteria present in the intestine can be improved by using the first agent containing the stems and leaves of the plant alone.

The oral intake method of the agent of the present invention is not limited, and may be taken as is, or may be taken after dispersing or dissolving the agent in water, hot water, milk, yogurt, etc. Furthermore, when the agent of the present invention is a multi-drug type, the multiple agents may be taken simultaneously or at different times. When taken at different times, for example, either the lactic acid bacteria-containing agent or the plant stem and leaf-containing agent may be taken first, but the time difference between the intake of the two is preferably within 24 hours, more preferably within 18 hours, and particularly preferably within 15 hours. Furthermore, the intake methods of the two agents may be the same or different. When they are different, for example, the lactic acid bacteria-containing agent is taken as is, and the plant stem and leaf-containing agent is taken after dispersing in water, hot water, milk, yogurt, etc., or vice versa.

In addition to the stems and leaves of the plant and the lactic acid bacteria, the agent of the present invention may contain other commonly used ingredients to the extent that the effect of the present invention is not impaired. Examples of such ingredients include various excipients, binders, gloss agents, lubricants, stabilizers, diluents, bulking agents, thickeners, emulsifiers, antioxidants, pH adjusters, colorants, fragrances, additives, etc. The content of the other ingredients can be appropriately selected depending on the form of the agent of the present invention. Furthermore, when the agent of the present invention is a multi-form, which other ingredients are contained in which agent is also appropriately selected depending on the form of the agent.

The daily oral dose of the agent of the present invention is preferably 10 mg or more as the dry mass of the stems and leaves of the plant. The agent of the present invention can be administered continuously, for example, every day, and can be administered continuously for a long period of time, for example, for one month or more.

Regardless of whether the agent of the present invention contains lactic acid bacteria or not, the daily oral dose of lactic acid bacteria is preferably 1 x ¹⁰ or more, particularly 1 x ¹⁰ or more and 1 x ¹⁰ or less, based on the oral dose of the stems and leaves of the plant.

As will be apparent from the description of the Examples below, the agent of the present invention can enhance the intestinal adhesiveness of lactic acid bacteria by the action of the stems and leaves of the plant. The intestinal adhesiveness referred to here preferably means the adhesiveness to the intestinal epithelium (intestinal epithelial cells).
Therefore, the agent of the present invention can enhance the action of lactic acid bacteria in the intestinal tract by orally taking it, and can be used as an intestinal immunity enhancer, for example, and can prevent or improve allergic symptoms such as atopic dermatitis, allergic rhinitis, hay fever, eczema, urticaria, and other rashes, diarrhea, and vomiting. The action of the intestinal immunity enhancer includes activating the action of Th1 cells and promoting IgA antibody production. The agent of the present invention is preferably applied to humans, but can also be applied to animals other than humans (e.g., mice, rats, hamsters, dogs, cats, cows, pigs, monkeys, etc.) as long as each action and effect is exerted. The allergy agent of the present invention excludes those that exert anti-allergic action by suppressing interleukin 4 production.

Examples of lactic acid bacteria whose intestinal adhesiveness (attachment) is enhanced by the agent of the present invention include the same lactic acid bacteria as those listed above as lactic acid bacteria that may be contained in the agent of the present invention. The present invention provides foods, medicines, quasi-drugs, etc. that contain the agent of the present invention. The present invention provides a method (excluding medical procedures) for promoting lactic acid bacteria adhesion in the intestinal tract by ingesting the agent of the present invention together with lactic acid bacteria.

The present invention will be described in more detail below with reference to examples. However, the scope of the present invention is not limited to these examples. In the following, unless otherwise specified, "%" means "mass %" and "parts" means "parts by mass."

For barley stalks and leaves, we used crushed powder (manufactured by Toyo Shinyaku Co., Ltd.) obtained by drying and crushing stalks and leaves cut before ear emergence. For sweet potato stalks and leaves, we used crushed powder (manufactured by the same company) obtained by drying and crushing sweet potato stalks and leaves cut at a length of 30 to 80 cm from the tip of the stalk and leaves. For kale, we used crushed powder (manufactured by the same company) obtained by drying and crushing kale stalks and leaves. For mulberry, we used crushed powder (manufactured by the same company) obtained by drying and crushing mulberry stalks and leaves. For Peony peony, we used crushed powder (manufactured by the same company) obtained by drying and crushing Peony peony stems and leaves. For all of these crushed powders, 90% by mass or more passed the 200 mesh section, and the moisture content was 5% by mass or less. These crushed powders were used as test substances in the following adhesion ability test.

(Adhesion Ability Test)
(1) Preparation of monolayer using 96-well plate In a 37°C, 5% by volume _CO2 incubator, intestinal epithelial cell line Caco-2 cells were cultured in a normal medium using a 75 ^cm2 flask. This normal medium was prepared by adding 5 mL FBS (Japan Bio Serum) and 0.5 mL NEAA (Non-Essential Amino Acids, SIGMA) to 44.5 mL DMEM (containing 1% penicillin-streptomycin). The cells suspended by trypsin treatment were seeded from the 75 ^cm2 flask to a 96-well clear plate at a cell density of 4.0 x ¹⁰⁴ cells/well. The cells were cultured at 37° C. in a 5% by volume CO ₂ incubator for 72 hours to prepare monolayers.

(2) Fluorescent labeling of B. coagulans Bacillus coagulans stocked in glycerol at -80°C was returned to room temperature (25°C) and inoculated into MRS liquid medium warmed to 37°C. The cultured cells were cultured at 37°C for 24 hours and used in the test.
The pre-cultured B. coagulans was collected and centrifuged at room temperature and 1000g for 3 minutes. The pellet obtained by removing the supernatant was suspended in 1mL of phosphate buffered saline (PBS) and centrifuged at room temperature and 1000g for 3 minutes. The same suspension and centrifugation were repeated once more, the supernatant was removed, the pellet was suspended in 1mL of PBS, and 15μL of carboxyfluorescein diacetate (CFDA, Dojindo Laboratories) was added. After incubation at room temperature for 30 minutes in the dark, the mixture was centrifuged at room temperature and 1000g for 3 minutes. The operation of removing the supernatant, suspending in 1mL of PBS, and centrifuging at room temperature and 1000g for 3 minutes was repeated twice. The supernatant was then removed and suspended in the test medium. Note that a blank was prepared by carrying out the same operation except that CFDA was not added. The test medium used was 45 mL RPMI1640 medium (containing 1% penicillin-streptomycin) to which 5 mL FBS (Japan Bio Serum) was added. 100 μL of the suspension was transferred to a black plate and the fluorescence intensity was measured. The excitation wavelength was 495 nm and the fluorescence wavelength was 515 nm. The blank value was subtracted from the measured fluorescence intensity, and the sample was diluted with the test medium so that the fluorescence intensity was 200, and this was used as the B. coagulans sample.

(3) Evaluation of B. coagulans adhesion ability by measuring fluorescence intensity The medium of the monolayer of Caco-2 cells prepared in (1) was replaced from the normal medium to the test medium. The medium was removed, and the dispersion in which the test substance was dispersed in the test medium and the B. coagulans sample prepared in (2) were added to a 96-well plate in an amount of 100 μL each (final fluorescence intensity of B. coagulans: 100). After 2 hours of culture in a 5% by volume _CO2 incubator at 37°C, 150 μl of formalin was added to each well, and the wells were fixed for 30 minutes at 4°C in the dark. After washing three times with PBS and adding 100 μl of PBS, the fluorescence intensity was measured with a Varioskan (excitation wavelength: 495 nm, fluorescence wavelength: 515 nm). Based on the measured values, the relative value (%) to the fluorescence intensity of the sample to which only lactic acid bacteria was added was calculated, and this was taken as the adhesion rate of lactic acid bacteria to intestinal cells (monolayer). The results are shown in Figure 1. The concentrations of the test substances in the Caco-2 cell culture medium during incubation are as shown in Table 1 below (the units of values in Table 1 are μg/mL).

The results in Figure 1 show that the stems and leaves of plants that are the raw materials for green juice increase the intestinal adhesion rate of lactic acid bacteria. Furthermore, it can be seen that the intestinal adhesion rate of lactic acid bacteria is further increased when the stems and leaves of two or more plants, including barley stems and leaves, are used.

Claims (2)

An oral preparation for maintaining lactic acid bacteria in the intestines, containing barley stems and/or leaves as active ingredients. The oral agent according to claim 1, which is a food product.