WO2024100755A1 - Composition for improving intestinal barrier function and strengthening intestinal epithelial tight junctions - Google Patents

Abstract

[Problem] The present invention addresses the problem of providing a composition for improving the intestinal barrier function and/or strengthening intestinal epithelial tight junctions that contains Lactobacillus plantarum L-137 or a processed product thereof. [Solution] This problem is solved by means of the abovementioned composition.

Description

Composition for improving intestinal barrier function and enhancing intestinal epithelial tight junctions

The present invention relates to a composition for improving intestinal barrier function and enhancing intestinal epithelial tight junctions.

Tight junctions expressed in intestinal epithelial cells are an important component of the intestinal barrier that prevents various foreign substances such as intestinal bacteria, pathogens, and toxins from entering cells. When the formation of tight junctions becomes incomplete due to aging or stress, the intestinal barrier function is reduced or damaged, allowing foreign substances to enter the body. The invading foreign substances trigger an immune response and induce inflammation, causing diseases such as food allergies, irritable bowel syndrome, inflammatory bowel disease, and skin diseases. It is known that yeast with a high polyamine content improves intestinal barrier function (Patent Document 1). However, it is not known that the lactic acid bacteria Lactobacillus plantarum L-137 strain has the effect of improving intestinal barrier function or enhancing intestinal epithelial tight junctions.

Type I diabetes is thought to develop through some mechanism caused by increased antigen exposure to intestinal immunity. Celiac disease, which type I diabetes patients are at risk of developing, is a disease caused by gluten contained in wheat and other foods, which causes an inflammatory reaction in the small intestinal mucosa. Intestinal permeability of allergens also affects food allergies, hay fever, atopic dermatitis, and other conditions. Therefore, improving the intestinal barrier function in the body and enhancing tight junctions in the intestinal epithelium is thought to be useful in treating, preventing, and improving various immune diseases.

JP 2021-69331 A

The present invention aims to provide a novel composition that has an effect of improving intestinal barrier function and/or enhancing intestinal epithelial tight junctions. In addition, the present invention aims to provide a composition for treating, preventing, or improving one or more immune diseases selected from the group consisting of: (i) type I diabetes; (ii) celiac disease; (iii) inflammatory bowel disease; (iv) irritable bowel syndrome; (v) atopic dermatitis; and (vi) food allergies.

The inventors have examined a large number of materials in search of one that has the effect of improving intestinal barrier function or enhancing intestinal epithelial tight junctions, and have surprisingly found that the lactic acid bacteria Lactobacillus plantarum L-137 strain (hereinafter sometimes referred to as "L-137 strain") has such desirable effects. This was a surprising finding made by the inventors. After further investigations, the inventors have completed the present invention.

That is, the present invention is as follows.
[1] A composition for improving intestinal barrier function, comprising Lactobacillus plantarum L-137 or a processed product thereof.
[2] A composition for enhancing intestinal epithelial tight junctions, comprising Lactobacillus plantarum L-137 or a processed product thereof.
[3] A composition for treating, preventing, or ameliorating one or more immune diseases selected from the group consisting of the following (i) to (vi), characterized by containing Lactobacillus plantarum L-137 or a processed product thereof: (i) type I diabetes; (ii) celiac disease; (iii) inflammatory bowel disease; (iv) irritable bowel syndrome; (v) atopic dermatitis; and (vi) food allergy.
[4] The composition for administration to humans, animals, or seafood described in any one of [1] to [3], which is a food, beverage, or feed composition.
[5] The composition described in [4], wherein the food or beverage is a food additive or a supplement.
[6] Use of Lactobacillus plantarum L-137 or a processed product thereof for the manufacture of a medicine for improving intestinal barrier function or enhancing intestinal epithelial tight junctions.
[7] Use of Lactobacillus plantarum L-137 or a processed product thereof for the manufacture of a medicine for treating, preventing, or ameliorating one or more immune diseases selected from the group consisting of the following (i) to (vi): (i) type I diabetes; (ii) celiac disease; (iii) inflammatory bowel disease; (iv) irritable bowel syndrome; (v) atopic dermatitis; and (vi) food allergy.
[8] A method for improving intestinal barrier function or enhancing intestinal epithelial tight junctions, comprising administering Lactobacillus plantarum L-137 or a processed product thereof to a subject.
[9] A method for treating, preventing, or ameliorating one or more immune diseases selected from the group consisting of the following (i) to (vi), comprising administering to a subject Lactobacillus plantarum L-137 or a processed product thereof: (i) type I diabetes; (ii) celiac disease; (iii) inflammatory bowel disease; (iv) irritable bowel syndrome; (v) atopic dermatitis; and (vi) food allergy.

According to the present disclosure, a composition that improves intestinal barrier function and/or enhances intestinal epithelial tight junctions and a method for producing the same can be provided. The composition of the present invention also has an anti-inflammatory effect. Because the composition of the present invention has these effects, it is useful for treating, preventing, or ameliorating various immune diseases, such as, for example, type I diabetes, celiac disease, inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), atopic dermatitis, and food allergies.

Figure 1 shows a graph evaluating the amount of FD-4 that passed from the upper to lower layers of IEC-6 cells. TTEST (a Black bar vs Gray bars) *p<0.05 FIG. 2 shows a graph evaluating the expression level of tight junction-related gene (ZO-1). FIG. 3 shows a graph evaluating the expression level of a tight junction-related gene (Occludin). FIG. 4 shows a graph evaluating the expression level of a tight junction-related gene (Claudin-1).

[Composition for improving intestinal barrier function and/or enhancing intestinal epithelial tight junctions]
The composition of the present invention is characterized by having an intestinal barrier function improving effect and/or intestinal epithelial tight junction enhancing effect. Intestinal epithelial tight junction is an important component of the intestinal barrier to prevent various foreign substances from entering cells, but it also creates a physical barrier against foreign substances that attempt to pass through the intercellular space. In the present invention, preferred examples of foreign substances that can be blocked from entering or passing through include, but are not limited to, various types of intestinal bacteria, pathogenic bacteria, toxins, allergens, etc. Specific examples of these include, for example, antigen proteins derived from food (such as foods containing eggs, dairy products, wheat, barley, and rye), mites, dust, endotoxins, lipopolysaccharide (LPS), etc., and preferably, lipopolysaccharide (LPS) and antigen proteins derived from food are included, but are not limited to these. In addition, in the present specification, the intestine is the small intestine, large intestine, duodenum, cecum, jejunum, ileum, and colon, and preferably, the small intestine, but are not limited to these.

Furthermore, the composition of the present invention preferably has an anti-inflammatory effect, more preferably an inhibitory effect on increased intestinal permeability due to the inhibitory effect on inflammatory cytokines such as IL (interleukin)-1, IL-6, IL-8, TNF (tumor necrosis factor)-α, and INF-γ. Incidentally, an increase in TNF-α may be observed in patients with inflammatory bowel disease. In addition, the composition of the present invention is preferably for preventing, improving, or treating (i) type I diabetes; (ii) celiac disease; (iii) inflammatory bowel disease; (iv) irritable bowel syndrome; (v) atopic dermatitis; and (vi) food allergies, but is not limited to these cases. Incidentally, inflammatory bowel disease includes ulcerative colitis and Crohn's disease. Specific examples of food allergies include, but are not limited to, allergies to food allergens such as chicken eggs, dairy products, wheat, walnuts, nuts such as peanuts and cashew nuts, fruits such as kiwi fruit and bananas, fish eggs such as salmon roe and cod roe, beans such as buckwheat and soybeans, and fish such as mackerel. Examples of atopic dermatitis include, but are not limited to, symptoms caused by dust, mites, and other pests, as well as the above-mentioned food allergens.

[Lactobacillus plantarum L-137]
The composition of the present invention is characterized by containing the lactic acid bacterium Lactobacillus plantarum L-137 strain (Accession No.: FERM BP-08607) or a processed product thereof.

The lactic acid bacteria Lactobacillus plantarum L-137 strain used in the present invention has been deposited at the National Institute of Advanced Industrial Science and Technology (currently the National Institute of Technology and Evaluation (NATEST) Patent Organism Depositary, address: Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan, 292-0818) under deposit number FERM BP-08607 (transferred from FERM P-15317 deposited on November 30, 1995). Mutant strains of Lactobacillus plantarum L-137 that have the characteristics of Lactobacillus plantarum L-137 are also included in the category of Lactobacillus plantarum L-137. In addition, other lactic acid bacteria may be contained in the composition of the present invention in addition to the Lactobacillus plantarum L-137 strain.

In the composition of the present invention, the Lactobacillus plantarum L-137 or a processed product thereof is preferably contained in an amount of about 0.0001 to 10% by weight, more preferably about 0.001 to 1% by weight, and even more preferably about 0.002 to 0.2% by weight, based on the total amount of the composition, but is not limited to these ranges.

Furthermore, the intake amount of the Lactobacillus plantarum L-137 strain of the present invention or a processed product thereof, when administered orally or by injection, can be determined depending on the age and weight of the person taking it, symptoms, administration time, dosage form, administration method, combination of drugs, etc. For example, it is preferable to set the intake amount of the Lactobacillus plantarum L-137 strain per day per adult (about 60 kg) to be preferably about 0.5 to 200 mg, more preferably about 1 to 100 mg, and even more preferably about 2 to 50 mg in terms of dried killed cells, but is not limited to these ranges. Alternatively, it is preferable to set the amount of Lactobacillus plantarum L-137 to be ingested per day by an adult (about 60 kg) in terms of viable bacteria, preferably about 5×10 ⁸ to 2×10 ¹¹ cfu (colony forming unit), more preferably about 1×10 ⁹ to 1×10 ¹¹ cfu, but this range is not limited. The number of times of ingestion can be once or several times a day. The above-mentioned dosage may be administered or applied once to several times a day.

[Cultivation of lactic acid bacteria]
In the present invention, Lactobacillus plantarum L-137 and other lactic acid bacteria may be cultured in any medium such as a natural medium, a synthetic medium, a semi-synthetic medium, etc. In the present invention, the lactic acid bacteria may be cultured according to a known method, a method known per se, or a method similar thereto.

The medium is not particularly limited, and preferably contains, for example, a nitrogen source and/or a carbon source. The nitrogen source is not particularly limited, and examples thereof include meat extract, peptone, gluten, casein, yeast extract, and amino acids. The carbon source is not particularly limited, and examples thereof include glucose, xylose, fructose, inositol, maltose, starch syrup, koji soup, starch, bagasse, bran, molasses, and glycerin. These may be used alone or in combination of two or more. In addition to the nitrogen source and/or carbon source, the medium may further contain an inorganic substance. The inorganic substance is not particularly limited, and examples thereof include ammonium sulfate, potassium phosphate, magnesium chloride, salt, iron, manganese, molybdenum, and various vitamins. These may be used alone or in combination of two or more.

The culture temperature and culture time of Lactobacillus plantarum L-137 and other lactic acid bacteria are not particularly limited as long as the culture can be carried out efficiently. In one embodiment of the present invention, the culture temperature may be, for example, usually about 25 to 40 degrees (°C), preferably about 27 to 35°C. The culture time may be, for example, about 12 to 48 hours. In one embodiment of the present invention, the lactic acid bacteria may be cultured by aeration and shaking. In one embodiment of the present invention, the pH of the medium is not particularly limited, but in one embodiment of the present invention, the pH may be usually about pH 3 to 6, preferably about pH 4 to 6.

[Processed product of lactic acid bacteria]
The "processed product" of the lactic acid bacteria Lactobacillus plantarum L-137 strain is preferably a processed product of the L-137 strain, and examples thereof include, but are not limited to, its culture solution or culture supernatant, residues obtained by filtering or centrifuging the same, ultrasonically disrupted solutions of bacteria, etc. In addition, the processed product of the present invention also includes, but is not limited to, solutions in which cell walls have been removed by enzymatic or physical treatment, protein or peptide complexes obtained by chemical or salting-out treatment, and concentrates, dried products, or dilutions thereof.
The L-137 strain may be in the form of live cells, dried cells, centrifuged cells, disrupted cells, or killed cells, but killed cells are preferred from the standpoints of stability, ease of handling, and the like.

In the present invention, the above-mentioned processed products may be used as they are, or may be freeze-dried, low-temperature dried, spray-dried, L-dried, or a combination of these to form a powder. These processed products may also be diluted with an appropriate solvent (water, alcohol, organic solvent, etc.) or may be made into a gel or solid by adding appropriate additives.

A method for preparing killed cells of Lactobacillus plantarum L-137 and other lactic acid bacteria will be specifically described below.
In the present invention, the method for preparing the killed cells is not particularly limited as long as the effects of the present invention are not lost, and may be, for example, any of the following methods: (I) a method in which live cells of lactic acid bacteria are separated from the culture solution after the completion of culture, and then the live cells are sterilized or sterilized to kill the cells, (II) a method in which live cells of lactic acid bacteria are sterilized in the culture solution to kill the cells, and then the killed cells are separated from the culture solution, etc. Sterilization can be carried out, for example, by filter filtration, but it may also be carried out by other known methods, such as gas sterilization with ethylene oxide or hydrogen peroxide, heat sterilization with gamma rays, electron beam irradiation, high frequency, etc.

The method for separating the bacterial cells from the culture liquid may be any of various methods commonly used in this field, and is not particularly limited. In one embodiment of the present invention, specifically, for example, a method may be adopted in which the culture liquid and the bacterial cells are separated by removing the supernatant from the culture liquid by means of centrifugation or the like. In this embodiment, distilled water is added to the culture liquid, centrifuged, the supernatant is removed, and then, if desired, distilled water is added to the residue from which the supernatant has been removed and the centrifugation is repeated several times. In one embodiment of the present invention, a filtration step may be included as a separation operation. The above-mentioned bacterial cells are dried using a spray drying device to obtain dried bacteria. As an example of the device, a spray drying device equipped with an atomizing device capable of forming spray droplets of about 1 to 10 μm can be preferably used, but is not limited to this.

The sterilization method is not particularly limited, and examples thereof include heating, ultraviolet irradiation, and formalin treatment. The sterilization may be performed on the harvested live bacterial cells, or on a culture solution containing the live bacterial cells.

When the heat treatment is performed, the heating temperature is not particularly limited, but may be, for example, usually about 60 to 100 degrees (°C), preferably about 70 to 90 degrees. The heating means may be a known method, but is not particularly limited, and may be, for example, a heater or other means. The heating time is not particularly limited as long as the sterilization treatment can be sufficiently completed, but, for example, the heating time after the desired temperature is reached may be usually about 5 to 40 minutes, preferably about 10 to 30 minutes.

The killed bacteria obtained as described above may be further subjected to grinding, crushing, spray drying, low-temperature drying, or freeze drying, or may be mixed with other raw materials (e.g., vitamins, amino acids, oligopeptides, etc.) to obtain a processed product of killed bacteria. In the present invention, the processed product of killed bacteria can also be suitably used as killed bacteria.

A preferred example of the composition of the present invention is for food and beverages and/or pharmaceuticals (including veterinary medicines). Another preferred example is that the composition of the present invention is used as an additive for food and beverages. For veterinary medicines, the animal is preferably an animal having an intestinal tract, more preferably, but not limited to, a mammal or seafood. Examples of mammals include humans, rats, mice, rabbits, dogs, cats, cows, horses, pigs, monkeys, etc., and examples of seafood include salmon, salmon, mackerel, pacific saury, sardines, herring, tuna, Alaska pollock, yellowtail, pangasius, eel, tilapia, squid, octopus, crab, shrimp, crayfish, sea cucumber, etc., but are not limited to these.
The composition for food and beverages, food and beverage additives, or pharmaceuticals can be formulated by appropriately mixing the above-mentioned L-137 strain of the present invention or a processed product thereof with pharma- ceutically acceptable carriers, additives, and the like. Formulation methods and techniques for this purpose have been well established, so they may be followed. For example, in the case of pharmaceuticals, the composition of the present invention can be a solid formulation or liquid formulation, specifically, an oral preparation such as a tablet, coated tablet, pill, powder, granule, capsule, liquid, syrup, emulsifier, suspension, or emulsion. The blending ratio of the carrier or additive may be appropriately set based on the range normally adopted in the fields of food and beverages, pharmaceuticals, or veterinary medicine.

There are no particular limitations on the pharma- ceutically acceptable carriers or additives, and examples of carriers include various carriers such as aqueous or oily bases. Examples of aqueous carriers include water, physiological saline, ethanol, glycerin, polyethylene glycol, propylene glycol, methylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, polyvinylpyrrolidone, polyacrylic acid, polysaccharide gum-based natural polymers, and the like. Examples of oily carriers include, but are not limited to, appropriate oils and waxes such as petrolatum, squalane, and paraffin.
Examples of additives include, but are not limited to, enzymes, pH adjusters, preservatives, bactericides, antioxidants, antifungal agents, shelf life improvers, bleaching agents, glossing agents, flavors, sweeteners, acidulants, seasonings, bittering agents, emulsifiers, thickeners, stabilizers, gelling agents, thickening agents, excipients, binders, disintegrants, lubricants, colorants, flavoring agents, etc. Technologies related to these have been well established in the past, so they may be used in the present invention.

Furthermore, when the composition of the present invention is for use in food and beverage products, the food and beverage products include health foods, functional foods, foods for specified health uses, and foods for the sick. The form of the food and beverage products is not particularly limited, but specific examples include tablets, granules, powders, drinks, etc., as so-called nutritional supplements or supplements. Other than these, examples include beverages such as tea, soft drinks, carbonated drinks, nutritional drinks, fruit drinks, and lactic acid drinks; noodles such as soba, udon, Chinese noodles, and instant noodles; sweets and breads such as candy, gum, chocolate, snacks, biscuits, jellies, jams, creams, baked goods, and breads; processed seafood and livestock foods such as ham, sausages, hanpen, and chikuwa; dairy products such as processed milk and fermented milk; oils and fats and oil-processed foods such as salad oil, tempura oil, margarine, mayonnaise, shortening, whipped cream, and dressings; seasonings such as sauces and sauces; retort pouch foods such as curry, stew, rice bowls, porridge, and zosui; and cold desserts such as ice cream, sherbet, and shaved ice, but are not limited to these. The technologies related to these have been well established in the past, so they may be followed in the present invention.

Furthermore, the composition of the present invention may contain any component known in the fields of, for example, medicine, pharmacology, veterinary medicine, livestock, feed, or food, as long as the effect of the present invention is not lost.
The feed is, for example, preferably, for mammals such as rats, mice, dogs, cats, rabbits, horses, cows, pigs, monkeys, etc., birds such as chickens, ducks, parrots, pigeons, sparrows, owls, etc., and/or for seafood such as salmon, chum salmon, mackerel, saury, sardines, herring, tuna, Alaska pollock, yellowtail, pangasius, eel, tilapia, squid, octopus, crab, shrimp, crayfish, sea cucumber, etc., but is not limited thereto.

[Methods for confirming improvement of intestinal barrier function, enhancement of intestinal epithelial tight junctions, and anti-inflammatory effects]
An example of a method for confirming the effect of the composition of the present invention is a method for confirming that a composition containing the Lactobacillus plantarum L-137 strain of the present invention or a processed product thereof has a superior intestinal barrier function improving effect, intestinal epithelial tight junction enhancing effect, and/or anti-inflammatory effect, compared to a composition not containing either the L-137 strain or a processed product thereof.

Specifically, the above-mentioned effects of the present invention may be confirmed by, for example, a method of seeding cells in a multiwell plate equipped with a porous culture support, a Transwell, adding a fluorescent substance to the upper layer of the formed monolayer and measuring the amount of the fluorescent substance that permeates to the lower layer, a method of using both sides of the formed monolayer separately, a method of measuring the transepithelial electric resistance (TER or TEER) value, or a method of evaluation based on interleukin or TNF production, which are well established in the field, as described in the Examples below. In addition, since tight junctions are composed of transmembrane proteins, such as Occludin and Claudin, and an intracellular lining protein, Zonula occludens (ZO), it is considered that an effect of enhancing the tight junctions of the intestinal epithelium has been obtained if the expression levels of these genes are increased or the decrease in the expression levels is suppressed. The expression levels of these genes can be evaluated by, for example, real-time PCR, DNA arrays, and other methods, but are not limited to these.

When the composition of the present invention is prepared in the form of a food or drink, animal feed, medicine (including veterinary medicine), or quasi-drug, the food or drink, feed, medicine, or quasi-drug, or the accompanying instructions or packaging box, etc., can be labeled to the effect of improving intestinal barrier function and/or enhancing intestinal epithelial tight junctions, in consideration of the action of the composition of the present invention.

[Production method of the composition]
The present invention preferably includes a method for producing a composition having an effect of improving intestinal barrier function and/or enhancing intestinal epithelial tight junction, characterized by including a step of mixing Lactobacillus plantarum L-137 or a processed product thereof with a carrier and/or an excipient.

Preferred carriers for use in the above steps have been well established in the food or pharmaceutical fields, and the present invention may follow suit. Examples of suitable carriers include aqueous or oily bases. Examples of aqueous carriers include water, saline, ethanol, glycerin, polyethylene glycol, propylene glycol, methylcellulose, hydroxypropylmethylcellulose, hydroxypropylcellulose, polyvinylpyrrolidone, polyacrylic acid, polysaccharide gum-based natural polymers, and the like. Examples of oily carriers include, but are not limited to, suitable oils and waxes such as petrolatum, squalane, and paraffin.

Furthermore, the preferred excipients used in the above steps have been well established in the food and pharmaceutical fields, and the present invention may follow them. For example, lactose, sucrose, mannitol, corn starch, powdered cellulose, calcium hydrogen phosphate, calcium carbonate, etc. can be preferably used, but are not limited to these.

The composition of the present invention can be appropriately processed and manufactured by a general method for manufacturing a composition, except for adding Lactobacillus plantarum L-137 or a processed product thereof to the composition. In other words, the present invention encompasses a method for manufacturing a composition that includes a step of mixing Lactobacillus plantarum L-137 or a processed product thereof with other ingredients, as desired.

The present invention includes various combinations of the above configurations within the technical scope of the present invention, as long as the effects of the present invention are achieved. In addition, appropriate modifications are possible as long as they fall within the technical scope of the present invention.

The present invention will be explained in more detail below with reference to examples and test examples, but the present invention is not limited to these.

[Test Example 1] Evaluation of the effect of Lactobacillus casei L-137 strain in suppressing the decline in intestinal barrier function In this test system, a transwell and a 24-well plate, together with rat small intestinal epithelial cells, are used to mimic the in vivo intestinal barrier. If LPS (Lipopolysaccharide) is added to the system as a foreign substance, the intestinal barrier function is reduced or destroyed by LPS. Whether the decline or destruction of the intestinal barrier function can be suppressed by adding the L-137 strain in advance to this experimental system was evaluated by measuring the fluorescent molecule FD-4 that had migrated from the upper layer to the lower layer of the cells.

Exam Preparation:
The rat small intestinal epithelial cell line (IEC-6) was purchased from RIKEN. The culture conditions were 37°C/9 days, and the medium was DMEM (1 g/L glucose, 10% FBS).
The killed bacteria of the L-137 strain (HK L-137) were made by our own company. The culture conditions and the method for producing the killed bacteria are also described in JP 2019-216712 A, but the Lactobacillus plantarum L-137 (Accession No. FERM BP-08607) strain was inoculated into an MRS (de Man, Rogosa, Sharpe) modified liquid medium and cultured at 32 ° C. for 18 hours. After culture, the bacteria were killed by heating at 80 ° C., the culture liquid was removed, and the bacteria were collected. The obtained bacteria were thoroughly dispersed in water, and 20% by mass of the bacteria and 80% by mass of dextrin were mixed and spray-dried to obtain dried killed bacteria.
LPS (product number: L3129) and FD-4 (Fluorescein isothiocyanate-dextran, product name: 60842-46-8) were both purchased from Sigma.

Test method: A rat small intestinal epithelial cell line (IEC-6) was seeded (seeding density: 1.8 x ¹⁰⁴ cells per well) on a transwell (product number: 83.3932.040, manufactured by SARSTEDT) placed on a 24-well plate (product number 3526, manufactured by Corning), and the following test was performed on the 7th day after confluence. Note that confluence was confirmed by confirming under a microscope that the seeded cells had proliferated to occupy approximately 80% or more of the bottom surface of the transwell.
(1) The medium in the transwell was removed, and a new medium containing 100 μg/mL killed cells of the L-137 strain was added. After 24 hours, the medium in the transwell was removed, and a medium containing 100 μg/mL LPS (Lipopolysaccharide) was added.
(2) 26 hours after LPS stimulation, 5 μL of 2.0 mg/mL FD-4 was added to the transwell, and 5 hours later, the lower layer medium was collected and the amount of FD-4 (μg/mL) that had passed into the IEC-6 cells was measured using a microplate reader (excitation wavelength: 490 nm, fluorescence wavelength: 520 nm, PerkinElmer Japan). The measurement results are shown in FIG. 1 and Table 1 below.

Results: The addition of L-137 reduced the measured value (fluorescence amount) of FD-4 that passed from the upper to lower layers of rat small intestinal epithelial cells, demonstrating that the L-137 strain of lactic acid bacteria of the present invention has the effect of suppressing the decline of intestinal barrier function.

Test Example 2 Evaluation of the effect of Lactobacillus casei L-137 strain on enhancing tight junctions in intestinal epithelium Test method: The same IEC-6 cells as in Test Example 1 were seeded (seeding density: 1 x ¹⁰ cells per well) in a 24-well plate (product number: 3256, Corning Corporation), and the following test was performed on the 8th day after confluence. The method for confirming confluence was the same as in Test Example 1.
(1) The medium in the 24-well plate was removed, and new medium containing 1, 10, or 100 μg/mL of L-137 strain was added. After 24 hours, the medium in the 24-well plate was removed, and medium containing 100 μg/mL of LPS (Lipopolysaccharide) was added.
(2) IEC-6 cells were harvested 6 hours after LPS stimulation, and total RNA was harvested using Maxwell® RSC simpleRNA Cells kit. The expression levels of each gene (ZO-1, Occludin, Claudin-1, and internal standard GAPDH) were measured using One Step TB Green PrimeScript RT-PCR Kit II (Takara Bio Inc.) and Thermal Cycler Dice® Real Time System III (TP970, Takara Bio Inc.). The expression levels of each gene were corrected using the internal standard GAPDH. The measurement results are shown in Figures 2 to 4 and Table 2 below. The values in the table are relative values of the expression levels when the absence of LPS stimulation is set to "1".

Results: The addition of L-137 strain suppressed the decrease in gene expression levels of ZO-1 and Occludin due to LPS stimulation, indicating that L-137 strain has the effect of enhancing intestinal epithelial tight junctions. Furthermore, for ZO-1 and Occludin, the effect of enhancing intestinal epithelial tight junctions was improved in a dose-dependent manner by increasing the amount of L-137 strain added.
Unlike the above two genes, the amount of Claudin-1 gene expression increased with stimulation at the amount of LPS added this time. Furthermore, the amount of gene expression increased further by adding L-137 strain and increasing the amount added. Although it showed a different tendency from ZO-1 and Occludin, Claudin is a relatively newly discovered protein, and there is still much that is not known about it. In addition to Claudin-1, it is known that there are at least 27 claudins, including Claudin-2, so further investigations will be conducted in the future by changing the type of claudin, cells and foreign substances (compounds that stimulate cells), and the amount added.

The composition containing the Lactobacillus casei L-137 strain of the present invention has the effect of improving the intestinal barrier function and/or enhancing the intestinal epithelial tight junction. The composition also has an anti-inflammatory effect. Therefore, the composition of the present invention is useful for treating, preventing, or improving various immune diseases such as type I diabetes, celiac disease, inflammatory bowel disease, irritable bowel syndrome, atopic dermatitis, food allergies, etc., and is also useful as a food or drink, feed, and pharmaceutical or quasi-drug.

Claims (9)

A composition for improving intestinal barrier function, characterized by containing Lactobacillus plantarum L-137 or a processed product thereof. A composition for enhancing intestinal epithelial tight junctions, characterized by containing Lactobacillus plantarum L-137 or a processed product thereof. A composition for treating, preventing or improving one or more immune diseases selected from the group consisting of the following (i) to (vi), characterized by containing Lactobacillus plantarum L-137 or a processed product thereof: (i) type I diabetes; (ii) celiac disease; (iii) inflammatory bowel disease; (iv) irritable bowel syndrome; (v) atopic dermatitis; and (vi) food allergy. The composition for administration to humans, animals, or fish and shellfish described in any one of claims 1 to 3, which is a food, beverage, or feed composition. The composition according to claim 4, wherein the food or drink is a food additive or a supplement. Use of Lactobacillus plantarum L-137 or a processed product thereof for the manufacture of a medicine for improving intestinal barrier function or enhancing intestinal epithelial tight junctions. 　Use of Lactobacillus plantarum L-137 or a processed product thereof for the manufacture of a medicine for the treatment, prevention, or amelioration of one or more immune diseases selected from the group consisting of the following (i) to (vi): (i) type I diabetes; (ii) celiac disease; (iii) inflammatory bowel disease; (iv) irritable bowel syndrome; (v) atopic dermatitis; and (vi) food allergy. A method for improving intestinal barrier function or enhancing intestinal epithelial tight junctions, comprising administering Lactobacillus plantarum L-137 or a processed product thereof to a subject. A method for treating, preventing, or ameliorating one or more immune diseases selected from the group consisting of the following (i) to (vi), characterized by administering Lactobacillus plantarum L-137 or a processed product thereof to a subject: (i) type I diabetes; (ii) celiac disease; (iii) inflammatory bowel disease; (iv) irritable bowel syndrome; (v) atopic dermatitis; and (vi) food allergy.