WO2025033535A1 - Composition for protecting tight junction - Google Patents

Abstract

The present invention addresses the problem of providing a novel use of EPSs of lactic acid bacteria. Moreover, the present invention addresses the problem of providing a composition for protecting a tight junction or a composition for promoting gene expression in a tight junction component molecule. The present invention provides a composition for protecting a tight junction or a composition for promoting the expression of a Zonula occludens-1 (ZO-1) gene, the compositions containing EPSs of lactic acid bacteria.

Description

Tight junction protection composition

The present invention relates to a composition for protecting tight junctions.

Tight junctions are intercellular adhesion mechanisms present in epithelial cells and vascular endothelial cells. They are mainly composed of the transmembrane proteins Occludin and Claudin, as well as the intracellular lining protein Zonula occludens (ZO). Tight junctions form a barrier by mechanically connecting cells together, and play an important role in preventing the invasion of foreign substances such as pathogens and toxins.

Regarding the strengthening of the barrier function, Patent Document 1 describes a composition containing Lactobacillus species grown as a biofilm and/or an extract of the biofilm, where the biofilm and/or the extract strengthen the barrier function so that when the composition is applied to a surface, the surface is less susceptible to microbial, viral, and/or chemical contamination. Patent Document 3 describes a skin quality improving agent containing Lactobacillus rhamnosus bacteria or an extract of the bacteria as an active ingredient, and shows that the application of a bactericidal powder cream of Lactobacillus rhamnosus KO3 strain reduces the amount of transepidermal water loss from the skin. Non-Patent Document 1 describes that lactic acid bacteria act on the intestinal immunity and on intestinal epithelial cells to increase the expression of tight junctions and mucin production, thereby improving the intestinal barrier function. Non-Patent Document 2 suggests that Lactobacillus rhamnosus OLL2838 improves intestinal barrier dysfunction. Non-patent literature 3 suggests that Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus salivarius, or Lactobacillus plantarum improves intestinal epithelial barrier dysfunction. Patent literature 4 describes the Lactiplantibacillus plantarum LOC1 strain, and shows that the strain increases the expression of specific tight junction-related genes in intestinal cells.

On the other hand, it has been confirmed that exopolysaccharides (EPS) produced by lactic acid bacteria have various health benefits. Patent Document 2 describes a composition for preventing or reducing the risk of developing secondary infections after viral infection, which contains exopolysaccharides of lactic acid bacteria. This document describes that the prevention or reduction in the risk of developing secondary infections after viral infection is due to the suppression of the expression of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM-1), and also describes that under the experimental conditions, EPS did not affect the mRNA expression of tight junction molecules.

Special table 2023-503350 International Publication WO2023/120547 Patent Publication 2022-182870 Patent Publication 2022-73988

Sachi Tanaka, Immunomodulatory Effects of Food-derived Components: Can Foods that Modulate Immune Function Prevent Disease?, Chemistry and Biology, 56(7), 2018, 469-474 E Miyauchi, H Morita, S Tanabe, Lactobacillus rhamnosus alleviates intestinal barrier dysfunction in part by increasing express ion of zonula occludens-1 and myosin light-chain kinase in vivo. J Dairy Sci. 2009 Jun;92(6):2400-8. doi: 10.3168/jds.2008-1698. Ruchi Jariwala, Hemanti Mandal and Tamishraha Bagchi, Indigenous lactobacilli strains of food and human sources reverse enteropathogenic E. coli O26:H11-induced damage in intestinal epithelial cell lines: effect on redistribution of tight junction proteins. Microbiology 2017;163:1263-1272

The present invention aims to provide a new use for EPS of lactic acid bacteria. It also aims to provide a composition for protecting tight junctions, or a composition for promoting gene expression of tight junction component molecules.

That is, the present invention relates to the following.
[1] A composition for protecting tight junctions, comprising an exopolysaccharide of a lactic acid bacterium.
[2] The composition described in [1], wherein the tight junction protection is promoting the recovery of damaged tight junctions or inhibiting damage to tight junctions.
[3] The composition described in [1] or [2], wherein the tight junction is a tight junction of a lung cell.
[4] The composition described in [2] or [3], wherein the injury is caused by a viral infection.
[5] The composition described in [2] or [3], wherein the injury is a decrease in Zonula occludens-1 (ZO-1) gene expression.
[6] The composition described in [4], wherein the virus is an influenza virus.
[7] The composition according to any one of [1] to [6], wherein the lactic acid bacteria are exopolysaccharide-producing bacteria.
[8] The composition according to any one of [1] to [7], wherein the lactic acid bacteria are selected from bacteria classified as Lactobacillus delbrueckii subsp. bulgaricus, bacteria classified as Lactococcus lactis subsp. lactis, and bacteria classified as Lactococcus lactis subsp. cremoris.
[9] The composition according to any one of [1] to [8], wherein the lactic acid bacterium is Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1 (FERM BP-10741).
[10] The composition according to any one of [1] to [9], for administration to a person selected from the group consisting of persons aged 65 or older, young children, infants, newborns, persons suffering from chronic respiratory diseases, and smokers.
[11] The composition described in any one of [2] to [10], wherein the promotion of tight junction recovery or the inhibition of tight junction damage is achieved by promoting ZO-1 gene expression.
[12] A composition for promoting ZO-1 gene expression, comprising exopolysaccharides of lactic acid bacteria.
[13] The composition described in [12], which is for promoting ZO-1 gene expression in damaged tight junctions.
[14] The composition described in [12] or [13], wherein the lactic acid bacteria are exopolysaccharide-producing bacteria.
[15] The composition according to any one of [12] to [14], wherein the lactic acid bacterium is classified as Lactobacillus delbrueckii subsp. bulgaricus.
[16] The composition according to any one of [12] to [14], wherein the lactic acid bacteria are selected from bacteria classified into Lactococcus lactis subsp. lactis and bacteria classified into Lactococcus lactis subsp. cremoris.
[17] A method for producing fermented milk for protecting tight junctions, comprising adding lactic acid bacteria classified as Lactobacillus delbrueckii subsp. bulgaricus and lactic acid bacteria classified as Streptococcus thermophilus to a milk preparation containing raw milk and fermenting the mixture.
[18] A method for producing fermented milk for protecting tight junctions, comprising adding lactic acid bacteria classified as Lactococcus lactis subsp. lactis or lactic acid bacteria classified as Lactococcus lactis subsp. cremoris to a milk preparation containing raw material milk and fermenting the mixture.
[19] An inhalant comprising exopolysaccharide of lactic acid bacteria.
[20] The inhalant described in [19], wherein the lactic acid bacteria are exopolysaccharide-producing bacteria.
[21] The inhalant described in [19] or [20], wherein the lactic acid bacteria are selected from bacteria classified as Lactobacillus delbrueckii subsp. bulgaricus, bacteria classified as Lactococcus lactis subsp. lactis, and bacteria classified as Lactococcus lactis subsp. cremoris.
[22] The inhalant according to any one of [19] to [21], wherein the lactic acid bacterium is Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1 (FERM BP-10741).

[23] An exopolysaccharide of a lactic acid bacterium, or a composition comprising an exopolysaccharide of a lactic acid bacterium, for use in a method for protecting tight junctions. Use of an exopolysaccharide of a lactic acid bacterium in the manufacture of a composition for protecting tight junctions for therapeutic or non-therapeutic purposes. A method or non-therapeutic method for protecting tight junctions, comprising a step of administering a composition comprising an exopolysaccharide of a lactic acid bacterium or an exopolysaccharide of a lactic acid bacterium to a subject. Use or non-therapeutic use of a composition comprising an exopolysaccharide of a lactic acid bacterium, or of an exopolysaccharide of a lactic acid bacterium, for protecting tight junctions.
[24] An exopolysaccharide of a lactic acid bacterium, or a composition comprising an exopolysaccharide of a lactic acid bacterium, for use in a method for promoting the recovery of damaged tight junctions or a method for suppressing damage to tight junctions. Use of an exopolysaccharide of a lactic acid bacterium in the manufacture of a composition for promoting the recovery of damaged tight junctions or a composition for suppressing damage to tight junctions. A method for promoting the recovery of damaged tight junctions or a method for suppressing damage to tight junctions, or a non-therapeutic method, comprising a step of administering a composition comprising an exopolysaccharide of a lactic acid bacterium or an exopolysaccharide of a lactic acid bacterium to a subject. Use or non-therapeutic use of a composition comprising an exopolysaccharide of a lactic acid bacterium, or of an exopolysaccharide of a lactic acid bacterium, for promoting the recovery of damaged tight junctions or suppressing damage to tight junctions.
[25] The exopolysaccharide, composition, use, method or non-therapeutic method, or use or non-therapeutic use of a lactic acid bacterium described in [23] or [24], wherein the tight junction is a tight junction of a lung cell.
[26] The exopolysaccharide, composition, use, method or non-therapeutic method, or use or non-therapeutic use of a lactic acid bacterium described in [24] or [25], wherein the injury is caused by a viral infection.
[27] The exopolysaccharide, composition, use, method or non-therapeutic method, or use or non-therapeutic use of a lactic acid bacterium described in [24] or [25], wherein the injury is a decrease in ZO-1 gene expression.
[28] The exopolysaccharide, composition, use, method or non-therapeutic method, or use or non-therapeutic use of the lactic acid bacteria described in [26], wherein the virus is an influenza virus.
[29] The exopolysaccharide, composition, use, method or non-therapeutic method, or use or non-therapeutic use of the lactic acid bacterium according to any one of [23] to [28], wherein the lactic acid bacterium is an exopolysaccharide-producing bacterium.
[30] The exopolysaccharide, composition, use, method or non-therapeutic method, or use or non-therapeutic use of a lactic acid bacterium according to any one of [23] to [29], wherein the lactic acid bacterium is selected from bacteria classified as Lactobacillus delbrueckii subsp. bulgaricus, bacteria classified as Lactococcus lactis subsp. lactis, and bacteria classified as Lactococcus lactis subsp. cremoris.
[31] The exopolysaccharide, composition, use, method or non-therapeutic method, or use or non-therapeutic use of the lactic acid bacterium according to any one of [23] to [30], wherein the lactic acid bacterium is Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1 (FERM BP-10741).
[32] The exopolysaccharide, composition, use, or use or non-therapeutic use of the lactic acid bacteria according to any one of [23] to [31] for ingestion by a person selected from the group consisting of persons aged 65 years or older, young children, infants, newborns, persons suffering from chronic respiratory diseases, and smokers. The method or non-therapeutic method according to any one of [23] to [31], wherein the subject is a person selected from the group consisting of persons aged 65 years or older, young children, infants, newborns, persons suffering from chronic respiratory diseases, and smokers.
[33] The exopolysaccharide, composition, use, method or non-therapeutic method, or use or non-therapeutic use of a lactic acid bacterium described in any one of [24] to [32], wherein the promotion of recovery of tight junctions or the inhibition of damage to tight junctions is due to the promotion of ZO-1 expression.
[34] An exopolysaccharide of a lactic acid bacterium, or a composition comprising an exopolysaccharide of a lactic acid bacterium, for use in a method for promoting ZO-1 gene expression. Use of an exopolysaccharide of a lactic acid bacterium in the manufacture of a composition for promoting ZO-1 gene expression for therapeutic or non-therapeutic purposes. A method or non-therapeutic method for promoting ZO-1 gene expression, comprising a step of administering a composition comprising an exopolysaccharide of a lactic acid bacterium or an exopolysaccharide of a lactic acid bacterium to a subject. Use or non-therapeutic use of a composition comprising an exopolysaccharide of a lactic acid bacterium, or of an exopolysaccharide of a lactic acid bacterium, for promoting ZO-1 gene expression.
[35] An exopolysaccharide of a lactic acid bacterium or a composition comprising an exopolysaccharide of a lactic acid bacterium for use in a method for promoting ZO-1 gene expression in damaged tight junctions. Use of an exopolysaccharide of a lactic acid bacterium in the manufacture of a composition for promoting ZO-1 gene expression in damaged tight junctions. A method or non-therapeutic method for promoting ZO-1 gene expression in damaged tight junctions, comprising a step of administering a composition comprising an exopolysaccharide of a lactic acid bacterium or an exopolysaccharide of a lactic acid bacterium to a subject. Use or non-therapeutic use of a composition comprising an exopolysaccharide of a lactic acid bacterium or of an exopolysaccharide of a lactic acid bacterium for promoting ZO-1 gene expression in damaged tight junctions.
[36] The exopolysaccharide, composition, use, method or non-therapeutic method, or use or non-therapeutic use of the lactic acid bacterium described in [34] or [35], wherein the lactic acid bacterium is an exopolysaccharide-producing bacterium.
[37] The exopolysaccharide, composition, use, method or non-therapeutic method, or use or non-therapeutic use of the lactic acid bacterium described in any one of [34] to [36], wherein the lactic acid bacterium is classified as Lactobacillus delbrueckii subsp. bulgaricus.
[38] The composition according to any one of [23] to [29] and [32] to [37], wherein the lactic acid bacteria are selected from bacteria classified into Lactococcus lactis subsp. lactis and bacteria classified into Lactococcus lactis subsp. cremoris.

According to the present invention, tight junctions are protected.

Figure 1 shows the change in intracellular virus count due to each treatment. non-IFV: non-infected with influenza virus (IFV), IFV: IFV infected with no EPS added, IFV+EPS: IFV infected with EPS added, IFV+BXA: IFV infected with BXA (baloxavir acid) added. *p<0.05, **p<0.01. Figure 2 shows the relative expression level of the ZO-1 gene by each treatment. *p<0.05. Figure 3 shows the results of the FITC-dextran permeability test for each treatment. *p<0.05.

The present invention relates to a composition containing exopolysaccharide (EPS) of lactic acid bacteria. More specifically, the present invention relates to a composition for protecting tight junctions that contains lactic acid bacteria EPS as an active ingredient.

[Active ingredient]

The composition of the present invention contains EPS of lactic acid bacteria as an active ingredient. Lactic acid bacteria is a general term for microorganisms that assimilate glucose to produce lactic acid at a rate of 50% or more based on the sugar. They are gram-positive cocci or bacilli with physiological properties such as no motility, no spore formation in many cases (some lactic acid bacteria such as Bacillus coagulans have the ability to form spores), and catalase negative. Lactic acid bacteria have been consumed throughout the world since ancient times through fermented milk, etc., and can be said to be extremely safe microorganisms. Lactic acid bacteria are classified into multiple genera. The EPS of lactic acid bacteria contained in the composition of the present invention is preferably produced by lactic acid bacteria classified into the genus Leuconostoc, lactic acid bacteria classified into the genus Lactococcus, or lactic acid bacteria classified into the genus Lactobacillus.

In addition, "containing an active ingredient" means that the ingredient is used in the composition in an effective amount to exert the intended function, and that it is specified in the labeling as an ingredient that contributes to the intended purpose. In foods with functional claims, active ingredients are sometimes called functional ingredients (ingredients that contribute to specific health purposes (excluding those related to reducing the risk of disease)).

The EPS used in the composition of the present invention is not particularly limited as long as it has the intended effect. Structurally, EPS produced by lactic acid bacteria are classified into homopolysaccharides and heteropolysaccharides (e.g., those composed of galactose and glucose) and may be modified by phosphorylation or sulfation, but any of them can be used as an active ingredient in the composition of the present invention. One preferred example of EPS is one that contains at least one of neutral polysaccharides and acidic polysaccharides in which phosphate groups are added to neutral polysaccharides. Such EPS is known to be produced by Lactobacillus delbrueckii subsp. bulgaricus, Lactococcus lactis subsp. lactis, Lactococcus lactis subsp. cremoris, etc. The EPS used in the present invention may be one type or a combination of two or more types.

Examples of particularly preferred EPS-producing lactic acid bacteria (exopolysaccharide-producing bacteria) for use in the composition of the present invention include Lactobacillus lactic acid bacteria, Lactococcus lactic acid bacteria, etc.

(Lactobacillus lactic acid bacteria)

Examples of Lactobacillus lactic acid bacteria include Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus acidophilus, Lactobacillus plantarum, etc. In this specification, "lactic acid bacteria belonging to the genus Lactobacillus" refers to the lactic acid bacteria described in the paper "A taxonomic note on the genus Lactobacillus: Description of 23 novel genera, emended description of the genus Lactobacillus Beijerinck 1901, and union of Lactobacillaceae and The 25 genera newly established by the reorganization of lactic acid bacteria published in the "Leuconostocaceae" are Lactobacillus, Paralactobacillus, Holzapfelia, Amylolactobacillus, Bombilactobacillus, Companilactobacillus, Lapidilactobacillus, Agrilactobacillus, Schleiferilactobacillus, Loigolactobacilus, Lacticaseibacillus, Latilactobacillus, Delactobacillus, and Lacticaceibacillus. Dellaglioa genus, Liquorilactobacillus genus, Ligilactobacillus genus, Lactiplantibacillus genus, Furfurilactobacillus genus, Paucilactobacillus genus, Limosilactobacillus It refers to lactic acid bacteria belonging to any of the genera Lactobacillus, Fructilactobacillus, Acetilactobacillus, Apilactobacillus, Levilactobacillus, Secundilactobacillus, and Lentilactobacillus. Among these Lactobacillus lactic acid bacteria, in the present invention, it is preferable that the lactic acid bacteria is classified as Bulgaricus (also called Bulgaricus bacteria). Furthermore, it is more preferable that the lactic acid bacteria is classified as Lactobacillus delbrueckii subsp. bulgaricus. That is, one particularly preferred example of the EPS used in the composition of the present invention is the EPS of a lactic acid bacterium classified as Lactobacillus delbrueckii subsp. bulgaricus.

(Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1)

In a particularly preferred embodiment, the lactic acid bacterium is Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1 (accession number: FERM BP-10741) (sometimes referred to as "Lactobacillus bulgaricus R-1 strain"). That is, one particularly preferred example of the EPS used in the composition of the present invention is the EPS of Lactobacillus bulgaricus R-1 strain.

bulgaricus strain R-1 has been internationally deposited at the National Institute of Technology and Evaluation (IPOD, NITE) (Room 120, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) in accordance with the Budapest Treaty (depositor: Meiji Co., Ltd., date of deposit: November 29, 2006, deposit number: FERM BP-10741).

A strain taxonomically equivalent to a certain strain (hereinafter referred to as strain S) is, for example, any of the following:
- a strain belonging to the same genus as strain S, preferably belonging to the same species as strain S, in which the entire sequence or a characteristic part of the 16S rRNA gene (such as the V1 region, the V2 region, or all or a part of the V1 region and the V2 region, or a part including the V1 region and the V2 region, etc.) has a sequence identity of 90% or more, preferably 95% or more, more preferably 98% or more, even more preferably 98.5% or more, even more preferably more than 98.7%, even more preferably 99% or more, even more preferably 100% with the sequence of strain S;
A strain belonging to the same genus as strain S, preferably the same species as strain S, which has the same mycological properties as strain S.

With regard to criteria for determining species similarity based on 16S rRNA gene sequences, those skilled in the art can refer to Stackebrandt E, Ebers J. Taxonomic parameters revisited: tarnished gold standards. Microbiol Today 2006;33:152-155.

The nucleotide sequence of the V1 to V3 regions of the 16S rRNA gene of OLL1073R-1 (corresponding to the nucleotide sequence from 34th to 535th in the full-length sequence of the 16S rRNA gene) is shown in SEQ ID NO:1 in the sequence listing.
16S rRNA gene. Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1 (SEQ ID NO:1)
GCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGAGCTGAATTCAAAGATYCCTTCGGGRTGATTTGTTGGACGCTAGCGGCGGATGGGTGAGTAACACGTGGGCAATCTGCCCTAAAGACTGGG ATACCACTTGGAAACAGGTGCTAATACCGGATAACAACATGAATCGCATGATTCAAGTTTGAAAGGCGGCGYAAGCTGTCACTTTAGGATGAGCCCGCGGCGCATTAGCTAGTTGGTGGGGTAAAG GCCTACCAAGGCAATGATGCGTAGCCGAGTTGAGAGACTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGAT GGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTGGTGAAGAAGGATAGAGGCAGTAACTGGTCTTTATTTGACGGTAATCAACCAGAAAGTCACGGCTAACTACGT

The mycological properties of OLL1073R-1 are shown below.
(1) Morphological characteristics Cell shape: rod-shaped Motility: none Presence or absence of spores: none Gram staining: positive

(2) Growth state on medium The strain was spread on a BL agar medium (Eiken Chemical) plate and cultured at 37° C. for 48 hours by the steel wool method, resulting in the appearance of opaque rough colonies.

(3) Physiological properties Nitrate reduction: negative Indole production: negative Gelatin liquefaction: negative Catalase: negative Attitude towards oxygen: facultative anaerobic Produces D(-) lactic acid from glucose by homolactic fermentation, and in BL liquid medium that does not produce gas, growth at 10°C is negative, and growth at 45°C is positive Arginine decomposition: negative Gas production from malic acid: negative Decomposition of various carbohydrates (positive +, negative -)
Arabinose -
Xylose -
Rhamnose -
Ribose -
Glucose +
Mannose +
Fructose +
Galactose -
Sucrose -
Maltose -
Cellobiose -
Lactose +
Trehalose -
Melibiose -
Raffinose -
Melezitose -
Dextrin -
Starch -
Glycogen -
Inulin -
Mannitol -
Sorbitol -
Inositol -
Esculin -
Salicin -

(4) EPS productivity: The present strain is characterized by its EPS productivity and its extracellular production of polysaccharides containing galactose and glucose as constituent sugars and containing phosphorus.

(EPS)

In addition to the present strain, other lactic acid bacteria of the Lactobacillaceae family that extracellularly produce polysaccharides containing galactose and glucose as constituent sugars are known, such as the following lactic acid bacteria.
Lactiplantibacillus plantarum C88 (Non-Patent Document 4); Lactobacillus. johnsonii strain 151 (Non-Patent Document 5); Lactobacillus delbrueckii subsp. bulgaricus NCFB2074 (Non-Patent Document 6); Lacticaseibacillus casei CG11 (Non-Patent Document 7); Lacticaseibacillus rhamnosus E/N (Non-Patent Document 8); Lactobacillus fermentum TDS030603 (Non-Patent Document 9); Lactobacillus gasseri FR4 (Non-Patent Document 10).

For this reason, it is believed that the effects of the present invention can be achieved with EPS produced by Lactobacillaceae lactic acid bacteria that produce polysaccharides containing galactose and glucose as constituent sugars outside the cells, preferably EPS produced by Lactobacillaceae lactic acid bacteria that produce polysaccharides containing galactose and glucose as constituent sugars and phosphorus outside the cells. Lactobacillaceae lactic acid bacteria include the genera Lactobacillus, Paralactobacillus, Forzapferia, Amyloactobacillus, Bombylactobacillus, Companilactobacillus, Rapidilactobacillus, Agriclottacillus, Schreiferilactobacillus, Leugolactobacillus, Lacticasebacillus, Latilactobacillus, Delagrioa, Liquorilactobacillus, Ligilactobacillus, Lactiprantibacillus, Furfurilactobacillus, and the like. Lactobacillus, Paucillactobacillus, Rimosilactobacillus, Fructilactobacillus, Acetylactobacillus, Apilactobacillus, Leviractobacillus, Secundilactobacillus and Lentilactobacillus, Fructobacillus, Leuconostoc, Oenococcus and Weissella are classified.

(Lactococcus genus lactic acid bacteria)

Examples of Lactococcus lactic acid bacteria include lactis, plantarum, and Raffinolactis. Among these Lactococcus lactic acid bacteria, the present invention prefers lactic acid bacteria classified as lactis. Furthermore, among Lactococcus lactic acid bacteria, it is more preferred to use Lactococcus lactis subsp. lactis or Lactococcus lactis subsp. cremoris. That is, one of the particularly preferred examples of EPS used in the composition of the present invention is the EPS of lactic acid bacteria classified as Lactococcus lactis subsp. lactis or Lactococcus lactis subsp. cremoris.

It is known that EPS produced by Lactococcus lactic acid bacteria also contains galactose and glucose. For example, the repeating units of EPS produced by Lactococcus lactis NIZO B40 consist of glucose, galactose, rhamnose, and phosphoric acid in a ratio of 2:2:1:1 (Non-Patent Document 11, Non-Patent Document 12). Furthermore, Lactococcus lactis subsp. cremoris FC (which can be isolated from Fujicco Caspian Sea Yogurt (registered trademark)) produces a phosphate polysaccharide containing rhamnose, galactose, and glucose in a molar ratio of 1:1:3 (Non-Patent Document 13). Lactococcus lactis subsp. lactis YZ1 produces a polysaccharide composed of mannose, galactose, and glucose (Non-Patent Document 14). From these findings, it is believed that the effects of the present invention can be achieved because EPS produced by Lactococcus lactic acid bacteria also contains galactose and glucose.

(Sugars contained in EPS, etc.)
In one embodiment, the EPS of a lactic acid bacterium contained in the composition of the present invention may include an acidic exopolysaccharide having a repeating structure in which repeating units represented by the following formula (I) are linked together.

In formula (I),
n is 0 or 1;
α-D-Galp represents an α-D-galactose residue in pyranose form;
β-D-Galp represents a β-D-galactose residue in pyranose form;
β-D-Galf represents a β-D-galactose residue in furanose form;
Gro3P represents glycerol 3-phosphate;
(1-2), (1-3), (1-5), and (1-6) represent the 1-2 bond (i.e., carbon-1-carbon bond-carbon-2 bond), the 1-3 bond, the 1-5 bond, and the 1-6 bond between residues, respectively.

In one embodiment, in the acidic bacterial exopolysaccharide, in the repeating structure in which the repeating units represented by formula (I) are linked together, it is preferable that an average of about one glycerol 3-phosphate group is added per repeating unit (for example, one (n=1) or 0 for each repeating unit, and about one per repeating unit on a weighted average for the entire acidic EPS), but this is not limited thereto.

The EPS of the lactic acid bacteria contained in the composition of the present invention may be contained as a lactic acid bacteria fermentation product. The lactic acid bacteria fermentation product includes not only the fermentation product by the lactic acid bacteria itself, but also processed products thereof. The lactic acid bacteria fermentation product itself includes, for example, fermented milk (specifically, yogurt, etc.). Processed products include, for example, crude products, culture filtrates and culture supernatants obtained by removing bacteria from the fermentation product by filtration, centrifugation, or membrane separation, concentrates obtained by concentrating the culture filtrates and culture supernatants, and dried concentrates. In one embodiment, the composition of the present invention does not contain bacteria.

　Conventional techniques can be used for the preparation of lactic acid bacteria EPS, and if more detailed conditions are required, the Examples in this specification can be referred to. When preparing lactic acid bacteria EPS as a lactic acid bacteria fermentation product, fermented milk containing EPS can be produced by adding EPS-producing lactic acid bacteria as a starter to raw milk, fermenting it, and producing EPS in the fermentation product. There are no particular limitations on the fermentation conditions, such as raw milk, fermentation temperature, and fermentation time, as long as the lactic acid bacteria used can produce EPS, and a person skilled in the art can set them appropriately.

[Application]
(function)

In one embodiment, the composition is a composition for protecting tight junctions. The term "tight junction" refers to the apical-most intercellular junction that connects adjacent cells (particularly epithelial cells and endothelial cells). The tight junction physically connects cells to each other to form a barrier, which can prevent the intrusion of foreign substances such as pathogens and toxins. The tight junction to be protected can be the tight junction of any cell (particularly epithelial cells or endothelial cells), but is preferably the tight junction of a lung cell, and more preferably the tight junction of an alveolar epithelial cell. Tight junctions include various constituent molecules. Examples of constituent molecules of tight junctions include occludin, claudin, Zonula occludens (ZO), and the like.

The composition may also be used to aid in tight junction protection, assist in tight junction protection, regulate tight junction protection, maintain tight junction protection, regulate tight junction function, maintain tight junction function, etc. The composition may also be used to alleviate a decline in tight junction protection, reduce the risk of a decline in tight junction protection, alleviate a decline in tight junction function, reduce the risk of a decline in tight junction function, etc.

　Tight junction protection includes promoting the recovery of damaged tight junctions, inhibiting tight junction injury, maintaining tight junctions, inhibiting the progression of tight junction destruction, inhibiting increased tight junction permeability, strengthening tight junctions, and reducing the risk of tight junction disorders. Damage to tight junctions can be, for example, a decrease in the expression of genes of molecules that constitute tight junctions in cells, and increased tight junction permeability. The decrease in the expression of genes of molecules that constitute tight junctions and increased tight junction permeability can be, for example, compared to healthy cells, cells obtained from a healthy subject, cells to which external factors such as viral infection have not been added, cells obtained from a subject to which external factors such as viral infection have not been added, etc.

Protection of tight junctions also includes regulating or helping to regulate one or more of the following: promoting the recovery of damaged tight junctions, inhibiting injury to tight junctions, maintaining tight junctions, inhibiting the progression of tight junction destruction, inhibiting increased permeability of tight junctions, strengthening tight junctions, and reducing the risk of tight junction disorders.

Promotion of recovery of damaged tight junctions means, for example, increasing the expression of genes of molecules that constitute tight junctions that have decreased in cells. Maintenance of tight junctions, inhibition of the progression of destruction of tight junctions, and inhibition of increased permeability of tight junctions means, for example, decreasing the permeability of tight junctions or inhibiting increased permeability of damaged tight junctions. Permeability of tight junctions can be evaluated, for example, by the fluorescein isothiocyanate (FITC)-dextran (4 kDa) permeability test described in Example 2. Inhibition of injury to tight junctions means, for example, inhibition of the decrease in the expression of genes of molecules that constitute tight junctions in cells, or when the fluorescein isothiocyanate (FITC)-dextran (4 kDa) permeability test described in Example 2 is performed, the permeability of tight junctions decreases or the increased permeability of damaged tight junctions is inhibited. The strengthening of tight junctions means, for example, that the function of tight junctions (e.g., barrier function, foreign body invasion suppression function) is strengthened or the formation of tight junctions is promoted by increasing the expression of genes of constituent molecules of tight junctions, or that the permeability of tight junctions is decreased or the increase in the permeability of damaged tight junctions is suppressed when a fluorescein isothiocyanate (FITC)-dextran (4 kDa) permeability test described in Example 2 is performed. In other words, tight junction protection can be the promotion of recovery of damaged tight junctions, the suppression of injury to tight junctions, and the strengthening of tight junctions by affecting the structure and function of tight junctions. In one embodiment, the promotion of recovery of tight junctions, the suppression of injury to tight junctions, or the strengthening of tight junctions is the promotion of recovery of expression of ZO-1 gene, or the suppression of the decrease of ZO-1 gene. In one embodiment, the composition of the present invention is a composition for promoting the expression of the ZO-1 gene or a composition for reducing the permeability of tight junctions, and in a preferred embodiment, the composition is a composition for promoting the expression of the ZO-1 gene in damaged tight junctions or a composition for reducing the permeability of damaged tight junctions.

The compositions of the present invention can also be used to aid, support, improve, or maintain one or more of the promotion of ZO-1 gene expression and the decrease in tight junction permeability. The compositions can also be used to alleviate one or more of the decrease in ZO-1 gene expression and the increase in tight junction permeability, or to reduce the risk of one or more of these.

Damage to tight junctions can occur due to various external factors, such as infection with fungi, bacteria, viruses, etc., smoking, ultraviolet light, and fine particulate matter, as well as internal factors, such as oxidative stress and the action of cytokines. The composition can protect tight junctions from damage caused by any factor. In other words, the composition can promote the recovery of tight junctions damaged by any factor, and can suppress damage to tight junctions caused by any factor, such as a decrease in gene expression of tight junction component molecules, but in a preferred embodiment, the damage is caused by viral infection. The virus can be any virus that can damage tight junctions, but is preferably an influenza virus, and more preferably an influenza A virus (A/H1N1).

The degree of infection of cells can be expressed, for example, by the multiplicity of infection (MOI). MOI represents the ratio of infectious substances to the target to be infected, for example, the ratio of virus particles to cultured cells in a well. For example, when 10 million viruses are added to 1 million cells, the MOI is 10, which represents the probability that 10 virus particles infect one cell. In this embodiment, the MOI can be calculated by the following formula:
MOI = number of virus particles/number of cells

For example, if tight junction damage is due to influenza virus infection, the MOI may be 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less, or may be 1 or more.

Those skilled in the art can appropriately evaluate whether a certain component has the effect of protecting the tight junctions of cells. For example, when epithelial cells are treated to damage the tight junctions by, for example, viral infection, the target component is added and the expression of the molecules that constitute the tight junctions is analyzed to evaluate. The expression of the molecules that constitute the tight junctions may be analyzed by quantifying the expression of the genes of the molecules that constitute the tight junctions, or the expression amount of the molecules may be analyzed as the amount of protein.

(subject)

The composition of the present invention is suitable for ingestion or administration to a subject for whom it is preferable to protect tight junctions. There are no particular limitations on such subjects (preferably humans), and the subject may be any subject (including healthy and sick individuals) who desires or requires the effect of protecting pulmonary tight junctions. Examples of subjects include those who are aware that their lungs or bronchi are weak, those aged 65 or older, young children (1-6 years old), infants (under 1 year old), newborns (under 28 days after birth), those suffering from chronic respiratory diseases (e.g., chronic obstructive pulmonary disease, bronchial asthma, pulmonary fibrosis, interstitial pneumonia, etc.), those infected with influenza virus, and smokers.

[Composition]
(Food compositions, pharmaceutical compositions)

The composition of the present invention may be a food composition or a pharmaceutical composition. Food and pharmaceutical compositions include those for humans as well as those for animals other than humans, unless otherwise specified.

Food, unless otherwise specified, includes general foods, functional foods, nutritional compositions, and also therapeutic foods (foods that serve the purpose of treatment; prepared based on a menu prepared by a nutritionist etc. in accordance with a doctor's prescribed meal plan), dietary therapy foods, ingredient-adjusted foods, nursing care foods, foods for medical support, and precision nutrition (individualized nutrition; appropriate meals (nutrition) suggested to suit an individual's constitution).Unless otherwise specified, food includes not only solid foods but also liquid foods, such as beverages, energy drinks, liquid foods, and soups. Functional foods refer to foods that can provide a specific functionality to the living body, and include all types of health foods, such as foods for specified health uses (including conditional FOSHUs [specified health foods]), foods with functional claims, health functional foods including foods with nutrient functions, foods for special dietary uses, dietary supplements, health supplements, dietary supplements, food supplements, medical foods (following the definition of the U.S. Food and Drug Administration (FDA)), supplements (for example, tablets, coated tablets, sugar-coated tablets, capsules, liquids, and other dosage forms), beauty foods (for example, diet foods), etc. In addition, in the present invention, "functional foods" includes health foods to which health claims based on the food standards of Codex Alimentarius (the Joint FAO/WHO Food Standards Commission) are applied. Food supplements are foods that supplement the normal diet and are concentrated with nutrients or other substances that have nutritional or physiological effects, either alone or in combination, and are labeled as "food supplements." Dietary supplements are products (other than tobacco) intended to supplement the diet, that contain one or more of the targeted ingredients, and that are labeled as a dietary supplement.

Unless otherwise specified, pharmaceuticals include those that fall under the category of medical drugs, prescription drugs, over-the-counter drugs, and quasi-drugs as defined in the "Law on Ensuring Quality, Efficacy, and Safety of Pharmaceuticals, Medical Devices, etc."

(Route of administration, etc.)

The composition of the present invention may be administered orally, parenterally, for example by inhalation, through a tube (gastrostomy, enterostomy), or intranasally, but is preferably administered orally.

In one embodiment, the composition is preferably used before or immediately after tight junction damage. For example, when damage is due to infection (e.g., viral infection), it is believed that the tight junction protective effect of EPS can be obtained by ingesting or administering the composition before the damage to the tight junction progresses due to viral proliferation over time. The composition can be expected to be more effective by ingesting or administering the composition immediately when there is a high risk of tight junction damage (e.g., infection with fungi, bacteria, viruses, etc., suffering from chronic respiratory disease, exposure to smoking, dust, chemicals, gas, or environmental pollutants, etc.) or when tight junction damage is suspected (e.g., infection with fungi, bacteria, viruses, etc., or suffering from chronic respiratory disease).

(Active ingredient content and dosage)

The content of the lactic acid bacteria EPS in the composition of the present invention may be any amount that exerts the desired effect. The dosage or intake amount of the composition can be appropriately set in consideration of various factors such as the age, weight, symptoms, metabolic and excretory functions, and concomitant medications of the subject, and the amount of the lactic acid bacteria EPS per daily dose can be, for example, 0.1 mg or more, preferably 0.6 mg or more, more preferably 1 mg or more, and particularly preferably 3 mg or more. The upper limit of the amount of EPS per daily dose can be 500 mg or less, preferably 300 mg or less, and particularly preferably 250 mg or less, regardless of the lower limit.

The amount of lactic acid bacteria EPS per administration or meal, i.e., per serving, can be, for example, 0.03 mg or more, preferably 0.2 mg or more, and more preferably 1 mg or more. Whatever the lower limit, the upper limit of the amount of EPS per serving can be 200 mg or less, preferably 100 mg or less, more preferably 70 mg or less, and particularly preferably 30 mg or less.

When the EPS of lactic acid bacteria in the composition of the present invention is used as a composition such as fermented milk, the daily amount of the composition can be, for example, 30 g or more, preferably 50 g or more, more preferably 60 g or more, and particularly preferably 100 g or more. Regardless of the lower limit, the upper limit of the daily amount of fermented milk can be, for example, 1500 g or less, preferably 1200 g or less, more preferably 900 g or less, and even more preferably 600 g or less.

The amount of the composition per serving can be, for example, 10 g or more, preferably 20 g or more, and more preferably 30 g or more. Whatever the lower limit, the upper limit of the amount of the composition per serving can be, for example, 500 g or less, preferably 400 g or less, more preferably 200 g or less, and particularly preferably 125 g or less.

The composition may be administered/ingested once a day, or multiple times a day, for example, three times with each meal. The composition contains as its active ingredient the EPS of lactic acid bacteria with a long history of consumption. Therefore, the composition of the present invention is suitable for long-term consumption, as its active ingredient is EPS with a long history of consumption. Therefore, it may be taken repeatedly or over a long period of time, for example, it can be administered/ingested continuously for three days or more, preferably one week or more, more preferably four weeks or more, and particularly preferably one month or more.

(Other ingredients, additives)

The composition of the present invention may contain other active ingredients or nutritional ingredients that are acceptable as foods or pharmaceuticals. Examples of such ingredients include amino acids (e.g., lysine, arginine, glycine, alanine, glutamic acid, leucine, isoleucine, valine), carbohydrates (glucose, sucrose, fructose, maltose, trehalose, erythritol, maltitol, palatinose, xylitol, dextrin), electrolytes (e.g., sodium, potassium, calcium, magnesium), vitamins (e.g., vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin B12, vitamin C, vitamin D, vitamin E, vitamin K, biotin, folic acid, pantothenic acid, and nicotinic acids), minerals (e.g., copper, zinc, iron, cobalt, manganese), antibiotics, dietary fiber, proteins, lipids, etc.

The composition may further contain additives that are acceptable for use as food or medicine. Examples of such additives include inert carriers (solid or liquid carriers), excipients, surfactants, binders, disintegrants, lubricants, solubilizers, suspending agents, coating agents, colorants, preservatives, buffers, pH adjusters, isotonicity agents, emulsifiers, stabilizers, sweeteners, antioxidants, flavors, acidulants, and natural products. More specifically, these include water, other aqueous solvents, pharma- ceutical acceptable organic solvents, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymers, sodium alginate, water-soluble dextran, water-soluble dextrin, sodium carboxymethyl starch, pectin, xanthan gum, gum arabic, casein, gelatin, agar, glycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sodium chloride, propylene glycol, glycerin, benzalkonium chloride, methyl paraoxybenzoate, lactose, starch, maltose, sorbitol, lactose, sucralose, stevia, aspartame, acesulfame potassium, citric acid, lactic acid, malic acid, tartaric acid, phosphoric acid, acetic acid, fruit juice, vegetable juice, etc.

(Dosage form/shape)

The food composition of the present invention may be prepared in any form, such as solid, liquid, mixture, suspension, powder, granule, paste, jelly, gel, capsule, etc. The food composition of the present invention may be in any form, such as dairy products, supplements, confectionery, beverages, drinks, seasonings, processed foods, side dishes, soups, etc. More specifically, the composition of the present invention may be in the form of fermented milk, lactic acid bacteria beverage, dairy beverage, milk drink, soft drink, ice cream, tablet, cheese, bread, biscuit, cracker, pizza crust, modified milk powder, liquid food, food for sick people, nutritional food, frozen food, processed food, etc., and may be in the form of granule, powder, paste, thick liquid, etc., to be mixed with beverages or foods for ingestion. Fermented milk is fermented milk and lactic acid bacteria beverage defined in the "Ministerial Ordinance on the Compositional Standards of Milk and Dairy Products (hereinafter abbreviated as "Milk, etc. Ordinance"). Fermented milk, as defined in the Milk and Milk Products Ordinance, is milk or milk containing an equivalent or greater amount of non-fat milk solids that has been fermented with lactic acid bacteria or yeast to form a paste or liquid, or these products that have been frozen.

One preferred embodiment of the food composition is fermented milk obtained by fermenting raw milk using EPS-producing lactic acid bacteria as a starter. The fermented milk may contain microorganisms such as yeast other than the target lactic acid bacteria. In one preferred embodiment, the fermented milk contains one or more types of lactic acid bacteria, but may or may not contain other microorganisms, such as yeast. The raw milk includes milk of animal origin and its processed products, such as cow's milk, skim milk, skim milk powder, skim concentrated milk, filtered concentrate or permeate of milk, condensed milk, whey, milk protein concentrate (MPC), whey protein concentrate (WPC), buttermilk, and fresh cream. The raw milk may or may not contain plant-based milk, such as soy milk, almond milk, oat milk, coconut milk, rice milk, and hemp milk.

In one embodiment, the pharmaceutical composition of the present invention is an inhalant. By using the pharmaceutical composition of the present invention as an inhalant, it is expected that the composition will have an effect on the tight junctions of lung cells. The form of the inhalant may be, for example, a dry powder suitable for dry powder metered dose inhalation (DPI) or the like, or may be a liquid suitable for pressurized metered dose inhalation (pMDI), soft mist metered dose inhalation (SMI), nebulizer, etc., and is not particularly limited as long as it can be administered to the lungs or airways.

Inhalants can be prepared by various methods known to those skilled in the art. When the inhalant is in liquid form, the preparation method may include, for example, dissolving the active ingredient and other ingredients such as excipients, isotonicity agents, and preservatives in an appropriate solvent such as purified water, water for injection, or sterile purified water. When the inhalant is in dry powder form, the preparation method may include, for example, freeze-drying a liquid mixture containing the active ingredient and other ingredients such as excipients by spray freeze-drying.

The inhalant may further contain one or more pharmacologically active ingredients in addition to the active ingredient of the present invention. Alternatively, the inhalant may be a kit formulation containing the active ingredient of the present invention and one or more pharmacologically active ingredients other than the active ingredient of the present invention. Examples of pharmacologically active ingredients other than the active ingredient of the present invention include, but are not limited to, bronchodilators such as salmeterol, indacaterol, tiotropium, glycopyrronium, etc., antiviral agents such as zanamivir, laninamivir, etc.

The pharmaceutical composition of the present invention can also be in any dosage form suitable for oral administration, including solid preparations such as tablets, granules, powders, pills, and capsules, liquid preparations such as solutions, suspensions, and syrups, gels, and aerosols.

(others)

In the production of the composition of the present invention, the stage of blending the EPS of the lactic acid bacteria can be appropriately selected. The stage of blending is not particularly limited as long as the characteristics of the EPS of the lactic acid bacteria are not significantly impaired. For example, a culture containing EPS obtained by culturing a lactic acid bacterium that produces EPS, or a crude product or purified product thereof can be mixed and blended with a raw material. Alternatively, when the composition of the present invention is implemented as fermented milk, a culture containing EPS, or a crude product or purified product thereof can be mixed and blended with a raw material or fermented fermented milk, or an EPS-producing lactic acid bacterium can be added as a starter to raw milk, fermented, and allowed to produce EPS, thereby producing fermented milk containing EPS.

The composition of the present invention can be labeled with its intended use (application), and can also be labeled with a recommendation that it be taken by a specific subject.

　The composition of the present invention may be labeled to the effect that it can be used for the protection of tight junctions, etc., and may also be labeled to recommend ingestion to a specific subject (including notifications for dietary management apps, nutritional management apps, health care apps, etc.). Note that a period such as "temporary" or "long-term" may be indicated at the beginning of each statement as appropriate. Labeling may be direct or indirect. An example of direct labeling is inscription on tangible objects such as the product itself, packaging, containers, labels, tags, etc., and an example of indirect labeling includes advertising and promotional activities by place or means such as websites, storefronts, pamphlets, exhibitions, media seminars, etc., books, newspapers, magazines, television, radio, mailings, e-mails, sales talks, voice, etc.

The present invention provides a method for protecting tight junctions (particularly pulmonary tight junctions) comprising the step of administering an exopolysaccharide of a lactic acid bacterium or a composition containing the exopolysaccharide, and such a method can include a step of testing the strength of tight junctions in the lungs of a subject after the step of administering the active ingredient. Such tests include confirmation of physical condition, medical interview, spirometry, chest X-ray, and interviews and questionnaires about lifestyle habits (smoking habits, allergies, medical history of respiratory diseases, etc.). The tests may be performed by the subject himself or herself, or by someone other than the subject.

[Method of producing fermented milk]

In one embodiment, the present invention provides a method for producing fermented milk for protecting tight junctions, which comprises adding lactic acid bacteria classified as Lactobacillus delbrueckii subsp. bulgaricus and lactic acid bacteria classified as Streptococcus thermophilus to a milk preparation containing raw milk and fermenting the mixture. Note that Streptococcus thermophilus is used for producing yogurt in accordance with the Codex standard.

In another aspect, the present invention provides a method for producing fermented milk for protecting tight junctions, which comprises inoculating (1%) lactic acid bacteria classified as Lactococcus lactis subsp. cremoris or lactic acid bacteria classified as Lactococcus lactis subsp. lactis into a milk preparation containing raw milk, and fermenting the mixture at its optimum temperature (approximately 25°C to 30°C).

The raw milk is the milk used as the raw material for processing, and may be, for example, raw milk, or raw milk mixed with skim milk powder, cream, water, etc. In addition to the above, the raw milk may further contain pasteurized milk, whole milk, skim milk, whole fat concentrated milk, skim milk concentrated milk, whole milk powder, buttermilk, salted butter, unsalted butter, whey, whey powder, whey protein concentrate (WPC), whey protein isolate (WPI), α-La (alpha-lactalbumin), β-Lg (beta-lactoglobulin), lactose, etc. The raw milk may also contain pre-warmed gelatin, agar, thickeners, gelling agents, stabilizers, emulsifiers, sucrose, sweeteners, flavorings, vitamins, minerals, etc. as appropriate.

In these manufacturing methods, fermented milk can be obtained by adding specific lactic acid bacteria as a starter to a milk preparation containing the above-mentioned raw milk and fermenting it. As starters, lactic acid bacteria classified as Lactobacillus delbrueckii subsp. bulgaricus, lactic acid bacteria classified as Lactococcus lactis subsp. lactis, and lactic acid bacteria classified as Lactococcus lactis subsp. cremoris, and lactic acid bacteria classified as Streptococcus thermophilus can be used. The lactic acid bacteria classified as Lactobacillus delbrueckii subsp. bulgaricus are not limited to a specific strain and may be various, but preferably the bulgaricus strain R-1 is used. The lactic acid bacteria classified as Streptococcus thermophilus are not limited to a specific strain and may be, for example, Streptococcus thermophilus 1131. Streptococcus thermophilus 1131 can be isolated from Meiji Bulgaria Yogurt LB81 (Meiji Co., Ltd.) and is commercially available. Lactic acid bacteria classified as Lactococcus lactis subsp. lactis are not limited to a specific strain and may be various, but preferably Lactococcus lactis subsp. lactis JCM5805 or JCM20101. Lactococcus lactis subsp. lactis JCM5805 or JCM20101 can be obtained from the RIKEN BioResource Center. Lactic acid bacteria classified as Lactococcus lactis subsp. cremoris are not limited to a specific strain and may be various, but preferably Lactococcus lactis subsp. cremoris FC. Lactococcus lactis subsp. cremoris FC strain can be isolated from "Fujicco Caspian Sea Yogurt (registered trademark)" manufactured and sold by Fujicco Co., Ltd., and is available commercially. The amount of lactic acid bacteria added may be in accordance with known methods for producing fermented milk, and may be, for example, 0.1 to 5% by weight, 0.5 to 4% by weight, or 1 to 3% by weight of the milk preparation.

Fermentation can be carried out according to methods known to those skilled in the art, for example, by leaving the milk preparation to which the lactic acid bacteria has been added at 30°C to 50°C, preferably 33°C to 47°C, more preferably 35°C to 44°C, for 1 hour to 30 hours, preferably 2 hours to 24 hours, more preferably 3 hours to 12 hours.

In addition to the above, the method for producing fermented milk may further include removing foreign matter using a filter, sterilizing the product by heating at, for example, 75 to 95°C for 5 to 15 minutes, stirring, etc.

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

Example 1
Materials and Methods

(cells, viruses, EPS)
A549 cells (human alveolar basal epithelial adenocarcinoma cells) were purchased from the RIKEN BioResource Center Cell Bank (Tsukuba, Japan). The cells were cultured in DMEM containing 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin (Sigma, MO, USA), and MEM non-essential amino acids (Thermo Fisher Scientific, MA, USA) at 5% CO ₂ and 37°C. Influenza virus A/Puerto Rico/8/34 (H1N1) was obtained from the University of Tokyo.

The EPS produced by Lactobacillus delbrueckii spp. bulgaricus OLL1073 R-1 was purified from the culture obtained by culturing Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1 in a 10% by mass skim milk medium. Specifically, trichloroacetic acid was added to the culture to a final concentration of 10% by mass to remove denatured proteins, and cold ethanol was added to the culture, which was cultured for 18 hours at 37°C, and the culture was left to stand at 4°C for 2 hours to obtain a precipitate containing EPS. This was dialyzed against MilliQ water using a dialysis membrane (molecular weight cutoff 6,000 - 8,000) to enzymatically decompose nucleic acids and proteins, and then ethanol precipitation was performed again to obtain a precipitate. This was dissolved in MilliQ water, dialyzed again, and freeze-dried to purify the EPS. This was then dissolved in autoclave-sterilized distilled water. The EPS solution was filtered through a 0.22 μm syringe filter, and the filtered solution was frozen at -80°C until use.

(Influenza virus infection of A549 cells)
A549 cells were cultured at ^1x105 cells/200μl/well in a 96-well flat-bottom plate 12 hours before virus infection. After 12 hours of culture, the A549 cell culture medium was replaced with fresh medium, and EPS was added to each well at a concentration of 400μg/ml. At the same time, A549 cells were infected with influenza virus at MOI=1 ( ^1x105 pfu) (per well) for 1 hour.

The cells were washed three times with DMEM and further cultured in EPS-free DMEM at 5% CO ₂ and 37° C. for 12 hours. RNA was then extracted from each A549 cell.

In some experiments, 250 nM baloxavir acid (BXA) (Shionogi, Osaka, Japan) was added to the wells as a positive control for a viral replication inhibitor. After 12 h of incubation, RNA was extracted from each infected A549 cell.

(RNA purification and quantitative RT-PCR)
RNA was purified from infected A549 cells and cDNA was synthesized using the Power SYBR® Green Cell-to CT ^™ kit according to the kit instructions (Thermo Fisher Scientific).

Influenza virus in virus-infected cells was quantified by quantitative RT-PCR using primers for the influenza virus M gene region (5'-GGCAAATGGTACAGGCAATG-3' (SEQ ID NO: 4) and 5'-AGCAACGAGAGGATCACTTG-3' (SEQ ID NO: 5)) (Non-Patent Document 15). cDNA prepared from influenza virus RNA determined based on the virus titer obtained by the 50% tissue culture infectious dose (TCID ₅₀ ) method was used as the standard DNA for quantitative RT-PCR.

The quantitative RT-PCR primers for ZO-1 (tight junction gene) in A549 cells used in this study are shown in Table 1 below.

Quantitative RT-PCR was performed using LightCycler 480 ProbeMaster and LightCycler 480 instruments and accompanying software programs (Roche Diagnostics, Mannheim, Germany). In some experiments, each sample was calibrated with the level of an internal standard (β-actin) and normalized to the mean value of the control samples.

[result]

When A549 cells were infected with influenza virus and cultured for 12 hours, the expression of the ZO-1 gene, one of the main molecules that constitute the tight junction, was significantly decreased in a virus number-dependent manner.

When EPS was added during viral infection, the number of intracellular viruses was significantly reduced compared to when EPS was not added (Figure 1), and a significant recovery in ZO-1 gene expression was observed (Figure 2).

[Discussion]

Thus, when A549 cells (human alveolar basal epithelial adenocarcinoma cells) were infected with a virus, the expression of the ZO-1 gene was decreased, whereas when EPS produced by Lactobacillus delbrueckii spp. bulgaricus OLL1073 R-1 was added during the infection, the expression of the ZO-1 gene was restored. Therefore, the EPS of lactic acid bacteria can be used as an active ingredient of a composition for protecting tight junctions, such as promoting the recovery of tight junctions, and as an active ingredient of a composition for promoting the expression of a component molecule of tight junctions, such as ZO-1. In addition, such a composition is suitable for ingestion by subjects (including subjects with subjective symptoms) whose lung cell (or alveolar epithelial cell) barrier is considered to be relatively weak, for example, those aged 65 years or older, infants, babies, newborns, those suffering from chronic respiratory diseases, and any person selected from the group consisting of smokers.

Example 2
Materials and Methods

The cells, viruses, and EPS used were the same as those used in Example 1.

(Influenza virus infection of A549 cells)
A549 cells were cultured at ^1x105 cells/200μl/well in a 96-well flat-bottom plate 12 hours before virus infection. After 12 hours of culture, the A549 cell culture medium was replaced with fresh medium, and EPS was added to each well at a concentration of 400μg/ml. At the same time, A549 cells were infected with influenza virus at MOI=1 ( ^1x105 pfu) (per well) for 1 hour.

(Fluorescein isothiocyanate (FITC)-dextran permeability test using a transwell system)
The cells were washed three times with DMEM and incubated in DMEM without EPS for 12 h at 37°C _in 5% CO2. After 12 h of incubation, 50 μg of FITC-dextran (4KDa) (Chondrex, WA, USA) was added to each apical compartment and the cells were incubated for another 3 h. After mixing the basolateral medium, 50 μl of medium from the basolateral compartment was sampled in triplicate in a black microplate (PerkinElmer, MA, USA) and the fluorescence intensity was measured by a microplate reader at 490 nm for excitation and 520 nm for emission (TECAN, Mennedorf, Switzerland).

[result]
As shown in Figure 3, the increased permeability of tight junctions caused by influenza virus infection tended to be reduced in infected cells to which EPS had been added during influenza virus infection.

[Discussion]

Thus, when A549 cells (human alveolar basal epithelial adenocarcinoma cells) are infected with a virus, tight junction permeability is enhanced, whereas when EPS produced by Lactobacillus delbrueckii spp. bulgaricus OLL1073 R-1 is added during virus infection, the enhancement of tight junction permeability is suppressed. This suggests that lactic acid bacteria EPS reduces tight junction permeability or inhibits the enhancement of tight junction permeability due to the destruction of tight junctions, and can be used in treatments for maintaining the barrier function of epithelial cells such as alveolar epithelial cells. Therefore, lactic acid bacteria EPS can be used as an active ingredient in compositions for protecting tight junctions, such as inhibiting the progression of destruction of tight junctions, promoting the recovery of tight junctions, and inhibiting the enhancement of tight junction permeability. Furthermore, such a composition is suitable for administration to subjects (including subjects with symptoms) whose lung cell (preferably alveolar epithelial cells) barrier is considered to be relatively weak, such as anyone selected from the group consisting of persons aged 65 or older, young children, infants, newborns, persons suffering from chronic respiratory diseases, and smokers.

[Production of fermented dairy products (plain yogurt) 1]

A yogurt base mix is prepared by mixing milk, dairy products (derived from milk), and water so that the final product has a non-fat milk solid content of 9.5% and a milk fat content of 3.0%. The prepared yogurt base mix is then homogenized, heat-sterilized at 95°C for 5 minutes, and then cooled to about 40°C. To this sterilized yogurt base mix, polysaccharide-producing bacteria Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1 and a lactic acid bacteria strain belonging to Streptococcus thermophilus are added as starters and fermented, or a strain of Lactococcus lactis subsp. cremoris and a lactic acid bacteria strain belonging to Streptococcus thermophilus are added as starters and fermented at 40 to 50°C to produce fermented milk. The produced fermented milk can be used for tight junction protection. Streptococcus thermophilus is added as a starter to promote fermentation in order to meet the Codex standard.

[Production of fermented dairy products (plain yogurt) 2]

A yogurt base mix is prepared by mixing milk, dairy products (derived from milk), and water so that the final product has a non-fat milk solid content of 9.5% and a milk fat content of 3.0%. The prepared yogurt base mix is then homogenized, heat sterilized at 95°C for 5 minutes, and then cooled to about 20-30°C. A polysaccharide-producing strain of Lactococcus lactis subsp. cremoris or a polysaccharide-producing strain of Lactococcus lactis subsp. lactis is added as a starter to this sterilized yogurt base mix, and fermented at 20-30°C to produce fermented milk. The produced fermented milk can be used for tight junction protection.

[Production of inhalant (liquid for inhalation) 1]

The EPS obtained by purifying it using the method described in Example 1 is dissolved in purified water to give concentrations of 0.1, 0.3, and 0.5% by mass to produce liquid preparations for inhalation. The inhalants produced can be used mainly for aerosol-type preparations.

[Production of inhalant (liquid for inhalation) 2]

EPS is purified by the method described in Example 1. EPS, sodium chloride, and benzalkonium chloride are dissolved in purified water to concentrations of 0.3 mass%, 0.9 mass%, and 0.01 mass%, respectively, to produce an inhalation liquid. The inhalation liquid produced can be used mainly for aerosol-type preparations.

[Production of inhalant (powder for inhalation) 3]
EPS is purified by the method described in Example 1. EPS and lactose hydrate are dissolved in purified water to a concentration of 0.3% by mass and 10% by mass, respectively, and the resulting solution is sprayed into liquid nitrogen to instantly freeze the droplets, and then the solvent is sublimated in a freeze dryer to obtain fine powder particles, thereby producing a powder for inhalation. The produced inhalant can be mainly used for dry powder type preparations.

The present invention can provide a food composition and a method for producing a food that are useful for protecting tight junctions. The present invention can also provide a food composition and a method for producing a food that supports the maintenance and improvement of people's health. The present invention can support the maintenance and improvement of people's health. Furthermore, the present invention can improve the nutrition of various people, ensure healthy lives, and promote welfare.

(References cited in the specification)

Non-patent document 4: Zhang, L., Liu, C., Li, D., Zhao, Y., Zhang, X., Zeng, X., Yang, Z., Li, S., 2013. Antioxidant activity on exopolysaccharide isolated from Lactobacillus plantarum C88. Int. J. Biol. Macromol. 54, 270-275.
Non-patent document 5: Gorska-Froczek, S., Sandstrom, C., Kenne, L., Paociak, M., Brzozowska, E., Strus, M., Heczko, P., Gamian, A., 2013. The structure and immunoreactivity of exopolysaccharide isolated from Lactobacillus johnsonii strain 151. Carbohydr. Res. 378, 148-153.
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(Sequence listed in the sequence listing)

SEQ ID NO:1 16S rRNA gene. Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1
SEQ ID NO:2 ZO-1 RT-PCR Forward Primer
SEQ ID NO:3 ZO-1 RT-PCR Reverse Primer
SEQ ID NO:4 influenza virus M gene region RT-PCR Forward Primer
SEQ ID NO:5 influenza virus M gene region RT-PCR Reverse Primer

Claims (17)

A composition for protecting tight junctions, comprising an exopolysaccharide of lactic acid bacteria, the tight junctions being those of lung cells. The composition according to claim 1, wherein the tight junction protection is promotion of recovery of damaged tight junctions or inhibition of damage to tight junctions. The composition of claim 2, wherein the injury is caused by a viral infection. The composition of claim 2, wherein the injury is a decrease in Zonula occludens-1 (ZO-1) gene expression. The composition according to claim 3, wherein the virus is an influenza virus. The composition according to claim 1, wherein the lactic acid bacteria are exopolysaccharide-producing bacteria. The composition according to claim 1, wherein the lactic acid bacteria are selected from bacteria classified as Lactobacillus delbrueckii subsp. bulgaricus, bacteria classified as Lactococcus lactis subsp. lactis, and bacteria classified as Lactococcus lactis subsp. cremoris. The composition according to claim 1, wherein the lactic acid bacterium is Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1 (FERM BP-10741). The composition according to claim 1, intended for ingestion by any person selected from the group consisting of persons aged 65 years or older, young children, infants, newborns, persons suffering from chronic respiratory diseases, and smokers. The composition according to claim 2, wherein the promotion of tight junction recovery or the inhibition of tight junction damage is achieved by promoting ZO-1 gene expression. A composition for promoting ZO-1 gene expression, containing exopolysaccharides of lactic acid bacteria. The composition according to claim 11, which is for promoting ZO-1 gene expression in damaged tight junctions. The composition according to claim 11, wherein the lactic acid bacteria are exopolysaccharide-producing bacteria. The composition according to claim 11, wherein the lactic acid bacteria is classified as Lactobacillus delbrueckii subsp. bulgaricus. The composition according to claim 11, wherein the lactic acid bacteria are selected from bacteria classified as Lactococcus lactis subsp. lactis and bacteria classified as Lactococcus lactis subsp. cremoris. A method for producing fermented milk for protecting tight junctions, comprising adding lactic acid bacteria classified as Lactobacillus delbrueckii subsp. bulgaricus and lactic acid bacteria classified as Streptococcus thermophilus to a milk preparation containing raw milk and fermenting the mixture. An inhalant containing exopolysaccharides of lactic acid bacteria.