Intestinal barrier protective agent and prophylactic agent for intestinal barrier disruption-related disease

Abstract

To find an intestinal bacterium for protecting intestinal barrier function that exhibits higher efficacy than conventional ones.SOLUTION: An intestinal barrier protective agent characterized by including Bifidobacterium adolescentis as an active ingredient, and a prophylactic agent for intestinal barrier disruption-related diseases containing the same.SELECTED DRAWING: Figure 7

Description

[Technical Field]

[0001] The present invention relates to an agent for protecting the intestinal barrier and an agent for preventing diseases associated with intestinal barrier breakdown, which contain Bifidobacterium adolescentis as an active ingredient. [Background technology]

[0002] In recent years, attention has been drawn to the influence that intestinal bacteria have on the pathology of metabolic disorders in the host, particularly the pathogenesis of obesity and diabetes. Currently, intestinal bacteria are thought to be a cause of obesity, along with dietary habits and lack of exercise, and novel treatments for obesity are being sought that involve changing the intestinal flora of obese patients and further improving the intestinal environment.

[0003] Dysbiosis is caused by various factors (stress, aging, alcohol, smoking, diet, antibiotics, lack of physical activity, etc.), and as a result, the intestinal barrier function is disrupted by three stimuli: lipopolysaccharide (LPS), a toxin derived from intestinal bacteria, inflammatory cytokines in the intestinal tract, and oxidative stress. This disruption allows LPS and inflammatory cytokines to enter the bloodstream and circulate, reaching the adipose tissue and liver, which determine insulin sensitivity, and causing chronic inflammation and insulin resistance (Non-Patent Documents 1 and 2).

[0004] Therefore, it is believed that protecting the intestinal barrier function can also prevent diseases associated with the breakdown of the intestinal barrier, such as obesity and diabetes.

[0005] Although there have been many attempts to find intestinal bacteria that can protect the intestinal barrier function, reports have so far shown that Bifidobacterium bifidum and Bifidobacterium animalis have the intestinal barrier protective effect against cytokine stimulation (Non-patent documents 3-6), and Bifidobacterium longum and Bifidobacterium animalis have the intestinal barrier protective effect against LPS stimulation (Non-patent documents 7-8), but no intestinal bacteria have been reported that have the intestinal barrier protective effect against multiple stimuli. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Coppe. JP, et al., PLoS Biol. 6. 2853. 2008 [Non-patent document 2] Winer. D. A, et al., Cell Metab. 23. 413. 2016 [Non-patent document 3] Al-Sadi. R, et al., Int. J. Mol. Sci. 22. 8070. 2021 [Non-patent document 4] Hsieh. C. Y, et al., Physiol Rep. 3. 3. e12327. 2015 [Non-Patent Document 5] Wang. X, et al., Biomedicines. 9. 641. 2011 [Non-patent document 6] Castro-Herrera. V. M, et al., Nutrients. 12. 1719. 2020 [Non-Patent Document 7] Zhao. L, et al., J. Funct. Foods . 92. 105030. 2022 [Non-patent document 8] Kim. JY, et al., Front Microbiol. 13. 817591. 2022 Summary of the Invention [Problem to be solved by the invention]

[0007] The objective of the present invention is to find an intestinal bacterium that protects the intestinal barrier function and that is more effective than conventional bacteria. [Means for solving the problem]

[0008] As a result of intensive research to solve the above problems, the present inventors discovered that a specific bifidobacterium has an excellent protective effect on the intestinal barrier function, and thus completed the present invention.

[0009] That is, the present invention is an intestinal barrier protecting agent characterized by containing Bifidobacterium adolescentis as an active ingredient.

[0010] The present invention also relates to an agent for preventing diseases associated with intestinal barrier breakdown, which contains the intestinal barrier protecting agent. [Effects of the Invention]

[0011] The intestinal barrier protecting agent of the present invention contains Bifidobacterium adolescentis, which is present in the intestine, as an active ingredient, and can therefore protect the intestinal barrier safely for a long period of time.

[0012] Furthermore, since the intestinal barrier protecting agent of the present invention can protect the intestinal barrier, it is also useful as a preventive agent for diseases associated with breakdown of the intestinal barrier. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 1 shows the relationship between TEER and time in LPS-stimulated monolayers of Example 1. [Figure 2] FIG. 1 shows the TEER after 48 hours in LPS-stimulated monolayers of Example 1. [Figure 3]Figure 1 shows the relationship between TEER and time for inflammatory cytokine-stimulated monolayers from Example 1. TI represents TNF-alpha+IFN-γ. [Figure 4] 1 shows the TEER after 48 hours in the inflammatory cytokine-stimulated monolayers of Example 1. TI represents TNF-alpha+IFN-γ. [Figure 5] FIG. 1 shows the relationship between TEER and time in the monolayer membrane stimulated with oxidative stress in Example 1. [Figure 6] FIG. 1 shows the TEER after 6 hours of oxidative stress stimulation in the monolayer membrane of Example 1. [Figure 7] FIG. 1 shows the effect of Example 1 on the localization of tight junction proteins (+: YIT 13021 added, −: YIT 13021 not added). [Figure 8] FIG. 1 shows the relationship between TEER and time for the monolayer film without stimulation in Example 2. [Figure 9] FIG. 1 shows the TEER after 6 hours in the monolayer film without stimulation in Example 2. [Figure 10] FIG. 1 shows the TEER after 48 hours in LPS-stimulated monolayers of Example 3. [Figure 11] Figure 1 shows TEER after 48 hours in inflammatory cytokine stimulated monolayers of Example 3. TI represents TNF-alpha + IFN-γ. [Figure 12] FIG. 1 shows the TEER after 6 hours of oxidative stress stimulation in the monolayer membrane of Example 3. [Figure 13] FIG. 1 shows the TEER after 24 hours in the monolayer membrane without stimulation in Example 4. [Figure 14] FIG. 1 shows the TEER after 24 hours for the monolayer membrane of Example 5 (heat-killed bacteria) without stimulation. [Figure 15] FIG. 1 shows the TEER after 24 hours in the monolayer membrane of Example 5 (live cells) without stimulation. [Figure 16] FIG. 1 shows the TEER after 24 hours for unstimulated monolayers of Example 6 (live cells cultured in combination with starch granules). DETAILED DESCRIPTION OF THE INVENTION

[0014] The intestinal barrier protecting agent of the present invention (hereinafter referred to as "the protecting agent of the present invention") contains Bifidobacterium adolescentis as an active ingredient.

[0015] The Bifidobacterium adolescentis used in the protective agent of the present invention may be any of the strains known as Bifidobacterium adolescentis or those isolated by conventional methods (for example, (1) Research on Bifidobacteria, edited by Mitsuoka Tomotari, Japan Bifidobacteria Center, (2) Methods for Identifying Intestinal Bacteria and Intestinal Constituent Bacteria, Fujisawa Michihiko, Japanese Journal of Bacteriology 2014, (3) PMID: 28394924 (Bifidobacterium adolescentis is isolated from mouse feces, but is applicable to humans)). For example, Bifidobacterium adolescentis YIT 13021, Bifidobacterium adolescentis YIT 13609, Bifidobacterium adolescentis YIT 12828, Bifidobacterium adolescentis type strain (ATCC15703), Bifidobacterium adolescentis YIT 13022, Bifidobacterium adolescentis YIT 11033, Bifidobacterium adolescentis YIT 11034, Bifidobacterium adolescentis YIT 11040, etc. These Bifidobacterium adolescentis can be used alone or in combination of two or more types.

[0016] Among these Bifidobacterium adolescentis, Bifidobacterium adolescentis YIT 13021, Bifidobacterium adolescentis YIT 13609, and Bifidobacterium adolescentis YIT 12828 are preferred, and Bifidobacterium adolescentis YIT 13021 is more preferred.

[0017] The Bifidobacterium adolescentis used in the protective agent of the present invention includes not only the above-mentioned strains but also strains that have been artificially genetically manipulated while maintaining the properties of Bifidobacterium adolescentis. Furthermore, although both live and dead Bifidobacterium adolescentis are effective, live bacteria are preferred.

[0018] Of the above-mentioned Bifidobacterium adolescentis, Bifidobacterium adolescentis YIT 13021 has been internationally deposited as Bifidobacterium adolescentis YIT 13021 (NITE BP-03808, date of deposit: January 24, 2023) at the Patent Microorganisms Depositary, National Institute of Technology and Evaluation (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818, Japan), the international depositary authority of the Budapest Treaty.

[0019] Bifidobacterium adolescentis YIT 13609 has been deposited domestically at the National Institute of Technology and Evaluation, Patent Microorganisms Depositary Center (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, 292-0818) as YIT 13609 (NITE P-04064, date of deposit: January 15, 2024).

[0020] Bifidobacterium adolescentis YIT 12828 has been deposited domestically at the above-mentioned Patent Microorganisms Depositary Center as YIT 12828 (NITE P-04065, date of deposit: January 15, 2024).

[0021] Bifidobacterium adolescentis YIT 13022 has been internationally deposited with the above-mentioned international depositary authority as Bifidobacterium adolescentis YIT 13022 (NITE BP-03809, date of deposit: January 24, 2023).

[0022] Bifidobacterium adolescentis YIT 11033 has been internationally deposited with the above-mentioned international depositary authority as Bifidobacterium adolescentis YIT 11033 (NITE BP-03810, date of deposit: January 24, 2023).

[0023] Bifidobacterium adolescentis YIT 11034 has been internationally deposited with the above-mentioned international depositary authority as Bifidobacterium adolescentis YIT 11034 (NITE BP-03811, date of deposit: January 24, 2023).

[0024] Bifidobacterium adolescentis YIT 11040 has been internationally deposited with the above-mentioned international depositary authority as Bifidobacterium adolescentis YIT 11040 (NITE BP-03812, date of deposit: January 24, 2023).

[0025] The protective agent of the present invention contains the above-mentioned Bifidobacterium adolescentis as an active ingredient, and can protect the intestinal barrier from one or more stimuli selected from LPS stimulation, inflammatory cytokine stimulation, and oxidative stress stimulation, preferably from two or more of the above stimuli, and more preferably from the three above stimuli.

[0026] Here, LPS stimulation refers to the action of lipopolysaccharide (LPS), a toxin derived from enterobacteria, on intestinal epithelial cells.

[0027] Inflammatory cytokine stimulation refers to the action of inflammatory cytokines such as TNF-α, IFN-γ, and IL-6 on intestinal epithelial cells.

[0028] Oxidative stress stimulation refers to the action of reactive oxygen species such as hydrogen peroxide generated by oxidative stress on intestinal epithelial cells.

[0029] Here, protection of the intestinal barrier means that the expression or localization of tight junction proteins present in the intestinal tract is not disrupted or disrupted even when subjected to one or more stimuli selected from LPS stimulation, inflammatory cytokine stimulation, and oxidative stress stimulation, preferably two or more of the above stimuli, and more preferably three of the above stimuli, or that a decrease in the index value of intestinal barrier strength (transepithelial electrical resistance; TEER) is suppressed, as will be shown in the Examples described below.

[0030] The form of the protective agent of the present invention is not particularly limited, and, like conventionally known bifidobacteria, examples include foods and beverages such as fermented milk, yogurt, and bacterial powder supplements, as well as pharmaceuticals such as lactic acid bacteria and bifidobacterial preparations. Of these, fermented foods such as fermented milk, and pharmaceuticals such as lactic acid bacteria and bifidobacterial preparations are preferred.

[0031] The protective agent of the present invention can protect the intestinal barrier by administering it to mammals, including humans. In this case, the protective agent of the present invention contains 100 mg of Bifidobacterium adolescentis per day. 5 ~10 10 It is sufficient to include it in an amount that will result in an intake of about cfu.

[0032] As described above, the protective agent of the present invention can protect the intestinal barrier and therefore can be used as a preventive agent for diseases associated with intestinal barrier disruption (hereinafter referred to as the "preventive agent of the present invention"). Since the preventive agent of the present invention can also protect against intestinal barrier disruption, diseases associated with intestinal barrier disruption also include diseases associated with intestinal barrier disruption. Examples of diseases associated with intestinal barrier disruption include diabetes, obesity, inflammatory bowel disease (Crohn's disease, ulcerative colitis, etc.), fatty liver, Parkinson's disease, cancer, irritable bowel disease, dyslipidemia, atopy, asthma, dementia, insomnia, depression, rheumatoid arthritis, etc. Among these diseases associated with intestinal barrier disruption, obesity and diabetes are preferred.

[0033] The preventive agent of the present invention can prevent diseases associated with intestinal barrier disruption by administering it to mammals, including humans. In this case, the preventive agent of the present invention contains 100 mg of Bifidobacterium adolescentis per day.5 ~10 10 It is sufficient to include it in an amount that will result in an intake of about cfu.

[0034] The fact that the above-mentioned diseases are associated with breakdown of the intestinal barrier is described in the following literature. <Inflammatory bowel disease, obesity, fatty liver> Lee. B, et al., J Immunol Res. 2018. 2645465. 2018 <Parkinson's disease> Van Ijzendoorn. SC D, et al., J Parkinsons Dis. 9. S323. 2019 <Cancer, irritable bowel disease> Oshima. T, et al., J Gastroenterol. 51. 768. 2016 <Dyslipidemia> Flaig. B, et al., Nutrients. 15. 228. 2023 <Atopic dermatitis, asthma> Niewiem. M, et al., Nutrients. 14. 1893. 2022 <Dementia> Stadlbauer. V, et al., BMC Geriatr. 20. 248. 2020 <Insomnia, depression> Li. Y, et al., Front Psychiatry. 9. 669. 2018 <Rheumatoid arthritis> Matei. D. E, et al., Med. 2. 864. e9. 2021

[0035] Among the Bifidobacterium adolescentis strains used in the protective agent and preventive agent of the present invention, Bifidobacterium adolescentis YIT 13021, Bifidobacterium adolescentis YIT 13022, Bifidobacterium adolescentis YIT 11033, Bifidobacterium adolescentis YIT 11034, Bifidobacterium adolescentis YIT 11040, and the like, have the property of adhering to and assimilating starch granules. This property is not present in the Bifidobacterium adolescentis type strain (ATCC 15703). This property, along with test methods for confirming this property, are described in the international application (PCT / JP2023 / 3018) filed January 31, 2023.

[0036] Therefore, when Bifidobacterium adolescentis having the above-mentioned properties is used in the protective agent and preventive agent of the present invention, it is preferable to contain starch granules together with the Bifidobacterium adolescentis. This inhibits the adhesion of enterobacteria such as Eubacterium lectare, which compete with them in the intestine, to the starch granules, allowing the Bifidobacterium adolescentis to adhere and colonize, thereby enhancing the effectiveness. Here, to make it easier to obtain the above-mentioned properties, it is preferable to culture Bifidobacterium adolescentis in a medium supplemented with soluble starch, starch granules, dextrin, etc.

[0037] Here, starch granules refer to starches derived from potatoes (e.g., potatoes, sweet potatoes), grains (e.g., wheat, rice, corn), legumes (e.g., soybeans, adzuki beans), tapioca, bananas, etc., which have not undergone any heating process involving water (e.g., boiling, steaming) except for the drying process during production, or have undergone a heating process involving water but have not gelatinized, retaining their natural granular state and being insoluble in room-temperature water. More specifically, starch granules refer to uncooked starches derived from potatoes (e.g., potatoes, sweet potatoes), grains (e.g., wheat, rice, corn), legumes (e.g., soybeans, adzuki beans), tapioca, bananas, etc., which have granular shapes (e.g., spherical, ovoid, polyhedral), and typically have diameters of 1 to 120 μm, depending on the plant species. This diameter can be measured using a microscope or particle size measuring device.

[0038] When starch granules are contained in the protective agent and preventive agent of the present invention, they may be contained so that the intake amount is about 0.5 to 100 g per day.

[0039] The protective agent and preventive agent of the present invention described above can safely protect the intestinal barrier and prevent diseases associated with intestinal barrier breakdown for a long period of time.

[0040] Furthermore, the protective agent of the present invention not only protects the intestinal barrier but can also enhance the intestinal barrier in the absence of any irritation. Therefore, the protective agent of the present invention can also be used as an intestinal barrier enhancer. Here, enhancing the intestinal barrier means increasing the index value of intestinal barrier strength (transepithelial electrical resistance; TEER). [Example]

[0041] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. In all of the following examples, significant differences were tested by t-test, with * indicating p<0.05, ** indicating p<0.01, and *** indicating p<0.001.

[0042] Example 1 Intestinal Barrier Protection: The test strain used was Bifidobacterium adolescentis YIT 13021 (hereafter referred to as "YIT 13021"). The control strain used was Bifidobacterium breve type strain (ATCC15700) (hereafter referred to as "YIT 4014"). These strains were cultured in mGAM medium supplemented with 1% glucose, and the intestinal barrier protective effects against LPS stimulation, inflammatory cytokine stimulation, or oxidative stress stimulation were tested using an unstimulated control. The effect of YIT 13021 on the localization of tight junction proteins was also tested.

[0043] (1) LPS stimulation 1.1 Cells Cryopreserved human colon epithelial cell line T84 was used. T84 was subcultured in DMEM / F-12 medium (10% FBS / F-12) containing 10% FBS and 100 μg / mL streptomycin at 37°C in 5% CO2.

[0044] 1.2 Preparation of monolayer film T84 monolayers were prepared using 24-well cell culture insert plates (Millipore). T84 cells were harvested from 80% confluent culture dishes and seeded at 60,000 cells / well in 400 μL of 10% FBS / F-12 on the apical side of the wells. 800 μL of 10% FBS / F-12 was added to the basal side, and the cells were cultured at 37°C in 5% CO2. Medium was replaced every 2–3 days, with 400 μL of medium on the apical side and 800 μL on the basal side. T84 monolayers were cultured for 10 days to form monolayers.

[0045] 1.3 Evaluation of protective effect against LPS-stimulated monolayers LPS (WAKO, #128-05171) derived from Escherichia coli O55 was used and dissolved in 10% FBS / F-12 to prepare a test concentration of 10 ng / mL. Stimulation with LPS was performed by adding an LPS-containing medium to both the apical and basal sides of the prepared monolayer membrane. Heat-killed cells of YIT 13021 or YIT 4014 were prepared by a known method and added to a 100 μg / mL (approximately 2.4 × 10 8 The protective effect against damage to the monolayer membrane due to stimuli was evaluated by multiplying the actual resistance (Ω) measured by a Millicell MRS-2 (Millipore) by the culture area (CM) of the cell culture insert plate. 2 ) divided by the transepithelial electrical resistance (TEER) value (Ω / cm 2 The TEER value of the monolayer membrane at each time point after the start of stimulation was divided by the TEER value at time 0, which was immediately after the start of stimulation, to calculate the percentage change in barrier breakdown from the start of stimulation.

[0046] 1.4 Results To examine the effects of YIT 13021 or YIT 4014 on intestinal barrier function, T84 cells were seeded onto cell culture insert plates and cultured for 10 days to form monolayers. LPS and heat-killed bacterial cells of the strains were added to the monolayers, and TEER values ​​were measured every 24 hours for up to 48 hours (Fig. 1). 48 hours after addition, no decrease in TEER was observed in monolayers treated with YIT 13021, and the decrease in TEER due to LPS stimulation was significantly suppressed. For YIT 4014, the decrease was the same as that observed with LPS stimulation alone (Fig. 2).

[0047] (2) Inflammatory cytokine stimulation 2.1 Cells The same cells as those used in 1.1 of (1) LPS stimulation above were used.

[0048] 2.2 Preparation of monolayer film Monolayer membranes were prepared in the same manner as in 1.2 of (1) LPS stimulation above.

[0049] 2.3 Evaluation of protective effect against inflammatory cytokine-stimulated monolayers The inflammatory cytokines used were TNF-α (R&D Systems, #210-TA-005) and IFN-γ (R&D Systems, #285-IF-100). Equal amounts of both cytokines were dissolved in 10% FBS / F-12 to a test concentration of 1 ng / mL. Stimulation with TNF-α and IFN-γ was performed by adding cytokine-containing medium only to the basal side of the prepared monolayer. Measurement of the TEER value of the monolayer after each elapsed time from the start of stimulation and calculation of the percentage change (%) were performed as described in 1.3 of (1) LPS stimulation above.

[0050] 2.4 Results The inflammatory cytokines TNF-α and IFN-γ were added to T84 monolayers, and YIT 13021 or YIT 4014 was added simultaneously to evaluate their protective effects against inflammatory cytokine-induced barrier disruption. TEER values ​​were assessed after 24 and 48 hours (Figure 3). TI indicates TNF-α + IFN-γ. Monolayers stimulated with inflammatory cytokines alone or with YIT 4014 showed little change after 24 hours, but TEER values ​​decreased to approximately 50% after 48 hours. In contrast, monolayers treated with YIT 13021 showed a 20% increase in TEER compared to the control group after 24 hours. The decrease after 48 hours was limited to approximately 70%, demonstrating a significant suppression of the decrease in TEER due to inflammatory cytokine stimulation (Figure 4).

[0051] (3) Oxidative stress stimulation 3.1 Cells The same cells as those used in 1.1 of (1) LPS stimulation above were used.

[0052] 3.2 Preparation of monolayer film Monolayer membranes were prepared in the same manner as in 1.2 of (1) LPS stimulation above.

[0053] 3.3 Evaluation of protective effect against oxidative stress-stimulated monolayers Oxidative stress was achieved using HO (Mitsubishi, #K-8230) diluted in 10% FBS / F-12 to a test concentration of 1500 μM. HO stimulation was performed by adding HO-containing medium to both the apical and basal sides of the prepared monolayer. Measurement of the TEER value of the monolayer after each elapsed time from the start of stimulation and calculation of the percent change (%) were performed as described in 1.3 of (1) LPS stimulation above.

[0054] 3.4 Results We evaluated the protective effects of YIT 13021 or YIT 4014 on barrier breakdown due to oxidative stress by adding HO to T84 monolayers. TEER values ​​were measured every hour for up to 6 hours after addition (Fig. 5). While YIT 4014 did not protect against HO stimulation, monolayers treated with YIT 13021 significantly suppressed the decrease in TEER compared to those treated with HO alone, demonstrating their protective effect against HO stimulation (Fig. 6).

[0055] (4) Effect on the localization of tight junction proteins 4.1 Cells The same cells as those used in 1.1 of (1) LPS stimulation above were used.

[0056] 4.2 Preparation of monolayer film Monolayer membranes were prepared in the same manner as in 1.2 of (1) LPS stimulation above.

[0057] 4.3 Immunostaining of monolayers The monolayers were washed twice with PBS and then fixed in 10% neutral buffered formalin in a 24-well cell culture insert plate for 15 minutes. After two washes, the cells were permeabilized with 0.25% Triton X-100 / PBS for 5 minutes. After two more washes, the cells were blocked with 10% BSA / PBS for 60 minutes at 37°C. After removal of the blocker, anti-ZO-1 antibody (Abcam, #ab216880) diluted 1:100 in PBS was added and incubated overnight at 4°C. After two washes, anti-rabbit Alexa 546 antibody (Invitrogen, #A11010) diluted 5:500 in PBS was added and incubated at 37°C in the dark for 60 minutes. Finally, the plate was washed three times, and the membrane was removed from the cell culture insert plate and placed on a glass slide. A drop of the mounting medium, VECTASHIELD with DAPI (VECTOR LABORATORIES), was placed on the plate, and a cover glass was placed on top to prepare the slide for observation. The cells were observed using an all-in-one fluorescence microscope, BZ-X700 (Keyence).

[0058] 4.4 Results Monolayers were subjected to the same stimuli as described above (1) to (3). After 48 hours of inflammatory cytokine and LPS stimulation and 6 hours of oxidative stress stimulation, the monolayers were immunostained for ZO-1, a protein that constitutes tight junctions, and observed (Fig. 7, -). Alternatively, ZO-1 immunostained monolayers were simultaneously stimulated as described above (1) to (3), and YIT 13021 was added. The protective effect of YIT 13021 on barrier function was evaluated (Fig. 7). The protective effect of YIT 13021 against LPS, inflammatory cytokine, and oxidative stress stimulation was confirmed by the disruption of the ZO-1 network, with ZO-1 present not only in the cell membrane but also in the cytoplasm, indicating disruption of tight junctions. On the other hand, in monolayers to which YIT 13021 was added simultaneously with stimulation, the meshwork localization of ZO-1 was disrupted and its presence in the cytoplasm was reduced, indicating that YIT 13021 suppressed the disruption of tight junctions. These results confirmed the protective effect of YIT 13021 on the intestinal barrier from the perspective of the localization of tight junction proteins.

[0059] Example 2 Strengthening the intestinal barrier: The test strains used were YIT 13021 and YIT 4014. To examine their effects on intestinal barrier function, T84 cells prepared as in Example 1 were seeded onto cell culture insert plates and cultured for 10 days to prepare monolayer membranes. Heat-killed YIT 13021 or YIT 4014 cells were added to the apical side of the monolayer membrane (no stimulation), and the TEER value was measured every hour for up to 6 hours (Figure 8). As a result, a significant increase in the TEER value of the monolayer membrane 6 hours after addition of YIT 13021 was observed compared to the untreated control (Figure 9). On the other hand, the TEER value of the monolayer membrane to which YIT 4014 was added was similar to that of the untreated control (Figure 9).

[0060] These results demonstrate that YIT 13021 has the effect of strengthening the intestinal barrier.

[0061] Example 3 Intestinal Barrier Protection: Bifidobacterium adolescentis strains other than YIT 13021 were also tested for their intestinal barrier protective effect similar to that of YIT 13021. The Bifidobacterium adolescentis strains used were YIT 13609 and YIT 12828.

[0062] (1) LPS stimulation Heat-killed cells of each strain were added to the apical side of the monolayer membrane in the same manner as in (1) LPS stimulation in Example 1, and TEER values ​​were measured 48 hours later. The results showed that 48 hours after addition, YIT 13609 and YIT 12828 had the same intestinal barrier protective effect against LPS stimulation as YIT 13021 (Figure 10).

[0063] (2) Inflammatory cytokine stimulation Heat-killed cells of each strain were added to the apical side of the monolayer membrane in the same manner as in (2) Inflammatory cytokine stimulation in Example 1, and TEER values ​​were measured 48 hours later. As a result, it was found that 48 hours after addition, YIT 13609 and YIT 12828 had the same intestinal barrier protective effect against inflammatory cytokine stimulation as YIT 13021 (Figure 11). (3) Oxidative stress stimulation Heat-killed cells of each strain were added to the apical side of the monolayer membrane in the same manner as in (3) Oxidative stress stimulation in Example 1, and the TEER value was measured 6 hours later. As a result, it was found that YIT 13609 and YIT 12828 had the same intestinal barrier protective effect against oxidative stress stimulation as YIT 13021 6 hours after addition (Figure 12).

[0064] Example 4 Strengthening the intestinal barrier: The test strains used were YIT 13021, YIT 13609, and YIT 12828. To examine their effects on intestinal barrier function, T84 cells prepared as in Example 1 were seeded onto cell culture insert plates and cultured for 10 days to produce monolayer membranes. Heat-killed cells of YIT 13021, YIT 13609, or YIT 12828 were added to the apical side of the monolayer membrane, and the TEER value was measured 24 hours later. The results showed that YIT 13609 and YIT 12828 had the same intestinal barrier enhancing effect as YIT 13021 ( FIG. 13 ).

[0065] Example 5 Strengthening the intestinal barrier: To examine the effect of live YIT 13021 bacteria on intestinal barrier function, T84 cells prepared as in Example 1 were seeded onto cell culture insert plates and cultured for 10 days to produce monolayer membranes. Live or heat-killed YIT 13021 bacteria were added to the apical side of the monolayer membrane at various concentrations, and TEER values ​​were measured and compared 24 hours later. A significant increase in TEER value was observed with heat-killed bacteria starting at an added concentration of 10 μg / mL ( FIG. 14 ), while a significant increase in TEER value was observed with live bacteria starting at an added concentration of 1 μg / mL, demonstrating that live bacteria exert a barrier-enhancing effect even at low concentrations ( FIG. 15 ).

[0066] Example 6 Strengthening the intestinal barrier: YIT 13021 has the characteristic of adhering to and assimilating starch granules, and the presence or absence of starch granules during culture may affect the properties of the bacterial cells. Therefore, we investigated the effect of live YIT 13021 bacteria cultured in mGAM medium, where the carbon source was changed from glucose to starch granules, on the intestinal barrier function. T84 cells prepared as in Example 1 were seeded onto cell culture insert plates and cultured for 10 days to produce monolayer membranes. Live YIT 13021 bacteria cultured in the presence of starch granules were added to the apical side of the monolayer membrane at various concentrations, and TEER values ​​were measured and compared after 24 hours. As a result, a significant increase in TEER value was confirmed for live YIT 13021 cells cultured in the presence of starch granules, starting at an added concentration of 0.1 μg / mL, indicating that live YIT 13021 cells cultured in combination with starch granules exhibited a barrier-enhancing effect even at low concentrations (Figure 16) compared to live YIT 13021 cells cultured using glucose as a carbon source (Figure 15). [Industrial Applicability]

[0067] The intestinal barrier protecting agent of the present invention can be used to protect the intestinal barrier and as a preventive agent for diseases associated with the breakdown of the intestinal barrier.

Claims

1. An intestinal barrier protection agent characterized by containing Bifidobacterium adolescentis as an active ingredient.

2. The intestinal barrier protecting agent according to claim 1, wherein the intestinal barrier protection is protection of the intestinal barrier from one or more stimuli selected from LPS stimulation, inflammatory cytokine stimulation, and oxidative stress stimulation.

3. A preventive agent for diseases associated with intestinal barrier breakdown, comprising the intestinal barrier protecting agent according to claim 1 or 2.

4. The preventive agent for intestinal barrier breakdown-associated diseases according to claim 3, wherein the intestinal barrier breakdown-associated diseases are selected from diabetes, obesity, inflammatory bowel disease, fatty liver, Parkinson's disease, cancer, irritable bowel disease, dyslipidemia, atopy, asthma, dementia, insomnia, depression, and rheumatoid arthritis.

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