JP5046684B2 - Intestinal barrier function recovery agent and intestinal barrier permeability enhancement inhibitor - Google Patents

Description

  The present invention relates to a function recovery agent for intestinal barrier function and an inhibitor for enhancing intestinal barrier permeability. That is, the present invention relates to the use of a lactic acid bacterium or a bacterial cell component of a lactic acid bacterium that acts on the intestinal barrier function and is a medicament or food for treating or preventing intestinal barrier permeability permeation or unwanted permeability.

  The intestinal tract functions as a barrier function that protects the body from harmful microorganisms, toxins, and allergens. This breakdown of the intestinal barrier function may cause disordered entry of foreign substances into the living body, which may cause diseases such as inflammatory bowel disease and food allergy. At present, it is thought that intracellular transport such as tight junction and endocytosis existing between epithelial cells of the intestine is involved in the permeability change of substances in the intestine. Low-molecular substances such as amino acids are absorbed through tight junctions, but in some cases, macromolecules such as bacteria, viruses, and proteins are absorbed through these tight junctions, causing infections and food allergies. It is considered. From such a viewpoint, an intestinal permeation model using Caco-2 cells has been proposed (Non-Patent Document 1). As a result of investigating the influence on the allergen permeability using this intestinal permeation model, various substances have been found to suppress the intestinal permeation and have been proposed as anti-food allergic agents and the like. For example, it is found that milk components such as peptides obtained from Edam cheese (Non-patent Document 1), whey proteins (Patent Documents 1 and 2), and casein degradation products (Patent Documents 2 and 3) suppress such intestinal permeability. It is.

  By the way, inflammatory bowel disease is a disease in which the intestine is inflamed and repeats diarrhea and severe abdominal pain, and the symptom calms down and recurs over a long period of time. Is designated as a specific disease (intractable disease). There are many unexplained details about the detailed cause, and anti-inflammatory drugs are only administered as symptomatic treatments. In this patient, an increase in inflammatory cytokines (IL-1, IL-6, IL-8, TNF-α, INF-γ) in the intestinal tract, T cell activation in the mucosa, or abnormal intestinal flora ( Inflammatory bowel disease is considered to be caused by the breakdown of the intestinal barrier function and the overexpression of inflammatory cytokines (Non-patent Documents 2 and 3). Therefore, the present inventors considered that if the intestinal barrier function can be improved by administration of lactic acid bacteria, it can contribute to the fundamental treatment of inflammatory bowel disease, and focused on the intestinal protective effect of lactic acid bacteria.

So far, as a report that lactic acid bacteria or products or degradation products of lactic acid bacteria have improved or improved the permeability of the intestinal tract, for example, Patent Document 4 discloses that adhesion of lactic acid bacteria (Lactobacillus) to intestinal cells is caused by minerals such as calcium. There is a report that the permeability is improved, and there is a report in Patent Document 5 that a culture of lactic acid bacteria (Lactobacillus reuteri) and glutamate derived from lactic acid bacteria (L. reuteri) suppress the intestinal permeability increase. Only.
Japanese Patent Laid-Open No. 08-73375 JP 09-241177 A JP 2002-257814 A Special Table 2002-507997 Special table 2003-522136 gazette Kobayashi et al., Evaluation system for allergen intestinal permeation inhibitory activity and active ingredients, New Food Industry, 2003, 45, 33-38 Edited by Hiroshi Seino, Hiromichi Ishikawa, and Hiroshi Nakura, "Mucosal immunity: The intestine is an immune control tower", published by Nakayama Shoten, pp. 144 and 152 (2001) Guamer F, Casellas F, Borruel N, AntolinM, Videla S, Vilaseca J, Malagelada JR, Role of microecology in chronic inflammatory bowel diseases., Eur J Clin Nutr, 56 (Suppl 4), S34-38 (2002)

  The present invention has been made in view of the above background art, and the present invention aims to improve the destroyed intestinal barrier function, and is effective for drugs and foods effective for diseases caused by the function of the intestinal barrier such as inflammatory bowel disease, Alternatively, it is an object to provide a drug or food effective for protecting the intestinal barrier. Therefore, the present inventors artificially added intestinal barrier function to the normal intestinal permeation model using Caco-2 cells by adding TNF-α, which is said to be a factor for enhancing intestinal permeability. An attempt was made to achieve the above object by finding a lactic acid bacterium or an active ingredient thereof capable of improving the state.

  In order to achieve the above object, lactic acid bacteria or lipid components derived from lactic acid bacteria (especially lipoteichoic acid) are used as active ingredients to enhance or promote the recovery or recovery of unwanted permeability, or to enhance unwanted permeability. Provided is a pharmaceutical or food composition for inhibiting or preventing.

  According to the present invention, recovery from the breakdown of the intestinal barrier function due to increased inflammation or the like is promoted and normalized, and can contribute to the treatment of various diseases considered to be caused by the breakdown of the intestinal barrier function such as inflammatory bowel disease. In addition, by preventing the increase in permeability of the intestinal barrier, the intestinal barrier function is maintained in a normal state, contributing to prevention of diseases based on the breakdown of the intestinal barrier function.

The present inventors selected a strain that contributes to recovery of intestinal barrier function and inhibition of enhanced intestinal barrier permeability from a number of lactic acid strains uniquely isolated from humans. As a lactic acid bacterium exhibiting high activity, Lactobacillus rhamnosus The OLL2838 strain (Lactobacillus rhamnosus OLL2838, accession number: NITE P-313 , hereinafter also referred to as L. rhamnosus OLL2838) was found. As a result of further investigations, it was suggested that lipoteichoic acid, which is a lipid component of L. rhamnosus OLL2838, is one of its active ingredients. Therefore, by using the lactic acid bacterium, its microbial lipid component, or lipoteichoic acid derived from microbial cells, therapeutic agents for various diseases that are thought to be caused by the breakdown of the intestinal barrier function, and prevention of diseases based on the breakdown of the intestinal barrier function It became possible to provide an agent.

  In the present invention, lactic acid bacteria is a general term for bacteria that assimilate glucose and produce lactic acid with a yield of sugar of 50% or more. As a physiological property, it is a gram-positive bacterium cocci or gonococci with no motility and no sporulation ability. And catalase negative. Lactic acid bacteria are isolated from various environments, such as plant epidermis, mammalian intestinal tract, ocean, soil, and fermented foods. In fermented foods such as pickles and soy sauce, not only contribute to the formation of taste and texture, but also lactic acid and bacteriocin. Since it has the ability to produce antibacterial substances such as, it has been eaten all over the world through fermented milk since ancient times. In addition, it is a well-known fact that the mammal's intestinal tract has various physiological effects on the host, and it can be said to be an extremely safe microorganism. To date, lactic acid bacteria have been classified into 11 genera of Lactococcus, Lactobacillus, Leuconostoc, Pediococcus, Streptococcus, Wissella, Tetragenococcus, Oenococcus, Enterococcus, Vagococcus, and Carnobacterium. In the present invention, these lactic acid bacteria can be used. Among these, preferred lactic acid bacteria include the genus Lactobacillus, in particular, Lactobacillus rhamnosus species, and more preferably L. rhamnosus OLL2838, but are not limited to these examples.

The Lactobacillus rhamnosus OLL2838 strain was deposited by the Incorporated Administrative Agency, Product Evaluation Technology Foundation, Patent Microorganism Deposit Center. The contents specifying the deposit are described below.
(1) Depositary Institution: National Institute of Technology and Evaluation, Patent Microorganism Depositary Center (2) Contact: 2-5-8 Kazusa Kamashi, Kisarazu City, Chiba Prefecture 292-0818
Phone number 0438-20-5580
(3) Accession number: NITE P-313
(4) Display for identification: Lactobacillus rhamnosus OLL2838
(5) Deposit date: February 14, 2007

Lactobacillus rhamnosus OLL2838 strain ( Accession number: NITE P-313 ) is a Gram-positive bacilli, and the colony form when cultured anaerobically on BL agar medium (Nissui Pharmaceutical) is round, white, sm
Ooth-shaped and raised in a conical shape. Physiological characteristics include homolactic fermentation format, growth at 15 ° C., rhamnose, ribose, glucose, mannose, fructose, galactose, sucrose, cellobiose, lactose, trehalose, melittose, mannitol,
Has fermentability to sorbitol.

  The intestinal barrier function-restoring agent (food composition) and the intestinal barrier permeability enhancement inhibitor (food composition) of the present invention can contain the above-mentioned lactic acid bacteria in various states. For example, not only bacterial bodies of lactic acid bacteria (whether live or dead), but also lactic acid bacteria fermented products (lactic acid bacteria beverages, sour milk, yogurt, etc.), and processed products (crushed, crushed) obtained by subjecting lactic acid bacteria to some treatment Used.

  Lactic acid bacteria can be cultured not only in the Lactobacillus rhamnosus OLL2838 strain but in a culture medium that satisfies the auxotrophy required by the strain. An example of such a medium is MRS medium. As the microbial cells, not only the microbial cells taken out from the lactic acid bacterium culture solution cultured using such a medium, but also the lactic acid bacterium culture solution after culturing can be used as it is or as a concentrate obtained by concentrating the medium. Lactic acid bacteria may be either live or dead, and are used as live cells, wet cells, and dry cells (obtained by spray drying, freeze drying, vacuum drying, drum drying, and the like). From the viewpoint of action and effect, it is preferable to use viable bacteria. However, the removed cells may be sterilized, that is, subjected to radiation sterilization treatment, heat treatment, etc., and used as dead bacteria. It can also be used as a suspension obtained by suspending a cell, a culture solution, or a concentrate taken out of a live cell or a dead cell again in an appropriate medium. Examples of the medium include a culture medium, water, and physiological saline.

  Furthermore, you may use as a fermented material fermented using lactic acid bacteria. Examples of fermented products include lactic acid bacteria beverages, sour milk, fermented milk, and yogurt as described above. Moreover, in this invention, you may include as a lactic-acid-bacteria processed material which performed a certain process to lactic acid bacteria. Examples of processed lactic acid bacteria include crushing lactic acid bacteria cells and bacteria-containing materials as described above, such as lactic acid bacteria cells, lactic acid bacteria-containing materials, fermented milk concentrates, pasted products, and dried products. Examples of the crushed material and the pulverized material are shown. In addition, the lactic acid bacteria processed material here means what used the whole microbial cell rather than the processed material which took out the specific component part of the microbial cell.

  The function-restoring agent for intestinal barrier function and the inhibitor for enhancing intestinal barrier permeability of the present invention include not only those containing lactic acid bacteria as cells (treated cells) as described above, but also lipid fractions from cells. You may use what was obtained as a microbial cell lipid component which extracted. The present inventors focused on the recovery of the function of the intestinal tract barrier function and the inhibition of the enhancement of the intestinal tract barrier permeability to the lipid component constituting lactic acid bacteria, and it was shown that lipoteichoic acid exhibits its activity among the lipid components. It is based on what has been done.

It is well known that lipoteichoic acid (LTA) is widely distributed in nature and constitutes the cell membrane of Gram-positive bacteria. In general, lipoteichoic acid has a structure of a glycerol phosphate polymer having diacylglycerol, but the structure of lipoteichoic acid differs depending on its origin (origin). In the present invention, lipoteichoic acid derived from the lactic acid bacteria, particularly lipoteichoic acid derived from L. rhamnosus OLL2838 ( accession number: NITE P-313 ) or Staphylococcus aureus (hereinafter also referred to as S. aureus) among lactic acid bacteria. Is not limited to these specific lactic acid bacteria and S. aureus, and other lactic acid bacteria such as B. longum, L. delbrueckii subsp. Bulgaricus, L. casei, L. gasseri, etc. Of course, lipoteichoic acid derived from bacteria other than lactic acid bacteria may be used.

  Of course, lipoteichoic acid can be used as a chemically pure compound, but it is not a single compound, but a crude extract of lactic acid bacteria as long as it contains lipid components, particularly lipoteichoic acid that has shown its activity. It does not prevent the use of crushed or pulverized lactic acid bacteria as active ingredients.

  In the present invention, recovery of the intestinal barrier function means recovery from a state in which the permeability in the intestinal tract has increased beyond the normal range to a pathological or undesirable level for some reason, and returns to the normal state. It is used not only for the purpose but also to bring it close to a qualitatively normal state. In addition, for recovery, promotion of recovery that shortens the recovery period until recovery from a state where the permeability in the intestinal tract exceeds a normal range for some reason and is pathologically or undesirably increased, in other words, usually Compared to the case of taking the food, it is used in a sense including restoring to a normal state earlier than natural healing.

  Further, in the present invention, inhibition of enhanced permeability of the intestinal barrier means that the barrier function in the intestinal tract is destroyed due to an exogenous or intrinsic cause, and the permeability of the intestinal barrier changes from a normal state to a pathological state. It is used in the meaning including preventing or suppressing the above, and maintaining its permeability in a normal state. The enhanced permeability of the intestinal barrier referred to in the present invention includes not only epithelial cells but also destruction, breakdown, weakness, and lowering of the intestinal barrier related to biological mechanisms that constitute other intestinal barriers such as the intestinal mucosa. .

  In the present invention, the recovery agent and the inhibitor include a lipid component which is an active ingredient of the present invention, particularly lipoteichoic acid or a lactic acid bacterium containing the same (viable or dead) or a lipid component, particularly lipoteichoic acid. Means the use of the lactic acid bacterial cell component prepared in the above as a medicine, not only when used alone, but also by adding appropriate excipients etc. to those components, It is also intended to include use as a pharmaceutical composition. The form of the pharmaceutical composition is not particularly limited, and various dosage forms such as tablets, granules, capsules, injections and solutions, dry syrups, powders, syrups and the like are exemplified. In addition, anti-inflammatory agents, anti-analgesic agents, other main drugs such as vitamins, side drugs, and appropriate excipients, binders, disintegrants, lubricants as necessary in the formulation, as long as they do not adversely affect the action. Known adjuvants such as agents, flavoring agents, solubilizing agents, suspending agents, and coating agents can be added. Moreover, you may add to the pharmaceutical which has biological standards, such as powdered milk.

  In the present invention, the food composition is prepared so as to contain a lipid component which is an active ingredient of the present invention, in particular lipoteichoic acid or a lactic acid bacterium containing the same (viable or dead) or a lipid component, particularly lipoteichoic acid. It means processed food with artificially added bacterial components of lactic acid bacteria, and not only foods obtained using the lactic acid bacteria of the present invention, but also compared with those obtained in normal processes The lactic acid bacterium, lipid component or lipoteichoic acid content of the present invention means a food composition having the same or higher content. This food composition includes existing foods such as milk, yogurt, cheese, fermented milk, tofu, porridge, kuzuyu, tea and fruit juice, bread, biscuits, crackers, pizza crusts, formula milk Foods made from various edible materials such as liquid foods, foods for the sick, infant milk powders, infant milk powders (including those with biological standards such as milk powder), nutritional foods, etc. Exemplified, not only those with the above-mentioned active ingredients added during the production of these foods, but also various proteins such as enteral nutrients (whole milk powder, skim milk powder, partially skimmed milk powder, casein, whey powder, whey protein , Whey protein concentrate, whey protein isolate, α-casein, β-casein, κ-casein, β-lactoglobulin, α-lactalbumin, lactoferrin Animal and vegetable proteins such as soybean protein, chicken egg protein, meat protein, and vegetable proteins such as lecithin and soybean protein), various sugars (monosaccharides such as glucose and fructose, disaccharides such as sucrose, xylitol and glycerin) Polyhydric alcohol, dextrin, processed starch (in addition to dextrin, soluble starch, British starch, oxidized starch, starch ester, starch ether, etc.), polysaccharides such as dietary fiber), various lipids (lard, fish oil, etc.) Animal oils such as oil, hydrogenated oil and transesterified oil, and plants such as soybean oil, coconut oil, safflower oil, corn oil, rapeseed oil, coconut oil, fractionated oils, hydrogenated oil and transesterified oil Oils and fats), various vitamins (vitamin A, carotene, vitamin B group, vitamin C, vitamin D group) Vitamin E, vitamin K group, vitamin P, vitamin Q, niacin, nicotinic acid, pantothenic acid, biotin, inositol, choline, folic acid, etc. and various minerals (calcium, potassium, magnesium, sodium, copper, iron, manganese, zinc, Selenium, etc.), organic acids (malic acid, citric acid, lactic acid, tartaric acid, etc.) and various other nutrients are mixed in any proportions, and a gelling agent is added to the mixture as it is, and the viscosity is easily swallowed. Examples of the food composition prepared are as follows. In addition, the food composition of the present invention is a so-called food for specified health use, that is, effects based on the effects of the present invention, such as protection of the intestinal barrier function, suppression of intestinal permeability enhancement, and recovery of enhanced intestinal permeability. Needless to say, foods that can be labeled as well as foods that are publicly permitted to specifically display health indications (expressions that show health benefits) and functional nutritional foods are included.

In the present invention, when used as bacterial cells of lactic acid bacteria, it is not particularly limited because it varies depending on age, sex, weight, symptom or purpose of use. If it is a living bacterium, 1 × 10 ⁴ to 1 × 10 ¹⁵ , preferably 1 × 10 ⁷ to 1 × 10 ¹³ can be used. If it is dead bacteria, 1 × 10 ⁴ to 10 ¹⁶ , preferably 1 × 10 ⁷ to 1 × 10 ¹⁴ can be used. Further, the cell concentration varies depending on the use mode, that is, whether it is used as the cell itself or as a suspension, and is not particularly limited. However, if it is intentionally mentioned, 0.001 to 100% (w / w ), Preferably 0.01 to 100% (w / w), more preferably 0.1 to 100% (w / w). Moreover, when using as a lactic-acid-bacteria fermented material, a processed material, etc., converting into a microbial cell and using as said range is preferable.

  In addition, the intake of lipid components of lactic acid bacteria, particularly lipoteichoic acid, varies depending on age, sex, weight, symptom or purpose of use and is not particularly limited. As an amount, 0.1 to 100 mg / kg can be taken, and preferably 0.1 to 10 mg / kg can be taken. The concentration of the lipid component, particularly the lipoteichoic acid concentration varies depending on the mode of use, that is, whether it is used as the lipid component or the lipoteichoic acid of the compound, and is not particularly limited. -100% (w / w), preferably 0.01-100% (w / w), more preferably 0.1-100% (w / w).

  The intake of these cells, lipid components, etc. may be increased or decreased as appropriate depending on age, sex, weight, symptom, or intended use (whether it is treatment or prevention of enhancement), and the type and intake of the pharmaceutical composition or food composition. It can also be adjusted as appropriate. The intake route is most preferably oral, but is not limited to oral, and examples include tube administration and enteral administration. The subject of administration is not limited to humans, and can be applied to various animals such as dogs, monkeys, mammals such as cows and horses as long as they have the same intestinal permeation mechanism.

  The functional recovery agent for intestinal barrier function and the food composition for functional recovery of the present invention are used for the treatment and improvement of a person whose function of the intestinal barrier is reduced due to some cause and intestinal permeability is increased. . When ingesting, it is between meals before and after meals if it is a medicine, and as a food composition if it is a meal, or as a material for meals, and as a snack, supplement, dietary supplement, etc. Ingestion is preferred, but this is not a limitation.

  In addition, an intestinal barrier permeability enhancement inhibitor or a food composition for inhibiting enhancement may be used by those who maintain a normal state of intestinal permeability, such as when a decrease in intestinal barrier function is expected. Used to maintain the barrier function or prevent its degradation. When ingesting, it is between meals before and after meals if it is a medicine, and as a food composition if it is a meal, or as a material for meals, and as a snack, supplement, dietary supplement, etc. Ingestion is preferred, but this is not a limitation.

Hereinafter, the present invention will be described in more detail based on Examples (Experiments 1 to 3). However, it goes without saying that the present invention is not limited to the following examples.
[Experiment 1: Selection of strains that affect intestinal permeation]
1) Preparation of test strain (Test strain)
Lactobacillus L. rhamnosus OLL2838 (shown as No. 34 in FIGS. 2 and 3), L. delbrueckii subsp. Bulgaricus MEP190901 (shown in FIG. 2) were used for the test. No. 3 in Fig. 3), L. casei MEP190902 (indicated by No. 9 strain in Figs. 2 and 3), L. gasseri MEP190903 (indicated by No. 10 strain in Figs. 2 and 3) .) These lactic acid bacteria were cultured in MRS medium for 18 hours, washed 3 times with PBS, and a medium obtained by removing antibiotics from the following medium for culturing Caco-2 cells was added to obtain 2 × 10 ⁵ cells / mL. A test strain suspension was prepared and used for the following intestinal permeation test.

2) Action test on intestinal permeation using Caco-2 cells (Creation of intestinal permeation model using Caco-2 cells)
Caco-2 cells (ATCC HTB-37, American Type Culture Collection (ATCC)) were used at passage 40-60. As a medium for cell culture, 10% fetal bovine serum (hereinafter also referred to as “FCS”, ICN Biochemicals, Inc.), 1% non-essential amino acid (Gibco Life Technologies), 100 IU / ml penicillin (Gibco Life Technologies), 100 μg / Dulbecco's Modified Eagle's Medium (hereinafter also referred to as “DMEM”, Gibco Life Technologies) containing ml streptomycin (Gibco Life Technologies) and 50 μg / ml gentamicin (Gibco Life Technologies) was used. First, Caco-2 cells were cultured in a 75 cm ² tissue culture flask until they were about 70-80% confluent. Next, Caco-2 cells were inoculated at a concentration of 2 × 10 ⁵ cells / cm ² into a 12-well Transwell cell culture chamber (diameter 12 mm, pore diameter 0.4 μm, with a permeable membrane), and 5% CO 2. Caco-2 monolayer cells were obtained by culturing at 37 ° C. for 14 days under _two atmospheres. The apical (lumen) side of the Caco-2 monolayer cells obtained in this way has many fine soft processes similar to the characteristics of the brush border and tight junction of the small intestine. In addition, since metabolic enzymes such as transporters and peptidases are also expressed during the culture period, they can be used as small intestinal membrane models (Hidalgo IJ, Raub TJ, Borchardt RT, Characterization of the human colon carcinoma cell line (Caco-2 ) as a model system for intestinal epithelial permeability, Gastroenterology, 96 (3), pp.736-749 (1989)).

(Verification of intestinal permeation model)
In order to verify whether a tight junction was sufficiently formed in the Caco-2 monolayer cells prepared by the above method, the transepithelial electrical resistance (TER) of the Caco-2 monolayer cells was measured. A resistance value measurement system (Millicell-ERS, Millipore) using an Ag / AgCl electrode was used for the assay with a transepithelial electrical resistance of about 300 Ω · cm ² or more. Each well was placed on a cluster plate and filled with an outer culture (basal side, 1.5 ml) and an inner culture (luminal side, 0.5 ml) (see FIG. 1). Caco-2 monolayer cells were cultured by replacing with fresh medium every 24 hours.

(Addition of test bacteria to Caco-2 cells and stimulation with TNF-α)
Next, 500 μL of the test strain suspension at a concentration of 2 × 10 ⁵ cells / mL was added to the inner culture of each well of the prepared Caco-2 monolayer cells, and after 1 hour, the method of Son et al. , Satsu H, Shimizu M., Histidine inhibits oxidative stress- and TNF-alpha-induced interleukin-8 secretion in intestinal epithelial cells., FEBS Lett., 579 (21), 4671-4677 (2005)) The stimulus was done. That is, TNF-α was added to the outer culture solution so that the final concentration was 100 ng / mL, and the culture was further performed for 48 hours. Thereafter, the following intestinal barrier protective effect was evaluated. At this time, a well to which only the TNF-α was added without adding the test strain suspension and a well to which neither the test strain suspension nor TNF-α was added (hereinafter also referred to as a control) were provided.

(Evaluation of intestinal barrier protection effect)
The intestinal barrier protective effect of each lactic acid bacterium was compared using the transepithelial electrical resistance (TER) value (Ω · cm ² ) and IL-8 production (pg / ml) as indicators. The TER value was measured at 0, 24, and 48 hours after addition of TNF-α using a resistance value measurement system (Millicell-ERS, Millipore) using an Ag / AgCl electrode. Further, the TER relative value (Relative TER) was calculated by dividing the TER value of each well by the TER value of the control. Each IL-8 production amount was measured for 48 hours after the addition of TNF-α by subjecting the outer culture solution collected after the culture to ELISA.

3) Results
The measurement result of TER relative value is shown in FIG. 2, and the measurement result of IL-8 is shown in FIG. Note that the measurement data shows mean ± standard error (SE) (n = 3) (the same applies to the following measurement results). Addition of TNF-α to the Caco-2 monolayer cell culture system decreased the TER relative value and increased the IL-8 concentration in the culture solution. In those to which the test strain suspension was added, many of the test strains were found to suppress the decrease in TER relative value and IL-8 production. In particular, L. rhamnosus OLL2838 (No. 34 strain) was excellent in the ability to suppress the decrease in TER value and the ability to suppress IL-8 production.

[Experiment 2: Activity test of heat-treated strains having an effect on intestinal permeation]
1) Heat treatment of test strain (test strain)
The test was L. rhamnosus OLL2838 (No. 34 strain), which was found to have a high inhibitory effect on TER relative value decrease in Experiment 1. According to Example 1, L.rhamnosus OLL2838 cells are prepared (live cells), and L.rhamnosus OLL2838 is heat-treated at 100 ° C. for 10 minutes (dead cells), 2 × 10 ⁵ cells / mL each. Was prepared and used for the following intestinal permeation inhibition test. In addition, the culture supernatant (extracellular metabolite) of L. rhamnosus OLL2838 was also tested in the same manner.

2) Action test on intestinal permeation using Caco-2 cells The test method was carried out in accordance with Example 1, 500 μL of 2 × 10 ⁵ cells / mL viable cell suspension, and 2 × 10 ⁵ cells / mL of the prepared suspension. The test was evaluated using 500 μL of the dead cell suspension and 500 μL of the culture supernatant obtained by culturing viable cells through a 0.22 μm filter. Both live and dead bacteria were suspended in a medium obtained by removing antibiotics from the Caco-2 cell culture medium described in Example 1.

3) Results The results are shown in FIG. 4 and FIG. In this experiment, recovery of the TER relative value of L. rhamnosus OLL2838 was observed, and the recovery effect or the inhibitory effect on the decrease of the TER relative value was attenuated by heat treatment (dead bacteria). The culture supernatant alone (metabolite) of this bacterium had little effect on the suppression of TER relative decrease or recovery. On the other hand, although IL-8 production suppression ability was attenuated by heat treatment (dead bacteria), it was found that the culture supernatant of this bacterium alone (metabolite) was maintained. It was suggested that the active ingredient is mainly present in the microbial cells as satisfying both recovery of TER relative value and suppression of IL-8 production.

[Experiment 3: Activity test of various bacterial treated products of strains having an action on intestinal permeation]
1) Preparation of various bacterial cell processed products of the test strain (Test strain)
The test was L. rhamnosus OLL2838, which was found to have a high TER relative value decrease inhibitory effect in Experiment 1. The cells were treated by the various treatment methods described below, washed twice with PBS and once with DMEM, and a medium obtained by removing antibiotics from the Caco-2 cell culture medium described in Example 1 was added. 2 × 10 ⁵ cells / mL suspension of treated cells was prepared and used for the following intestinal permeation inhibition test.

(Enzymatic treatment of bacterial cells)
Lipase treatment: For the purpose of lipid degradation. The enzyme used was derived from pig (Type II (Crude), EC 3.1.1.3, 30-90 units / mg protein, SIGMA). The solution was treated at a final concentration of 0.5 mg / mL at 37 ° C. for 2 hours.
Actinase treatment: for the purpose of protein degradation. The enzyme used was Streptomyces griseus (Kaken Pharmaceutical). The solution was treated with a 2 mg / mL final concentration solution at 37 ° C. for 2 hours.

2) Action test on intestinal permeation using Caco-2 cells The test method was carried out according to Experiment 1, and 500 μL of the prepared 2 × 10 ⁵ cells / mL suspension of treated cells was evaluated.

3) Results The results are shown in FIG. 6 and FIG. L. rhamnosus OLL2838's TER relative value recovery promoting effect and IL-8 production inhibitory activity observed in Experiment 2 were reduced by lipase treatment. On the other hand, no change was observed in L. rhamnosus OLL2838 cells treated with actinase. This suggested that the active ingredient having an action on intestinal permeation is a lipid component.

[Experiment 4: Activity test of lipoteichoic acid]
The cell wall of Gram-positive bacteria is rich in tycoic acid and tycuronic acid. Tychoic acid has lipoteichoic acid (LTA) bound to cell membrane glycolipid and wall teichoic acid bound to cell wall peptidoglycan, and since it is involved in various biological reactions, it is known to be a physiologically important component for bacteria. Yes. Experiment 3 suggested that the active component of L. rhamnosus OLL2838 cells acting on the intestinal permeation was a lipid component. Therefore, as one of the active components, we focused on lipoteichoic acid, which is a cell wall component of lactic acid bacteria. Changes in expression of MLCK (myosin light chain kinase) protein, an enzyme responsible for permeation and tight junction barrier function, were evaluated.

1) Preparation of lipoteichoic acid The lipoteichoic acid derived from Staphylococcus aureus (SIGMA) was used for the test.

2) Intestinal Permeation Inhibition Test Using Caco-2 Cells The test method was performed according to Experiment 1, and lipoteichoic acid 0.1, 1, 10, 100 ng / mL of 500 μL was used for evaluation. Moreover, the well which added 500 microliters of lipoteichoic acid 0.1 and 100 ng / mL to the well which does not add TNF- (alpha) was also provided.

3) Western analysis of expression change of MLCK enzyme related to tight junction
In addition to the intestinal permeation inhibition test using Caco-2 cells, Western analysis was performed on changes in protein expression of MLCK, which is an enzyme responsible for tight junction barrier function, upon addition of lipoteichoic acid. MLCK (myosin light chain kinase) is known as an enzyme that regulates tight junctions of the intercellular pathway in intestinal permeation.

    The test method was performed according to Experiment 1, and 100 ng / mL lipoteichoic acid was used for the evaluation. After 48 hours, Caco-2 cells were washed 3 times with PBS, and added with RIPA buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 1% SDS) Extracted. Further, the supernatant was collected by centrifugation (50,000 rpm, 30 min), and the supernatant was used as a test solution for Western blotting to analyze protein expression (n = 3 for each sample). Protein detection uses the ECL (chemiluminescence) method. The intensity of the band thus obtained was measured. Each band intensity value was divided by the control band intensity to obtain a relative intensity level.

4) Results (Test on intestinal permeation)
The test results when TNF-α is added to the wells are shown in FIGS. TER relative value decrease inhibitory action or recovery promoting action (FIG. 8) and IL-8 production inhibitory effect (FIG. 9) showed concentration dependency on lipoteichoic acid, and showed the highest effect especially when 100 ng / ml was added. . Moreover, the comparison test result with the case where TNF- (alpha) is not added to a well is shown to FIG. 10 and FIG. The effect of lipoteichoic acid was not observed in Caco-2 cells that were not stimulated with TNF-α, against the inhibitory effect on TER relative value decrease (FIG. 10) and IL-8 production inhibitory effect (FIG. 11). From this, the action on the intestinal permeation of the intestinal epithelium increased by TNF-α stimulation is particularly related to lipoteichoic acid among the bacterial components of L. rhamnosus OLL2838, and lipoteichoic acid is intestinal permeation of normal intestinal epithelium Was shown to have no effect.

(Western analysis of protein expression change of MLCK related to tight junction)
When TNF-α was added to a Caco-2 cell monolayer cell model, MLCK protein expression was increased, but co-culture with lipoteichoic acid suppressed the increase in MLCK protein expression (FIG. 12). MLCK has the function of phosphorylating myosin light chain in the presence of calcium-calmodulin (Ca-CaM), thereby contracting actin filaments. In the Caco-2 cells used in the test, tight junctions open when MLCK is activated. That is, the protein expression of MLCK increased by the addition of TNF-α means that the Tyco junction is opened and intestinal permeation is enhanced. From these results, it was suggested that lipoteichoic acid derived from lactic acid bacteria increases the TER relative value via MLCK.

  According to the above experiment, the lactic acid bacterium of the present invention has an action of protecting the intestinal barrier function, and recovers or promotes the recovery of inflammatory intestinal permeability caused by TNF-α and the like. It was understood that the enhancement of intestinal permeability caused by α and the like was inhibited. That is, it can be expected that intestinal permeability is restored to normal function by ingesting lactic acid bacteria or bacterial components of lactic acid bacteria, particularly lipid components such as lipoteichoic acid, to patients with excessively enhanced intestinal permeability. . In addition, by taking these lipoteichoic acid and lactic acid bacterium components in a normal intestinal permeability state, abnormal enhancement of intestinal permeability due to TNF-α and the like can be prevented.

It is a schematic block diagram of an intestinal tract penetration model. It is a figure which shows the effect | action which various lactic acid bacteria exert on intestinal tract penetration, Comprising: It is a figure which used TER value as the parameter | index. (A) is a graph showing the time lapse of the TER relative value, (b) is a graph showing the TER relative value after 48 hours. It is a figure which shows the effect | action which various lactic acid bacteria have on intestinal permeation, Comprising: It is a figure which used the production amount of IL-8 as a parameter | index. It is a figure which shows the effect | action which the living microbe of the lactic acid bacterium heat-processed, the dead microbe, and the microbe metabolites have exerted on intestinal permeation | transmission, and is a figure which used TER value as the parameter | index. (A) is a graph showing the time lapse of the TER relative value, (b) is a graph showing the TER relative value after 48 hours. It is a figure which shows the effect | action which the live bacteria, dead bacteria, and fungal metabolite of heat-treated lactic acid bacteria have on intestinal permeation, and is a figure using the production amount of IL-8 as an index. It is a figure which shows the effect | action which the lactic acid bacterium treated with the enzyme has on the intestinal permeation, and is a figure using the TER value as an index. (A) is a graph showing the time lapse of the TER relative value, (b) is a graph showing the TER relative value after 48 hours. It is a figure which shows the effect | action which the lactic acid bacterium treated with the enzyme has on the intestinal permeation, and is a figure using the production amount of IL-8 as an index. It is a figure which shows the effect | action which LTA exerts on intestinal permeation in the presence of TNF-α, and is a figure using the TER value as an index. (A) is a graph showing the time lapse of the TER relative value, (b) is a graph showing the TER relative value after 48 hours. It is a figure which shows the effect | action which LTA exerts on intestinal permeation in the presence of TNF-α, and is a figure using the production amount of IL-8 as an index. It is a figure which shows the effect | action which LTA exerts on intestinal permeation in the absence of TNF-α, and is a figure using the TER value as an index. (A) is a graph showing the time lapse of the TER relative value, (b) is a graph showing the TER relative value after 48 hours. It is a figure which shows the effect | action which LTA exerts on intestinal permeation in the absence of TNF-α, and is a figure using the production amount of IL-8 as an index. It is a figure which shows the expression of MLCK protein, Comprising: (a) is a figure which shows an example of the result of the Western blot which shows the expression, (b) is a figure which shows the comparison of the expression level. The arrow in the figure (a) shows the band of MLCK protein.

Claims (5)

Accession number: Lactobacillus rhamnosus deposited at NITE P-313 . A functional recovery agent for intestinal barrier function or a food composition for functional recovery, comprising the lactic acid bacterium according to claim 1 as an active ingredient. An intestinal barrier permeability-enhancing inhibitor or a food composition for enhancing inhibition comprising the lactic acid bacterium of claim 1 as an active ingredient. Using the serial mounting of lactic acid bacteria to claim 1 for the manufacture of a nutritional or pharmaceutical preparations for hyperpermeability or undesired permeability of the treatment or prevention of intestinal tract. A method for producing a nutritional or pharmaceutical preparation for the treatment or prevention of intestinal hyperpermeability or unwanted permeability, comprising:
The manufacturing method which has the process of mix | blending the lactic acid bacteria of Claim 1 .