JP6590330B2 - Oral immune tolerance enhancing substance screening method and oral immune tolerance enhancing composition - Google Patents

Description

  The present invention relates to a screening method for a substance / lactic acid bacterium that enhances oral tolerance and a composition thereof.

  Food allergy refers to “a phenomenon in which symptoms that are disadvantageous to the living body are induced through an antigen-specific immunological mechanism caused by food” (see, for example, Non-Patent Document 1).

  If the protein in food is fully digested and the protein is degraded, no immune response will occur even if it is absorbed. There are various barriers in the intestinal tract in the process of absorbing orally ingested food, such as digestion of proteins by the action of digestive enzymes, physical barrier by mucus on mucous membranes, and secretion of IgA antibodies that bind to proteins. Exists. In the intestinal tract, these barriers prevent entry of undigested food into the body, but actually undigested proteins are also routinely absorbed.

  However, it is controlled so that food allergy does not occur by oral tolerance, which is a mechanism that does not cause an immune reaction against such foreign proteins (see, for example, Non-Patent Document 2). In other words, food allergy is considered to be a condition in which immune tolerance to specific foods does not work well.

  At present, the development of food allergies is being prevented by removing specific food allergens, but this is not a fundamental treatment unless oral tolerance is induced in the body for the causative allergen. In addition, severely ill patients may cause anaphylactic shock to trace amounts of allergens attached to dishes, etc., and there is a limit to dietary elimination. Currently, “removal of minimum necessary causal food based on correct diagnosis” in which causal allergen is ingested within a range determined by an oral tolerance test in a hospital is instructed (for example, see Non-Patent Document 3).

  However, this method is not a fundamental treatment for food allergies as well as a removed diet. As a therapeutic method aiming at fundamental treatment, oral immunotherapy that induces acquisition of resistance to a causative allergen by ingesting a small amount of food containing the causative allergen has been clinically studied. At present, this treatment method is not recommended as general practice because anaphylactic shock often occurs during the course of treatment and a serious side reaction may occur (see Non-patent Document 4, for example).

  Therefore, if it becomes possible to efficiently induce oral tolerance, the fundamental treatment of food allergy can be safely performed, and even for those who have not yet developed food allergy, It can be prevented in advance.

  Bacteria that live in the body are expected to induce an appropriate immune response and suppress the occurrence of allergies. In particular, lactic acid bacteria have a rich dietary experience and are known to cause various immune responses depending on the bacterial species, and are expected to play some role in the induction of oral tolerance. On the other hand, it is not certain what kind of lactic acid bacteria are involved in the normal function of oral tolerance.

  It is known that intake of lactic acid bacteria alleviates symptoms of type I allergy such as perennial allergic rhinitis, hay fever and bronchial asthma (see, for example, Patent Documents 1 and 2).

  However, there are still many unclear points about the mechanism of food allergy, and treatments targeting only type I allergies are merely symptomatic treatments and are not a fundamental solution. Therefore, it is required not only to suppress type I allergy but also to achieve oral solution of food allergy by enhancing oral immune tolerance and causing it to function firmly. That is, if the enhancement factor of oral immune tolerance is specified and a substance that enhances oral immune tolerance can be selected, the above-described problems are solved.

“Guidelines for Medical Treatment of Food Allergy 2011” 2011, p2 “Food Allergy Clinical Practice Guidelines 2012” 2012, p20-25 “Guidelines for Nutrition Guidance for Food Allergies 2011” 2011, p4 “Food Allergy Guidelines 2012” 2012

JP 2011-254773 A JP 2010-209015 A

  An object of the present invention is to provide a method for screening an oral tolerance enhancer and a composition containing the same.

The present inventors have identified a factor involved in enhancing oral immune tolerance, found a composition that enhances oral immune tolerance based on the factor, and completed the present invention. That is, the present invention relates to the following.
(1) A method for screening an oral tolerance enhancer,
A screening method comprising a step of evaluating a test substance that promotes production of interferon β as an oral tolerance enhancer.
(2) The screening method according to (1), including a step of selecting a culture of lactic acid bacteria, bacterial cells, or bacterial cell components as a test substance before the evaluation step.
(3) The screening method according to (2), wherein the lactic acid bacterium is a lactic acid bacterium of the genus Pediococcus, Lactobacillus, Lactococcus or Tetragenococcus.
(4) The method according to any one of (1) to (3), which comprises a step of bringing interferon β-producing cells into contact with the test substance and measuring interferon β production promoting activity in the cells before the evaluation step. Screening method.
(5) The screening method according to (4), wherein the interferon β-producing cells express toll-like receptor 3 (TLR3) and / or toll-like receptor 9 (TLR9).
(6) The screening method according to (4) or (5), wherein the interferon β-producing cells are dendritic cells.
(7) A composition for enhancing oral tolerance comprising a culture of lactic acid bacteria, bacterial cells, or bacterial cell components as active ingredients.
(8) The composition for enhancing oral tolerance according to (7), wherein the lactic acid bacterium is a lactic acid bacterium of the genus Pediococcus, Lactobacillus, Lactococcus or Tetragenococcus.
(9) The composition for enhancing oral tolerance according to (8), wherein the lactic acid bacterium is Pediococcus acidilactici.
(10) The composition for enhancing oral tolerance according to (9), wherein the lactic acid bacterium is Pediococcus acidilactici K15 strain.

According to the method for screening an oral tolerance enhancer of the present invention, a candidate substance is selected using the presence or absence of interferon β production promoting action as an index, so that the candidate substance can be conveniently administered in vitro without direct administration to humans or animals. It becomes possible to screen. Lactic acid bacteria are promising candidates for oral immune tolerance enhancing substances, which include Toll-like receptor 3 (TLR3) and / or Toll-like receptor 9 (in which RNA and DNA of lactic acid bacteria are expressed in dendritic cells and the like. This is probably because TLR9) is involved in the enhancement of oral tolerance. However, the relationship between RNA / DNA and enhanced oral tolerance is not intended to be bound by theory. The obtained oral immune tolerance enhancing composition can be suitably used as an anti-IV type allergy agent, a food allergy therapeutic agent, a treatment adjuvant for oral immunotherapy, or a food allergy preventive agent.

It is a figure which shows the result of the delayed type hypersensitivity reaction when oral tolerance is induced | guided | derived to the mouse | mouth which administered the control antibody or the anti-interferon beta neutralizing antibody. It is a figure which shows the result of the OVA specific antibody titer in serum when a mouse | mouth which administered the control antibody or the anti-interferon beta neutralizing antibody induced | guided | derived oral tolerance. It is a figure which shows the result of the OVA-specific cytokine production of a spleen cell when an oral tolerance is induced | guided | derived to the mouse | mouth which administered the control antibody or the anti-interferon beta neutralizing antibody. It is a figure which shows the result of the interferon beta production promotion test of the bone marrow origin dendritic cell at the time of stimulating with various lactic acid bacteria. It is a figure which shows the result of a delayed type hypersensitivity reaction when investigating the oral immune tolerance enhancement effect of Pediococcus acidilactici K15. It is a figure which shows the result of an OVA specific antibody titer when investigating the oral tolerance tolerance effect of Pediococcus acidilactici K15. It is a figure which shows the result of the antigen-specific cytokine production of a spleen cell when investigating the oral tolerance tolerance effect of Pediococcus acidilactici K15. It is a figure which shows the result of the K15 viable count in feces at the time of K15 gnotobiotic mouse production. It is a figure which shows the result of the mRNA expression level of each cytokine of the Peyer's patch cell of a sterile mouse and K15 gnotobiotic mouse. It is a figure which shows the result of each cytokine mRNA expression level of the spleen dendritic cell of an aseptic mouse and K15 gnotobiotic mouse. It is a figure which shows the measurement result of the viable count in the stool at the time of K15 gnotobiotic mouse and K162 gnotobiotic mouse production. It is a figure which shows the OVA specific antibody titer in serum when oral tolerance is induced | guided | derived to K15 gnotobiotic mouse | mouth and K162 gnotobiotic mouse | mouth. It is a figure which shows the OVA specific cytokine production of a spleen cell when an oral immune tolerance is induced | guided | derived to a K15 gnotobiotic mouse and a K162 gnotobiotic mouse. It is a figure which shows the onset rate of the diarrhea at the time of investigating the oral immune tolerance enhancement effect of K15 in a food allergy model. It is a figure which shows the mRNA expression level of the inflammatory cytokine of a small intestine at the time of investigating the oral immune tolerance enhancement effect of K15 in a food allergy model. It is a figure which shows the OVA specific cytokine production of the mesenteric lymph node cell when investigating the oral immune tolerance enhancement effect of K15 in a food allergy model. It is a figure which shows the OVA specific antibody titer in serum at the time of investigating the oral immune tolerance enhancement effect of K15 in a food allergy model. It is a figure which shows the time-dependent change of the number of bacteria of K15 in a carrot fermentation liquid.

  Hereinafter, the present invention will be described in more detail. In addition, this invention is not limited to the following embodiment, It can implement by changing variously within the range of the summary.

(1) Screening Method for Oral Immune Tolerance Substance In the screening method of the present invention, whether or not a candidate substance has an interferon β production promoting action is evaluated as one of the indexes for determining the oral tolerance tolerance enhancing action. . As used herein, “oral tolerance” means that a state in which an immune response is suppressed against an antigen taken orally (ie, an immune tolerance state) is induced. The state of immune tolerance can be evaluated by methods known to those skilled in the art, for example, measurement of T cell reactivity that plays an important role in immune tolerance.

  As used herein, “enhanced oral tolerance” refers to oral tolerance that is significantly greater than the test substance, eg, 5% or more, 10% or more, 20% or more, or 50% or more, compared to the control substance. Means enhanced. The test substance to be evaluated for the effect of enhancing oral immune tolerance is not limited to a specific chemical substance or a mixture thereof, and may be a bacterial culture, a bacterial cell, or a bacterial cell component. By selecting bacteria whose safety is ensured by eating experience as test substances, efficient screening becomes possible. From the viewpoint of food applicability and safety, the bacterium as the test substance is preferably a lactic acid bacterium. More preferred is a lactic acid bacterium of the genus Bacillus, Lactococcus or Tetragenococcus.

  Bacterial cells can be obtained from a fermented product of bacteria. For example, when lactic acid bacteria are used, they can be obtained by fermentation using a material capable of growing lactic acid bacteria such as carrot juice and tomato juice. The bacterial cell component is, for example, a nucleic acid held by a bacterium, and specifically DNA, single-stranded RNA, or double-stranded RNA. Bacterial nucleic acid components can be obtained by conventional fractionation methods.

  When bacteria are used as test substances, the amount of double-stranded RNA contained in the bacteria may be evaluated in advance as an additional screening index. This is because bacteria possessing a large amount of double-stranded RNA may have an interferon production promoting action (results not shown). The number of double-stranded RNAs contained in bacteria can be determined by comparison with those contained in pathogenic bacteria such as Listeria, Salmonella, Clostridium perfringens, Helicobacter pylori, and Salmonella typhi.

  The selected test substance is brought into contact with an interferon β-producing cell, and the interferon β production promoting activity in the cell is measured. The interferon β production promoting activity of the test substance can be evaluated by a conventional method. Although not intended to be limited, interferon production promoting activity may be measured by co-culturing interferon-producing cells and a test substance and measuring the interferon β concentration in the culture supernatant. The co-cultivation conditions are appropriately determined by those skilled in the art. For example, the culture time may be 6 to 8 hours. In addition, when the interferon β-producing cells are bone marrow-derived dendritic cells, the ratio of the cells and lactic acid bacteria is 1:10 to 1: 100 (bone marrow-derived tree) in order to ensure a sufficient production amount of interferon β. It may be added to the culture solution so as to form symptomatic cells: lactic acid bacteria. Alternatively, the production of interferon β can be confirmed if at least 10 μg to 100 μg of lactic acid bacteria are present per 1 ml of the culture solution.

  When the production of interferon β in the interferon β-producing cells added with the test substance is significantly increased, for example, 5% or more, 10% or more, 20% or more, or 50% or more compared to the control substance, the test substance is administered orally It can be evaluated as an immunological tolerance enhancer. Interferon β is produced by methods known to those skilled in the art, for example, methods using an antigen-antibody reaction with an interferon β antibody (enzyme immunoassay (ELISA), radioimmunoassay (RIA), etc.) You may measure through the utilized method (CPE (cytopathogenic effect) method etc.).

  Examples of interferon β-producing cells used in the present invention include cells expressing a receptor that recognizes double-stranded RNA, such as TLR3, TLR9, etc., particularly dendritic cells.

(2) Oral immune tolerance enhancing composition The oral immune tolerance enhancing composition of the present invention comprises an oral tolerance enhancing substance obtained by the above screening method, such as a culture of lactic acid bacteria, bacterial cells, or bacterial cell components as active ingredients. . Lactic acid bacteria are lactic acid bacteria of the genus Pediococcus, Lactobacillus, Lactococcus, Tetragenococcus, for example. Lactic acid bacteria belonging to the genus Pediococcus are preferable, for example, Diococcus acidilactici, particularly Pediococcus acidilactici K15 strain is more preferable.

  As long as the oral immune tolerance enhancing action is exhibited, the lactic acid bacteria culture, microbial cells, or microbial components that are active ingredients may be prepared by any method. Lactobacillus cells can be obtained by fermentation using a material capable of growing lactic acid bacteria such as carrot juice and tomato juice. The bacterial cell component is, for example, a nucleic acid possessed by lactic acid bacteria, and specifically DNA, single-stranded RNA, or double-stranded RNA. The nucleic acid component of lactic acid bacteria can be obtained by a normal fractionation method.

  When ingesting the oral immune tolerance enhancing composition of the present invention for the purpose of preventing the development of food allergies or assisting in the treatment of oral immunotherapy, the intake may be appropriately set according to the symptoms and physique of the ingestor. When a lactic acid bacterium belonging to the genus Pediococcus is used as an active ingredient, the microbial cell intake is, for example, 1 to 1000 mg / body weight 60 kg / day.

  Lactic acid bacterial cells or bacterial cell components, which are active ingredients, may be used alone or in combination with other components, for example, added to foods and beverages and pharmaceuticals.

(3) Method for producing oral immune tolerance enhancing composition The oral immune tolerance enhancing composition of the present invention is obtained by culturing lactic acid bacteria and collecting the culture, bacterial cells, or bacterial cell components. As long as an active ingredient that exhibits oral immune tolerance enhancing activity is obtained, there are no particular limitations on the culture conditions of lactic acid bacteria, the culture, the fungus body, or the method of collecting the fungal ingredient as the active ingredient.

  Below, the manufacturing method of the oral tolerance tolerance composition which uses the microbial cell of Pediococcus acidilactici K15 strain as an active ingredient is illustrated.

  First, for example, carrots boiled for 10 minutes are crushed, and the supernatant obtained by centrifugation is used as carrot juice. Pediococcus acidiractis is inoculated into the carrot juice and cultured at 25-37 ° C. for 12-72 hours. By boiling for 10 minutes after culturing, a carrot fermentation broth of lactic acid bacteria is obtained.

  The carrot fermentation broth obtained as described above can be used as the composition for enhancing oral tolerance of the present invention.

  EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

[Humoral factors related to oral tolerance]
It was shown that interferon β is involved in the induction of oral tolerance.

1. Administration of Antibody to Mice Rat IgG control antibody (Sigma) or anti-interferon β-neutralizing antibody (Yamasa) was applied 7 times every other day to 5-week-old female BALB / c mice (CLEA Japan). It was administered intravenously at 50 μg / animal.

2. Induction of oral tolerance in mice During the fourth intravenous administration, 25 mg of ovalbumin (OVA, Sigma) was orally administered to induce oral tolerance.

3. OVA sensitization Nine days after the seventh intravenous administration, 300 μg of OVA and 100 μl of Complete Freund's Adjuvant were administered subcutaneously on the back surface in order to induce allergy.

4). Measurement of delayed type hypersensitivity reaction Delayed type hypersensitivity reaction was examined by administering 50 μg of OVA subcutaneously on the back of the sole of the foot 14 days after dorsal subcutaneous immunization, and measuring the size of the swelling 24 hours later. The result is shown in FIG. In addition, the significant difference test was performed by the t test.

  As shown in FIG. 1, in the control antibody-administered mice, the delayed hypersensitivity reaction was suppressed by oral administration of OVA (oral tolerance induction), whereas in the anti-interferon β neutralizing antibody-administered mice, the delayed hypersensitivity reaction was observed. It was not suppressed.

5. Measurement of OVA-specific antibody titer in serum After measurement of delayed type hypersensitivity reaction, blood was collected under isoflurane anesthesia. Centrifugation was performed at 1500 rpm for 30 minutes to obtain serum. The amount of OVA-specific IgG and the amount of OVA-specific IgE in serum were measured by enzyme immunoassay. Specifically, anti-mouse IgG antibody and anti-mouse IgE antibody (manufactured by eBioscience) were added to 1M sodium bicarbonate buffer (pH 8.2) at a dilution of 500 times, and coated on a 96-well plate at 50 μl / well. Thereafter, the serum sample was diluted 2-fold to 50,000-fold, dispensed 50 μl onto the plate, and incubated for 1 hour. After washing, biotin-labeled OVA was dissolved in a phosphate buffer containing 0.05% Tween containing 1% BSA to a concentration of 2 μg / ml, and 50 μl was dispensed onto the plate. The biotin-labeled OVA was produced using OVA manufactured by Sigma and a biotinylation kit manufactured by Dojindo Laboratories. After incubating for 1 hour, washing was performed, and a peroxidase enzyme (manufactured by Vector) labeled with streptavidin was added to bind to biotin. Color development was performed by adding 50 μl per well of TMB substrate solution (Moss / Cosmo Bio) and reacting at room temperature for 20 minutes. The reaction was stopped with 0.5N hydrochloric acid, the absorbance at 450 nm was measured with a microplate reader (manufactured by TECAN), and the amounts of OVA-specific IgG and IgE were quantified relative to the physiological saline intake group. The result is shown in FIG. In addition, the significant difference test was performed by the t test.

  As shown in FIG. 2, in the mice administered with the control antibody, both OVA-specific IgG and IgE were suppressed by induction of oral tolerance. In the anti-interferon β-neutralizing antibody administration group, OVA-specific IgE and OVA-specific IgG were suppressed by induction of oral tolerance, confirming that interferon β was not involved in suppression of antibody production. .

6). Measurement of OVA-specific cytokine production of spleen cells After blood collection, the spleen was collected and cells were prepared. After collecting the spleen, the spleen was crushed with a mesh, the cells were suspended, and the spleen cells were collected. Then, using a 96-well round bottom plate (BD FALCON), the spleen cells become 1 × 10 ⁶ cells / well in 10% FBS (JRH) -added RPMI1640 medium containing 100 μg / ml of OVA (manufactured by Sigma). And suspended for 3 days. After culturing, centrifugation was performed at 1500 rpm for 5 minutes, the supernatant was collected, and the concentrations of IL-6, IL-17, IFN-γ, and IL-4 were measured by enzyme immunoassay. IL-6, IFN-γ, and IL-4 were measured using an ELISA Kit manufactured by BD farming, and IL-17 was measured using an ELISA Kit manufactured by eBioscience. The results are shown in FIG. In addition, the significant difference test was performed by the t test.

  As shown in FIG. 3, all cytokine production was suppressed by induction of oral tolerance in the control antibody-treated mice. On the other hand, cytokines other than IL-4 were not suppressed in anti-interferon β neutralizing antibody-administered mice. From the above, it was shown that interferon β is involved in the induction of oral tolerance in the living body. In addition, since OVA-specific IgE in serum and OVA-specific IL-4 production in spleen cells are not suppressed, it was shown that interferon β does not significantly affect type I allergy involving them. However, it was confirmed that it is strongly related to delayed type IV allergy via T cells and the like.

[Interferon β production promotion test of various lactic acid bacteria using mouse bone marrow-derived dendritic cells]
Using the lactic acid bacteria shown in Table 1, an interferon β production promotion test was conducted.

1. Preparation of Lactic Acid Bacteria Suspension MRS medium was inoculated with various lactic acid bacteria at 1 × 10 ⁷ cells / ml. After stationary culture at 30 ° C. for 24-48 hours, the medium was removed by a centrifugal concentrator to collect the cells. Tetragenococcus was collected after culturing in an MRS medium containing 10% sodium chloride. Thereafter, the cells were washed with physiological saline, sterilized by boiling at 95 ° C. for 10 minutes, and lyophilized.

2. Interferon β production promotion test (1) Preparation of bone marrow-derived dendritic cell suspension Bone marrow-derived dendritic cells were dislocated from BALB / c mice (8-12 weeks old, female, Nippon Claire) under isoflurane inhalation anesthesia. After euthanasia, the femur and tibia were removed from the lower limbs, and a 6 cm dish for cell culture containing RPMI 1640 medium (manufactured by Sigma) supplemented with ice-cold 1% fetal calf serum (FCS, non-immobilized) (Made by BD FALCON). RPMI 1640 supplemented with 1% FCS was injected to extrude the bone marrow, and then suspended. The obtained cell suspension was filtered with a cell strainer (40 μm, BD FALCON) and then centrifuged at 440 × g for 5 minutes.

Add hemolysis buffer (5 mL, 0.155 M NH ₄ Cl, 0.01 M Tris, pH 7.5), place on ice for 5 minutes, add 1% FCS-added RPMI 11640 (5 mL), centrifuge, add 1% FCS Washed twice more with RPMI 1640. phycoerythrin (PE) -labeled anti-IA antibody (Clone M5 / 114.14.2, BD Pharmingen, 0.2 mg / mL), PE-labeled anti-CD4 antibody (Clone GK1.5, BD Pharmingen, 0. 2 mg / mL) and PE-labeled anti-CD8 antibody (Clone 53-6.7, manufactured by BD Pharmingen, 0.2 mg / mL) diluted with a MACS running buffer 1000 times each (100 μL / 10 ⁷ cells) and rabbit IgG (50 μg / mL, Zymed) was added and allowed to stand on ice for 30 minutes.

After washing once with MACS running buffer, anti-PE magnetic beads (20 μL / 10 ⁷ cells, manufactured by Miltenyi) and MACS running buffer (80 μL / 10 ⁷ cells) were added and allowed to stand at 4-8 ° C. for 15 minutes. After washing once with MACS running buffer 20 times the amount of the reaction solution, the suspension was suspended in MACS running buffer (0.5 mL / 10 ⁸ cells), and negative fraction was obtained using an automatic magnetic separation system (Auto MACS, manufactured by Miltenyi). Separated. The separated cells were washed once with RPMI 1640 medium supplemented with 1% FCS and then suspended in a basic medium supplemented with granulocyte monocyte colony stimulating factor (GM-CSF). The basic medium was 100,000 U / L penicillin / 100 mg / L streptomycin (Sigma), 2-mercaptoethanol (50 μM, manufactured by Wako Pure Chemical Industries), L-glutamic acid (2 mM, manufactured by Nacalai Tesque), HEPES ( 20 mM, manufactured by Dojindo Laboratories) supplemented RPMI1640 medium (Gibco) and 10% of FCS (Hyclone) deactivated were used.

For GM-CSF, 10% of the culture supernatant of plasmacytoma X63-Ag8 (J558L-GM-CSF) into which the mouse GM-CSF gene was introduced was added to the basal medium. The cell was suspended in Trypan blue (Gibco Co.), after the number of cells was counted using a hemocytometer (manufactured by BD FALCON Corp.) 6-well plate for cell culture 1.2 × 10 ⁶ / 4mL / well This was dispensed and cultured. On the 3rd and 6th days after the start of the culture, 2 mL of the medium was aspirated and removed, 2 mL of a new basic medium supplemented with GM-CSF was added, and the floating cells were recovered as immature dendritic cells on the 8th day after the start of the culture (CD11c positive) Cells> 85%). The cells were washed 3 times with 1% FCS-added RPMI 1640 medium and then suspended in a basic medium to obtain a bone marrow-derived dendritic cell suspension.

(2) Measurement of interferon β production promoting activity The cells obtained as described above and lactic acid bacteria were used for co-culture. Bone marrow-derived dendritic cells were added to a 96-well flat bottom plate (BD FALCON) at 2 × 10 ⁵ / well, and lactic acid bacteria (other than Tetragenococcus) were added at 100 μg / well. Tetragenococcus genus (K15 was also used as a control) was added so that bone marrow-derived dendritic cells: lactic acid bacteria = 1: 100. After culturing for 6 hours, the supernatant was collected, and the interferon β concentration in the supernatant was measured using an interferon β measurement kit (manufactured by PBL). The results are shown in FIG.

  As shown in FIG. 4, the amount of interferon β produced varied depending on the genus and species among lactic acid bacteria. On the other hand, it was confirmed that lactic acid bacteria belonging to the genus Pediococcus acidilactici, Pediococcus pentosaceus and Tetragenococcus have very high activity.

[Enhanced oral tolerance of lactic acid bacteria belonging to the genus Pediococcus]
Lactic acid bacteria Pediococcus acidilactici K15 were continuously orally administered to 5-week-old BALB / c mice (female, CLEA Japan, Inc.) to examine the effect of enhancing oral tolerance. By sensitizing ovalbumin (OVA, Sigma) and inducing allergies, the effects of lactic acid bacteria intake on delayed-type hypersensitivity reaction, serum OVA-specific antibody titer, and antigen-specific cytokine production of spleen cells were examined. . The test was conducted according to the following method.

1. Administration of Lactic Acid Bacteria Lactic acid bacteria K15 suspension was cultured in MRS (Difco) culture for 24 hours, then washed twice with physiological saline and boiled for 10 minutes to give 5 × 10 ⁹ cells / ml. It was prepared as follows. In the untreated (allergy non-induction) control group, allergy induction control group, and oral tolerance tolerance induction group, physiological saline 0.2 ml / day was started, and in the K15 intake group, lactic acid bacteria suspension 0.2 ml / day was started. It was ingested for 15 days from day 14 to day 14.

2. Induction of oral tolerance To the oral tolerance induction group and the K15 intake group, OVA 20 mg / 0.2 ml physiological saline was administered on the 7th, 9th, 11th, and 14th days of the test, and oral immunization was performed. Induced tolerance. 0.2 ml of physiological saline was administered to the untreated control group and the allergy induction control group.

3. Allergy induction On the 21st day from the start of the test, 300 μg of OVA (Sigma) and 100 μl of Complete Freund's Adjuvant were administered subcutaneously on the back surface in order to induce allergy.

4). Measurement of delayed type hypersensitivity reaction 14 days after dorsal subcutaneous immunization (35 days after the start of the test), 50 μg of OVA was administered subcutaneously on the dorsal surface of the sole of the foot, and the size of the swelling was measured 24 hours later. Examined. The result is shown in FIG. In addition, the significant difference test was performed by the t test.

  As shown in FIG. 5, the delayed hypersensitivity reaction was suppressed by induction of oral tolerance, but it was confirmed that the suppression effect was enhanced by ingestion of K15.

5. Measurement of OVA-specific antibody titer in serum After measurement of delayed type hypersensitivity reaction, blood was collected under isoflurane anesthesia. Centrifugation was performed at 1500 rpm for 30 minutes to obtain serum. The amount of OVA-specific IgG1, the amount of OVA-specific IgG2a, and the amount of OVA-specific IgE in serum were measured by enzyme immunoassay. Specifically, OVA (manufactured by Sigma, Grade V) was dissolved in 1 M sodium hydrogen carbonate buffer (pH 8.2) so as to be 50 μg / ml, and a 96-well plate was coated with 50 μl / well. Thereafter, the serum sample was diluted 2 to 50000 times, dispensed 50 μl onto the plate, and incubated for 1 hour. After washing, an anti-mouse IgG1 antibody, an anti-mouse IgG2a antibody, and an anti-mouse IgE antibody (manufactured by BD Pharmingen) were incubated for 1 hour in a solution diluted with a 1% BSA-added 0.05% Tween-containing phosphate buffer 500 times. After washing, a peroxidase enzyme (manufactured by Vector) labeled with streptavidin was added to bind to biotin. Color development was performed by adding 50 μl per well of TMB substrate solution (Moss / Cosmo Bio) and reacting at room temperature for 20 minutes. The reaction was stopped with 0.5N hydrochloric acid, the absorbance at 450 nm was measured with a microplate reader (manufactured by TECAN), and each OVA-specific antibody titer was quantified by a relative value with respect to the allergy induction control group. The result is shown in FIG. In addition, the significant difference test was performed by the t test.

  As shown in FIG. 6, there was no difference in the amount of OVA-specific IgE between the oral tolerance group and the K15 intake group. It was confirmed that it was strongly suppressed.

6). Measurement of OVA-specific cytokine production of spleen cells After blood collection, the spleen was collected and cells were prepared. Cell preparation and cytokine measurement were performed in the same manner as in Example 1. The results are shown in FIG. In addition, the significant difference test was performed by the t test.

  As shown in FIG. 7, it was confirmed that the production amount of each cytokine was most suppressed in the K15 intake group. From these results, it was shown that oral tolerance was enhanced by K15 intake, and delayed hypersensitivity reaction, antigen-specific antibody titer in serum, and antigen-specific cytokine production in spleen cells were suppressed.

[Test of gnotobiotic mice for lactic acid bacteria belonging to the genus Pediococcus]
It was confirmed that IFN-β was actually induced in the living body by producing a K15 gnotobiotic mouse using a sterile mouse. The test was conducted as follows.

1. Preparation of K15 gnotobiotic mouse A suspension of lactic acid bacteria K15 was administered to a sterile mouse (4-5 weeks old, Sankyo Lab Service Co., Ltd.). The lactic acid bacteria suspension is cultured in MRS (Difco) culture solution for 24 hours, then centrifuged and collected. After discarding the supernatant medium, the suspension becomes 5 × 10 ⁹ cells / ml in sterilized physiological saline. So that it was suspended. 0.4 ml of lactic acid bacteria suspension was administered to a sterile mouse. Then, after raising for 4 to 5 weeks, cervical dislocation was performed under isoflurane anesthesia, and the spleen and Peyer's patch were collected, and then each tissue was analyzed. One group consisted of 5 animals.

2. Confirmation of fixation of lactic acid bacteria In order to confirm the fixation of lactic acid bacteria, feces were collected 1 day, 3 days and 28 days after administration of lactic acid bacteria, and the number of lactic acid bacteria was measured. The number of lactic acid bacteria was determined by suspending feces in physiological saline, applying to MRS agar medium, culturing at 30 ° C. for 48 hours, and counting the number of colonies. The results are shown in FIG.

As shown in FIG. 8, it reached 1 × 10 ⁹ cells / g stool 3 days after administration of lactic acid bacteria, and it was confirmed that the number of bacteria was maintained during the test period thereafter.

3. Analysis of Peyer's patches After collecting Peyer's patches, 10% FCS, 10 mM EDTA, 20 mM HEPES, 10 μg / ml polymyxin B (Sigma), 100,000 U / L penicillin / 100 mg / L streptomycin (Sigma), 1 mM Sodium Pyruvate Incubated in phosphate buffer containing (Gibco) for 20 minutes. Thereafter, the cells were washed with a phosphate buffer to remove epithelial cells, and thereafter, 400 U / L collagenase D (Roche), 50 μg / ml DNaseI, 100,000 U / L penicillin / 100 mg / L streptomycin containing 10% FCS ( Sigma), L-glutamic acid (2 mM), and HEPES (20 mM) -added RPMI1640 medium (Gibco) were used to obtain Peyer's patch cells. Thereafter, RNA was extracted with RNeasy Mini Kit (Qiagen), and reverse transcription was performed with PrimeScript RT Reagent (Takara) to obtain cDNA.

  Using this, quantitative real-time PCR was performed by Sybr Premix Ex Taq (manufactured by Takara). As specific primers for each mRNA, interferon β is sense 5′-GCACTGGGGTGGAATGAGACT-3 ′ (SEQ ID NO: 1), antisense 5′-AGTGGAGAGCAGTTGAGGAACA-3 ′ (SEQ ID NO: 2), and interleukin 6 is sense 5 ′. -AGTTGCCCTTCTTGGGACTGA-3 '(SEQ ID NO: 3), antisense 5'-TCCACGATTTCCAGAGAAC-3' (SEQ ID NO: 4), interleukin 12p40 is sense 5'-AAGAAGGAAAATGGATTTTGGTCC-3 '(SEQ ID NO: 5), antisense 5'- ATGTCACTGCCCGAGAGCTCAG-3 ′ (SEQ ID NO: 6), interleukin 10 is sense 5′-GAGAAGCATGGCCCA GAAATC-3 ′ (SEQ ID NO: 7), antisense 5′-CGCATCCCTGAGGGTCTTCA-3 ′ (SEQ ID NO: 8), β-actin is sense 5′-GCTACAGCTTCACCACCACAG-3 ′ (SEQ ID NO: 9), antisense 5′-GGTCTTTACGGATGTCAACGTC- Using 3 ′ (SEQ ID NO: 10), measurement was performed with Mx3000P (manufactured by Stratagene). Each expression level was standardized by the expression level of β-actin, and the relative expression level for sterile mice was determined. The results are shown in FIG.

  As shown in FIG. 9, it was confirmed that only the mRNA expression level of interferon β in Peyer's patch cells was increased in K15 gnotobiotic mice. This indicates that K15 enhances interferon β relative to the living body.

4). Analysis of splenic dendritic cells After collection of spleen cells, 400 unit / ml collagenase D (Roche), 100,000 U / L penicillin / 100 mg / L streptomycin (Sigma) containing 10% FCS, L-glutamic acid ( 2 mM) and HEPES (20 mM) added RPMI 1640 medium (manufactured by Gibco) to obtain spleen cells. Then, it was made to react with PE labeled anti-mouse CD11c antibody and PE-Cy7 labeled anti-mouse CD11b antibody to carry out labeling. Then, sorting was performed using FACSAria to obtain CD11b ⁻ CD11c ⁺ cells and CD11b ⁺ CD11c ⁺ cells. For each, RNA was extracted with RNeasy Mini Kit (Qiagen), and then reverse transcription reaction was performed with PrimeScript RT Reagent (manufactured by Takara) to obtain cDNA. Using this, quantitative real-time PCR was performed by Sybr Premix Ex Taq (manufactured by Takara). The results are shown in FIG. In addition, the significant difference test was performed by the t test.

As shown in FIG. 10, it was confirmed that the mRNA expression level of interferon β was increased in CD11b ⁺ CD11c ⁺ cells in the spleen of K15 gnotobiotic mice. On the other hand, no significant change was observed in the expression levels of other cytokines. From the above, it was shown that K15 causes increased expression of interferon β in the living body.

[Oral immune tolerance induction test using gnotobiotic mice of lactic acid bacteria belonging to the genus Pediococcus]
After producing gnotobiotic mice as in Example 4, it was confirmed whether oral immune tolerance was induced. As a control, Lactobacillus plantarum K162 that does not induce interferon β was used.

1. Preparation of gnotobiotic mice Gnotobiotic mice were prepared in the same manner as in Example 4 for both K15 and K162. The establishment of each lactic acid bacterium was confirmed by the same method as in Example 4, and it was confirmed that both strains were established as shown in FIG.

2. Induction of oral tolerance Four weeks after the administration of lactic acid bacteria, OVA 50 mg / 250 μl physiological saline was orally administered to induce oral tolerance. 250 μl of physiological saline was administered to the group not induced by oral tolerance. OVA to be administered was sterilized with gamma rays at Funabashi Farm.

3. OVA sensitization To induce allergy one week after oral tolerance, OVA (Sigma) 300 μg and Complete Freund's Adjuvant 100 μl were administered subcutaneously on the back surface.

4). Measurement of OVA-specific antibody titer The OVA-specific antibody titer was measured in the same manner as in Example 1. K15 gnotobiotic mice were quantified as relative values to the control (no oral tolerance tolerance induction) group. The results are shown in FIG. In addition, the significant difference test was performed by the t test.

  In K15 gnotobiotic mice, the antigen-specific IgE and IgG levels were significantly suppressed by induction of oral tolerance, whereas in K162 gnotobiotic mice, induction of oral tolerance was weak.

5. Measurement of OVA-specific cytokine production of spleen cells OVA-specific cytokine production of spleen cells was measured in the same manner as in Example 1. The results are shown in FIG. In addition, the significant difference test was performed by the t test.

  In the K15 gnotobiotic mouse, production of each cytokine was significantly suppressed by induction of oral tolerance, whereas in K162 gnotobiotic mice, the suppression by induction of oral tolerance was weak. This indicates that the induction of interferon β via the intestinal tract is related to the enhancement of oral tolerance.

[Food allergy test of lactic acid bacteria belonging to the genus Pediococcus]
It was confirmed in a food allergy model that the development of food allergy is strongly suppressed by enhancing oral tolerance.

1. Administration of lactic acid bacteria A K15 lactic acid bacteria suspension was prepared in the same manner as in Example 3. From the 0th day to the 20th day from the start of the test, 0.2 ml of the lactic acid bacteria suspension was administered. Test in untreated (allergy non-induction) control group, allergy induction / non-onset group, allergy induction / onset group, allergy induction / oral immune tolerance induction / onset group, allergy induction / K15 administration + oral immune tolerance induction / onset group This was carried out, and 0.2 ml of physiological saline was administered except for the K15 administration group.

2. Induction of oral immune tolerance Induction of oral immune tolerance was performed on the 14th day of the test for allergy induction / oral immune tolerance induction / onset group, allergy induction / K15 administration + oral immune tolerance induction / onset group, OVA 25 mg / 0.2 ml physiological It was performed by administering saline. The other groups received 0.2 ml of physiological saline.

3. Allergy induction On the 21st and 35th days from the start of the test, OVA (Sigma) 10 μg / 0.1 ml physiological saline and aluminum hydroxide gel (Wako Pure Chemical Industries) 0.1 ml were mixed, and 0 2 ml was administered.

4). Onset Onset On the 51st day, the 53rd day, the 55th day, the 57th day, the 59th day, the 61st day, and the 63rd day, OVA 50 mg / 250 μl physiological saline was orally administered once to develop the onset. After observation of diarrhea on day 63, blood was collected under isoflurane anesthesia, and then mesenteric lymph nodes and small intestine tissue were collected.

5. Observation of diarrhea Diarrhea was observed 1 hour after oral administration of OVA on day 63 of the test. The results are shown in FIG. Although the onset of diarrhea was suppressed by induction of oral tolerance, it was confirmed that the onset of diarrhea was most suppressed in the K15 intake group.

6). Inflammatory cytokine mRNA expression level in small intestine tissue After extracting RNA from small intestine tissue using TRIzol, reverse reaction and quantitative real-time PCR were performed in the same manner as in Example 4. As specific primers for each mRNA, mouse KC (Keratinocyte Chemoattractant) is sense 5′-CTGCCACCCAAACCGAAGTC-3 ′ (SEQ ID NO: 11), antisense 5′-AGCTTCAGGGTCAAGGCAAG-3 ′ (SEQ ID NO: 12), and TNF-α is , Sense 5′-CTGGGACAGTGACCTGGACT-3 ′ (SEQ ID NO: 13), antisense 5′-GCACCTCAGGGGAAGAGTCTG-3 ′ (SEQ ID NO: 14), interleukin 1β is sense 5′-AAATGCCTCGTGCTGTCTGACC-3 ′ (SEQ ID NO: 15), The sense 5′-CTGCTTGAGAGGTGCTGATGTACC-3 ′ (SEQ ID NO: 16) was used. It measured by Mx3000P (made by Stratagene). Each expression level was normalized by the expression level of β-actin and quantified by a relative value with respect to the untreated group. The results are shown in FIG. In addition, the significant difference test was performed by the t test.

  As shown in FIG. 15, the expression level of each inflammatory cytokine was suppressed by induction of oral immune tolerance. Among them, it was confirmed that it was most strongly suppressed in the K15 intake group.

7). OVA-specific cytokine production in mesenteric lymph nodes After collecting mesenteric lymph nodes for each group, the mesenteric lymph nodes were crushed with a mesh, cells were suspended, and mesenteric lymph node cells were collected. Then, using a 96-well round bottom plate (BD FALCON), 1 × 10 ⁶ mesenteric lymph node cells / in 10% FBS (JRH) -added RPMI 1640 medium containing 100 μg / ml of OVA (manufactured by Sigma) / It was suspended in a well and cultured for 3 days. After culturing, centrifugation was performed at 1500 rpm for 5 minutes, the supernatant was collected, and the amount of each cytokine produced was measured by enzyme immunoassay. The measurement method was the same as in Example 1. The results are shown in FIG.

  As shown in FIG. 16, it was confirmed that OVA-specific cytokine production was most suppressed in the K15 intake group.

8). Measurement of serum OVA-specific antibody titer In the same manner as in Example 1, serum OVA-specific antibody titer was measured. OVA-specific IgM was measured by coating with an anti-mouse IgM antibody (BD pharmingen). The results are shown in FIG. In addition, the significant difference test was performed by the t test.

  As shown in FIG. 17, it was confirmed that OVA-specific IgG and IgM were strongly suppressed in the K15 intake group. From this, it was shown that the induction of allergy was suppressed by the enhancement of oral tolerance.

[Production of carrot fermentation liquor using lactic acid bacteria belonging to the genus Pediococcus]
An oral immune tolerance enhancing composition was prepared using pediococcus lactic acid bacteria and carrots.

1. Preparation of carrot juice juice A commercially available carrot was cut into pieces, and 200 ml of water was added to 200 g of carrot, followed by boiling for 10 minutes. Then, it grind | pulverized with the mixer and produced the carrot crushing liquid. Then, the solid substance was removed by performing centrifugation. Then, the carrot juice was collected by concentrating with an evaporator.

2. Cultivation of Pediococcus acidilactici K15 using carrot juice as a medium The carrot juice was adjusted so that the Brix values were 5, 10, 15, and 20, and then the pH was 6.8-7.0. It adjusted with 5N sodium hydroxide so that it might become. And what was heat-processed for 10 minutes was used as the culture medium. Pediococcus acidilactici K15 was ingested to about 1 × 10 ⁶ cfu / ml for each medium and cultured at 30 ° C. The change in the number of bacteria at that time is shown in FIG. The number of bacteria was measured by applying to MRS agar medium.

  As shown in FIG. 18, Pediococcus acidilactici K15 grew well in the medium of each Brix value. This carrot fermentation broth can be used as a composition for enhancing oral immune tolerance.

Claims (1)

Pediococcus reed di Lacty sheet culture, as an active ingredient the bacteria or bacterial components, by enhancing oral tolerance obtained by orally administering the food allergen before, the food allergy A composition for treating or preventing.

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