JP5144186B2 - Antiallergic agent - Google Patents

Description

  The present invention relates to an antiallergic agent having an enhanced effect of β-glucan useful for allergic diseases and an antiallergic food containing the same, and more specifically, to β-glucan as an active ingredient, to Pediococcus pentosaseus (Pediococcus pentosaseus) The present invention relates to an antiallergic agent containing the lactic acid bacteria to which it belongs and an antiallergic food containing the antiallergic agent.

  The immune system is a system that protects the living body from bacteria and viruses that enter the human body or tumors that occur in the body. On the other hand, nutritional components necessary for a living body such as food can cause tolerance and can be actively taken into the living body. However, the immune system produces an allergic reaction as a result of an oversensitive response. Allergic diseases are roughly classified into four types, from type I to type IV, depending on the mechanism of action. Pollen allergic rhinitis, perennial rhinitis, bronchial asthma and the like are said to be type I allergy mediated by immunoglobulin E (IgE). Atopic dermatitis is also called a combined type of type I and type IV. In addition, type I, type II, and type IV are considered to be involved in the development of food allergies, and allergic diseases related to type I are increasing in Japan.

Type I allergic symptoms are triggered by antigen-presenting cells biasing T cell differentiation in response to the invasion of antigens such as pollen, and inducing IgE antibody production by B cells. Thereafter, when specific lymphocytes to which antibodies are bound are subjected to antigen invasion again, it is believed that allergic symptoms occur due to release of granules from these cells and release of inflammatory substances such as histamine. Therefore, for the purpose of preventing and treating allergies, there are a wide range of target action points from the initial antigen presentation stage to the final degranulation stage. Allergic foods have been proposed. A target for suppressing antigen presentation is a hawthorn hot water extract (see Patent Document 1). In addition, colostrum (refer to Patent Document 2), lactic acid bacteria KW3110 strain (refer to Patent Document 3), and seeds of Amaranth Hippochondriacus, which have improved the balance of helper T cell differentiation (Th1 / Th2 balance) as the action point. A large number of foods and drinks typified by (see Patent Document 4) have been proposed. Further, strictinin, a tea leaf component (see Patent Document 5), has been proposed as a material for suppressing the IgE class switch. These are intended to suppress allergic reaction by suppressing IgE antibody production as a result. However, none of them has been directly evaluated for the ability to produce IgE antibody in a living body, or even if it has been evaluated, no statistical evaluation has been performed, so it has not been confirmed whether the final purpose has been achieved. The lactic acid bacteria KW3110 strain has been statistically evaluated for its suppression of IgE production in the living body, and shows a significant effect. However, it is assumed that oral administration for a considerably long period of 100 days or more is usually more effective in mice. New material is anxious.
In addition, all of these food materials that are effective against allergies have a large effective dose compared to pharmaceutical products, and in fact, an effective dose is not ingested in the commercial product form.

  In addition, it is known that β-glucan has antiallergic activity, and it has also been proposed to use β-glucan in combination with lactic acid bacteria. For example, a culture containing β-glucan obtained by culturing a bacterium belonging to the genus Aureobasidium, an immunostimulant containing lactic acid bacteria (see Patent Document 6), a material containing β-glucan, An infection control composition containing a heat-treated body of lactic acid-producing bacteria (see Patent Document 7) has been proposed. However, in any case, β-glucan and lactic acid bacteria do not exhibit a synergistic effect, and the effect is not fully satisfactory.

JP 09-143090 A JP 2006-96752 A JP 2005-137357 A JP 2003-63972 A JP 2005-198664 A Japanese Patent Laid-Open No. 2005-220065 JP 2003-40785 A

  An object of the present invention is to provide an antiallergic agent capable of synergistically enhancing the antiallergic activity of β-glucan and exhibiting sufficient effects even with a small amount of ingestion, and an antiallergic food containing the antiallergic agent There is.

  It is thought that the anti-allergic activity of β-glucan involves a mechanism that modifies the immune function originally held by the living body and suppresses an excessive immune response. On the other hand, in type I allergy, it is considered that the helper T cells among the lymphocytes are predominantly in the Th2 type cell population, and the Th2 cytokine concentration such as interleukin 4 is increased in vivo. When β-glucan is taken orally into a living body, it is recognized by immune cells such as dendritic cells, macrophage cells, and M cells in the intestinal tract, and secretes inflammatory cytokines such as interleukin-12. It is believed that the cell population differentiates into Th1-type helper T cells that produce cytokines such as interferon γ. It is also known that interleukin 12 has an action of suppressing differentiation of Th2-type helper T cells. When a patient with an allergic disease ingests β-glucan, interleukin 12 production is induced as a result, and the helper T cell population of the Th2 subset is reduced, which is considered to suppress the allergic disease. Although the action of β-glucan is known to enhance foreign body phagocytosis and the like, the mechanism that acts on allergic diseases seems to be the action in which the production of interleukin 12 is most closely involved.

Therefore, the present inventor has conducted intensive studies focusing on substances that enhance the interleukin 12 production-inducing action.
The present inventor first constructed an in vitro evaluation system that is an index of β-glucan anti-allergic activity, and searched for an increase in activity by adding a sample in the presence of β-glucan in the evaluation system. Went.
As a result, it was found that specific lactic acid bacteria have an activity to increase the anti-allergic activity of β-glucan synergistically, and the present invention has been completed.

That is, this invention solves the said subject by providing the anti-allergic agent characterized by including ( beta) glucan and Pediococcus pentosaceus MIX strain | stump | stock (accession number FERM P-21300). is there.

The anti-allergic agent of the present invention has a low-volume β-glucan compared to the conventional one, and can obtain an effect comparable to a high-capacity anti-allergic action (prevention and treatment effect of allergic diseases). Therefore, the antiallergic food of the present invention containing the antiallergic agent of the present invention can take an effective dose that exhibits a sufficient effect in the commercial product form.
In addition, since the additive component used in the present invention is a lactic acid bacterium derived from a natural food material with experience of eating, the antiallergic agent and antiallergic food of the present invention are also excellent in safety.

  The β-glucan used in the present invention is not particularly limited as long as it can be used in foods, and β-glucan conventionally used in antiallergic agents can be used. Derived β-glucan is preferred.

Lactic acid bacteria belonging to Pediococcus pentosaceus used in the present invention have a high IL-12 (interleukin 12) production-inducing effect, and this action provides an antiallergic action and also exhibits a synergistic effect with β-glucan. , Synergistically enhance the anti-allergic activity of β-glucan.
As the lactic acid bacteria belonging to the above-mentioned Pediococcus pentosaceus, Pediococcus pentosaceus MIX strain is preferable. The strain has been deposited with the National Institute of Advanced Industrial Science and Technology, the Patent Biological Deposit Center, and the deposit number is FERM P-21300.

The mycological properties of the lactic acid bacterium Pediococcus pentosaceus MIX are shown below.
Bacterial morphology when cultured for 18 hours at 30 ° C. using MRS liquid medium (DIFCO) (1) Bacterial morphology Cocci (2) Gram staining Positive (3) Motility None (4) Spore None (5) Catalase None (6) facultative anaerobic (7) metabolism of glucose Convert to 50% or more lactic acid (8) Growth temperature range Growth is observed at 15 ° C, 30 ° C and 35 ° C, but growth is not observed at 45 ° C ( 9) Lactic acid fermentation Homo type (10) Light application of lactic acid DL
(11) Carbohydrate fermentability Glycerol is negative, D-arabinose is negative, L-arabinose is positive, ribose is positive, D-xylose is positive, galactose is positive, glucose is positive, fructose is positive, mannose is positive, rhamnose Is negative, mannitol is negative, sorbitol is negative, α-methyl D-glucoside is negative, amygdalin is positive, esculin is positive, salicin is positive, cellobiose is positive, maltose is positive, lactose is positive, melibiose is negative, sucrose is negative , Positive for trehalose, negative for inulin, negative for melezitose, positive for raffinose, negative for starch, positive for gluconic acid

  Lactic acid bacteria Pediococcus pentosaceus MIX strain is isolated from a natural food material (sake moromi) with abundant food experience and can be safely used as an antiallergic agent.

  Lactic acid bacteria Pediococcus pentosaceus MIX strains may be mixed with the culture as it is as an antiallergic agent, or the cells are collected from the culture, concentrated and dried as necessary, and then combined with the antiallergic agent. May be. Moreover, you may mix | blend the various animal and plant substances fermented using lactic-acid-bacteria Pediococcus pentosaceus MIX strain | stump | stock in the antiallergic agent as said lactic acid bacteria.

The antiallergic agent of the present invention preferably contains β-glucan so that the daily intake of β-glucan per adult is 1 to 10,000 mg, particularly 100 to 2,000 mg.
Further, the content ratios of β-glucan and lactic acid bacteria of the antiallergic agent of the present invention are not particularly limited, but preferable content ratios are as follows.
β-glucan 1-99% by mass and lactic acid bacteria 99-1% by mass, particularly preferably β-glucan 50-90% by mass and lactic acid bacteria 50-10% by mass, with the total content of β-glucan and lactic acid bacteria being 100% by mass.

  The anti-allergic agent of the present invention was formulated by blending the β-glucan, the lactic acid bacteria, and various pharmaceutically acceptable carriers, excipients, other additives, and other components as necessary. Is. The dosage form of the antiallergic agent of the present invention is an oral preparation such as a tablet, powder, granule or capsule, and can be formulated by a conventional method. As other components, other antiallergic components, anti-inflammatory drugs, various vitamins, herbal medicines, minerals, and the like can be appropriately blended.

  The antiallergic food of the present invention is a food containing the above-described antiallergic agent of the present invention. Examples of foods that contain the antiallergic agent of the present invention include confectionery such as breads, noodles, tablets, and candy, and beverages such as soft drinks, juices, and nutritional drinks, but are not limited thereto. is not. The timing of addition to food is not particularly limited, and may be added during the production process of the food, or may be added to the produced food.

  The content of the antiallergic agent in the antiallergic food of the present invention is not particularly limited, but the daily intake of β-glucan per adult is 1 to 10,000 mg, particularly 100 to 2,000 mg. Thus, it is preferable to contain the antiallergic agent of the present invention.

  EXAMPLES The present invention will be specifically described below with reference to examples. However, the present invention is not limited to these examples, and various embodiments are possible. The present invention is based on the idea disclosed in the present specification and drawings. It is to be understood that all embodiments are included as long as they are followed.

(Lactic acid bacteria used)
Among lactic acid bacteria isolated from plant materials with abundant Japanese dietary experience, such as pickled pickles, pickled bitter gourd, pickled kimchi, yeast, etc. 5 typical strains shown in Table 1 were selected on the basis of the fact that a plurality of strains were found and that the growth was high in a normal medium. MIX shown in Table 1 is Pediococcus pentosaceus MIX strain which is a lactic acid bacterium used in the present invention. RIE in the table is Leuconostoc mesenteroides RIE strain, which is deposited under the accession number FERM P-21110 at the National Institute of Advanced Industrial Science and Technology and the Patent Biological Depositary Center. Similarly, AYA is a Lactobacillus plantarum AYA strain, which is deposited under the accession number FERM P-21106 at the National Institute of Advanced Industrial Science and Technology and the Patent Biological Depositary Center.

(Preparation of lactic acid bacteria sample)
Each lactic acid strain shown in Table 1 cultured for 48 hours at 37 ° C. in MRS medium containing 10 μg / ml cycloheximide was collected by centrifugation, washed 3 times with sterile water, and then suspended in sterile water. And autoclaving at 121 ° C. for 30 minutes. These were freeze-dried to obtain lactic acid bacteria samples.

Example 1 (Evaluation of lactic acid bacteria using THP-1 cells)
About each lactic acid strain shown in Table 1, the response with respect to a human was evaluated as follows with a THP-1 cell.
When THP-1 cells are undifferentiated, IL-12 production is not induced, and β-glucan responsiveness is not confirmed. Since the response of β-glucan was confirmed by the addition of LPS (lipopolysaccharide), the LPS addition system was used. In order to confirm the optimal amount of LPS added, the LPS concentration-dependent effect was examined. The result is shown in FIG. From this, it was decided to use a system to which 20 ng / ml LPS was added, which most reflects the responsiveness of β-glucan.
Next, for the purpose of confirming the optimal addition amount of β-glucan, the concentration dependency of β-glucan in the presence or absence of peptidoglycan as a positive control was confirmed. As a result, as shown in FIG. 2, concentration dependency was confirmed regardless of the presence or absence of peptidoglycan. However, when peptidoglycan was present, the rise of the concentration dependency curve was quicker, and the peptidoglycan was compared to β-glucan. A synergistic effect was confirmed. The optimum maximum glucan concentration was 20 μg / ml, which is the Half maximum in the presence of peptidoglycan.

Medium preparation Basal medium: SIGMA RPMI1640, Code No .; R0883, Lot. No .: 80K2350 was used as the basal medium.
Additives: Fetal calf serum (FCS) as additive; ICN Cellect Gold; Lot. 500 ml) Na-Pyruvate in RPMI1640 pH 7.2 prepared with NaOH and then filter sterilized Preparation method: Prepared as follows.
425 ml of RPMI1640 was dispensed into a 500 ml bottle ↓ 50 ml of inactivated FCS was added ↓ 25 ml of 20 times added solution was added ↓ 500 ml of 50 mM β-mercaptoethnol (in RPMI1640) was added ↓ Mixed storage was performed at 4 ° C.
Thaw the THP-1 cell stock. Store the stock cells stored in liquid nitrogen in 37 ° C warm water. ↓ Add the total volume of cell suspension (1 ml) to a 50 ml centrifuge tube containing 9 ml of medium. ↓ Centrifuge at 1240 rpm for 5 minutes. ↓ Remove supernatant ↓ + Suspend cells in 25 ml FCS medium ↓ Incubator with 75 cm ³ flask
Culture conditions: 37 ° C, 5% CO ₂ , 100% wet conditions

Cell count
(1) 10 ml of trypan blue solution was placed in a microcentrifuge tube, and an equal volume of cell suspension was added and pipetted.
(2) The suspension was placed on a blood cell counter and the number of viable cells was counted.
The number of cells was calculated by the following calculation.
Cell number concentration = number of cells per compartment x dilution rate (2 times) x 10 ⁴ cells / ml
Passage
(1) Transfer the appropriate passage volume of the culture solution to a new flask and add a new medium.
(2) Cultivation at 37 ° C under 5% CO ₂ Cell storage solution for cryopreservation: manufactured by Nippon Zenyaku Kogyo Co., Ltd., Cell Banker Code No., ZCB-101, Lot No., 309180
Preparation of cells 1. Centrifuge the cells grown on the mid log face (1240 rpm, 5 min) and remove the supernatant. Suspend cells in a cell banker.
3. Dispense cell suspension into cryotube.
4). Place the cryotube in a slow cooling container and move the container to -80 ° C for 5.2 to 3 days, then transfer it from the slow cooling container to the storage container and store at -80 ° C.

IL-12 production inducing additive using THP-1 cells:
β-glucan; LPS sterilized by lyophilization after suspension of stock solution concentration 10 mg / ml EtOH; stock solution 20 mg / ml
Lactic acid bacteria: Lactic acid bacteria samples were suspended in RPMI 1640 and used at 1 mg / ml after weighing.
Suspend THP-1 cells in medium (10% FCS RPMI1640 medium with Glucose, HEPES, Pyruvate, 2-mercaptoethanol) to 3 × 10 ⁵ cells / ml ↓
Add 25 μl of 20 μg / ml LPS and 100 μl of 10 mg / ml βglucan to 50 ml cell suspension ↓
Dispense the suspension into a 24-well plate at 1.0 ml / well each ↓
Add 10 μg / ml lactic acid bacteria sample (1 mg / ml 10 μl).
↓
24 hr 37 ℃ 5% CO ₂ culture atmosphere ↓
0.5 ml of the culture supernatant was sampled and subjected to hIL-12 p40 ELISA.
Table 2 shows the quantification results of IL-12 by ELISA.

Example 2 (Evaluation of lactic acid bacteria using J774.1 cells)
About each lactic acid strain shown in Table 1, the response with respect to a mouse | mouth was evaluated as follows with J774.1 cell.
IL-12 production induction additive using J774.1 cells:
Lactic acid bacteria: Lactic acid bacteria samples were suspended in RPMI 1640 and used at 1 mg / ml after weighing.
Suspend J774.1 cells in medium (10% FCS RPMI1640 medium) at 3 × 10 ⁵ cells / ml ↓
Dispense the suspension into a 24-well plate at 1.0 ml / well each ↓
Add 10 μg / ml lactic acid bacteria sample (1 mg / ml 10 μl).
↓
24 hr 37 ℃ 5% CO ₂ culture atmosphere ↓
0.5 ml of the culture supernatant was sampled and subjected to hIL-12 p40 ELISA.
Table 2 shows the quantification results of IL-12 by ELISA.

Details of the quantification of IL-12 by ELISA are as follows.
Use Plate: Nunc MaxiSorb
Primary antibody:
Anti-human IL-12 p40 antibody; Mouse monoclonal; R & D systems Inc. MAB609; Lot. ZD054121
For mouse IL-12; Monoclonal Anti-mouse IL-12 / IL-23 p40 Antibody R & D systems Inc. MAB499; LotAGN05
Concentration: Stock solution 500 μg / ml in PBS (Aliquot; 20 μl / tube) 325 times diluted with PBS Wash buffer used: 0.05% Tween20 in PBS
Reaction buffer: 0.1% Block A, PBS 0.01% NaN ₃ or 0.1% Block A, PBS (for HRP-Streptavidin)
Blocking buffer: × 4 Block A (YUKIJIRUSHI) with 0.01% NaN ₃
Standard substance for human IL-12; hIL-12 p40 R & D systems Inc .; Lot. JB054111 Conc .; 100 ng / ml
Dilution: 1st; × 10 (70 μl + 630μl) with reaction buffer → 10000 pg / ml
2nd; × 3.16 (200μl + 432 μl) with reaction buffer → 3162pg / ml
Each dilution × 10 (50μl + 450μl) with reaction buffer → 316, 100, 31.6pg / ml
For mouse IL-12; mIL-12 / IL-23 p40 Homodimer R & D systems Inc .; 499ML
Secondary antibody:
For human IL-12; Biotinylated anti human IL-12 antibody; Mouse monoclonal; ENDOGEN Inc. M-121-B; Lot. DB53895
Concentration: Stock solution 500 μg / ml in 0.1% Block A, TBS (Aliquot; 10 μl) Diluted 650 times with reaction buffer for mouse IL-12; Biotinylated Anti-mouse IL-12 / IL-23 p40 Antibody
R & D Systems. BAF499; Lot. AKD11
Concentration: Stock solution 500μg / ml in 0.1% Block A, TBS (Aliquot; 10μl) Diluted 650 times with reaction buffer
Streptavidin; HRP conjugated Streptavidin; ENDOGEN N100 Lot.EB60509
Concentration: Stock solution 1.25mg / ml 0.1% Block A, PBS NaN ₃ free HRP diluted 10,000 times
HRP substrate: TMB Moss Inc.
Reaction stop solution: 2N H ₂ SO ₄
Equipment used: Beckman Coulter plate washer
Plate Reader: ： ARVO MAX (Perkin Elmer.)

The same primary antibody for humans and mice is dispensed on a plate (100 μl / well), incubated at 4 ° C for 20 hours ↓
Remove the solution, dispense blocking buffer (250 ml / well), and incubate at 25 ° C for 1 hr ↓
Wash plate 3 times with wash buffer ↓
Dispense Standard hIL-12 p40 and sample (culture supernatant) (100 μl / well) and incubate at 25 ℃ for 2 hr ↓
Wash plate 3 times with wash buffer ↓
Dispense primary antibody to plate (100 μl / well), incubate for 2 hr at 25 ℃ ↓
Wash plate 3 times with wash buffer ↓
Dispense HRP-streptavidin (100 μl / well), incubate at 25 ℃ for 30 min ↓
Wash plate 3 times with wash buffer ↓
Dispense TMB substrate (100 μl / well) at 25 ° C, incubate for 5-10 min ↓
Add stop solution (100 μl / well)
↓
450nm absorbance measurement

Example 3 (Concentration dependence test of Pediococcus pentosaceus MIX strain)
About the Pediococcus pentosaceus MIX strain | stump | stock, the addition density | concentration was changed and IL-12 production induction using the THP-1 cell as described in Example 1 was implemented in presence of β-glucan and absence. The result is shown in FIG. The curve gradient changed depending on the presence or absence of β-glucan, and a synergistic effect on β-glucan was confirmed. On the other hand, a concentration dependence test was also performed in which the addition concentration of Pediococcus pentosaceus MIX strain was fixed and the concentration of β-glucan was changed. The result is shown in FIG. From this result, a synergistic effect of β-glucan on Pediococcus pentosaceus MIX strain was also confirmed.
J774.1 cells were also subjected to a concentration dependency test of Pediococcus pentosaceus MIX strain. The result is shown in FIG.

Example 4
(Evaluation of the effects of feeding lactic acid bacteria and β-glucan on the suppression of nasal discharge in allergic rhinitis model guinea pigs)
1. Animal Experiment Schedule 20 male guinea pigs aged 4 weeks were weighed and divided into groups so that the average body weight and variance of 10 animals in each group were approximately equal by random sampling. After grouping, solid feed LRC4 (manufactured by Oriental Yeast Co., Ltd.) within 5 months after production was placed in a feeder and allowed to ingest freely. β-glucan 500 mg / kg / day + Lactic acid bacteria Pediococcus pentosaceus MIX strain 500 mg / kg / day was orally administered twice a day from the next day for 28 consecutive days.
In addition, a thin-shaft swab soaked in a 10% TDI solution was applied to both nasal vestibules of all guinea pigs for 10 seconds, and this operation was repeated once a day for 5 days from the next day of grouping for sensitization. I did it. Thereafter, breeding was continued without sensitization, and nasal discharge was induced 4 weeks after the final sensitization. Nasal discharge was induced by applying a thin-shaft swab soaked in 5% TDI solution in contact with both nasal vestibules for 10 seconds.
2. Measurement of nasal discharge The nasal discharge for 15 minutes from the start of nasal discharge was absorbed into absorbent cotton, placed in a sealed microtube, the weight thereof was measured, and the average value of the amount of nasal discharge for each group of 10 animals was calculated. Further, the value obtained by subtracting the average value of the nasal discharge of the β-glucan and lactic acid bacteria MIX strain administration group from the average value of the nasal discharge of the control group was divided by the average value of the nasal discharge of the control group as the nasal discharge suppression value.
3. Experimental results As shown in Table 3, the amount of nasal discharge associated with the development of allergic rhinitis was significant in the group administered with β-glucan 500 mg / kg + lactic acid bacteria Pediococcus pentosaceus MIX strain 500 mg / kg (risk rate 1). Anti-allergic agent containing β-glucan and Pediococcus pentosaceus MIX strain has the effect of alleviating allergic symptoms by oral administration, that is, exhibits anti-allergic action It was confirmed.

It is a figure which shows the response of (beta) glucan by addition of LPS. It is a figure which shows the density | concentration dependence of (beta) glucan in the presence or absence of peptidoglycan. It is a figure which shows the result of the density | concentration dependence test (when changing the addition density | concentration of a MIX strain | stump | stock) of the Pediococcus pentosaceus MIX strain | stump | stock about the THP-1 cell in Example 3. It is a figure which shows the result of the density | concentration dependence test (when changing the addition density | concentration of (beta) glucan) of the Pediococcus pentosaceus MIX strain | stump | stock about the THP-1 cell in Example 3. It is a figure which shows the result of the density | concentration dependence test (when changing the addition density | concentration of a MIX strain | stump | stock) of the Pediococcus pentosaceus MIX strain | stump | stock about J774.1 cell in Example 3. FIG.

Claims (2)

An antiallergic agent comprising β-glucan and Pediococcus pentosaceus MIX strain (Accession No. FERM P-21300) .   The antiallergic agent according to claim 1, wherein the β-glucan is yeast-derived β-glucan.

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