JP2008099632A - Lactic acid bacterium having immunostimulatory activity/anti-allergic activity - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a lactic acid bacterium of the genus Lactobacillus that is useful for various uses such as food and beverage, feed, medicine, cosmetic, etc., and exhibits an immunologically high function. <P>SOLUTION: The lactic acid bacterium of the genus Lactobacillus has immunostimulatory activity/anti-allergic activity. The lactic acid bacterium is selected from the group consisting of Lactobacillus paracasei NHH2 strain (NITE P-262), Lactobacillus plantarum NHT4 strain (NITE P-264), Lactobacillus gasseri NHMT2 strain (NITE P-265) and Lactobacillus salivarius subsp. salivarius NHMF1 strain (NITE P-263). The composition having an immunostimulatory activity/anti-allergic activity comprises the lactic acid bacterium as an active ingredient. The food and beverage, the feed, the cosmetic or the medicine contains the lactic acid bacterium or the composition. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a lactic acid bacterium having an immunostimulatory action / antiallergic action, and more specifically to a novel Lactobacillus lactic acid bacterium having an immunostimulatory action / antiallergic action and use thereof.

Lactic acid bacteria are contained in feces and various fermented foods, and have been used for a long time as fermented foods such as yogurt. In recent years, utilization as a raw material for various functional foods has been promoted, and in particular, Lactobacillus lactic acid bacteria are positioned as representatives of probiotics.
Patent Documents 1 and 2 describe that the cells of various lactic acid bacteria and their cytoplasmic fractions have an immunostimulatory action. Patent Document 3 describes use of Lactobacillus lactic acid bacteria as an antiallergic agent. However, there has been a demand for lactic acid bacteria that exhibit higher immune functions such as an immunostimulatory action and an antiallergic action than these conventionally reported lactic acid bacteria.

JP-A-5-252900 JP-A-7-228536 Japanese Patent Laid-Open No. 2004-26729

  An object of the present invention is to provide a Lactobacillus genus lactic acid bacterium that exhibits an immunologically high function and is useful for various uses such as foods and drinks, feeds, pharmaceuticals, and cosmetics.

  As a result of intensive studies to achieve the above-mentioned object, the inventors of the present invention have a higher immunostimulatory effect than the conventionally reported Lactobacillus lactic acid bacteria and at the same time have a high antiallergic effect. I found lactic acid bacteria. That is, it was confirmed that a novel Lactobacillus lactic acid bacterium isolated from human feces exerted an immunostimulatory effect on human peripheral blood lymphocytes and an antiallergic effect on human basophils. . The present invention is based on such knowledge.

That is, the present invention provides the following [1] to [4].
[1] Lactobacillus genus lactic acid bacteria having immunostimulatory action and antiallergic action.
[2] Lactobacillus paracasei NHH2 strain (NITE P-262), Lactobacillus plantarum NHT4 strain (NITE P-264), Lactobacillus gasseri NHMT2 strain (NITE P-265), and Lactobacillus salivaius sub [1] The lactic acid bacterium according to [1], which is selected from the group consisting of species Salivarius NHMF1 strain (NITE P-263).
[3] A composition having an immunostimulatory action and an antiallergic action, comprising the lactic acid bacterium according to [1] or [2] as an active ingredient.
[4] A food, drink, feed, cosmetic or pharmaceutical comprising the lactic acid bacterium according to [1] or [2] or the composition according to [3].

  ADVANTAGE OF THE INVENTION According to this invention, the Lactobacillus genus lactic acid bacteria which exhibit a high immunostimulatory action and antiallergic action in a living body are provided. Such lactic acid bacteria are useful in various applications such as foods and drinks, feeds, cosmetics, and pharmaceuticals.

  The lactic acid bacterium of the present invention exhibits an immunostimulatory action / antiallergic action. The immunostimulatory action (adjuvant action) means an action that activates the immune function of the living body. Specifically, it means an effect of enhancing production of antigen-specific antibodies and cytokines after antigen sensitization in lymphocytes such as mononuclear cells, and an effect of enhancing cytokine production when antigens are not sensitized. Here, there are various types of cytokines, and examples include those classified as inflammatory cytokines. Inflammatory cytokines mean cytokines that cause inflammation in a living body, such as TNF, IL-6, IL-1β, and IL-8. In the present invention, when it has an immunostimulatory effect, it may be anything that enhances the production of at least one of these cytokines.

On the other hand, the antiallergic action means an action that suppresses the allergic reaction of the living body. Specifically, the action of suppressing the intracellular accumulation and extracellular release of histamine in immune cells such as basophil cells, and the simple action. It means an action to enhance cytokine production after sensitizing and non-sensitizing lymphocytes such as nuclei. Examples of the cytokine used herein include T _H 1 cytokine. T _H 1 cytokine is a cytokine produced by T _H 1 cells, such as IL-12p70 and IFNγ.

  The lactic acid bacteria of this invention should just exhibit at least one of an immunostimulatory effect and an antiallergic effect, and may exhibit both. In addition, the lactic acid bacterium of the present invention may be a live cell or a dead cell. Dead cells can be prepared by heating (for example, about 20 minutes to 1 hour at 90 to 120 ° C.), lyophilization, or the like.

  The lactic acid bacteria of the present invention belong to the genus Lactobacillus. Examples of Lactobacillus microorganisms include Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus gasseri, Lactobacillus salivarius, Subspecies salivarius.

  The lactic acid bacteria of the present invention can be collected from feces, fermented milk products, pickles and the like of various mammals including humans. For example, an appropriate amount of these is planted in a medium such as MRS medium (for example, 37 ° C. for 48 hours in the case of MRS medium), and the culture is collected by centrifugation, etc., by a conventional method. It can be obtained by purification and lyophilization.

  The present inventors are Lactobacillus paracasei NHH2 strain, Lactobacillus plantarum NHT4 strain, Lactobacillus gasseri NHMT2 strain, Lactobacillus salivarius subtilis as Lactobacillus lactic acid bacteria exhibiting immunostimulatory action and antiallergic action. The S. sarivarius NHMF1 strain was isolated from a natural product and newly identified from the results of bacteriological properties, 16S-rDNA sequence and the like.

Specifically, human feces were appropriately diluted, and modified LBS agar medium (LBS agar medium (Pancreatic Digest of Casein 10.0 g, Yeast Extract 5.0 g, Monopotassum Phosphate 6.0 g, Ammonium Citrate 2.0 g, 0.0 g, 2.0 g) , Polysorbate80 1.0g, Sodium Acetate Hydrate 25.0g , Magnesium Sulfate 0.575g, Manganese Sulfate 0.12g, Ferrous Sulfate 0.034g, Agar 15.0g) 84g, Lab-lemco powder 8g, Sodium acetate · 3H 2 O 15 g, purified water 1000 ml, acetic a cid (3.7 ml). The modified LBS medium was prepared as a flat plate by dissolving each component other than the acidic acid while heating and then cooling to 50 ° C. and adding the acidic acid (adjusted to pH 4.3). After culturing at 37 ° C. for 72 hours under anaerobic conditions, 4 types of NHH2, NHT4, NHMT2 and NHMF1 were isolated from the formed colonies. Table 1 shows the bacteriological properties of each isolated bacterium.

  In addition to the above bacteriological properties, the results of 16S-rDNA sequence and other genetic studies were finally classified into the following four types.

1. Lactobacillus paracasei NHH2 strain DNA was extracted from the NHH2 strain, and 16S-rDNA sequencing was performed using primers 16S-27f (SEQ ID NO: 9) and 16S-1525r (SEQ ID NO: 10). The procedure and conditions of the 16S-rDNA sequence are as follows. First, DNA was extracted from the bacterial cells, and PCR was performed using 16S-27f and 16S-1525r primers. PCR conditions were 94 ° C. · 2 minutes, followed by 30 cycles of 94 ° C. · 1 minute−50 ° C. · 1 minute−68 ° C. · 1 minute 30 seconds, and finally 68 ° C. · 7 minutes. Next, the obtained PCR product was purified using QIAquick PCR Purification Kit (QIAGEN). Then, PCR was performed using BigDye (registered trademark) Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) using purified DNA as a template and primers of 16S-27f and 16S-1525r. PCR conditions were 96 ° C. for 2 minutes, followed by 30 cycles of 96 ° C., 10 seconds-50 ° C., 5 seconds-60 ° C., 4 minutes. After purification of the PCR product thus obtained, the sequence was decoded with a DNA sequencer. The decoding sequence was homology searched with BLASTN.

As a result of the 16S-rDNA sequence, 16S-rDNA (SEQ ID NO: 1: NHH2-forward, SEQ ID NO: 2: NHH2-reverse) consisting of the base sequences described in SEQ ID NO: 1 and SEQ ID NO: 2 was obtained. As a result of homology search between such a base sequence and 16S rDNA of various Lactobacillus lactic acid bacteria, L. casei, L. paracasei, and L. rhamnosus (L. rhamnosus) I thought there were three possibilities. Therefore, L. J. et al. H. Ward and M.M. J. et al. Based on Timemins; Letters in Applied Microbiology 1999, 29, 90-92, PCR using primers specific to each of these three types of Lactobacillus was performed to confirm the degree of amplification. PCR is performed by L. paracasei detection primers (SEQ ID NO: 13 and SEQ ID NO: 14), L. casei detection primer (SEQ ID NO: 15 and SEQ ID NO: 16), L. Using the rhamnosus detection primers (SEQ ID NO: 17 and SEQ ID NO: 18), a reaction solution having the following composition was used: 5X buffer (promega) 10 μl, NTPs 4 μl, MgCl _{2 4} μl, template 1 μl, Primer 1 μl each, Taq poly 0 .1 μl, sterile DW to 50 μl. PCR conditions are 94 ° C for 3 minutes, then 45 ° C for 45 seconds-72 ° C for 1 minute, followed by a cycle of 94 ° C for 45 seconds-45 ° C for 45 seconds-72 ° C for 1 minute. For 30 times, followed by 94 ° C. for 45 seconds, 45 ° C. for 45 seconds, and 72 ° C. for 5 minutes.

  As a result, since amplification was confirmed only in the case of a primer specific for L. paracasei, this strain was identified as a kind of Lactobacillus paracasei.

  Lactobacillus paracasei NHH2 strain has been deposited on September 26, 2006 at the Japan Patent Evaluation Microorganism Depositary Center for Product Evaluation Technology (2-5-8, Kazusa Kamashi, Kisarazu, Chiba, Japan) The accession number is NITE P-262.

2. Lactobacillus plantarum NHT4 strain DNA was extracted from the NHT4 strain and subjected to 16S-rDNA sequencing in the same manner as the NHH2 strain described above. No. 7: NHT4-forward, SEQ ID NO. 8: NHT4-reverse). A homology search was performed between the nucleotide sequence and 16S-rDNA of various Lactobacillus lactic acid bacteria. As a result, it was identified as L. plantarum (L. plantarum).

  Lactobacillus plantarum NHT4 strain has been deposited on September 26, 2006 at the Japan Patent Evaluation Microorganism Depositary Center (2-5-8 Kazusa-Kamashita, Kisarazu-shi, Chiba, Japan) The accession number is NITE P-264.

3. Lactobacillus gasseri NHMT2 strain DNA was extracted from the NHMT2 strain and subjected to 16S-rDNA sequencing in the same manner as the NHH2 strain described above. As a result, 16S-rDNA consisting of the base sequences described in SEQ ID NO: 3 and SEQ ID NO: 4 (SEQ ID NO: 3: NHMT2-forward, SEQ ID NO: 4: NHMT2- reverse). A homology search was performed between the nucleotide sequence and 16S-rDNA of various Lactobacillus lactic acid bacteria. As a result, it was identified as Lactobacillus gasseri (L. gasseri).

  Lactobacillus gasseri NHMT2 strain has been deposited on September 26, 2006 at the Japan Patent Evaluation Microorganism Depositary Center for Product Evaluation Technology (2-5-8, Kazusa Kamashi, Kisarazu, Chiba, Japan) The accession number is NITE P-265.

4). Lactobacillus salivarius subspecies salivarius NHMF1 strain DNA was extracted from the NHMF1 strain and subjected to 16S-rDNA sequencing in the same manner as the NHH2 strain described above. As a result, 16S consisting of the nucleotide sequences set forth in SEQ ID NO: 5 and SEQ ID NO: 6 -RDNA (SEQ ID NO: 5: NHMF1-forward, SEQ ID NO: 6: NHMF1-reverse) was obtained. A homology search was performed between the nucleotide sequence and 16S-rDNA of various Lactobacillus lactic acid bacteria. As a result, it was inferred to be classified into L. salivarius. However, when the salicin (sugar) metabolism was confirmed using the “api50CH” kit manufactured by Biomelieu Japan, according to the instructions attached to the kit, it was negative for salicin metabolism. Therefore, Lactobacillus salivarius subspecies Identified as L. salivarius subsp. Salivarius.

  Lactobacillus salivarius sub-species salivarius NHMF1 strain is dated September 26, 2006 at the Japan Institute for Product Evaluation Technology Patent Microorganism Depositary Center (2-5-8 Kazusa Kamashita, Kisarazu, Chiba, Japan) It has been deposited and its deposit number is NITE P-263.

  The lactic acid bacteria of the present invention can be used as an active ingredient of the composition of the present invention that exhibits an immunostimulatory action / antiallergic action. In the composition of the present invention, the form of the lactic acid bacterium of the present invention can be determined according to the application on the condition that it exhibits the action as an active ingredient. For example, live cells and dead cells may be used, or a culture containing a culture medium or a purified product of the culture may be used.

  In the composition of the present invention, the above-mentioned lactic acid bacteria are combined with other components that can constitute the composition together with the lactic acid bacteria without impairing the immunostimulatory action and antiallergic action exhibited by the lactic acid bacteria of the present invention. Examples of such other components include nutrient components for maintaining and growing the function of the lactic acid bacteria of the present invention (for example, carbon source, nitrogen source, vitamins, minerals, etc.).

  Such a lactic acid bacterium of the present invention can be used as it is or in the form of the composition of the present invention as described above, as a food or drink, feed, cosmetics, pharmaceutical or the like or as a raw material thereof. In this case, the composition and lactic acid bacteria of the present invention can be used in combination with other components according to each use, for example, in the case of use taken orally, in combination with protein, lipid, carbohydrate, dietary fiber, vitamin and the like. . It can also be used in combination with an appropriate carrier such as an excipient, diluent, filler, extender, binder, disintegrant, surfactant, lubricant and the like. Particularly in the case of pharmaceuticals, it is generally used with a pharmacologically acceptable carrier among the above-mentioned carriers. Moreover, a coloring agent, a preservative, a stabilizer, a fragrance | flavor, a sweetener, etc. can also be added as needed.

  Examples of the foods and drinks include fermented milk, lactic acid bacteria beverages, health foods, lactic acid bacteria-containing foods such as bread and confectionery, soft drinks, food additives, pickles, and meat processed products such as hams and sausages. Examples of the feed include animal feeds such as laboratory animals, livestock, pets, and display animals. The form of pharmaceutical preparation is not particularly limited. Examples of forms suitable for oral administration include forms such as liquids, capsules, granules, tablets, pills and powders. Moreover, the form of external medicines, such as an ointment, may be sufficient. As cosmetics, for example, lotion, cream, milky lotion, face wash and the like can be mentioned.

  The blending ratio of the lactic acid bacterium of the present invention or the composition in the food, drink, cosmetics, and pharmaceuticals of the present invention is generally adjusted according to the type, form, and situation (age, health condition, physique, etc.) of the lactic acid bacterium used. Although it is difficult to make it, it can usually be in a range of 10 mg / day to 10 g / day in terms of daily lactic acid bacteria intake.

Hereinafter, the present invention will be described in detail with reference to examples.
〔reagent〕
Human interleukin-2 (IL-2) was purchased from Genzyme (Cambridge, MA) and human interleukin-4 (IL-4) from PEPRO TECH (Rocky Hill, NJ). L-Leucyl-L-leucine methyl ester (LLME) was purchased from BACHEM (Torrance, CA), and muramyl dipeptide (MDP) was purchased from CHEMICON (Temecula, CA).

[Lactic acid bacteria]
L31: Lactobacillus paracasei NHH2 strain (NITE P-262)
L32: Lactobacillus plantarum NHT4 strain (NITE P-264)
L33: Lactobacillus gasseri NHMT2 strain (NITE P-265)
L34: Lactobacillus salivarius subsp. Salivarius (Lactobacillus salivarius subsp. Salrivarius) NHMF1 strain (NITE P-263)
In the following examples, the above four types of bacteria may be represented by abbreviations L31, L32, L33, and L34, respectively.

Example 1 [Immune Stimulating Action of Lactic Acid Bacteria (1) Effect of Enhancement of Antigen-Specific Antibody Production of Lactic Acid Bacteria]
When sensitizing human peripheral blood lymphocytes by in vitro immunization, the presence of freeze-dried heat-killed lactic acid bacteria can enhance antigen sensitization and increase antigen-specific antibody-producing cells. It was verified by the law.

(Separation of human peripheral blood lymphocytes)
Blood was collected from a healthy person using a vacuum blood collection tube containing heparin, diluted with an equal volume of PBS, and mixed well. Lymphocyte Separation Medium (LSM, Organon Teknika, Durham, NC) is dispensed in advance in a 50 ml centrifuge tube, and 25 ml of diluted blood is gently added so as not to disturb the interface between LSM and blood. Layered. Subsequently, it was centrifuged at 400 × g for 30 minutes at room temperature. Peripheral blood mononuclear cell layers (T cells, B cells, NK cells and monocytes) floating in a white band at the boundary between LSM and plasma were collected using a Pasteur pipette in a 50 ml centrifuge tube coated with serum. Three times more ERDF medium (Invitrogen, Carlsbad, CA) of the collected mononuclear cell suspension was added, mixed well, and centrifuged at 200 × g for 5 minutes. The resulting cell pellet was resuspended in ERDF medium and centrifuged at 200 × g for 5 minutes. This washing operation was repeated 2-3 times until there was no platelet contamination.

The number of cells was measured with an automatic blood cell counter F-520 (Sysmex, Hygo, Japan).
In addition, blood collection was performed with the consent of the donor based on the Helsinki Accord and guidelines established by the Kyushu University Graduate School of Agricultural Research Ethics Committee.

(L-Leucyl-L-leucine methyl ester (LLME) processing)
After suspending peripheral blood mononuclear cells in ERDF medium to a cell density of 1 × 10 ⁷ cells / ml, LLME is added to a final concentration of 0.25 mM, and 20 times at room temperature with occasional stirring. Treated for minutes. The treated cells were washed 3 times with ERDF medium, and after removing LLME sufficiently, they were used for in vitro immunization.

(In vitro immunization of human peripheral blood lymphocytes and lactic acid bacteria treatment)
For antigen sensitization, peripheral blood mononuclear cells were seeded in a 48-well plate at 5 × 10 ⁶ cells / ml per well, and IL-2 (1 or 10 U / ml), IL-4 (1 or 10 ng) / Ml), 2-mercaptoethanol (50 μM) and β-lactoglobulin (β-LG; 10 μg / ml) as an antigen. A control (Ctrl) was subjected to antigen sensitization by this method. At this time, in the fraction specified as Dp + K, as a positive control, 1 μMCpG-D (ggTGCATCATGATGAGGGGGG (SEQ ID NO: 11), Sigma Genosys (Hokaido, Japan) uppercase letters are phosphodiester, lowercase letters are phosphodioxides, After 3 days, 1 μM CpG-K (tcgagcgrrcrcC (SEQ ID NO: 12), Sigma Genosys) was further added and cultured.

All cultures were carried out under conditions of 37 ° C. and 5% CO ₂ , 10% inactivated fetal bovine serum (Fetal Bovine Serum: FBS, TRACE SCIENTIFIC, Melbourn, Australia), penicillin (100 U / ml: MEIJI SEIKA, Tokyo, Japan), ERDF medium containing streptomycin (100 μg / ml: MEIJI SEIKA) was used.

  In addition, lactic acid bacteria L31, L32, and L34 were treated by adding 1 or 10 μg / ml of lyophilized lactic acid bacteria heated and killed at the time of antigen sensitization and culturing for 6 to 8 days. As the heat-killed cells of lactic acid bacteria, those prepared by lyophilizing each strain washed twice with milli-Q water after culturing at 100 ° C. for 30 minutes were used.

(Detection of antigen-specific antibody-producing cells (ELISPOT method))
The presence of antigen-specific antibody-producing cells was confirmed by ELISPOT (enzyme-linked immunosorbent spot assay) method.
First, 100 μl of antigen (10 μg / ml) diluted with PBS was dispensed into each well of a 96-well cellulose membrane bottom plate (MultiScreen-HA; Millipore Bedford, Mass.), And allowed to stand overnight at 4 ° C. Adsorbed on top. In order to remove the non-adsorbed antigen, 200 μl of PBC was dispensed into each well, allowed to stand for 3 minutes, and then removed by suction. This washing operation was repeated three times.

Next, in order to prevent non-specific antibody adsorption, 200 μl of 1% fish gelatin (FG) -PBS was dispensed into each well and allowed to stand at 37 ° C. for 2 hours for blocking. After performing the above washing operation three times, cells were prepared. That is, the cells on the 6th day from the start of in vitro immunization were collected in a 1.5 ml tube, the number of cells was measured, and then centrifuged at 6,000 rpm for 1 minute to collect the supernatant. The remaining cell pellet was added to ERDF containing 10% FBS containing IL-2 (1 U / ml), IL-4 (1 ng / ml) and 2-mercaptoethanol (50 mM) at a cell concentration of 1 × 10 ⁶ cells / cell. Adjusted to ml. 100 μl of this was dispensed into each well and cultured at 37 ° C. under 5% CO ₂ for 20 hours. After completion of the culture, the cell solution was removed by suction, and the above washing operation was performed three times.

  Next, 100 μl of peroxidase (POD) -labeled anti-human IgM antibody (same as the secondary antibody for ELISA) diluted 10,000-fold with 1% FG-PBS was dispensed into each well and reacted at 37 ° C. for 2 hours. I let you. After performing the same washing operation with TPBS three times as described above, the substrate solution TrueBlue Peroxidase Substrate (KPL, Gaithersburg, MD) was dispensed into each well in a volume of 50 μl, reacted at 37 ° C., and the antigen-specific antibody-producing cells were reacted. Detected as a blue spot. When the reaction was sufficiently advanced, each hole was washed with deionized water to stop the reaction. After sufficiently drying in a light-shielded state, the cellulose film was punched out using MELIPUNCH Kit (Millipore) and fixed by being sandwiched between transparent seals. This was captured in a computer as a scanner image, and the captured image was analyzed with image analysis software ImageJ to digitize the number of spots.

  FIG. 1 shows spot images and the number of spots of β-LG-specific antibody-producing cells and non-specifically bound antibody-producing cells. In FIG. 1, “β-LG” is a spot image and the number of spots corresponding to β-LG-specific antibody-producing cells. On the other hand, “FG” is a spot image and the number of spots corresponding to a cell producing an antibody specific to the blocking reagent FG, that is, a cell producing an antibody that binds nonspecifically. In addition, the numerical value of 1 or 10 in FIG. 1 represents the heat-killed cell density | concentration (microgram / ml) of the added lactic acid bacteria. From FIG. 1, a significant antigen-specific antibody production enhancing effect was observed in L31, L32, and L34.

Example 2 [Immune Stimulating Action of Lactic Acid Bacteria (2) Immune Response Enhancement Effect by Lactic Acid Bacteria]
By coexisting with heat-killed cells of lactic acid bacteria that have been lyophilized during antigen sensitization of human peripheral blood lymphocytes by in vitro immunity, the immune response of peripheral blood lymphocytes is enhanced, resulting in increased expression of inflammatory cytokines. Whether it was enhanced was verified by the CBA method.

(Measurement of cytokine production in culture supernatant)
The concentration of various cytokines secreted into the culture supernatant of the culture supernatant prepared with or without the addition of lactic acid bacteria under the same conditions as in Example 1 (control and DpK) was measured. In this example, four types of L31, L32, L33, and L34 were used as lactic acid bacteria.

The concentration of various cytokines secreted into the culture supernatant was determined by the Human Information Kit (IL-8, IL-1β, IL-6, IL-6) of BD ^™ Cytometric Bead Array System (CBA; Becton Dickinson, Franklin Lakes, NJ). 10, TNF, for measuring IL-12p70) and Human Flex Set (for measuring IL-2, IL-4, IFN-γ), and using BD FACSAria (Becton Dickinson). The measurement was performed based on the protocol attached to each kit.

  The measurement results of the concentrations of each inflammatory cytokine of TNF, IL-6, IL-1β, and IL-8 are shown in FIGS. 2-1, 2-2, 2-3, and 2-4. As is clear from FIGS. 2-1 to 2-4, the L31 to L34 lactic acid strains showed an effect of enhancing the expression of inflammatory cytokines such as TNF, IL-6, IL-1β, and IL-8.

Example 3
Next, when the freeze-dried lactic acid bacteria heat-killed cells coexist with peripheral blood lymphocytes collected from healthy individuals and allergic patients, the immune response of peripheral blood lymphocytes is enhanced, resulting in inflammatory Whether the expression level of cytokines was enhanced was verified by the CBA method.

  In Example 1, the collection targets were healthy individuals and allergic patients, lactic acid bacteria were added to untreated peripheral blood lymphocytes that were not subjected to in vitro immunity, and four types of L31, L32, L33, and L34 were used as lactic acid bacteria. A culture supernatant was obtained in the same manner except that it was used and heat-killed lactic acid bacteria were added at a concentration of 0.1 or 1 μg / mL. Then, with respect to the culture supernatant prepared with or without each lactic acid bacterium added (control and DpK), the concentrations of various cytokines secreted into the culture supernatant were measured based on the CBA method as in Example 2.

  The measurement results of the concentration of each inflammatory cytokine are shown in FIG. 3-1, FIG. 3-2, and FIG. 3-3 (healthy person); FIG. 3-4, FIG. 3-5, and FIG. Show. In addition, the numerical value of 0.1 or 1 in FIGS. 3-1 to 3-6 represents the heat-killed cell concentration (μg / ml) of the added lactic acid bacteria. As is apparent from FIGS. 3-1 to 3-6, strong TNF production enhancement was observed in L31 in healthy individuals, whereas in allergic patients, strong TNF production enhancement in L33 was strong against IL31 and L32. 6, IL-8 production enhancement, but weak IL-6 and IL-8 production enhancement effect was observed in L34.

Example 4 [Antiallergic Action of Lactic Acid Bacteria (1) Histamine Accumulation Inhibitory Effect of Lactic Acid Bacteria and (2) Histamine Release Inhibitory Effect]
The inhibitory effect on histamine accumulation and release in KU812F of lactic acid bacteria was verified by calculating β-hexosaminidase activity in supernatant samples and cell lysate samples.

(Induction of differentiation of human basophil-like cell line KU812F and treatment with lactic acid bacteria)
KU812F (obtained from Tohoku University Institute of Aging Medicine) was cultured in a 10% FBS-containing RPMI-1640 medium (Nissui Pharmaceutical Co., Ltd.) under conditions of 37 ° C., 5%, CO ₂ /95% air. Each of the heat-killed lactic acid bacteria L31, L32, L33, and L34 prepared by lyophilization under the same conditions as in Example 1 was added to KU812F at a concentration of 0.1 or 1 μg / ml, followed by treatment for 3 days. The change in histamine release ability was followed.

(Measurement of β-hexosaminidase activity)
The amount of histamine secreted from the cell or intracellular was estimated by measuring the enzyme activity of an intragranular enzyme called β-hexosaminidase released together with a chemical mediator such as histamine during degranulation.

KU812F cells treated with the above lactic acid bacteria were adjusted to 2 × 10 ⁶ cells / tube, and Tyrode's solution (137 mM NaCl, 2.7 mM KCl, 1.8 mM CaCl ₂ , 1.1 mM MgCl ₂ , 11. It was washed twice with ₉ mM NaHCO ₃ , 0.4 mM NaH ₂ PO ₄ , pH 7.2). After resuspending in 150 μl of Tyrode's solution, it was treated with 10 μM ionomycin (Sigma, St. Louis, MO) and reacted at 37 ° C. for 1 hour (ionomycin stimulation). On the other hand, a similar treatment was prepared except that no ionomycin was added (no stimulation).

  After 1 hour, the supernatant was collected by centrifugation and used as a supernatant sample. The obtained cell pellet was dissolved in 150 μl of 0.1% Triton X-100 to obtain a cell lysate sample. 40 μl each of the obtained supernatant sample and cell lysate sample were added to a 96-well immunoplate, and 100 μl of 2 mM p-nitrophenyl-N-acetyl-β-D-glucosaminide (NAG) was added thereto as a substrate, The mixture was allowed to stand at 37 ° C. for 30 minutes for color reaction. After completion of the reaction, the reaction was stopped by adding 150 μl of 0.2 M glycine, and then the absorbance at 405 nm was measured. On the other hand, a sample prepared and measured in the same manner except that lactic acid bacteria were not added was used as a control.

(1) Histamine accumulation inhibitory effect of lactic acid bacteria The histamine accumulation inhibitory effect of lactic acid bacteria on KU812F was evaluated by relative histamine accumulation amount. Relative histamine accumulation was calculated as β-hexosaminidase activity in the supernatant and cell lysate samples. The results of relative histamine accumulation are shown in Fig. 4-1 for no stimulation and Fig. 4-2 for ionomycin stimulation. As a result, a significant inhibitory effect on histamine accumulation was observed in L33 and L34 (FIGS. 4-1 and 4-2).

(2) Histamine release inhibitory effect of lactic acid bacteria The histamine release inhibitory effect of lactic acid bacteria on KU812F was evaluated by relative histamine release activity. The relative histamine releasing activity was produced as (β-hexosaminidase activity in the supernatant sample) / (β-hexosaminidase activity in the supernatant and cell lysate sample) × 100 upon stimulation with ionomycin. The result of relative histamine releasing activity is shown in FIG. As is clear from FIG. 5, suppression of relative histamine release activity was observed in L32.

Example 5 [Anti-allergic effect of lactic acid bacteria (3) Anti-allergic effect of lactic acid bacteria]
The effect of lactic acid bacteria on cytokine production of human peripheral blood lymphocytes was examined.
First, lactic acid bacteria were added at the time of antigen sensitization for in vitro immunization, and the influence of peripheral blood lymphocytes on cytokine production was verified. That is, were prepared and measurements of the sample in accordance addition to the to be measured cytokines and T _H 1 cytokine IL-12p70, IFN-γ is the conditions of Example 2. The measurement results of IL-12p70 and IFN-γ are shown in FIGS. 6-1 and 6-2. In addition, the numerical value of 1 or 10 in FIGS. 6-1 and FIGS. 6-2 represents the heat dead cell density | concentration (microgram / ml) of the added lactic acid bacteria. As is apparent from FIGS. 6-1 and Figure 6-2, the expression enhancing action of IL-12 p70 is a T _H 1-type cytokines in L32 is also the IFN-gamma is a T _H 1-type cytokines in L31~L34 An expression enhancing action was observed.

Next, lactic acid bacteria were added to the peripheral blood lymphocytes immediately after separation, and the effect on cytokine production was verified. That is, peripheral blood mononuclear cells were collected in the same manner as in Example 1 except that healthy subjects and allergic patients were collected, and heat-killed lactic acid bacteria were added at a concentration of 0.1 or 1 μg / mL. After adjustment in the same manner, heat-killed cells of lactic acid bacteria were added and cultured without antigen sensitization. Subsequently, IL-12p70 and IFN-γ, which are T _H 1 cytokines, were measured according to the conditions of Example 2. The measurement results of each cytokine are shown in FIGS. 7-1 and 7-2 (healthy people); FIGS. 7-3 and 7-4 (allergic patients). In addition, the numerical value of 0.1 or 1 in FIGS. 7-1 to 7-4 represents the heat-killed cell density | concentration (microgram / ml) of the added lactic acid bacteria. As apparent from FIGS. 7-1 to 7-4, L32 has a strong IL-12 p70 production enhancing effect on healthy human peripheral blood lymphocytes, and L31 to 34 has a weak IFN-γ production enhancing effect. Indicated. Furthermore, for allergic patient-derived peripheral blood lymphocytes, L31 and L32 showed a strong IL-12 p70 production enhancing effect, and L31, L32 and L33 showed a strong IFN-γ production enhancing effect.

Example 6 [Comparison with known lactic acid bacteria (immunostimulatory activity)]
A comparative test of immunostimulatory activity was performed between the lactic acid bacteria of the present invention and known lactic acid bacteria.
Known lactic acid bacteria include 4 strains of L. acidophilus (L1 (JCM1028), L2 (JCM1034), L3 (JCM1132), L4 (JCM1229)) and Lactobacillus amylovorus (L. amylovorus). 1 strain (L5 (JCM2125)), 3 strains of L. gasseri (L10 (JCM1130), L11 (JCM1131), L12 (JCM8787)), 2 strains of Lactobacillus johnsonii (L. johnsonii) (L16 (JCM1022), L17 (JCM8791)) were used.

  A sample was prepared and the number of spots was measured in the same manner as in Example 1 except that these known strains were used as heat-killed cells instead of L31, etc., and the relative value to the number of DpK spots (specific spot number). Was calculated. In addition, about L1, L2, L3, L4, and L5, the addition amount of the lactic-acid-bacteria dead body was made into three types, 0.1 microgram / ml, 1 microgram / ml, or 10 microgram / ml. The result of the specific spot number of each bacterium is shown in FIG. 8 together with the results of L31, L32, L33, and L34. In addition, the numerical value of 0.1, 1 or 10 in FIG. 8 represents the heat-killed cell density | concentration (microgram / ml) of the added lactic acid bacteria.

  As is clear from FIG. 8, the lactic acid bacteria of the present invention, in particular L31, L32, and L34, have been shown to exhibit higher immunostimulatory activity than other lactic acid bacteria.

Example 7 [Comparison with known lactic acid bacteria (cytokine production ability)]
The cytokine production ability between the lactic acid bacteria of the present invention and known lactic acid bacteria was compared in the same manner as in Example 6. For IL-12p70, the relative value of the measured value of each group to the measured value of DpK was calculated. The measured value of IL-6 was calculated as a relative value to the measured value of the control. The results of each cytokine of each bacterium are shown in FIG. 9-1 (IL-12p70) and FIG. 9-2 (IL-6) together with the results of L31 to 34. In addition, the numerical value of 0.1, 1 or 10 in FIGS. 9-1 and 9-2 represents the heat-killed cell density | concentration (microgram / ml) of the added lactic acid bacteria.
As is apparent from FIGS. 9-1 and 9-2, L31, L32, L33, and L34 of the lactic acid bacterium of the present invention showed higher immunostimulatory activity than other lactic acid bacteria.

FIG. 1 is a graph showing an antigen-specific antibody production enhancing effect in the in vitro immunization of the lactic acid bacterium of the present invention. 2-1 is a figure which shows the TNF production enhancement effect in the in vitro immunity of the lactic acid bacteria of this invention. 2-2 is a figure which shows the IL-6 production enhancement effect in the in vitro immunity of the lactic acid bacteria of this invention. 2-3 is a figure which shows the IL-1 (beta) production enhancement effect in the in vitro immunity of the lactic acid bacteria of this invention. 2-4 is a figure which shows the IL-8 production enhancement effect in the in vitro immunity of the lactic acid bacteria of this invention. 3-1 is a figure which shows the TNF production enhancement effect from the healthy human peripheral blood lymphocyte of the lactic acid bacteria of this invention. 3-2 is a figure which shows the IL-6 production enhancement effect from the healthy human peripheral blood lymphocyte of the lactic acid bacteria of this invention. 3-3 is a figure which shows the IL-1 (beta) production enhancement effect from the healthy human peripheral blood lymphocyte of the lactic acid bacteria of this invention. 3-4 is a figure which shows the TNF production enhancement effect from the allergic patient peripheral blood lymphocyte of the lactic acid bacteria of this invention. 3-5 is a figure which shows the IL-6 production enhancement effect from the allergic patient peripheral blood lymphocyte of the lactic acid bacteria of this invention. 3-6 is a figure which shows the IL-8 production enhancement effect from the allergic patient peripheral blood lymphocyte of the lactic acid bacteria of this invention. 4-1 is a figure which shows the histamine accumulation | storage suppression effect under the non-stimulation of the lactic acid bacteria of this invention. 4-2 is a figure which shows the histamine accumulation | storage suppression effect under the ionomycin stimulation of the lactic acid bacteria of this invention. FIG. 5 is a diagram showing the histamine release inhibitory effect of the lactic acid bacteria of the present invention. 6-1 is a figure which shows the IL-12p70 production enhancement effect at the time of the in-vitro immunization of the lactic acid bacteria of this invention. 6-2 is a figure which shows the IFN-gamma production enhancement effect at the time of the in-vitro immunization of the lactic acid bacteria of this invention. 7-1 is a figure which shows the IL-12p70 production enhancement effect from the healthy human peripheral blood lymphocyte of the lactic acid bacteria of this invention. FIG. 7-2 is a graph showing an effect of enhancing IFN-γ production from peripheral blood lymphocytes of healthy persons by the lactic acid bacteria of the present invention. 7-3 is a figure which shows the IL-12p70 production enhancement effect from the allergic patient peripheral blood lymphocyte of the lactic acid bacteria of this invention. FIG. 7-4 is a diagram showing an effect of enhancing IFN-γ production from peripheral blood lymphocytes of allergic patients of lactic acid bacteria of the present invention. FIG. 8 is a diagram showing a comparison of immunostimulatory activity between the lactic acid bacteria of the present invention and known lactic acid bacteria. FIG. 9-1 is a diagram showing a comparison of IL-12p70 production ability between the lactic acid bacteria of the present invention and known lactic acid bacteria. FIG. 9-2 is a diagram showing a comparison of IL-6 production ability between the lactic acid bacteria of the present invention and known lactic acid bacteria.

Claims (4)

  Lactobacillus lactic acid bacteria having immunostimulatory action and antiallergic action.   Lactobacillus paracasei NHH2 strain (NITE P-262), Lactobacillus plantarum NHT4 strain (NITE P-264), Lactobacillus gasseri NHMT2 strain (NITE P-265), and Lactobacillus salivaius subspecies Sarivarius The lactic acid bacterium according to claim 1, which is selected from the group consisting of NHMF1 strain (NITE P-263).   A composition having an immunostimulatory action and an antiallergic action, comprising the lactic acid bacterium according to claim 1 as an active ingredient.   A food, drink, feed, cosmetic or pharmaceutical comprising the lactic acid bacterium according to claim 1 or 2 or the composition according to claim 3.

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