JP2008061512A - Lactic acid bacterium of genus streptococcus normalizing immunological balance and food and drink using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a lactic acid bacterium having specific immunological functionality, in particular a lactic acid bacterium acting in the direction to normalize the immunological balance and resultantly having an ability to afford prophylactic or ameliorating effects on allergic symptoms or diseases, to further provide a food and drink such as a fermented dairy product using the lactic acid bacterium and to provide a method for producing the food and drink. <P>SOLUTION: The lactic acid bacterium Streptococcus thermophilus OH1 (AHU1838) is characterized as follows. The lactic acid bacterium promotes IFN-γ(interferon-γ) production for a murine spleen cell without increasing IL-4 (interleukin-4) production, induces IL-12 (interleukin-12) production for a murine intraperitoneal macrophage without increasing IL-10 (interleukin-10) production and has immunoregulatory actions. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a specific lactic acid bacterium having a function of normalizing immune balance, and a food or drink for normalizing immune balance or preventing or ameliorating allergic symptoms, characterized by containing this lactic acid bacterium. The present invention further relates to a method for producing these food and drink for normalization of immune balance and food or drink for preventing or improving allergic symptoms.

  Recently, helper T cells are divided into two subtypes, type 1 helper T cells (Th1) and type 2 helper T cells (Th2), and these control mice control diseases related to various immune responses. And we have learned about humans. Th1 and Th2 are balanced with respect to each other, and if this balance is maintained, they are in a healthy state. However, if the balance is lost, it is presumed that this will lead to the onset and development of various diseases. That is, if it is biased toward the Th1 side relative to the proper Th1 / Th2 balance, diabetes, liver damage, respiratory tract inflammation, host versus graft reaction, rheumatoid arthritis, multiple sclerosis, arteriosclerosis, psoriasis, nephritis, etc. will occur If it is biased to the Th2 side, cancer, immunodeficiency, asthma, dermatitis, allergic diseases, nephritis, infectious diseases, etc. are thought to develop.

  This state of Th1 / Th2 balance can now be estimated by examining specific cytokine production from helper T cells. For example, when production of Th1-type cytokines such as interleukin 2 (IL-2) and interferon γ (IFN-γ) is dominant, interleukin 4 (IL-4) and interleukin 5 (IL-5) are directed to the Th1 side. If the production of Th2-type cytokines such as) is dominant, it is said that the production is biased toward Th2.

  Lactic acid bacteria are known to have beneficial physiological activities including intestinal regulation and immunostimulatory effects. In clinical studies conducted in humans, lactic acid bacteria prevent the development of cancer by alleviating the symptoms of atopy and perennial allergic rhinitis and by enhancing the antitumor activity of the immune system It has been shown. Furthermore, lactic acid bacteria have been suggested to improve the immune balance biased toward the Th2 side of allergic patients to an appropriate state, and are expected to be effective in treating and improving allergic diseases.

  However, individual immune responses are completely different depending on lactic acid bacteria. For example, not all strains are effective in promoting a specific immune response of Th1 type even if they are lactic acid bacteria having an immunomodulating action. Therefore, the selection of the strain is extremely important for the proper use of lactic acid bacteria.

Some strains, such as Lactobacillus paracasei KW3110, increase the production of interleukin 12 (IL-12), which is an indicator of Th1 response activation, on mouse spleen-derived lymphocytes sensitized with ovalbumin, It is known to reduce the production of IL-4, which is an index of Th2 response activation (Patent Document 1).

In addition, as a result of examining various lactic acid strains last year, the present inventors have found a lactic acid bacterium that increases the production of INF-γ more when acting on mouse spleen cells (at the 2006 Annual Meeting of the Japanese Society for Agricultural Chemistry). Presentation).
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  The present invention provides a specific immunity-functional lactic acid bacterium, in particular, a lactic acid bacterium that acts in the direction of normalizing the immune balance and, as a result, has the ability to prevent or improve allergic symptoms or diseases. With the goal. Furthermore, this invention also aims at providing food-drinks, such as fermented milk products using such lactic acid bacteria, and its manufacturing method.

The present inventors have found that Bifidobacterium (Bifidobacterium) genus Lactobacillus (Lactobacillus) genus Streptococcus (Streptococcus) genus Lactococcus (Lactococcus) strains of various lactic acid bacteria belonging to the genus such as normalizing immune balance of the whole body The present invention was completed by examining in detail the action to be performed and selecting lactic acid bacteria having a higher effect on improving the immune balance.

The present invention
[1] Streptococcus thermophillus lactic acid bacteria characterized by having an immunomodulating action as defined below:
(1) Mouse spleen cells were co-cultured for 36 hours in 1 mL of lactic acid bacteria at a concentration of 1 μg / mL at 2 × 10 ⁶ cells / mL, followed by 0.5 μg / mL at 4 × 10 ⁶ cells / mL. When the amount of production of interferon γ (IFN-γ) and interleukin 4 (IL-4) in the supernatant was measured by culturing in 250 μL of the medium in the presence of α-CD3 for 36 hours,
(A) the ratio of IFN-γ production stimulated by lactic acid bacteria / control IFN-γ production is at least 2.0;
(B) the ratio of IL-4 production stimulated by lactic acid bacteria / IL-4 production of control is 2.0 or less; and (c) IFN-γ production stimulated by lactic acid bacteria / IL-4 production stimulated by lactic acid bacteria The ratio of is at least 20.0; and
(2) Mouse peritoneal macrophages were co-cultured with lactic acid bacteria at a predetermined concentration at 5 × 10 ⁵ cells / mL for 48 hours in 250 μL of medium, and interleukin 12 (IL-12) and interleukin 10 in the supernatant were cultured. When measuring the production amount of (IL-10),
(D) induces production of at least 3 ng / mL of IL-12 at a lactic acid bacteria concentration of 100 μg / mL;
(E) does not induce the production of IL-10 greater than 4 ng / mL at a lactic acid bacteria concentration of 1000 μg / mL; and (f) the ratio of IL-12 production / IL-10 production is at least 4.0;
[2] Furthermore,
(3) The mouse bone marrow-derived dendritic cells co-cultured with lactic acid bacteria at any concentration of 5 to 20 μg / mL were subjected to mixed lymphocyte reaction with spleen cells derived from mice having another MHC haplotype. The lactic acid bacterium according to the above [1], which shows significant cytotoxic activity as compared with the control when the latter is used as an effector cell and the mouse-derived tumor cell having the same MHC haplotype is used as a target cell.
[3] Lactic acid bacteria Streptococcus thermophillus OH1 (AHU1838) (FERM-);
[4] A food or drink comprising the lactic acid bacterium according to any one of [1] to [3] or a processed product thereof;
[5] The food or drink according to [4], which is used for normalizing immune balance or preventing or improving allergy,
I will provide a.

  The present invention further provides a method for producing a dairy product, comprising a step of fermenting milk or a raw material containing a dairy product using the lactic acid bacterium according to any one of [1] to [3].

According to the present invention, immunocompetent cells are stimulated to induce high production of INF-γ, and production of Th1 cytokines IFN-γ and IL-12 and Th2 cytokines such as interleukin 10 (IL-10 ) Lactic acid bacteria Streptococcus thermophillus having both functions of production suppression, particularly Streptococcus thermophillus OH1 (AHU1838) (FERM-) is provided. The lactic acid bacteria of the present invention contribute to maturation of immature dendritic cells and are extremely effective lactic acid strains.

  Since lactic acid bacteria are very safe, even if taken with foods and beverages, no adverse effects on the human body are expected. Moreover, since it can be easily used in manufacture of food-drinks and a processing method is also well-known, it has advantages, such as a wide application range.

  Furthermore, the lactic acid bacteria of this invention and the food-drinks using this can obtain evaluation more than equivalent to the conventional thing also in taste.

  The lactic acid bacteria of the present invention can be obtained by selecting those having desired activity according to the method described below from strains etc. isolated from fermented milk products or unsterilized milk according to a conventional method.

1. Mouse spleen cell stimulation test (IFN-γ measurement)
Spleens are aseptically removed from C57BL / 6 mice and spleen cell suspensions are prepared in RPMI-1640 medium supplemented with 10% (V / V) heat-inactivated fetal calf serum (FCS). The filtrate filtered through a nylon filter is subjected to centrifugation at 1,500 rpm (rotation / min), 4 ° C. for 5 minutes, and a spleen cell pellet is collected. This pellet is resuspended in 0.144 M sodium chloride-0.017 M Tris-HCl buffer (pH 7.65) and incubated at 37 ° C. for 2.5 minutes under 5% CO ₂ to hemolyze the red blood cells and finally Prepare spleen cells (4 × 10 ⁶ cells / mL) used in the experiment.

The spleen cells (5 × 10 ⁵ cells) obtained above are co-cultured with lactic acid bacteria having a final concentration of ⁵ to 0.625 μg / mL in a final volume of 250 μl at 37 ° C. and 5% CO ₂ for 48 hours.

  After the culture, IFN-γ in the supernatant is detected by ELISA, and its concentration is determined. Instead of lactic acid bacteria, spleen cells co-cultured with phosphate buffered saline (PBS; pH 7.4) are used as controls.

2. Mouse spleen cell stimulation test (measurement of IFN-γ and IL-4)
Spleens are aseptically removed from BALB / c mice and a spleen cell suspension is prepared in RPMI-1640 medium supplemented with 10% (V / V) heat inactivated FCS. The filtrate filtered through the nylon filter is subjected to centrifugation at 1,500 rpm, 4 ° C. for 5 minutes to collect a spleen cell pellet. This pellet was resuspended in 0.144 M sodium chloride-0.017 M Tris-HCl buffer (pH 7.65), incubated at 37 ° C. for 2.5 minutes under 5% CO ₂ and finally hemolyzed. The spleen cells (1 × 10 ⁷ cells / mL) used for the preparation are prepared.

The spleen cells obtained above are first co-cultured with lactic acid bacteria (final concentration 1 μg / mL) at a final volume of 1 mL for 36 hours at 37 ° C. and 5% CO ₂ . Next, spleen cells were washed to remove lactic acid bacteria, adjusted to 4 × 10 ⁶ cells / mL, and fresh RPMI-1640 containing anti-mouse CD3 antibody (α-CD3) (final concentration 0.5 μg / mL). Incubate again in medium at a final volume of 250 μL for 36 hours at 37 ° C., 5% CO ₂ .

  After culturing, IFN-γ and IL-4 in the supernatant are detected by ELISA and their concentrations are determined. As a control, spleen cells co-cultured with PBS (pH 7.4) instead of lactic acid bacteria are used.

3. Mouse peritoneal macrophage (PEC) stimulation test (measurement of IL-10 and IL-12)
PEC is collected by intraperitoneally administering 2 mL of 10% thioglycolate medium to C57BL / 6 mice and washing the peritoneal cavity of the mice with PBS after 4 days. Cells are washed with RPMI-1640 medium and then adjusted to 4 × 10 ⁶ cells / mL. This PEC (5 × 10 ⁵ cells) was co-cultured with lactic acid bacteria at a final concentration of 1-1000 μg / mL in a final volume of 250 μl in RPMI-1640 medium containing 10% FCS for 2 days at 37 ° C. under 5% CO _2. Incubate.

  After the culture, IL-10 and IL-12 in the supernatant are detected by ELISA, and their concentrations are determined. PEC co-cultured with PBS (pH 7.4) is used as a control instead of lactic acid bacteria.

  Macrophages are one of the most efficient producing cells of IL-12, and IL-12 is known to act on NK cells and T cells to induce production of IFN-γ. This is thought to activate Th1 and correct the Th1 / Th2 balance imbalance.

4). Examination of the effect of lactic acid bacteria on the maturation of bone marrow-derived dendritic cells (BMDC) Bone marrow cells (5 × 10 ⁶ ) were obtained from BALB / c mouse thighs and granulocyte macrophage colony stimulating factor (GM-CSF). ) (Final concentration 30 ng / mL), 5% in RPMI-1640 in a final volume of 1 mL in the presence of interleukin 3 (IL-3) (final concentration 30 ng / mL) and lactic acid bacteria (0-20 μg / mL) Incubate at 37 ° C. under CO ₂ . After 2.5 days from the start of culture, the medium was replaced with a fresh medium (added with cytokines and lactic acid bacteria), and after further culturing for 2 days (4.5 days after the start of culture), adherent cells (BMDC; Bone marrow-derived dendritic cells) Get.

The obtained BMDC is incubated in RPMI-1640 medium containing mitomycin C (MMC) having a final concentration of 120 μg / mL for 30 minutes at 37 ° C. under 5% CO ₂ to stop cell growth. After removing MMC, mixed lymphocyte reaction (MLR) was performed by culturing with spleen cells (5 × 10 ⁶ cells) prepared from C57BL / 6 mice at 37 ° C. under 5% CO ₂ for 4 days. Do.

Cytotoxicity is measured by the chromium release test method. The cell after MLR obtained above was used as an effector cell, and P815 cells (DBA / 2 mouse-derived primary tumor) into which [ ⁵¹ Cr] was incorporated were used as target cells, and the ratio of the number of effector cells: target cells (E / T) Ratio) is 80: 1, 40: 1, 20: 1 and incubated at 37 ° C., 5% CO ₂ for 4 hours. As a negative control, the same experiment is performed using MBL-2 cells (C57BL / 6 mouse-derived lymphoma). The supernatant after the culture is collected, and γ-rays emitted from [ ⁵¹ Cr] are measured with a liquid scintillation counter. The amount of [ ⁵¹ Cr] released spontaneously when only target cells are cultured in the medium (S), the maximum amount released (Max) when 1N hydrochloric acid is added to the target cells. Calculate the cytotoxic activity (%) by the following formula:
Cytotoxic activity (%) = (amount of [ ⁵¹ Cr] when co-cultured with effector cells−S) / (Max−S) × 100
5. Evaluation Method Based on the results of the above tests, the lactic acid bacteria of the present invention can be selected as follows. The most important factor in the selection of lactic acid bacteria is the high nature of inducing IFNγ production against cells of the immune system. Therefore, the above 2. The ratio of IFN-γ production stimulated by lactic acid bacteria / control IFN-γ production when measured by the above method (IFN-γ production stimulated by lactic acid bacteria ÷ IFN-γ production of control) was calculated, Choose a larger one. For the purposes of the present invention, desirable lactic acid bacteria have a ratio of at least 2.0, preferably 2.5 or more, more preferably 3.0 or more.

  In addition, since IL-4 has the property of promoting allergic reactions, lactic acid bacteria can keep IL-4 production low even when stimulating cells of the immune system, that is, at least do not promote IL-4 production. It is necessary to be a thing, Preferably it is what suppresses. Accordingly, the ratio of IL-4 production stimulated by lactic acid bacteria / IL-4 production of control (measured by the above method) (calculated IL-4 production stimulated by lactic acid bacteria ÷ IL-4 production of control) was calculated, Select one with a small number. For the purposes of the present invention, desirable lactic acid bacteria have the above ratio of 2.0 or less, preferably 1.6 or less, and more preferably 1.4 or less.

  Then, the ratio of IFN-γ production / IL-4 production (IFN-γ production ÷ IL-4 production) as measured by the above method is calculated. For the purposes of the present invention, desirable lactic acid bacteria have the above ratio of at least 20.0, preferably 24.0 or higher, more preferably 30.0 or higher.

  Furthermore, the ability to induce or suppress other cytokines is also important for the purpose of correcting the immune balance. IL-12 is an index indicating that Th1 immunity is activated. Therefore, lactic acid bacteria are selected to have a high property of inducing IL-12 production against cells of the immune system, that is, those that promote IL-12 production. In particular, those having a high ability to induce IL-12 production at low lactic acid bacteria concentrations are desirable. Therefore, for the purposes of the present invention, in the above-described test, at least 3 ng / mL, preferably 4 ng / mL or more of IL-12 production is induced at a concentration of lactic acid bacteria of 100 μg / mL or less, preferably 10 μg / mL or less. Lactic acid bacteria are desirable.

  In addition, IL-10 is known to negatively control macrophage activity and suppress the production of inflammatory cytokines (such as IL-12, IL-6, and TNF-α). Therefore, it is necessary to select lactic acid strains that can keep IL-10 production low even when stimulating cells of the immune system, that is, those that do not promote at least IL-10 production, and preferably those that suppress IL-10 production. is there. In particular, those having a low ability to induce IL-10 production even at a high concentration of lactic acid bacteria are desirable. For the purposes of the present invention, a desirable lactic acid bacterium is a lactic acid bacterium that does not induce IL-10 production of 4 ng / mL or more, preferably 3 ng / mL or more at a lactic acid bacterium concentration of 1000 μg / mL in the above-described test, and / or A lactic acid bacterium that does not induce IL-10 production at a concentration of lactic acid bacteria of 100 μg / mL is preferably 2 ng / mL or more, preferably 1 ng / mL or more.

  Then, the ratio of IL-12 production / IL-10 production (IL-12 production ÷ IL-10 production) as measured by the above method is calculated, and a large value is preferable. When the IL-10 production amount is less than the detection limit, for the sake of convenience, the above calculation of the ratio is performed assuming that the IL-10 production amount is “1”. For the purposes of the present invention, desirable lactic acid bacteria have the above ratio of at least 4.0, preferably 6.0 or higher, more preferably 8.0 or higher.

  Overall, according to the above evaluation criteria, IFN-γ production is high, IL-4 production is low, IFN-γ production / IL-4 production ratio is high, and IL-12 production is high. Select those having a low IL-10 production and a high ratio of IL-12 production / IL-10 production.

  In addition, for the purpose of the present invention, lactic acid bacteria that provide high cell-specific cytotoxic activity in the above-described cytotoxicity test are desirable. For the purposes of the present invention, desirable lactic acid bacteria have a cytotoxic activity of at least 10 when measured at an E / T ratio of 80: 1 or 40: 1 at any lactic acid bacteria concentration of 5-20 μg / mL. %, Preferably 15% or more, particularly preferably 20% or more.

The lactic acid bacteria of the present invention thus selected are Gram-positive facultative anaerobic mesophilic bacteria. Table 1 shows the mycological properties of Streptococcus thermophillus OH1 (FERM-), one of which.

このようにして選択した本発明の乳酸菌Streptococcus thermophillus ＯＨ１は、日本微生物資源学会（ＪＳＣＣ）において認められた公的保存機関である北海道大学大学院農学研究科応用生命工学専攻菌株保存室（ＡＨＵ）に「ＡＨＵ１８３８」として寄託されており、また、平成１８年８月２７日付で独立行政法人産業技術総合研究所 特許生物寄託センターに寄託されている。

The lactic acid bacterium Streptococcus thermophillus OH1 of the present invention selected in this way is stored in the strain storage room (AHU) of the Department of Applied Biotechnology, Graduate School of Agriculture, Hokkaido University, which is an official preservation organization recognized by the Japanese Society for Microbial Resources (JSCC). It has been deposited as “AHU1838” and has been deposited with the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology on August 27, 2006.

  The lactic acid bacteria of the present invention can be cultured by a known method.

  The cultured lactic acid bacterium can be used for ingestion of its microbial cells (which can be either live or dead) or processed products thereof (for example, microbial cell extracts, crushed materials, fractions, etc.) You may contain these in food-drinks.

  The food / beverage products of the present invention can be produced by appropriately incorporating the lactic acid bacteria of the present invention into the food / beverage products by methods known in the technical field such as the food manufacturing industry. As a method for containing lactic acid bacteria, for example, the lactic acid bacteria of the present invention are in the form of freeze-dried cells, spray-dried cells, wet cells, etc., or crushed products of these various cells, appropriate fractions, etc. And the method of adding food / beverage materials and manufacturing food / beverage products is mentioned.

  There is no restriction | limiting in particular about the kind and form, etc. of the food / beverage products of this invention. Preferably, confectionery (for example, tablet-shaped confectionery, ice cream, jelly, etc.), various powdered foods, capsules, beverages, fats and oils foods and the like are used. Moreover, most preferably, fermented milk products (for example, fermented milk, lactic acid bacteria beverages, etc.) manufactured using the lactic acid bacteria of this invention are mentioned.

  As a method for producing a dairy product, for example, a method in which a lactic acid bacterium of the present invention is used as a starter for a raw material such as skim milk powder, yeast extract, L-cysteine hydrochloride, calcium carbonate, sugar solution, etc. A method for producing a fermented milk beverage by diluting the fermented product thus produced with a syrup solution or the like is mentioned.

1. Testing the effect of various lactic acid strains on the ability of mouse spleen cells to induce cytokine production
1-1. The strains used for screening were lactic acid strains for fermented dairy products provided by companies participating in the dairy industry, isolates from commercial dairy products, and the RIKEN BioResource Center (JCM) and independent administrative agencies. The strains obtained from the Corporate Product Evaluation Technology Infrastructure (NBRC) were isolated from each single colony to confirm the purity. These test strains are shown in Table 2.

番号（＃）７〜９の菌株は、市販のヨーグルトから単離したものであり、Ｓ６、Ｂ１、Ｂ４は発明者らが任意につけた識別記号である。＃７〜９の菌株の１６ｓリボゾームＤＮＡ配列を解析したところ、全て同一であり、ＮＣＢＩデータベース検索を行なったところ、Accession No. NZ_AAGR01000053として登録されている配列と１００％一致した。また、＃７〜９と同一又は最も近い菌株は、Lactobacillus casei（ＡＴＣＣ３３４）であることが判明した。

The strains with numbers (#) 7-9 were isolated from commercially available yogurt, and S6, B1, and B4 are identification symbols arbitrarily given by the inventors. When the 16s ribosomal DNA sequences of strains # 7 to 9 were analyzed, they were all identical, and an NCBI database search was performed, which showed a 100% match with the sequence registered as Accession No. NZ_AAGR01000053. Moreover, it turned out that the same or the closest strain as # 7-9 is Lactobacillus casei (ATCC334).

All strains were cultured under anaerobic conditions of N ₂ , CO ₂ and H ₂ (8: 1: 1) at 37 ° C. until stationary growth. Lactobacillus delrueckii subsp. Bulgaricus, Lactobacillus paracasei subsp. Casei, Lactobacillus paracasei subsp. Paracasei, Lactobacillus plantarum, Lactobacillus acidophilus and Lactobacillus gasseri is, de Man, Rogosa, and Shape (MRS) medium (Becton, Dickinson Co, Sparks, Md, USA ). Each strain of Streptcoccus thermophillus was cultured in M17 medium (Becton, Dickinson Co, Sparks, Md, USA). Bifidobacterium breve was cultured in Gifu Anaerobic Medium (GAM) medium (Nissui Pharmaceutical, Tokyo, Japan). These cultures were collected by centrifugation at 3,000 × g, 10 minutes, 4 ° C. and washed 3 times with ice-cold sterile phosphate buffered saline (PBS, pH 7.4). The bacteria were sterilized by heat treatment at 105 ° C. for 5 minutes, lyophilized and stored at −80 ° C. At the time of use, it was suspended in PBS at a concentration of 10 mg / mL and used for the experiment.

1-2. Preparation of mouse spleen-derived lymphocytes C57BL / 6 mice and BALB / c mice (Charles River Japan, Yokohama) used were females of 5 to 8 weeks of age and maintained and maintained in an SPF environment.

RPMI-1640 medium (each final concentration 180 U / mL penicillin, 0.09 mg / min) with heat-inactivated fetal calf serum (FCS) added to 10% (V / V) was removed by aseptic treatment from mice. cell suspension using tweezers until it becomes cell unit in mL streptomycin, 2.16 mg / mL HEPES, 0.1 mg / mL sodium pyruvate; hereinafter referred to as “prepared RPMI-1640 medium”) A liquid was prepared. The obtained spleen cell suspension was passed through a nylon mesh filter to remove cell clusters and adipose tissue. The filtrate was subjected to centrifugation (1,500 rpm / minute, 4 ° C., 5 minutes) to obtain a cell pellet. About 2 mL of 0.144 M sodium chloride, 0.017 M Tris-HCl buffer (pH 7.65) was added to the pellet, resuspended, incubated at 37 ° C., 5% CO ₂ for 2.5 minutes, Hemolyzed. After washing by centrifugation, resuspended in the prepared RPMI-1640 medium, and after determining the number of viable cells by trypan blue staining method, mouse spleen cell-derived lymphocytes were adjusted to 4 × 10 ⁶ cells / mL. It was adjusted.

1-3. Preparation of lactic acid bacteria Lyophilized lactic acid bacteria powder was suspended in PBS to a concentration of 10 mg / mL and sterilized by heat treatment at 105 ° C. for 5 minutes. At the time of co-culture, this was diluted to the concentration used in the prepared RPMI-1640 medium and used.

1-4. Effect of lactic acid bacteria on IFN-γ production ability of mouse spleen cells By co-culturing mouse spleen cells and various lactic acid bacteria, and quantifying the produced IFN-γ, we compared the immunostimulatory ability of each lactic acid bacteria It was.

Spleen cells (5 × 10 ⁵ cells) prepared from C57BL / 6 mice were co-cultured so that the final concentration of lactic acid bacteria was ⁵ , 2.5, 1.25, 0.625 μg / mL. All samples were incubated in a 96-well flat bottom microtiter plate for 48 hours at 37 ° C., 5% CO ₂ atmosphere to a final volume of 250 μl. After culturing, the supernatant was collected, and the IFN-γ concentration was determined by ELISA. The ELISA was performed using an “OptEIA mouse IFN-γ” (trade name) kit (BD Biosciences Pharmingen, California, USA; Becton Dickinson, Japan).

The cytotoxicity to mouse spleen cells was examined by quantifying the amount of thymidine incorporated radiolabeled with tritium [ ³ H] using a scintillation counter. In all the lactic acid bacteria used, since the amount of [ ³ H] thymidine incorporated was a sufficient concentration, it was judged that no cytotoxicity of the lactic acid bacteria was observed at the concentration used. Therefore, it was shown that the difference in the production of INF-γ is not due to cytotoxicity but due to the difference in stimulating ability of lactic acid strains.

  The results are shown in FIG. In the following table, “ND” indicates that it was less than the detection limit.

Lactobacillus gasseri ＪＣＭ ８７８７（＃３）を除くほとんどの乳酸菌株は、マウス脾臓細胞によるＩＦＮ‐γ産生を誘導した。一方、ＩＦＮ‐γは、陰性対照試料コントロール（「Ｃｏｎｔｒｏｌ」；乳酸菌の代わりにＰＢＳを添加したもの）から得られた培養上清中ではほとんど検出されず、観察されたマウス脾臓細胞によるＩＦＮ‐γ産生が乳酸菌での刺激によるものであることが示された。

Most lactic acid strains except Lactobacillus gasseri JCM 8787 (# 3) induced IFN-γ production by mouse spleen cells. On the other hand, IFN-γ was hardly detected in the culture supernatant obtained from the negative control sample control (“Control”; PBS added instead of lactic acid bacteria), and IFN-γ by the observed mouse spleen cells Production was shown to be due to stimulation with lactic acid bacteria.

  In addition, it was shown that the stimulating ability was completely different depending on the lactic acid strain, and it was found that there was a difference of 10 times or more in the inducing ability between the strain having a high IFN-γ production inducing ability and the strain having a low ability.

1-5. Examination of the effect of lactic acid bacteria pretreatment on mouse spleen cells on cytokine production Lactic acid strains (# 8, 12, 14, 18, 22, 27, 32, 37, 38) with high IFN-γ production induction ability in the above results and Using a lactic acid strain (# 3) that did not show any inducing ability, co-cultured with mouse spleen cells as a preculture, and after culturing, the lactic acid bacteria were eliminated by washing, and an anti-mouse CD3 antibody (α-CD3) was used. Comparative studies were performed on cytokines produced by stimulation.

Spleen cells (2 × 10 ⁶ cells) prepared from BALB / c mice were pre-cultured in the presence of various lactic acid bacteria at a final concentration of 1 μg / mL. The first half was cultured in a 24-well flat bottom plate at 37 ° C. in a 5% CO ₂ atmosphere for 36 hours so that the final volume was 1 mL. Next, in order to remove lactic acid bacteria, the cultured cells were washed three times with the prepared RPMI-1640 medium, and after cell counting, adjusted to 4 × 10 ⁶ cells / mL.

The prepared cells were cultured in the presence of α-CD3 (final concentration 0.5 μg / mL). The latter half was cultured in a 96-well flat-bottomed microtiter plate for 36 hours in a 37 ° C., 5% CO ₂ atmosphere so that the final volume was 250 μl. After the culture, the supernatant was collected, and the IFN-γ and IL-4 concentrations were determined by ELISA. ELISA was performed using an “OptEIA mouse IFN-γ” (trade name) kit and an “OptEIA mouse IL-4” (trade name) kit (BD Biosciences Pharmingen, California, USA; Becton Dickinson, Japan).

The results are shown in FIGS. 2A and B and Table 4. L. gasseri JCM 8787 (# 3), L. paracasei subsp. Tolerans JCM 1173 (# 14) and L. casei subsp. Casei 027 (R-12) (# 32) produce IFN-γ and IL-4 Both were low. On the other hand, L. casei (# 8) and S. thermophillus OH1 (# 22) were shown to be high lactic acid strains with high IFN-γ production and relatively low IL-4 production.

表４には、測定値に加えて、ＩＦＮ‐γ産生量（ｐg/mL）とＩＬ‐４産生量（ｐg/mL）との比（ＩＦＮ‐γ産生量÷ＩＬ‐４産生量）を示す。本発明の目的からは、ＩＦＮ‐γ産生量が多く、ＩＬ‐４産生量が少ないもの、そして上記の比が大きいものが好ましい。この点に関して、試験した菌株のうち、＃８、＃１４、＃１８、＃２２、＃３８が特に好ましい結果を示していた。

Table 4 shows the ratio of IFN-γ production (pg / mL) to IL-4 production (pg / mL) (IFN-γ production ÷ IL-4 production) in addition to the measured values. . For the purpose of the present invention, those having a large amount of IFN-γ production, a small amount of IL-4 production, and those having a large ratio are preferred. In this regard, of the tested strains, # 8, # 14, # 18, # 22, and # 38 showed particularly favorable results.

  Overall, as a result of this test, # 8 and # 22 were selected as particularly preferred strains.

2. Measurement of IL-12 and IL-10 production inducing ability for intraperitoneal cells by lactic acid bacteria
2-1. Preparation of mouse peritoneal macrophages (PEC) 2 mL of 10% thioglycolate medium was intraperitoneally administered to C57BL / 6 mice, and 4 days later, PEC was collected from the mouse intraperitoneally using PBS. The collected PEC was washed twice with the above-prepared RPMI-1640 medium and adjusted to 4 × 10 ⁶ cells / mL.

The adjusted PECs (5 × 10 ⁵ ) were co-cultured so that the final concentration of lactic acid bacteria was 1000, 100, 10, 1 μg / mL. As the lactic acid bacteria, lactic acid strains (# 8, 12, 14, 18, 22, 27, 32, 38) selected as having high IFN-γ production-inducing ability in the above test 1-3 and some other strains (# 5, 10, 16, 23, 24, 28, 39, 40) and a lactic acid strain (# 3) that did not show any induction ability were used. All samples were cultured for 48 hours in a 96-well flat bottom microtiter plate at 37 ° C. in a 5% CO ₂ atmosphere to a final volume of 250 μl. After culturing, the supernatant was collected and IL-12 and IL-10 concentrations were determined by ELISA. For the ELISA, “OptEIA mouse IL-12p70” (trade name) kit and “OptEIA mouse IL-4” (trade name) kit (BD Biosciences Pharmingen, California, USA; Becton Dickinson, Japan) were used.

The results are shown in FIGS. 3A and 3B and Tables 5 and 6. Production ability to induce IL-12 is S. thermophillus OH1 (# 22), were higher in lactic acid bacteria S. thermophillus OJT101 (# 24) and L. paracasei subsp. Paracasei BZN2.12.4A (1HB ) (# 38). These lactic acid strains also suppressed IL-10 production from PEC. In contrast, S. thermophillus 21072 (# 12), L. delbrueckii subsp. Bulgaricus NBRC13953 (# 18) and L. delbrueckii subsp. Bulgaricus JCM 1002 (# 27) strongly enhanced IL-10 production from PEC. , IL-12 production was suppressed.

これらの結果から、S. thermophillus ＯＨ１（＃２２）及びL. paracasei subsp. paracasei ＢＺＮ２．１２．４Ａ（１ＨＢ）（＃３８）は高いＩＬ‐１２産生誘導能を有し、かつＩＬ‐１０の産生誘導能が低く抑えられた乳酸菌株であることが確認できた。これら乳酸菌株は、Ｔｈ２優位なアレルギー状態から、Ｔｈ１免疫を賦活化し、アレルギーの症状を緩和することができる乳酸菌として有力な株であることが示された。

These results, S. thermophillus OH1 (# 22) and L. paracasei subsp. Paracasei BZN2.12.4A (1HB ) (# 38) has a high IL-12 production-inducing activity and IL-10 production It was confirmed that the strain was a lactic acid strain with a low induction ability. These lactic acid strains have been shown to be effective strains of lactic acid bacteria that can activate Th1 immunity and alleviate allergic symptoms from an allergic state predominantly Th2.

3. Tests on the maturation ability of bone marrow-derived dendritic cells by lactic acid bacteria The effects of various lactic acid bacteria on the maturation of bone marrow-derived dendritic cells in BALB / c mice A sex test was conducted to investigate. In addition, the expression of costimulatory molecules was also examined for dendritic cells before MLR by flow cytometry (FACS) analysis.

In addition to the strain S. thermophillus OH1 (# 22) which was excellent in the above test, the lactic acid bacterium used in this test was a commercially available isolate from yogurt ( L. casei (# 8)).

3-1. Preparation of Bone Marrow-Derived Dendritic Cells (BMDC) Bone marrow cells (5 × 10 ⁶ cells) prepared from femur bone aseptically removed from BALB / c mice were treated with IL-3 (final concentration 30 ng / mL) and GM-CSF. Under conditions of dendritic cell maturation cytokine (final concentration 30 ng / mL), lactic acid bacteria were added and cultured so that the final concentration was 0, 5, 10, 20 μg / mL. After 2.5 days from the start of culture, the plate was gently shaken to remove non-adherent cells and then removed. After replacement with a fresh medium, cytokines and lactic acid bacteria were newly added. After further culturing for 2 days (4.5 days after the start of culture), only adherent cells were peeled off with trypsin to obtain bone marrow-derived dendritic cells (BMDC). All samples were cultured in 12-well flat bottom plates at 37 ° C., 5% CO ₂ atmosphere to a final volume of 1 mL.

3-2. Measurement of cytotoxicity BMDC obtained in RPMI-1640 medium supplemented with DNA replication inhibitor mitomycin C (MMC) (final concentration 120 μg / mL) at 37 ° C. under 5% CO ₂ atmosphere for 30 minutes Incubation stopped cell growth. MMC-treated BMDC (2 × 10 ⁵ cells) were washed 4 times with RPMI-1640 to completely remove MMC, and spleen cells (5 × 10 ⁶ cells) prepared from C57BL / 6 mice and mixed lymphocytes Reaction (MLR) was performed. MLR was performed by culturing in a 5 mL PP round tube (Falcon) for 4 days at 37 ° C. in a 5% CO ₂ atmosphere.

Cytotoxic activity (effector cell killing of target cells) was measured by the chromium release test method. Target cells were P815 cells (DBA / 2 mouse-derived primary tumor) and MBL-2 cells (C57BL / 6 mouse-derived lymphoma) that had been preincubated for 1 hour and incorporated [ ⁵¹ Cr], and the cells after MLR were effector cells. As a test. The effector cells and target cells were mixed at a ratio of 80: 1, 40: 1, and 20: 1, and incubated at 37 ° C. in a 5% CO ₂ atmosphere for 4 hours. The supernatant after the culture was collected, and γ rays emitted from the radioactive substance [ ⁵¹ Cr] released from the killed target cells into the medium were measured with a liquid scintillation counter. At this time, the amount (S) of spontaneously released [ ⁵¹ Cr] is a measured value obtained by culturing only the target cells in the medium, and the maximum released amount (Max) is obtained by adding 1N hydrochloric acid to the target cells. The measured value was used. Based on the measured values for these and each sample, the cytotoxic activity (%) was calculated by the above formula.

  The results are shown in FIG.

Ｐ８１５細胞はＭＨＣ（主要組織適合性遺伝子複合体）ハプロタイプがＨ‐２^ｄであるのに対して、ＭＢＬ‐２細胞はＨ‐２^ｂとなっている。ＢＭＤＣはＢＡＬＢ／ｃマウス（Ｈ‐２^ｄのＭＨＣハプロタイプを持つ）から調製したので、Ｐ８１５細胞に対する殺傷能力が高い方がよく、ＭＢＬ‐２細胞に対する殺傷能力はほとんどないのであれば細胞特異的が高いといえる。

P815 cells whereas MHC (major histocompatibility complex) haplotype is ^{H-2 d, MBL-2} cells has a ^{H-2 b.} Since BMDC was prepared from BALB / c mice (having the H-2 ^d MHC haplotype), it should have a higher killing ability against P815 cells and cell-specific if there is little killing ability against MBL-2 cells. It can be said that it is expensive.

# 22 induced the highest cytotoxic activity against P815 cells at a concentration of 20 μg / mL. On the other hand, cytotoxic activity to MBL-2 cells is a negative control was not detected at all, it was confirmed that the P815 cell-specific cytotoxic activity with H-2 ^d to MHC haplotypes. From this, it was shown that BMDC capable of activating immature mouse lymphocytes could be induced by addition of # 22.

  On the other hand, the commercially available yogurt isolated lactic acid strain (# 8) was not observed to be significantly different from the control at any concentration, and it was confirmed that # 22 had a high dendritic cell maturation ability.

3-3. Expression analysis of CD40 molecule and CD86 molecule About the BMDC before MLR prepared above, the expression of CD40 molecule and CD86 (B7.2) molecule, which are costimulatory molecules with T cells, was evaluated by flow cytometry analysis. PBS was used for control.

The obtained BMDC (2 × 10 ⁵ ) was subjected to FITC-labeled anti-mouse CD11c antibody (50-fold dilution, clone name: HL-3, catalog number 553802, Japan BD) and PE-labeled anti-mouse CD40 antibody (35 Double dilution, clone name: 3-23, catalog number 553790, Japan BD) or PE labeled anti-mouse CD86 antibody (35 times dilution, clone name: GL-1, catalog number 553691, Japan BD) Incubated at 4 ° C. for 10 minutes. CD11c is a marker molecule for dendritic cells. After washing the cells, CD40 and CD86 positive cells in CD11c positive cells were measured using a flow cytometer “FACScalibur” (registered trademark, Japan BD), and using analysis software “CellQuest” (trade name, Japan BD). Histogram data was created.

  The results are shown in FIG. CD40 and CD86 molecules are costimulatory molecules having CD40L and CD28 molecules as ligands, respectively, and the higher the expression intensity, the higher the activation ability for T cells. The commercial yogurt isolated lactic acid strain (# 8) did not show enhanced expression of CD40 and CD80 molecules at any concentration, whereas # 22 enhanced both molecules, especially against CD86 molecules. High expression enhancement was shown.

  The CD86 molecule (also known as B7.2) on dendritic cells binds to the CD28 molecule on T cells when the T cell receptor (TCR) and MHC transmit signals through the peptide. It is one of the costimulatory molecules that can activate T cells by transmitting signals. By enhancing the expression of this CD86 molecule, this BMDC can efficiently activate T cells. These results indicate that # 22 is a highly effective lactic acid strain in dendritic cell maturation.

5. Example of production of food and drink using lactic acid bacteria of the present invention
5-1. As a starter for manufacturing yogurt fermented yogurt was used Lactobacillus delbrueckii subsp. Bulgaricus JCM1002 are species of the type strain lactic acid bacteria # 22 obtained above, namely Steptococcus thermophillus OH1 (AHU1838), and as a standard bacteria.

The skim milk was dissolved in water at 126 g / L and subjected to path tooling (90 ° C., 15 minutes). The strain used was stored at −80 ° C. and cultured for 18 hours at the time of use. S. thermophillus was grown in M17 medium (Nippon Becton Dickinson Co., Ltd.) and L. delbrueckii was grown in MRS medium (OXOID). Next, each strain was inoculated into a fresh medium and cultured until the late stage of logarithmic growth (37 ° C., 18 hours). The cells were collected by centrifugation, washed with physiological saline, and suspended in the same solution.

To start the yogurt fermentation, each strain was added at the following concentrations: 2.5 × 10 ⁶ cfu / mL for S. thermophillus , 5.0 × 10 ⁶ cfu / mL for L. delbrueckii . Fermentation was performed at 43 ° C. Since the pH reached around 4.5 in about 4 and a half hours, the sample was cooled to stop the fermentation. Thereafter, it was stored at about 5 ° C. and evaluated.

  The yogurt produced by fermentation formed a soft and smooth card and exhibited a refreshing fragrance and a soft sour taste, and was able to produce an excellent yogurt. Even compared with commercially available yogurt, it had the same or better properties, taste and aroma.

2. The # 22 lactic acid bacterium ( Steptococcus thermophillus OH1 (AHU1838)) obtained above was used as a production starter for fermented milk beverages . The used strain stored at −80 ° C. was cultured for 18 hours in M17 medium at the time of use and grown. Next, this strain was inoculated into a fresh medium, cultured until late in logarithmic growth (37 ° C., 18 hours), and the cells were collected by centrifugation, washed with physiological saline, and suspended in the same solution.

To start fermentation of fermented milk, the above cells were added to raw milk at a concentration of 2.5 × 10 ⁶ cfu / mL. Fermentation was performed at 43 ° C., and the pH became around 5.0 in about 5 hours. Therefore, the sample was cooled to stop the fermentation. Thereafter, it was stored at about 5 ° C. and evaluated.

  The finished fermented milk obtained excellent fermented milk with a smooth acidity and plump taste.

FIG. 1 is a diagram showing the concentration of IFN-γ produced from spleen cells derived from C57BL / 6 mice by stimulation with various lactic acid bacteria. FIG. 2 (Panel A) is a diagram showing the concentration of IFN-γ produced from BALB / c-derived spleen cells by α-CD3 stimulation after pre-culture with various lactic acid bacteria. FIG. 2 (Panel B) is a graph showing the concentration of IL-4 produced from BALB / c-derived spleen cells by α-CD3 stimulation after pre-culture with various lactic acid bacteria. FIG. 3 (Panel A) is a view showing the concentration of IL-12 produced from C57BL / 6 mouse-derived peritoneal macrophages by stimulation with various lactic acid bacteria. FIG. 3 (Panel B) is a diagram showing the concentration of IL-10 produced from C57BL / 6 mouse-derived peritoneal macrophages by stimulation with various lactic acid bacteria. FIG. 4 is a diagram showing cytotoxic activity after MLR. Stimulation was performed with the reference strain (# 8) (panels A and C) and the lactic acid strain of the present invention (# 22) (panels B and D), respectively. Panels A and B are cytotoxic to P815 cells and panels C and D are cytotoxic to MBL-2 cells. FIG. 5 shows expression histograms of CD40 and CD80 molecules in BMDC (CD11c positive cells) induced by the reference strain (# 8) (panel A) and the lactic acid strain of the present invention (# 22) (panel B). It is.

Claims (5)

Streptococcus thermophillus lactic acid bacteria characterized by having an immunomodulating action as defined below:
(1) Mouse spleen cells were co-cultured for 36 hours in 1 mL of lactic acid bacteria at a concentration of 1 μg / mL at 2 × 10 ⁶ cells / mL, followed by 0.5 μg / mL at 4 × 10 ⁶ cells / mL. When the amount of production of interferon γ (IFN-γ) and interleukin 4 (IL-4) in the supernatant was measured by culturing in 250 μL of the medium in the presence of α-CD3 for 36 hours,
(A) the ratio of IFN-γ production stimulated by lactic acid bacteria / control IFN-γ production is at least 2.0;
(B) the ratio of IL-4 production stimulated by lactic acid bacteria / IL-4 production of control is 2.0 or less; and (c) IFN-γ production stimulated by lactic acid bacteria / IL-4 production stimulated by lactic acid bacteria The ratio of is at least 20.0; and
(2) Mouse peritoneal macrophages were co-cultured with lactic acid bacteria at a predetermined concentration at 5 × 10 ⁵ cells / mL for 48 hours in 250 μL of medium, and interleukin 12 (IL-12) and interleukin 10 in the supernatant were cultured. When measuring the production amount of (IL-10),
(D) induces production of at least 3 ng / mL of IL-12 at a lactic acid bacteria concentration of 100 μg / mL;
(E) does not induce the production of IL-10 above 4 ng / mL at a lactic acid bacteria concentration of 1000 μg / mL; and (f) the ratio of IL-12 production / IL-10 production is at least 4.0. further,
(3) The mouse bone marrow-derived dendritic cells co-cultured with lactic acid bacteria at any concentration of 5 to 20 μg / mL were subjected to mixed lymphocyte reaction with spleen cells derived from mice having another MHC haplotype. The lactic acid bacterium according to claim 1, which shows a significant cytotoxic activity as compared with the control when the cytotoxic activity is measured using the later cells as effector cells and the mouse-derived tumor cells having the same MHC haplotype as target cells. Lactic acid bacteria Streptococcus thermophillus OH1 (AHU1838) (FERM-).   Food-drinks containing the lactic acid bacteria of any one of Claims 1-3, or its processed goods.   The food or drink according to claim 4, which is used for normalizing immune balance or preventing or improving allergy.

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