JP2020074719A - Novel lactic acid bacterium with excellent immunostimulatory ability - Google Patents

Abstract

To provide a novel lactic acid bacterium having excellent immunological properties (for example, having higher IL-12 production inducing activity, IgE production suppressing activity, and/or anticancer activity).SOLUTION: The above subject has been solved by providing "Vine 5" strain belonging to Leuconostoc mesenteroides, a novel lactic acid bacterium (deposited at the International Patent Organism Depositary, National Institute of Technology and Evaluation (NITE-IPOD) on July 11, 2018 (accession number NITE P-02751)).SELECTED DRAWING: None

Description

  The present invention relates to a novel lactic acid bacterium having excellent immunological properties and a method of using the same.

  Lactic acid bacteria contribute to keeping the intestinal microflora in a good state, and lactic acid fermented foods obtained by fermenting milk with lactic acid bacteria are widely eaten as health-conscious foods and drinks. In addition, microbial cells obtained by heating lactic acid bacteria are also useful as foods or food additives. Some lactic acid bacteria, such as OK432 strain (Non-Patent Document 1), have immunological properties such as immunostimulation and physiologically active effects, but the effects are not sufficient.

  Among cytokines produced by lymphocytes, macrophages, etc., IL-12 induces differentiation of helper T progenitor cells (Th0) into helper T cell type 1 (Th1), activation of natural killer cells (NK cells), etc. Has the effect of. Helper T cells include Th1 cells and Th2 cells, and Th1 cells include interleukin-2 (IL-2), interferon-γ (suppresses IFN-γ and IgE antibody production), TNF-α, and TNF. By producing -β, granulocyte-macrophage colony stimulating factor (GM-CSF), and IL-3, the activity of phagocytic cells such as T cells and monocytes is increased, and resistance to infection by bacteria and the like is increased. Moreover, natural killer cells (NK cells) have the ability to specifically kill cancer cells, virus-infected cells and the like. Therefore, enhancement of IL-12 production can be expected to enhance resistance to viral infection, prevention of cancer, and the like. These actions are those in which immune cells such as white blood cells and lymphocytes directly kill and phagocytose pathogens, and are called cell-mediated immunity, and are distinguished from the immune action by antibody production (humoral immunity).

  On the other hand, there is a balance between cell-mediated immunity and humoral immunity, and when cell-mediated immunity is activated, humoral immunity is suppressed. It is said that modern human beings have a balance on the side of antibody production (humoral immunity), which is one of the causes of increase in pollinosis and allergies. Alleviation of these symptoms can be expected by the enhancement of IL-12. Therefore, lactic acid bacteria with high IL-12 production inducing activity have many uses.

  Further, it is expected that if the anti-IgE can be reduced while having desired immunological properties, it will be more useful in terms of anti-inflammatory.

Journal of Japanese Society of Oral Science, 2003, 52, No. 2, p. 51-62

  The present invention is a novel lactic acid bacterium having excellent immunological properties (for example, having higher IL-12 production inducing activity, IgE production suppressing activity, and / or anticancer activity) as compared with conventional lactic acid bacteria. The challenge is to provide. Furthermore, the present invention provides a method of utilizing such a novel lactic acid bacterium.

  The inventors of the present invention have solved the above-mentioned problems by providing a "vine 5" strain belonging to Leuconostoc mesenteroides, which is a novel lactic acid bacterium. The five vine strains have a 16S rDNA gene (SEQ ID NO: 1) containing the nucleic acid sequence shown in FIG. The "Vine 5" strain belonging to the Leuconostoc mesenteroides was deposited on July 11, 2018 at the Japan Biotechnology Center Biotechnology Center Patent Organism Depositary Center (NITE-IPOD) (accession number NITE P-02751). ).

For example, the present invention provides the following:
(Item 1)
Five strains belonging to Leuconostoc mesenteroides (accession number NITE P-02751).
(Item 2)
A mutant of 5 strains (accession number NITE P-02751) belonging to Leuconostoc mesenteroides, which has a 16S rDNA gene consisting of the nucleic acid sequence shown in SEQ ID NO: 1 and produces IL-12. A mutant strain having an inducing activity.
(Item 3)
It is a mutant strain of 5 strains (Accession No. NITE P-02751) belonging to Leuconostoc mesenteroides, which has a 16S rDNA gene consisting of the nucleic acid sequence shown in SEQ ID NO: 1 and has IgE inhibitory activity. Having a mutant strain.
(Item 4)
It is a mutant strain of 5 strains (accession number NITE P-02751) belonging to Leuconostoc mesenteroides, which has a 16S rDNA gene consisting of the nucleic acid sequence shown in SEQ ID NO: 1 and has an anticancer activity. A mutant strain having
(Item 5)
A method for producing a lactic acid bacterium heated bacterial cell, which comprises a step of culturing the vine 5 strain (accession number NITE P-02751) or a mutant strain thereof according to any one of Items 1 to 4.
(Item 6)
A heated lactic acid bacterium produced using the vine 5 strain (accession number NITE P-02751) or a mutant strain thereof according to any one of Items 1 to 4.
(Item 7)
A lactic acid bacterium heated bacterial cell is produced, which comprises a step of culturing the vine 5 strain (accession number NITE P-02751) or a mutant strain thereof according to any one of Items 1 to 4 in a medium containing Kuzu-derived dextrin. Method.
(Item 8)
A heated lactic acid bacterium produced by the method according to Item 7.
(Item 9)
A composition for inducing IL-12 production, comprising the heated lactic acid bacterium according to Item 6 or 8.
(Item 10)
A composition for suppressing IgE, comprising the heated lactic acid bacterium according to item 6 or 8.
(Item 11)
A composition for treating or preventing cancer, comprising the heated lactic acid bacterium according to Item 6 or 8.

(Mycological characteristics)
The novel vine 5 strain (accession number NITE P-02751) belonging to Leuconostoc mesenteroides of the present invention has the following mycological characteristics characteristic of Leuconostoc mesenteroides, and is used for fermented foods such as sauerkraut. This bacterium had the following mycological characteristics.
・ Gram-positive cocci, in the form of chains or diplococci.
-It is classified into lactic acid bacteria and heterolactic acid bacteria that produce acetic acid, ethanol, CO _{2 and the} like of short chain fatty acids other than lactic acid bacteria by decomposing sugar.

(Definition of terms)
Listed below are definitions of terms used particularly in the present specification.

  As used herein, the term “heated bacterial cell” refers to a bacterial cell that has been sterilized by heating after culturing. Although it is not limited, it typically refers to cells that have been sterilized by heating at 70 to 121 ° C for 10 to 60 minutes after culturing at 20 to 40 ° C for 10 to 40 hours.

  As used herein, the term "anti-cancer activity" refers to the inhibition (partial or total) or inhibition of unregulated cell growth and / or the inhibition of cancer (partial) as defined herein. Or overall) or deterrence. Anti-cancer activity includes, for example, the ability to reduce, arrest or repair gene damage, regulate unwanted cell proliferation, regulate misregulation of cell death, or mechanism of metastasis (e.g., migratory ability). Includes the ability to adjust. The cancer targeted by the present invention is not limited, for example, metastatic brain tumor, neuroendocrine tumor, melanoma, prostate cancer, head and neck cancer, ovarian cancer, lung cancer, liver cancer, breast cancer, genitourinary organ. Cancer, gastric cancer, colorectal cancer, cervical cancer, liposarcoma, rhabdomyosarcoma, choriocarcinoma, pancreatic cancer, retinoblastoma, multiple myeloma, and other types of cancer.

  The present invention provides a novel lactic acid bacterium having excellent immunological properties, particularly a lactic acid bacterium belonging to Leuconostoc mesenteroides. The lactic acid bacterium of the present invention has, for example, higher IL-12 production inducing activity, IgE production suppressing activity, and / or anticancer activity.

FIG. 1 shows the nucleic acid sequence of the 16S rDNA gene of the “Vine 5” strain. FIG. 2 shows the 16S rDNA gene (sequence of SID23149-01 in the upper row) of the “Vine 5” strain and RIB. This is an alignment in comparison with 9186 strain (obtained from Liquor Research Institute, sequence of SID23149-02 in the lower row). The 1243rd nucleic acid is "G" in SIID23149-01, whereas it is "A" in SIID23149-02. FIG. 2 shows the 16S rDNA gene (sequence of SID23149-01 in the upper row) of the “Vine 5” strain and RIB. This is an alignment in comparison with 9186 strain (obtained from Liquor Research Institute, sequence of SID23149-02 in the lower row). The 1243rd nucleic acid is "G" in SIID23149-01, whereas it is "A" in SIID23149-02. FIG. 2 shows the 16S rDNA gene (sequence of SID23149-01 in the upper row) of the “Vine 5” strain and RIB. This is an alignment in comparison with 9186 strain (obtained from Liquor Research Institute, sequence of SID23149-02 in the lower row). The 1243rd nucleic acid is "G" in SIID23149-01, whereas it is "A" in SIID23149-02. FIG. 3 is a simplified molecular phylogenetic tree based on the nucleic acid sequence of the 16S rDNA gene of the “Vine 5” strain. FIG. 4 is a graph comparing the IL-12 production inducing activities of the “Tsuru 5” strain and the OK432 strain. FIG. 5 is a graph showing excellent IL-12 production-inducing activity of the “Tsuru 5” strain cultured with Kuzu-derived dextrin. FIG. 6 is a graph showing excellent IL-12 production-inducing activity by the “Tsuru 5” strain cultured with different concentrations of kudzu-derived dextrin. FIG. 7 is a graph comparing the tumor weights when the “Tsuru 5” strain was used (test substance administration group) and when it was not used (untreated group). FIG. 8 shows a typical scheme for producing heated cells.

(1. Cultivation method of "Tsuru 5" strain)
Typical culture conditions for the “Vine 5” strain are static culture in MRS medium at 25 to 37 ° C. for 24 to 48 hours, but are not limited thereto.

(2. Preparation of mutant strain)
The mutant strain is prepared, for example, by subjecting it to general mutation treatment, or by adaptive or natural mutation by subculture.

  The mutagenesis process can be performed using a general mutagen. Examples of the mutagen include a drug having a mutagenic effect and ultraviolet rays. Examples of the drug having a mutagenic effect include streptomycin, ofloxacin, ethyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine, nucleotide base analogs such as bromouracil, and acridines. ..

  The mutant strain of the present invention preferably has an IL-12 production-inducing activity comparable to that of the “Vine 5” strain. For example, the mutant strain of the present invention has an IL-12 production-inducing activity of 70% to 130%, 80% to 120%, or 90% to 110%, as compared with the "Vine 5" strain. The mutant strain of the present invention is superior to the OK432 strain (for example, Streptococcus pyogenes (Group A type 3) Su strain penicillin-treated lyophilized powder commercially available from Chugai Pharmaceutical Co., Ltd. as “Picibanil”). Production inducing activity, at least 1.2 fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, or at least 1.7 fold IL-12 It has a production inducing activity.

  The mutant strain of the present invention preferably has an IgE inhibitory activity like the "Vine 5" strain. The mutant strain of the present invention preferably has anticancer activity similar to that of the “Vine 5” strain.

(3. Method for preparing kudzu-derived dextrin)
Any method for preparing dextrin from plants can be used for the production of kudzu-derived dextrin. A typical preferable production process of kudzu-derived dextrin is as follows.
・ Kudzu root is crushed → mashed → filtered with a bleaching cloth → removal of impurities → precipitation → drying → 0.1% to 0.3% enzyme is added and liquefied at the same time as cooking (liquefaction condition: pH 5.0 to 7.0, 50 to 75 ° C.) → sterilization (90 to 112 ° C., 10 to 60 minutes) → drying.

  Hereinafter, the present invention will be described based on examples, but the following examples are provided only for the purpose of illustration. Therefore, the scope of the present invention is not limited to the above detailed description of the invention or the following examples, but is limited only by the claims.

(Example 1: Isolation of "Vine 5" strain)
The “vine 5” strain belonging to Leuconostoc mesenteroides was isolated by the following procedure.
・ Kudzu vines are accumulated and cultured in a liquid medium for lactic acid bacteria to which antibiotics are added at 25 ° C to 37 ° C for 24 to 48 hours. Five new vines belonging to mesenteroides were selected.

  The “Vine 5” strain belonging to Leuconostoc mesenteroides obtained as described above was deposited on July 11, 2018 at the Japan Institute for Product Evaluation Technology, Biotechnology Center, Patent Organism Depositary (NITE-IPOD). (Accession number NITE P-02751).

(Example 2: Characterization of "Vine 5" strain (1) 16S rDNA gene analysis)
The nucleotide sequence of the 16S rDNA gene of the isolated "Vine 5" strain was determined. The results are shown in Figure 1 (SEQ ID NO: 1). FIG. 2 shows the 16S rDNA gene of the “Vine 5” strain (SID23149-01 in the upper row, SEQ ID NO: 1) and RIB. This is an alignment in comparison with 9186 strain (obtained from Liquor Research Institute, SID23149-02 in the lower row, SEQ ID NO: 2).

  FIG. 3 is a simplified molecular phylogenetic tree based on the nucleic acid sequence of the 16S rDNA gene of the “Vine 5” strain. The upper left line is a scale bar, the numerical value located at the branch of the phylogenetic branch is the bootstrap value, and the "T" at the end of the strain name indicates the reference strain (Type strain) of that species.

(Example 3: Characterization of "Vine 5" strain (2) IL-12 production-inducing activity)
Spleen cells were prepared from BALB / c mice, and dried lactic acid bacteria were added to a cell culture solution having a concentration of 2.5 × 10 ⁶ / ml so that the test lactic acid bacteria had a weight concentration of 1 μg / ml and 10 μg / ml. 200 μL / well was dispensed into a well culture plate, and the cells were cultured under 5% CO ₂ and 37 ° C. for 2 days, and then the IL-12 concentration in the culture solution was measured by the sandwich ELISA method. The BALB / c mice used were 11 weeks old, and 10% FBS (fetal bovine serum) -RPMI1640 + 100 μg / ml streptomycin + 100 U / ml penicillin + 50 μM 2-mercaptoethanol was used as the spleen cell culture medium. Six wells were used for one sample and one concentration. One-way analysis of variance was used for statistical analysis, Dunnett's test was used for comparison with the positive control, and Tukey's test was used for paired comparison between each sample to test for the presence or absence of a significant difference. The comparison between 1 μg / ml and 10 μg / ml of individual samples was tested by Welch's t-test.

  The lactic acid bacteria were subjected to neutralization culture, collected, washed and heat-sterilized before being used as a sample, and compared with the result of OK432.

  IL-12 was measured by a sandwich ELISA method using a specific antibody. Both the primary and secondary antibodies were manufactured by BioLegend Inc. Rat anti-mouse monoclonal antibody was used.

  The results are shown in Fig. 4. This result shows that the IL-12 production-inducing activity of the “Vine 5” strain of the present invention is remarkably superior to that of the OK432 strain.

(Example 4: Characterization of "Vine 5" strain (3) Culture using Kuzu-derived dextrin)
Instead of replacing glucose in the culture medium (MRS medium) of the "vine 5" strain of the present invention with kudzu-derived dextrin, the "vine 5" strain was cultured and tested for IL-12 production-inducing activity in spleen cells.

The kudzu-derived dextrin was produced by the following procedure.
・ Kudzu root is crushed → mashed → filtered with a bleaching cloth → removal of impurities → precipitation → drying → 0.1% to 0.3% enzyme is added and liquefied at the same time as cooking (liquefaction condition: pH 5.0 to 7.0, 50 to 75 ° C.) → sterilization (90 to 112 ° C., 10 to 60 minutes) → drying.

  In this Example 4, the liquefying enzyme T (BHI Co., Ltd.) originating from Bacillus subtilis was selected as the enzyme to be used. Enzymes used include, but are not limited to, amylases such as α-amylase. In addition, β-amylase and glucoamylase can also be used. Typically, "BAN" from Novozymes Japan KK, "Crystase E5CC" from Amano Enzymes, "Spitase CP40-FG" from Nagase Chemtex Co., Ltd., and "UNIASE" from Yakult Pharmaceutical Co., Ltd. BM8 ”, but is not limited thereto. The amount of enzyme to be added can be appropriately selected by those skilled in the art, and for example, it can be used at an enzyme concentration of 0.003 to 1%. The enzyme reaction conditions are not limited, but are typically pH 5.0 to 7.0 and 50 to 75 ° C.

  The “Vine 5” strain was cultured under the same conditions as in Example 3 using glucose (5%), and IL-12 was cultured in the same manner as in Example 3 except that 1 μg of lactic acid bacteria was added per 1 ml of the cell culture medium. The production inducing activity was tested. Furthermore, the lactic acid bacteria were grown using a culture medium in which glucose (5%) in the culture medium (MRS medium) used in the culture of the lactic acid bacteria used in this test was replaced with the kudzu-derived dextrin (5%) prepared by the above method. Then, the same experiment as in Example 3 was conducted using the cell suspension. When Kuzu-derived dextrin is used for culturing lactic acid bacteria, insoluble residues increase. Therefore, as the lactic acid bacterium to be added to the spleen cell culture solution, a suspension immediately after heating and stirring the whole lactic acid bacterium culture medium was used (shown as "Kudzu-derived dextrin (whole)" in FIG. After the culture in the lactic acid bacterium culture medium was completed and after heating, the mixture was allowed to stand for a while and only the bacterial cell layer in the supernatant was used (shown as "Kudzu-derived dextrin (bacteria)" in Fig. 5) for comparison. In addition, the results using OK432 strain cultured in a culture medium containing glucose are also shown in FIG. 5 as a comparison target.

  As is clear from the results of FIG. 5, when the kudzu-derived dextrin was used instead of glucose, the IL-12 production-inducing activity was further increased. It was also clarified that this increase was caused not by the components in the culture medium other than the bacterial cells but by the heated bacterial cells of the lactic acid bacteria.

  FIG. 6 shows the results of performing the above experiment while changing the weight concentration of the lactic acid bacteria used. When the amount of bacterial cell suspension per 1 ml of spleen cell culture medium was set to 10 μg / ml, the enhancing effect of the IL-12 production-inducing activity of the “Tsuru 5” strain in comparison with the OK432 strain became more remarkable and glucose was increased. When the kudzu-derived dextrin was used instead, the effect of enhancing the IL-12 production-inducing activity of the "Tsuru 5" strain was even more excellent.

(Example 5: Characterization of "Vine 5" strain (4) IgE inhibitory activity)
IgE in the measurement sample was measured using "Levis (registered trademark) IgE-ELISA kit (mouse)" (Shibayagi Co., Ltd.). The microplate spectrophotometer used was a Bio-rad product, and the standard curve was calculated using a cubic polynomial. The measurement was carried out at two points per sample and the average value was adopted. As for statistical processing, the variance was tested by F test, and Student's t-test was used for equal variance, and Welch's t-test was used for unequal variance.

  Table 1 shows the IgE in plasma in the non-administration and the administration of the "Vine 5" strain in each mouse species.

  In all of the mouse strains, the administration of the "Vine 5" strain showed a slight decrease tendency. Also, focusing on the mouse species, C57BL / 6N mice had significantly lower values than the other mouse species.

(Example 6: Characterization of "Vine 5" strain (5) Anticancer activity)
The antitumor effect when the test substance was orally administered to a tumor-bearing mouse model in which a mouse breast cancer 4T1 cell line was subcutaneously transplanted for 7 days before transplantation for 28 days was examined using tumor volume and tumor weight as indicators. This test was properly carried out in compliance with the animal experimentation regulations, animal experimentation committee regulations and animal experiment approval regulations of KAC Inc. (approval number: 17-1107).

  Regarding the condition of the tumor surface, crust formation was observed in Day 18 in 1 group of each of the untreated group and the test substance administration group. Crust formation was observed in all animals after Day 20. Body weight increased in each group from the grouping date to the sampling date. Regarding the tumor volume, in Day 17 and Day 24, the test substance administration group showed a significantly lower value than the untreated group. Regarding the tumor weight, the average tumor weight in the untreated group was 1.341 g, whereas the average tumor weight in the test substance administration group was 1.014 g, which was a significant low value. Regarding the lung weight, the untreated group was 0.202 g, whereas the test substance administration group was 0.186 g, and no significant difference was observed. The results are shown in Table 2 below.

  FIG. 7 is a graph showing tumor weight.

  From the above results, it was observed that the test substance-administered group showed a lower value in the mean tumor volume and the mean tumor weight than the untreated group. Therefore, the test substance was orally administered from 7 days before transplantation for 28 days. It was shown to have an antitumor effect.

(Example 7: Preparation of heated cells)
The production of heated cells is typically according to the scheme of FIG. Specifically, after culturing at 20 to 40 ° C for 10 to 40 hours, heat sterilization was performed at 70 to 121 ° C for 10 to 60 minutes. The heated bacterial cell of the present invention can be used as an active ingredient of a composition having immunopotentiating ability and a composition for treating and / or preventing cancer.

  Disclosed is a novel lactic acid bacterium having excellent immunological properties (for example, having higher IL-12 production inducing activity, IgE production suppressing activity, and / or anticancer activity) as compared with conventional lactic acid bacteria.

  The "Tsuru 5" strain belonging to Leuconostoc mesenteroides was deposited on July 11, 2018 at the Japan Biotechnology Center Biotechnology Center Patent Organism Depositary (NITE-IPOD) (accession number NITE P-02751). ..

SEQ ID NO: 1: Nucleic acid sequence of 16S rDNA gene of “Vine 5” strain SEQ ID NO: 2: RIB. Belonging to Leuconostoc mesenteroides Nucleic acid sequence of 16S rDNA gene of 9186 strain

Claims (11)

  Five strains belonging to Leuconostoc mesenteroides (accession number NITE P-02751).   A mutant of 5 strains (accession number NITE P-02751) belonging to Leuconostoc mesenteroides, which has a 16S rDNA gene consisting of the nucleic acid sequence shown in SEQ ID NO: 1 and produces IL-12. A mutant strain having an inducing activity.   It is a mutant strain of 5 strains (Accession No. NITE P-02751) belonging to Leuconostoc mesenteroides, which has a 16S rDNA gene consisting of the nucleic acid sequence shown in SEQ ID NO: 1 and has IgE inhibitory activity. Having a mutant strain.   It is a mutant strain of 5 strains (accession number NITE P-02751) belonging to Leuconostoc mesenteroides, which has a 16S rDNA gene consisting of the nucleic acid sequence shown in SEQ ID NO: 1 and has an anticancer activity. A mutant strain having   A method for producing a heated lactic acid bacterium, comprising the step of culturing the vine 5 strain (accession number NITE P-02751) or a mutant strain thereof according to any one of claims 1 to 4.   A lactic acid bacterium heated bacterial cell produced by using the vine 5 strain according to any one of claims 1 to 4 (accession number NITE P-02751) or a mutant strain thereof.   A lactic acid bacterium heated bacterium comprising a step of culturing the vine 5 strain (accession number NITE P-02751) or a mutant strain thereof according to any one of claims 1 to 4 in a medium containing Kuzu-derived dextrin. how to.   A heated lactic acid bacterium produced by the method according to claim 7.   A composition for inducing IL-12 production, which comprises the heated lactic acid bacterium according to claim 6 or 8.   A composition for suppressing IgE, comprising the heated lactic acid bacterium according to claim 6 or 8.   A composition for treating or preventing cancer, which comprises the heated lactic acid bacterium according to claim 6 or 8.