Bifidobacterium that inhibits the production of IL-17

Abstract

The present invention provides a Bifidobacterium derived from the infant intestine, which inhibits the production of IL-17 and is applicable to foods and beverages, as well as foods and beverages containing the Bifidobacterium. A Bifidobacterium longum species that inhibits the production of IL-17 is provided. The Bifidobacterium is Bifidobacterium longum strain N714 (NITE BP-03004).

Description

Technical Field

[0001] The present invention relates to a Bifidobacterium strain derived from the infant gut that suppresses the production of IL-17, and food and beverage products containing the strain. In particular, the present invention relates to Bifidobacterium longum. Background Art

[0002] Known cells involved in immunity include type 1 helper T (Th1) cells involved in cell-mediated immunity and Th2 cells involved in humoral immunity. In addition to Th1 and Th2 cells, recent studies have also discovered a new subset of T cells called Th17 cells. These Th17 cells have been found to play a central role in allergic reactions, autoimmunity, and some defense against infections. Th17 cells are named for their characteristic of producing interleukin 17 (IL-17).

[0003] Interleukins (ILs) are a general term for bioactive substances produced by immune cells such as lymphocytes, monocytes, and macrophages, and are protein substances that mediate intercellular interactions related to immune responses. Among them, IL-17 is mainly produced by Th17 cells and is known to induce various factors such as inflammatory cytokines, chemokines, and cell adhesion factors by acting on cells such as fibroblasts, epithelial cells, vascular endothelial cells, and macrophages, thereby causing inflammation (see NPL 1 and 2).

[0004] IL-17 contains several subsets, and Th17 cells produce IL-17A and IL-17F. IL-17A and IL-17F have 50% homology at the amino acid level and the corresponding receptors are the same, indicating many overlapping functions. It is also known that human Th17 cells produce more IL-17F than IL-17A.

[0005] Citation List

[0006] Non-Patent Literature

[0007] [NPL 1] Aggarwal S, Gurney AL. IL-17: prototype member of an emerging cytokine family. J Leukoc Biol 2002; 71: 1-8.

[0008] [NPL 2] Kolls JK, Linden A. Interleukin-17 family members and inflammation. Immunity 2004; 21: 467-76. Summary of the Invention

[0009] Technical problem

[0010] In this study, the inventors investigated whether the production of IL-17 could be inhibited by diet and found that Bifidobacterium isolated from infant feces contained Bifidobacterium that inhibited the production of IL-17, thus completing the present invention.

[0011] Solution to the problem

[0012] To solve the above problems, the present invention is characterized by Bifidobacterium belonging to the genus Bifidobacterium, which inhibits the production of IL-17.

[0013] To solve the above problems, the present invention is further characterized in that the Bifidobacterium is Bifidobacterium longum strain N714 (NITE BP-03004).

[0014] To solve the above problems, the present invention is further characterized by foods and beverages containing Bifidobacterium.

[0015] Advantageous effects of the invention

[0016] The Bifidobacterium of the present invention inhibits the production of IL-17. Description of the drawings

[0017] Figure 1 It is a graph comparing the inhibitory effects of the strain of the present invention and a comparative strain on the production of IL-17F.

[0018] Figure 2 It is a graph comparing the inhibitory effects of the strain of the present invention and a reference strain on the production of IL-17F. Detailed description of the invention

[0019] The present invention will now be described in detail.

[0020] 1. Bifidobacterium longum strain N714 (NITE BP-03004)

[0021] The Bifidobacterium in the present invention is Bifidobacterium longum. The symbol N714 in the present invention is the number independently assigned to the strain by Nissin Foods Holdings Co., Ltd. This strain was first isolated from infant feces by the inventors.

[0022] Bifidobacterium longum strain N714 was deposited under the following conditions:

[0023] (1) Name of the depositary institution: National Institute of Technology and Evaluation, Patent Microorganisms Depositary, Japan (NITE)

[0024] (2) Contact information: Room 122, 2-5-8 Kamigashirazume, Kisarazu-shi, Chiba-ken, Japan 292-0818

[0025] (3) Trustee Number: NITE BP - 03004

[0026] (4) Identification Mark: N714

[0027] (5) Deposit Date: July 10, 2019

[0028] The bacteriological characteristics of Bifidobacterium longum strain N714 are shown in Tables 1 and 2 below. The bacteriological characteristics were determined according to the methods described in Bergey's Manual of Systematic Bacteriology, Volume 2 (1986). Table 1 shows the shape and other characteristics related to this strain, and Table 2 shows the results of physiological and biochemical characterization using Api 50CH and Api CHL (Biomérieux). In Tables 1 and 2, the symbol "+" indicates positive and the symbol "-" indicates negative.

[0029] [Table 1]

[0030]

[0031] [Table 2]

[0032]

[0033] 2. Inhibitory test for IL-17 production

[0034] The Bifidobacterium longum strain N714 of the present invention has a high ability to inhibit the production of IL - 17, as shown in the experimental examples described below. The inhibition of IL - 17 production was confirmed by the following method.

[0035] <Preparation of Samples Used in the IL - 17 Production Inhibition Test>

[0036] The samples used in the IL - 17 production inhibition test were prepared by culturing Bifidobacterium for 24 hours at 37°C in the GAM medium (Nippon Suisan Kaisha, Ltd.) shown in Table 3. Then the proliferated bacteria were collected by centrifugation. The collected bacteria were washed with sterile water and collected by centrifuge. The washing and collection were repeated 3 times. Then heat sterilization was carried out, and the heat - sterilized samples were freeze - dried with a freeze - dryer to obtain dry bacterial powder.

[0037] [Table 3]

[0038]

[0039] <Inhibition Test of IL - 17 Production>

[0040] Human peripheral blood mononuclear cells (hPBMC, CTL) were used for the inhibition assay of IL-17 production. Using IMDM medium (Sigma) supplemented with 10% fetal bovine serum and 4 mM GlutaMAX (Gibco), cells were seeded at 1.0×10 6 cells / well, and the samples prepared above were inoculated at 10 ng / well on a 96-well plate. As a Th17 cell differentiation stimulant, 25 ng / mL rhIL-6 (BioLegend), 12.5 ng / mL rhIL-23 (BioLegend), 25 ng / mL rhIL-21 (BioLegend), 2 ng / mL rhTGF-β1 (BioLegend) and 25 ng / mL rhIL-1β (BioLegend) were added and incubated at 37 °C, 5% CO2 for 96 hours. After cultivation, the culture supernatant of hPBMC was collected, and IL-17F in the supernatant was measured using a commercially available immunoassay kit (R&D).

[0041] 3. Food and beverage products

[0042] The Bifidobacterium of the present invention can be used by being compounded in food and beverage products. The Bifidobacterium of the present invention is particularly suitable for beverages such as fermented milk and lactic acid bacteria beverages. According to the current "Ministerial Ordinance on Standard of Ingredients for Milk and Dairy Products", the ingredient standard requires a minimum content of 1.0×10 7 cfu / mL for fermented milk (solid content of skim milk is 8.0% or higher) and milk-based lactic acid bacteria beverages (solid content of skim milk is 3.0% or higher), and a minimum of 1.0×10 6 cfu / mL for lactic acid bacteria beverages (solid content of skim milk is less than 3.0%), but the above bacterial counts can be achieved by growing in a fermentation solution (such as milk) or in the form of the final product. In addition to fermented milk and lactic acid bacteria beverages containing Bifidobacterium, Bifidobacterium can also be used in dairy products such as butter, processed egg products such as mayonnaise, and pastries such as cream cakes. Bifidobacterium can also be appropriately used in processed foods such as instant noodles and biscuits. In addition to these applications, the food products of the present invention can be made into a preparation form of Bifidobacterium (for example, powder, granule, capsule and tablet) together with an appropriate carrier and necessary additives.

[0043] In addition to general beverages and foods, the bifidobacteria of the present invention can also be incorporated into specific health foods and nutritional supplements. In addition to foods, the bifidobacteria of the present invention can also be applied to the field of cosmetics, such as lotions, the field of medicine, such as intestinal regulators, the field of daily necessities, such as toothpaste, and the fields of animal feed and plant fertilizers, such as silage, animal feed, and plant liquid fertilizers.

[0044] Industrial Applicability

[0045] Bifidobacterium (Bifidobacterium longum strain N714) has a high activity in inhibiting the production of IL-17.

[0046] Examples

[0047] Examples of the present invention will now be described, but the present invention is not limited to the following examples.

[0048] <Example 1> Inhibition Test of IL-17 Production

[0049] An IL-17 production inhibition test was conducted on the Bifidobacterium longum strain N714 of the present invention and the Bifidobacterium longum control strain owned by the company.

[0050] The strain of the present invention, the following type strain and comparative strain were each cultured in GAM medium (Nippon Suisan Kaisha, Ltd.) shown in Table 3 at 37°C for 24 hours. AnaeroPac (Mitsubishi Gas Chemical Company, Inc.) was used to culture the bacteria under anaerobic conditions. Then the bacteria were collected by centrifugation. The collected bacteria were washed 3 times with sterile water, heat sterilized, and frozen. Then the bacteria were freeze-dried with a freeze dryer to produce dry bacterial powder. The obtained dry bacterial powder was suspended in PBS(-) to obtain a bifidobacteria suspension.

[0051] The hPBMC was adjusted to 1.0×10 6Cells per well were seeded in 96-well plates. Then each sample group was added to a final concentration of 10 ng / mL. Th17 cell differentiation stimulants of 25 ng / mL rhIL-6 (BioLegend), 12.5 ng / mL rhIL-23 (BioLegend), 25 ng / mL rhIL-21 (BioLegend), 2 ng / mL rhTGF-β1 (BioLegend), and 25 ng / mL rhIL-1β (BioLegend) were added and incubated at 37 °C and 5% CO2 for 96 hours. After incubation, the culture supernatants of hPBMC were collected and IL-17 in the supernatants was measured using a commercially available immunoassay kit (R&D). Samples without the Bifidobacterium suspension and Th17 cell differentiation stimulants were defined as "unstimulated", while samples containing only the Th17 cell differentiation stimulants were defined as "stimulated".

[0052] Figure 1 The concentration (pg / mL) of IL-17F in the culture supernatants in the presence of each sample group is shown.

[0053] Figure 1 Indicates that some Bifidobacterium strains inhibit the production of IL-17F while others do not. Compared with other Bifidobacterium strains, strain N714 has the highest activity in inhibiting IL-17F production, although the production of IL-17F is higher than that of the unstimulated samples. The low concentration of unstimulated IL-17F may be due to the failure to differentiate into Th17 cells. Although we are not sure about the reason for the inhibition of IL-17F production in strain N714, we speculate as follows at this stage: One possibility is that the addition of strain N714 inhibits Th17 cell differentiation itself, thus inhibiting the production of IL-17F. Another possibility is that although Th17 cells are differentiated, the production of IL-17 is inhibited for some reason. One or both of these reasons, or some other reasons, may be the cause of the inhibition of IL-17 production.

[0054] In this study, IL-17F was used as an indicator to evaluate IL-17 because IL-17F has the highest yield and is easy to detect among the IL-17 subgroups. However, the production of subgroups other than IL-17F is very low and difficult to detect. Therefore, IL-17F was used as the evaluation indicator in this study. Based on the above inferences, it is assumed that the production of subgroups other than IL-17F is also inhibited by Bifidobacterium longum subsp. longum N714.

[0055] <Example 2> Evaluation of the superiority of Bifidobacterium longum subsp. longum strain N714

[0056] Then, the Bifidobacterium longum strain N714 was compared with the reference strain Bifidobacterium longum JCM1217 to confirm the superiority of the Bifidobacterium longum strain N714. The experiment was conducted 3 times as in Example 1, and the average value was compared with the stimulated group. The results are as Figure 2 shown.

[0057] Figure 2 It was clearly shown that the production of IL-17 in the group containing the Bifidobacterium longum strain N714 was significantly inhibited compared with the stimulated group.

[0058] As described above, the Bifidobacterium longum strain N714 has the function of effectively inhibiting the production of IL-17.

Claims

1. Bifidobacterium, which is Bifidobacterium longum strain N714 with the deposit number of NITE BP-03004 Bifidobacterium longum ).

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