DNA damage-restoration promoter having genus bifidobacterium bacterium as active ingredient

Abstract

To provide a novel application of Bifidobacterium.SOLUTION: There is provided an application as a DNA damage-restoration promoter, an acid resistance-imparting agent of cell, a protein synthesis promoter, or a bacterium infection prophylactic agent which has the genus Bifidobacterium bacterium (e.g.BF-1 strain) having mucin adhesiveness as an active ingredient.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to uses of bacteria of the genus Bifidobacterium.

[0002] Bifidobacterium bacteria (hereinafter referred to as "bifidobacteria") are major bacteria in the human intestinal flora, and many bifidobacteria-containing fermented milk and beverage products, live bacterial preparations, and other products are commercially available.

[0003] For example, Patent Document 1 proposes a fermented milk food product that reduces the burden on the stomach after eating, taking advantage of the property that Bifidobacterium bifidum has excellent survival properties even when stored under aerobic conditions in fermented milk food products. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 4881304 Summary of the Invention [Problem to be solved by the invention]

[0005] However, Patent Document 1 only provides limited disclosure regarding the effects of specific bifidobacteria on gastric cells and their uses. Specifically, it only discloses that specific bifidobacteria have preventive and therapeutic effects on gastric cells against gastritis and ulcers, as well as against unidentified stomach complaints, hyperacidity, and gastroesophageal reflux disease. Therefore, there remains room for further research and development into the properties and uses of bifidobacteria.

[0006] Therefore, an object of the present invention is to elucidate new properties of bifidobacteria and to provide new uses based on those properties. [Means for solving the problem]

[0007] As a result of extensive research, the present inventors unexpectedly discovered new properties of Bifidobacterium bacteria having the ability to adhere to mucin, particularly the Bifidobacterium bifidum YIT 10347 strain (hereinafter referred to as "BF-1 strain"), which also has the ability to adhere to mucin. The method for analyzing these properties will be described in detail in the Examples section below. The inventors then discovered that these properties make the BF-1 strain and products containing the BF-1 strain suitable for the following uses, leading to the completion of the present invention.

[0008] [1] A DNA damage repair promoter whose active ingredient is Bifidobacterium, a bacterium with mucin-adhesive properties.

[0009] [2] The DNA damage repair promoter according to [1] above, wherein the Bifidobacterium bacterium having mucin-adhering properties has a sortase gene.

[0010] [3] The DNA damage repair promoter according to [1] or [2], wherein the mucin-adherent Bifidobacterium bacterium expresses a protein that is displayed on the cell surface by sortase.

[0011] [4] The DNA damage repair promoter according to [3] above, wherein the protein presented on the cell surface by sortase is a protein having the [L / I / V][S / A]XTG motif in its amino acid sequence.

[0012] [5] The DNA damage repair promoter according to any one of [1] to [3] above, wherein the Bifidobacterium bacterium has 20 or more [L / I / V][S / A]XTG motifs.

[0013] [6] The DNA damage repair promoter according to any one of [1] to [5] above, wherein the Bifidobacterium bacterium is Bifidobacterium bifidum.

[0014] [7] An agent for imparting acid resistance to cells, the active ingredient of which is a Bifidobacterium bacterium with mucin-adhering properties.

[0015] [8] The acid resistance imparting agent according to the above [7], wherein the Bifidobacterium bacterium having mucin-adhering properties has a sortase gene.

[0016] [9] The acid tolerance imparting agent according to [7] or [8], wherein the mucin-adherent Bifidobacterium bacterium expresses a protein that is presented on the cell surface by sortase.

[0017]

[10] The acid resistance imparting agent according to [9] above, wherein the protein presented on the cell surface by sortase is a protein having an [L / I / V][S / A]XTG motif in its amino acid sequence.

[0018]

[11] A protein synthesis promoter containing mucin-adherent Bifidobacterium bacteria as an active ingredient.

[0019]

[12] The protein synthesis promoter according to

[11] above, wherein the Bifidobacterium bacterium having mucin-adhering properties has a sortase gene.

[0020]

[13] The protein synthesis promoter according to

[11] or

[12] , wherein the mucin-adherent Bifidobacterium bacterium expresses a protein that is presented on the cell surface by sortase.

[0021]

[14] The protein synthesis promoter according to

[13] above, wherein the protein presented on the cell surface by sortase is a protein having the [L / I / V][S / A]XTG motif in its amino acid sequence.

[0022]

[15] A bacterial infection preventive agent containing as an active ingredient Bifidobacterium bacteria with mucin adhesive properties.

[0023]

[16] The bacterial infection preventive agent according to

[15] , wherein the Bifidobacterium bacterium having mucin-adhering properties has a sortase gene.

[0024]

[17] The bacterial infection preventive agent according to

[15] or

[16] , wherein the mucin-adherent Bifidobacterium bacterium expresses a protein that is presented on the cell surface by sortase.

[0025]

[18] The bacterial infection preventive agent according to

[17] , wherein the protein presented on the cell surface by sortase is a protein having the [L / I / V][S / A]XTG motif in its amino acid sequence. [Effects of the Invention]

[0026] According to the present invention, a DNA damage repair promoter containing a mucin-adhesive Bifidobacterium bacterium as an active ingredient can be provided. The present invention can also provide an agent for imparting acid resistance to cells, a protein synthesis promoter, or a bacterial infection preventive agent containing a mucin-adhesive Bifidobacterium bacterium as an active ingredient. [Brief explanation of the drawings]

[0027] [Figure 1] FIG. 1 is a diagram illustrating the property of mucin non-adherent strain #1476 not having adhesive properties to mucin. [Figure 2] FIG. 2 is a table illustrating the primer sets for CDS0427, which encodes sortase. [Figure 3] FIG. 3 shows electrophenograms obtained by a bioanalyzer when the sortase protein coding region was amplified for the BF-1 strain and a mucin-nonadherent strain. [Figure 4] FIG. 4 is a table showing the genomic information of bifidobacteria used for comparison of sortases and sortase-dependent proteins (SDPs). [Figure 5] FIG. 5 shows the total number of sortase-dependent proteins present in various bifidobacteria. [Figure 6] FIG. 6 shows the breakdown of sortase recognition sequences of sortase-dependent proteins present in various bifidobacteria. [Figure 7]FIG. 7 shows the adhesion of the BF-1 strain to GCIY cells and the acid resistance of GCIY cells. [Figure 8] FIG. 8 shows the acid resistance of GCIY cells treated with the BF-1 strain and a mucin-nonadherent strain. [Figure 9] FIG. 9 is a table listing some of the pathways in GCIY cells whose expression was altered and significantly affected by the adhesiveness of the BF-1 strain. [Figure 10] FIG. 10 is a table showing the abbreviations of various bifidobacteria and the breakdown of the various sortase recognition sequences. DETAILED DESCRIPTION OF THE INVENTION

[0028] The bifidobacterial analysis method and its uses of the present invention will be described in detail below. However, the description of the constituent elements described below is an example of one embodiment of the present invention, and the present invention is not limited to these contents.

[0029] The active ingredient of the DNA damage repair promoter, the agent for imparting acid resistance to cells, the protein synthesis promoter, and the agent for preventing bacterial infection of the present invention is a bacterium of the genus Bifidobacterium. First, the bacterium of the genus Bifidobacterium will be described.

[0030] Examples of the Bifidobacterium bacteria include Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium adolescentis, Bifidobacterium catenulatum, Bifidobacterium pseudocatenulatum, and Bifidobacterium animalis, and one or more of these bacterial species may be used. Among these, Bifidobacterium bifidum is preferred from the viewpoints of promoting DNA damage repair, imparting cellular acid resistance, promoting protein synthesis, and preventing bacterial infection.

[0031] A particularly preferred example of the Bifidobacterium bifidum is the Bifidobacterium bifidum YIT 10347 strain (BF-1 strain, internationally deposited as FERM BP-10613 on June 23, 2005, with the International Patent Organism Depositary of the National Institute of Advanced Industrial Science and Technology (6-1 Central, Higashi 1-chome, Tsukuba, Ibaraki Prefecture, Japan)). In the present invention, "having mucin adhesive properties" means that the target Bifidobacterium bacterium has the ability to adhere to mucin, regardless of the adhesion site or type. Specifically, this means that when measured at absorbance (OD) 490 nm using the following method, the absorbance (OD) 490 nm is 0.2 or higher when the mucin concentration is 0.01 μg / well or higher. (Method for measuring mucin adhesion ability) Various mucins serially diluted with carbonate buffer are immobilized overnight at 4°C on a 96-well plate for ELISA (Nunc MaxiSorp® flat-bottom 96-well plate or LIMBRO microplate). PBS containing 1% gelatin is layered on the mucin-coated plate, and blocking is performed at room temperature for 1 hour. A bacterial solution of Bifidobacterium bacteria diluted with PBS containing 1% gelatin to an optical density (OD) of 1 at 600 nm is then layered on the blocked mucin-coated plate and incubated at 37°C for 1 hour. After removing the bacterial solution, the plate is washed three times with PBS, and then incubated with anti-Bifidobacterium rabbit polyclonal antibody (formalin-treated killed Bifidobacterium bacteria are sent to Eurofin for contract manufacturing) at room temperature for 1 hour. After removing the primary antibody (anti-Bifidobacterium rabbit polyclonal antibody), the plate was washed three times with PBS and incubated with the secondary antibody (Goat anti-rabbit Ig, Human ads-HRP, SouthernBiotech) for 1 hour at room temperature. After removing the secondary antibody, the plate was washed three times with PBS and then subjected to a horse radish peroxidase color reaction. The absorbance (OD) at 490 nm was measured using a microplate reader. The buffer preparation and other procedures followed the protocol published by Sigma-Aldrich (http: / / www.sigmaaldrich.com / content / dam / sigma-aldrich / docs / SAJ / Brochure / 1 / j_recipeabelisa.pdf).

[0032] Furthermore, since the Bifidobacterium bacterium must have sortase activity, either live bacterial cells having sortase activity or a processed product of bacterial cells having sortase activity is preferred. Furthermore, from the viewpoint of mucin adhesiveness, the Bifidobacterium bacterium preferably expresses a protein that is presented on the cell surface by sortase, and the protein presented on the cell surface by sortase is preferably a protein having an [L / I / V][S / A]XTG motif in its amino acid sequence. Furthermore, from the viewpoint of mucin adhesiveness, the Bifidobacterium bacterium preferably expresses 10 or more proteins having the [L / I / V][S / A]XTG motif, more preferably 20 or more, and even more preferably 30 or more.

[0033] In the present invention, "promoting DNA damage repair" means promoting the repair of damaged DNA molecules. Bifidobacterium bacteria can be used as a DNA damage repair promoter and can also be used to produce a DNA damage repair promoter.

[0034] Furthermore, "imparting acid resistance to cells" means that acid resistance is imparted to cells, enabling the cells to survive in an acidic environment. "Promoting protein synthesis" means promoting protein synthesis in cells. "Preventing bacterial infection" means having a preventive effect against bacterial infection of cells. Bifidobacterium bacteria can also be used as preparations aimed at these effects, and can also be used to produce such preparations.

[0035] When used as a DNA damage repair promoter, an agent for imparting acid resistance to cells, a protein synthesis promoter, or an agent for preventing bacterial infection, the dosage form of an oral preparation may be, for example, a tablet, capsule, granule, sugar-coated tablet, pill, fine granule, powder, dust, sustained-release preparation, suspension, emulsion, syrup, lyophilized preparation, liquid, elixir, etc.

[0036] The preparations can be produced by conventional methods, and the Bifidobacterium may be used alone or in combination with a pharmaceutically acceptable carrier, such as an excipient, binder, disintegrant, surfactant, lubricant, flow enhancer, flavoring agent, colorant, fragrance, diluent, disinfectant, osmotic pressure adjuster, pH adjuster, emulsifier, preservative, stabilizer, absorption aid, antioxidant, UV absorber, humectant, thickener, glossing agent, activity enhancer, anti-inflammatory agent, isotonicity adjuster, soothing agent, and odor enhancer.

[0037] Examples of the binder include starch, dextrin, powdered gum arabic, gelatin, methyl cellulose, hydroxypropyl cellulose, crystalline cellulose, ethyl cellulose, polyvinylpyrrolidone, and macrogol.

[0038] Examples of the disintegrant include hydroxypropyl starch, sodium carboxymethylcellulose, calcium carboxymethylcellulose, carboxymethylcellulose, and low-substituted hydroxypropylcellulose.

[0039] Examples of the surfactant include sodium lauryl sulfate, soybean lecithin, sucrose fatty acid ester, polysorbate 80, and the like.

[0040] Examples of the lubricant include talc, waxes, hydrogenated vegetable oil, magnesium stearate, calcium stearate, aluminum stearate, polyethylene glycol, and the like.

[0041] Examples of the flow promoter include light anhydrous silicic acid, dried aluminum hydroxide gel, synthetic aluminum silicate, magnesium silicate, and the like.

[0042] Examples of the diluent include distilled water for injection, physiological saline, aqueous glucose solution, olive oil, sesame oil, peanut oil, soybean oil, corn oil, propylene glycol, polyethylene glycol, and the like.

[0043] Furthermore, from the viewpoint of the DNA damage repair promoting effect, cellular acid resistance imparting effect, protein synthesis promoting effect, or bacterial infection preventing effect of the present invention, the above preparations can be used not only as pharmaceuticals but also as foods and beverages, quasi-drugs, pet food, etc. In this case, the above Bifidobacterium bacteria can be incorporated into the above foods and beverages, either as they are or with the addition of various nutritional components. These foods and beverages can be used as health foods or food ingredients useful for providing the above effects, and these foods and beverages, etc. or their containers may be labeled to indicate that they have the above effects.

[0044] When a DNA damage repair promoter, a cellular acid resistance promoter, a protein synthesis promoter, or a bacterial infection preventive agent is incorporated into a food or beverage, additives that can be used in foods and beverages may be used as appropriate, and the additives may be formed into an edible form, such as granules, particles, tablets, capsules, or pastes, using conventional means. The additives may also be added to various foods, such as processed meat products such as ham and sausage, processed seafood products such as kamaboko and chikuwa, bread, confectionery, butter, powdered milk, and fermented foods and beverages, or to beverages such as water, fruit juice, milk, soft drinks, and tea drinks. Among these foods and beverages, fermented products containing Bifidobacterium bacteria as an active ingredient, such as fermented milk, lactic acid bacteria beverages, fermented soy milk, fermented fruit juice, and fermented plant liquid, are preferred.

[0045] These fermented foods and beverages can be produced according to conventional methods. For example, fermented milk can be produced by inoculating and culturing Bifidobacterium bacteria in a sterilized milk medium, followed by homogenization to obtain a fermented milk base. A separately prepared syrup solution is then added and mixed, homogenized using a homogenizer, or the like, and flavors are added to produce the final product. The fermented milk thus obtained can be made into any form, such as plain, soft, or fruit-flavored, solid, or liquid.

[0046] Next, the dosage of Bifidobacterium will be described. There is no strict limit to the dosage of Bifidobacterium bacteria, which are the active ingredients of the DNA damage repair promoter, cellular acid resistance promoter, protein synthesis promoter, or bacterial infection preventive agent of the present invention. Since the effects obtained vary depending on various modes of use such as the subject and the applicable disease, it is desirable to set the dosage appropriately. However, from the viewpoint of the DNA damage repair promoting effect, the effect of imparting cellular acid resistance, the effect of promoting protein synthesis, or the effect of preventing bacterial infection, it is preferable to set the dosage of Bifidobacterium bacteria to 1×10 3 ~1×10 11 The amount containing the CFU per day is preferably 1 x 10 8 ~1×10 10 A daily dose of CFU is more preferred. [Example]

[0047] (Test Example 1) <Evaluation of mucin adhesion and antibody reactivity> Various mucins serially diluted with carbonate buffer were immobilized overnight at 4°C on a 96-well plate for ELISA (Nunc MaxiSorp® flat-bottom 96-well plate or LIMBRO microplate). PBS containing 1% gelatin was overlaid on the mucin-coated plate, and blocking was performed at room temperature for 1 hour. A bacterial suspension of the BF-1 strain, diluted with PBS containing 1% gelatin to an optical density (OD) of 1 at 600 nm, was then overlaid on the blocked mucin-coated plate and incubated at 37°C for 1 hour. Furthermore, a bacterial suspension of the non-mucin-adherent strain Bifidobacterium bifidum #1476 (hereinafter referred to as the non-mucin-adherent strain), which is expected to lack the ability to adhere to mucin, was prepared as a control for the BF-1 strain, and the same procedure was performed. After removing the bacterial solution, the plates were washed three times with PBS and incubated with anti-BF-1 rabbit polyclonal antibody (formalin-treated BF-1 killed cells were sent to Eurofin for contract manufacturing) for 1 hour at room temperature. After removing the primary antibody (anti-BF-1 rabbit polyclonal antibody), the plates were washed three times with PBS and incubated with secondary antibody (Goat anti-rabbit Ig, Human ad-HRP, SouthernBiotech) for 1 hour at room temperature. After removing the secondary antibody, the plates were washed three times with PBS and incubated with horse radish peroxidase color reaction. The absorbance (OD) at 490 nm was measured using a microplate reader. The buffer preparation and other procedures followed the protocol published by Sigma-Aldrich (http: / / www.sigmaaldrich.com / content / dam / sigma-aldrich / docs / SAJ / Brochure / 1 / j_recipeabelisa.pdf).

[0048] The results of Test Example 1 are shown in Figure 1. In Figure 1, the horizontal axis represents the amount of mucin per well (µg / well), and the vertical axis represents the absorbance at OD 490 nm. Figure 1 confirms that the BF-1 strain adheres to mucin-coated plates when the mucin concentration in the well exceeds a certain level, whereas the mucin-nonadherent strain (#1476) does not. Therefore, Test Example 1 demonstrated that the mucin-nonadherent strain #1476 does not adhere to mucin.

[0049] (Test Example 2) <Strains used and cultivation method> The mucin-adherent strain used was Bifidobacterium bifidum YIT 10347 (FERM BP-10613), a BF-1 strain, and the mucin-nonadherent strain #1476.

[0050] Each strain was inoculated into 50 mL of m-ILS medium and cultured overnight at 37°C. The culture was then centrifuged (10,000 × g, 5 min) to recover the strains. After washing with PBS, the strains were washed again with HBS-P (10 mM HEPES pH 7.4, 150 mM NaCl, 0.05% (v / v) Surfactant P20) and suspended in 5 mL of HBS-P. This bacterial suspension was used for various tests.

[0051] PCR PCR was performed using the primer set for CDS0427, which encodes the sortase shown in Figure 2, and DNA from B. bifidum YIT 10347 (strain BF-1) as a template to amplify the protein-coding region of the sortase. The results are shown in Figure 3.

[0052] Figure 3 shows the electrophenogram of the bioanalyzer. In the BF-1 strain, CDS0427 (approximately 1.2 kbp), which encodes the sortase, was amplified, but in #1476, an approximately 2.1 kbp amplification product containing some insertion was obtained. This suggests that the sortase in the mucin-nonadherent strain #1476 is inactive.

[0053] 3 shows that the mucin-nonadherent strain lacks the sortase gene. In other words, it was confirmed that the mucin-adherent BF-1 strain has the sortase gene.

[0054] Sortase is a type of prokaryotic enzyme generally referred to as "sortase" or "housekeeping sortase." Sortases are known to be classified into six classes, A to F, based on the similarity of their amino acid sequences, but the sortase in the present application is classified into class E, and its function in relation to adhesiveness is unknown.

[0055] (Test Example 3) Extraction of protein information from genome information The genome information for the 17 bifidobacterial species listed in Figure 4 was downloaded from DDBJ (http: / / www.ddbj.nig.ac.jp / index-j.html). Protein-coding sequences (CDS) were extracted from the genome information using Genomics Workbench ver. 9.01 (Qiagen, Tokyo, Japan), and text searches were performed using Excel. Additionally, the free genome browser Artemis (http: / / www.sanger.ac.uk / science / tools / artemis) was used to search for protein-coding sequences with specific amino acid sequences (L (leucine) P (proline) X (arbitrary) T (threonine) G (glycine) or [L (leucine) / I (isoleucine) / V (valine)][S (serine) / A (alanine)] X (arbitrary) T (threonine) G (glycine)). The amino acid sequence of the target protein was subjected to a homology search using the genetic information processing software GENETYX Ver. 11 (Genetyx, Tokyo, Japan), and the locations of the genes hit by Artemis were confirmed to investigate whether there were any homologous genes (more than 50% at the amino acid level) in the genomes of various bifidobacteria. The results are shown in Figures 5 and 6.

[0056] Figures 5 and 6 show the total number of sortase-dependent proteins (hereinafter referred to as "SDP") present in various bifidobacteria (Figure 5) and a breakdown of sortase recognition sequences (Figure 6). In Figure 5, the horizontal axis shows the abbreviations of the various bifidobacteria disclosed in Figure 4, and the vertical axis shows the total number of proteins that have an LPXTG motif or an [L / I / V][S / A]XTG motif and contain oligomers of R (arginine), K (lysine), and H (histidine) that confer a positive charge at the C-terminus, i.e., SDP proteins.

[0057] In Figure 6, the horizontal axis shows the abbreviations of various bifidobacteria, and the vertical axis shows the total number of sortase recognition sequences. Figure 10 shows a breakdown of the abbreviations of various bifidobacteria and the sortase recognition sequences. Because one SDP may have multiple recognition sequences, the total number of SDPs in Figure 6 is greater than the total number shown in Figure 5. Figure 6 confirms that B. bifidum has significantly more SDPs with the [L / I / V][S / A]XTG motif than other bacterial species. Specifically, B. bifidum has 38 [L / I / V][S / A]XTG motifs, while other bifidobacteria have between 3 and 14 [L / I / V][S / A]XTG motifs.

[0058] Therefore, Test Example 3 demonstrated that the genome of B. bifidum contains coding regions for proteins that are presented on the cell surface by sortase. Furthermore, it was demonstrated that there are multiple coding regions for proteins that are presented on the cell surface by sortase, and that the amino acid sequences of the proteins contain the [L / I / V][S / A]XTG motif. As will be described later, bifidobacteria that contain many proteins with the [L / I / V][S / A]XTG motif are advantageous because they have high adhesiveness and strong DNA damage repair activity.

[0059] Example 1 Effects of bacterial adhesion on cultured cells Using GCIY cells, a cultured cell line derived from gastric cancer, the adhesive properties of the BF-1 strain and the mucin-nonadherent strain #1476 were compared.

[0060] <Adhesion of bacteria to GCIY cells and acid resistance of GCIY cells> GCIY cells were used to analyze the interaction between host cells and bacteria. For adhesion experiments, the BF-1 strain or the mucin-nonadherent strain #1476 was collected by centrifugation (3,000 × g, 4°C, 10 min), washed with MEM medium containing 10% FBS (f-MEM medium), and resuspended in the same medium. 2 × 10 3 Or 1×10 4 After rinsing the cell sheet (1 × 10 cells / well) with fresh f-MEM medium, the BF-1 strain, mucin-nonadherent strain #1476 (1 × 10 5 , 1×10 6 ,1×10 7 CFU / well) or f-MEM medium (negative control) was added and co-cultured for 30 minutes at 37°C in a CO2 incubator. The co-cultured cell sheet was rinsed three times with fresh f-MEM medium, and then acidified to pH 4.5 f-MEM medium was added and cultured for 4.5 hours. After this acid treatment, the cell sheets were rinsed three times with fresh f-MEM medium and then observed under a microscope for cell morphology. The acid-treated cell sheets were cultured overnight in fresh f-MEM medium, and viable cell counts as well as cell morphology were examined.

[0061] The results are shown in Figures 7(A) and (B). In Figure 7(A), the horizontal axis represents the BF-1 strain and the non-mucin-adherent strain (#1476), respectively. The vertical axis represents the cell count of the BF-1 strain or the non-mucin-adherent strain adhered to GCIY cells. The median, interquartile range, median, minimum, maximum, outlier, p-value, etc. are listed, but explanation is omitted as they are the same as standard notations. The number of adherent bacteria of the non-mucin-adherent strain #1476 was significantly lower than that of BF-1. Furthermore, this result was consistent with the results of mucin adhesion in "Test Example 1" (Figure 1).

[0062] In Figure 7(B), the horizontal axis represents the negative control (medium only), the BF-1 strain, and the mucin-nonadherent strain (#1476). The vertical axis represents the viability of GCIY cells. The effect of bacterial adhesion on the acid resistance of GCIY cells was examined in Figure 7(B). Treatment with the BF-1 strain significantly increased the viability of GCIY cells compared to the untreated negative control, but no such effect was observed in the mucin-nonadherent strain #1476.

[0063] Furthermore, Figure 8 shows photographs of cell morphology. (A) and (B) are photographs of GCIY cells cocultured with the BF-1 strain (A) and the non-mucin-adherent strain (B), respectively, immediately after acid treatment. (C) and (D) are photographs of GCIY cells cocultured with the BF-1 strain (C) and the non-mucin-adherent strain (D), respectively, after acid treatment and overnight incubation. It was confirmed that GCIY cells cocultured with the BF-1 strain were morphologically more active than those cocultured with the non-mucin-adherent strain #1476. Therefore, it was confirmed that GCIY cells cocultured with the BF-1 strain were conferred acid resistance.

[0064] Next, to explore the mechanism by which bacterial adhesion enhances cellular acid resistance, we performed microarray analysis of GCIY cells co-cultured with the BF-1 strain or the mucin-nonadherent strain #1476.

[0065] <Co-culture of GCIY cells and bacteria> GCIY cells were seeded into 6-well plates at a coenfluent concentration (4–5 × 10 5 The cell sheet was then rinsed with fresh f-MEM medium and cultured until the cell sheet reached 5 × 10 cells / ml. After rinsing, the BF-1 strain, mucin-nonadherent strain #1476 (5 × 10 6CFU / well) or f-MEM medium (negative control) was added and co-cultured for 1 or 30 minutes at 37°C in a CO2 incubator. The co-cultured cell sheet was rinsed three times with fresh f-MEM medium and then cultured for 2 or 12 hours. The cell sheet was detached from the plate by adding 0.25% Trypsin-EDTA (Gibco) and kept in a CO2 incubator at 37°C for 5 minutes before RNA extraction.

[0066] When co-cultured with BF-1 strain or the mucin-non-adherent strain #1476, the top 1,000 genes showing significant expression changes between the strains were subjected to pathway analysis using WikiPathways, and pathways involved in DNA damage repair, promotion of protein synthesis, and prevention of bacterial infection were identified.

[0067] Figure 9 shows a partial list of pathways in GCIY cells whose expression was significantly affected by the adhesiveness of the BF-1 strain. Groups A, B, and C are involved in (Group A) DNA damage repair, (Group B) protein synthesis promotion, and (Group C) bacterial infection prevention, respectively.

[0068] Therefore, it was revealed that co-culturing the mucin-adherent BF-1 strain with target cells significantly altered the expression of genes involved in DNA damage repair, protein synthesis promotion, and bacterial infection prevention in the target cells.

[0069] Furthermore, the pathway in Figure 9 includes genes that regulate the expression of genes involved in the DNA damage repair pathway, which is advantageous in that it can be expected to be effective not only in reducing DNA damage caused by UV rays, but also in reducing DNA damage caused by tobacco, radiation, active oxygen, drugs, etc. [Industrial Applicability]

[0070] The Bifidobacterium bacteria of the present invention have sortase activity and therefore mucin adhesiveness, and when co-cultured with gastric cells, they not only confer acid resistance to gastric cells but also significantly alter the expression of genes involved in cellular DNA damage repair, protein synthesis promotion, and bacterial infection prevention. Therefore, the Bifidobacterium bacteria of the present invention can be used as an agent for conferring acid resistance to cells, an agent for DNA damage repair, an agent for protein synthesis promotion, and an agent for preventing bacterial infection.

Claims

[Claim 1] A DNA damage repair promoter containing, as an active ingredient, a Bifidobacterium bacterium having mucin adhesive properties.

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