JP2022088787A - Bifidobacteria regulating expression of clock gene - Google Patents

Abstract

To provide a method for effectively regulating an expression of a clock gene, and in particular, to provide a method of promoting the transcription of Per2 gene which plays a key role in circadian rhythm formation.SOLUTION: The present invention discloses bifidobacteria that are longum species of the genus bifidobacterium which regulates an expression of a clock gene. The bifidobacteria are bifidobacterium longum strain N576 (NITE BP-03308). The present invention also discloses a food or beverage containing the bifidobacteria.SELECTED DRAWING: Figure 1

Description

The present invention relates to a strain of bifidobacteria derived from the intestine of an infant that controls the expression of a clock gene, and a beverage or food containing a treated product of the bifidobacteria.

Many organisms, including mammals, keep their biological clocks in a rhythm with a cycle of about 24 hours that matches the rotation of the earth. This biological clock is controlled by the clock genes possessed by living organisms, and its cycle fluctuations are closely related to in vivo phenomena such as sleep and wakefulness, core body temperature, hormone secretion, blood pressure, and metabolic function. On the other hand, it has been clarified that when the body clock is disturbed due to irregular life, the risk of mental illness such as insomnia and depression, lifestyle-related diseases such as metabolic syndrome and diabetes, and cancer and aging increases. .. For example, it has been reported that the risk of prostate cancer increases in shift work workers (see Non-Patent Document 1).

Bmal, Clock, Per, Cry, Dec, Rev-Erb, ROR, DBP, E4BP4, etc. are known as clock genes, and in mammals including humans, the protein encoded by the Bmal, Clock gene is responsible. Two mechanisms, the Positive Feedback mechanism and the Negative Feedback mechanism carried by the protein encoded by Per and Cry, play a central role in the biological clock. It is known that various in vivo phenomena occur by promoting and suppressing the transcription of other genes by this mechanism. In addition, in mice in which a part of the clock gene is knocked out and destroyed, the periodicity of body temperature disappears (see Non-Patent Document 2), and the clock gene does not function sufficiently, resulting in disturbance of the body clock, resulting in health. It has been reported that the risk increases.

Thus, it is a widely known fact that the clock gene is involved in the biological clock. Furthermore, it has been reported that an extract obtained from a specific plant and its components promote and suppress the expression of the clock gene itself (see Patent Document 1).

Japanese Unexamined Patent Publication No. 2018-43970

Rao D. et al. Onco Targets Ther. 8: 2817-26 (2015) Gerhart-Hines et al. Nature 503: 410-413 (2013)

By the way, if the promotion and suppression of the expression of the clock gene can be controlled, it can be expected to contribute to the normalization of the biological clock and the promotion of health. However, although not limited to the above substances, it may be difficult for some people to obtain the effect due to the influence of their constitution or the like. Therefore, a highly safe method capable of effectively controlling the expression of the clock gene is still desired.

The present invention has been made in view of the above problems. That is, an object of the present invention is to provide a method capable of effectively controlling the expression of a clock gene.

The present inventors focused on the transcription-promoting ability of the gene Per2, which plays a central role in the formation of circadian rhythm among clock genes, and screened a large number of bifidobacteria. As a result, it was found that among the bifidobacteria derived from the intestine of infants, there is a bifidobacteria having a clock gene expression control effect, and the present invention has been completed.

In order to solve the above problems, the present invention is characterized by being a bifidobacteria of the genus Bifidobacterium longum species that regulates the expression of clock genes.

Further, in order to solve the above-mentioned problems, it is preferable that the bifidobacteria are Bifidobacterium longum N576 strain (NITE BP-03308).

Further, in order to solve the above-mentioned problems, the present invention is characterized in that it is a food or drink containing the above-mentioned bifidobacteria.

The bifidobacteria of the present invention control the expression of clock genes. This is expected to form a circadian rhythm, contribute to the normalization of the body clock, and improve health. In addition, the intestinal environment can be adjusted by ingesting bifidobacteria.

FIG. 1 is a diagram comparing the Per2 transcription promoting effect of the strain of the present invention and the comparative strain using HT-29 cells. FIG. 2 is a diagram comparing the Per2 transcription promoting effect of the strain of the present invention and the comparative strain using POCA. FIG. 3 is a diagram comparing the effects of the strain of the present invention on expression control on other clock genes using HT-29 cells.

Hereinafter, the present invention will be described in detail.
1. 1. Bifidobacterium longum N576 strain (NITE BP-03308)
The bifidobacteria of the present invention is Bifidobacterium longum. The symbol N576 in the present invention is a number originally assigned to the strain by NISSIN FOODS HOLDINGS CO., LTD. The strain of the present invention was first isolated from infant feces by the present inventors.

The Bifidobacterium longum N576 strain of the present invention has been deposited under the following conditions.
(1) Depositary organization name: National Institute of Technology and Evaluation Patent Microorganisms Depositary Center (2) Contact: 292-0818 Room 2-5-8 Kazusakamatari, Kisarazu City, Chiba Prefecture (3) Deposit number: NITE BP -03308
(4) Display for identification: N576
(5) Contract date: October 30, 2020

The mycological properties of the Bifidobacterium longum N576 strain of the present invention are as shown in Tables 1 and 2 below. This mycological property is determined by colony observation with an actual microscope, morphological observation with an optical microscope, and the method described in Cowan and Steel's Manual for the Identification of Medical Bacteria, 3rd ed. (1993). Table 1 shows the shape of this strain, and Table 2 shows the results of physiological and biochemical property tests using API 20A (manufactured by BioMérieux). In Table 2, "+" indicates positive and "-" indicates negative.

*生化学試験、**発酵試験

* Biochemical test, ** Fermentation test

2. 2. Clock gene expression control ability test The Bifidobacterium longum N576 strain of the present invention has the clock gene Per2 transcription promoting ability and the transcription inhibitory ability of Bmal1, Cry1, E4BP4 as shown in the experimental examples described later. The expression control ability of the clock gene was confirmed by the following test method.

<Preparation of bifidobacteria sample>
The subject (Bifidobacterium sample) used for the evaluation of the expression control ability of the clock gene was cultured with Bifidobacterium in the medium shown in Table 3 at 35 ° C. for 13 hours. Next, the grown cells were centrifuged and collected. The collected cells were washed with sterile water three times, sterilized by heating, and then frozen. Then, it was freeze-dried using a freeze-dryer to obtain a dried cell powder. The obtained dried bacterial cell powder suspended in PBS to a concentration of 1 mg / mL was used as a bifidobacteria sample.

<Evaluation of clock gene Per2 transcription promoting ability by HT-29 cell line>
In the evaluation of the clock gene Per2 transcription promoting ability, HT-29 cells (ATCC), which are epithelial cell-like cells derived from human colon adenocarcinoma, were used. First, HT-29 cells were subjected to 37 ° C, 5% CO ₂ conditions using McCoy A5 medium (Gibco) containing 10% fetal bovine serum (Biological Industries) and 100 unit / mL penicillin-streptomycin (Gibco). Cultured below. Prior to the assay, HT-29 cells cultured in flasks were stripped using trypsin EDTA (Gibco) (incubate at 37 ° C for 3 minutes), suspended in fresh DMEM / F12 medium, and placed in a 24-well plate 1 × 10 ⁶ cells / mL HT-29 cells were seeded in 500 μL increments and incubated at 37 ° C. at 5% CO ₂ for 24 hours. A cell counter, Countess (invitrogen), was used to count the number of cells. Two hours prior to assaying, HT-29 cells were treated with 50% FBS (final concentration conversion) in the wells. Next, a 100-fold diluted bifidobacteria sample with fresh DMEM / F12 medium was used as the assay medium, and the medium was replaced with the assay medium, which was then cultured for 18 hours under 37 ° C. and 5% CO ₂ conditions. After culturing for 18 hours, RNA was extracted using the RNeasy mini kit (QIAGEN). From the extracted RNA, cDNA was synthesized using ReverTra Ace qPCR RT Master Mix (Toyobo Co., Ltd.). For gene expression measurement by RT-PCR, TB Green Premix Ex Taq II (Takara Bio Inc.) and LightCycler 96 System (Roche Diagnostics) were used. RNA extraction, cDNA synthesis and RT-PCR followed the protocol of each kit and instrument. The primer sequences used for the measurement are shown in Table 4. For relative quantification, qPCR was also performed on β-actin, which is a housekeeping gene.

<Evaluation of clock gene Per2 transcription promoting ability by POCA>
In the evaluation of the clock gene Per2 transcription promoting ability, a small intestinal absorption model (KAC, hereinafter POCA) using Caco-2 cells, which are epithelial cell-like cells derived from human colon adenocarcinoma, was used. POCA purchased from KAC was incubated with DMEM / F12 (Gibco) at 37 ° C for 24 hours at 5% CO ₂ . POCA was treated with 50% FBS (final concentration conversion) in the insert 2 hours prior to assaying. Next, a bifidobacteria sample multiplied 100 times with fresh DMEM / F12 medium was used as an assay medium, and the medium was exchanged with the assay medium, and then cultured at 37 ° C. under 5% CO ₂ conditions for 18 hours. After culturing for 18 hours, RNA was extracted using the RNeasy mini kit (QIAGEN). From the extracted RNA, cDNA was synthesized using ReverTra Ace qPCR RT Master Mix (Toyobo Co., Ltd.). For gene expression measurement by RT-PCR, TB Green Premix Ex Taq II (Takara Bio Inc.) and LightCycler 96 System (Roche Diagnostics) were used. RNA extraction, cDNA synthesis and RT-PCR followed the protocol of each kit and instrument. The primer sequence used for the measurement is the same as in Table 4. For relative quantification, qPCR was also performed on β-actin, which is a housekeeping gene.

<Regulatory evaluation of clock genes other than Per2 using HT-29 cell line>
The Bmal1, Cry1, and E4BP4 genes, which are genes other than the clock gene Per2, were also evaluated for their transactivation ability using HT-29 cells (ATCC). The evaluation method was the same as for the Per2 gene. The primer sequences used for the measurement are shown in Table 5. For relative quantification, qPCR was also performed on Gapdh, which is a housekeeping gene.

3. 3. Food and drink
The bifidobacteria of the present invention can be used by being contained in foods and drinks. For example, fermented milk containing bifidobacteria and lactic acid bacteria beverage containing bifidobacteria can be considered. The current ministry ordinance on ingredient standards for milk and dairy products does not specifically specify the number of bifidus bacteria as an ingredient standard, but fermented milk (non-fat milk solids content of 8.0% or more) and lactic acid bacteria beverages (non-fat milk). 1.0 x 10 ⁷ cfu / mL or more is preferable for solid content of 3.0% or more, and 1.0 x 10 ⁶ cfu / mL or more is preferable for lactic acid bacteria beverages (non-fat milk solid content of less than 3.0%), such as milk. The above-mentioned number of bacteria can be achieved by growing in a fermented solution or in the form of a final product. In addition to fermented milk containing bifidobacteria and lactic acid bacteria beverages containing bifidobacteria, it can also be used for dairy products such as butter, processed egg products such as mayonnaise, and sweet breads such as butter cake. Further, it can be suitably used for processed foods such as instant noodles and cookies. In addition to the above, the food product of the present invention may be in a form (for example, powder, granules, capsules, tablets, etc.) formulated by adding appropriate carriers and additives together with the bifidobacteria as needed. good.

It is also useful to include the bifidobacteria of the present invention in foods for specified health use, dietary supplements, etc. in addition to general beverages and foods.

In addition to foods, the bifizus bacterium of the present invention is used in the fields of cosmetics such as cosmetics, pharmaceuticals such as intestinal regulators, daily necessities such as toothpaste, animal feeds such as silage, animal feeds, and plant liquid fertilizers. Is also applicable.

The bifidobacteria of the present invention (Bifidobacterium longum N576 strain) have an effect of controlling the expression of a clock gene.

Hereinafter, examples of the present invention will be shown, but the present invention is not limited to the following examples.
<Example 1: Evaluation of clock gene Per2 transcription promoting ability using HT-29 cells>
The clock gene Per2 transcription promoting ability was evaluated for the Bifidobacterium longum N576 strain of the present invention and the in-house bifidobacteria.

First, the strain and the reference strain of the present invention were cultured at 35 ° C. for 13 hours in the medium shown in Table 3. Next, the grown cells were centrifuged and collected. The collected cells were washed with ultrapure water and freeze-dried. The obtained dried bacterial cell powder suspended in PBS to a concentration of 1 mg / mL was used as a bifidobacteria sample. As the reference strain, a reference strain of Bifidobacterium longum (JCM1217) and an allogeneic strain (BLN61) contained in the same library were used.

Next, HT-29 cells were seeded on a 24-well plate so as to have 5.0 × 10 ⁵ cells / well and cultured for 24 hours. Next, each bifidobacteria sample was added to a final concentration of 10 μg / mL, and cultured for 18 hours under the conditions of 37 ° C. and 5% CO ₂ . After culturing for 18 hours, RNA was extracted from the cells and gene expression was measured by RT-PCR. The non-treat was the one to which the bifidobacteria suspension was not added. In addition, as a positive control, caffeine, which has been reported as a per2 transcription promoting ability, was used.

The results when each sample group was added are shown in Table 6 and FIG. The results are shown by Per2 / β-Actin values and compared with non-treat.

As is clear from Table 6 and FIG. 1, the bifidobacteria (Bifidobacterium longum N576 strain) of the present invention have higher transcription-promoting ability than non-treat and slightly more than caffeine, which is a positive control. It was a low value. On the other hand, the reference strain has almost the same value as the non-treat, and it can be inferred that it has no transcription promoting ability. Furthermore, a significant difference was observed between the bifidobacteria of the present invention and non-treat, and between the bifidobacteria of the present invention and the reference strain.

<Example 2: Evaluation of clock gene Per2 transcription promoting ability using POCA>
Next, POCA was used to evaluate the clock gene Per2 transcription promoting ability. Since the test method is the same as that of the first embodiment, the description thereof will be omitted. The results are shown in Table 7 and FIG.

As is clear from Table 7 and FIG. 2, the bifidobacteria (Bifidobacterium longum N576 strain) of the present invention have higher transcription-promoting ability than non-treat and slightly more than caffeine, which is a positive control. It was a high value. In addition, a significant difference was observed between the bifidobacteria of the present invention and non-treat. On the other hand, as in Example 1 above, the reference strain had almost the same value as non-treat. This suggests that the reference strain has no transcription promoting ability regardless of the cells. However, the N576 strain according to the present invention promotes transcription of the Per2 gene regardless of the difference in cells. Therefore, it can be inferred that the N576 strain has a specific ability to promote transcription of the Per2 gene.

<Example 3: Control evaluation of clock genes other than Per2 using HT-29 cell line>
Finally, the effect of the Bifidobacterium longum N576 strain of the present invention on Bmal1, Cry1, E4BP4, which are clock genes other than the Per2 gene, was evaluated using HT-29 cells. Since the test method is the same as that of the first embodiment, the description thereof will be omitted. The results are shown in Table 8 and FIG.

As is clear from Table 8 and FIG. 3, the bifidobacteria (Bifidobacterium longum N576 strain) and the reference strain of the present invention are clock genes Bmal1, Cry1, E4BP4 as compared with non-treat except for a part. It can be seen that the expression of is suppressed. Furthermore, when the N576 strain and the reference strain are compared, it can be seen that the expression of the N576 strain is significantly suppressed. From the above, it was clarified that the N576 strain according to the present invention has an effect of controlling the expression of the clock gene.

Claims (3)

Bifidobacterium, a longum species of the genus Bifidobacterium that regulates the expression of clock genes. The bifidobacteria according to claim 1, wherein the bifidobacteria are Bifidobacterium longum N576 strain (NITE BP-03308). A food or drink containing the bifidobacteria according to claim 1 or 2.

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