WO2017169966A1 - Bifidobacterium having serotonin-secretion-promoting ability - Google Patents

Abstract

[Problem] The purpose of the present invention is to provide a strain of Bifidobacteria having a high serotonin-secretion-promoting ability, and a food and/or beverage containing a treated form of the Bifidobacterium. [Solution] The inventors focused on the fact that serotonin is indispensable for the biosynthesis of BDNF and, as a result of screening numerous Bifidobacteria, discovered a Bifidobacterium derived from the infant intestine having high serotonin production ability; specifically, Bifidobacterium longum N61 (NITE BP-02214).

Description

Bifidobacterium with ability to promote serotonin secretion

TECHNICAL FIELD The present invention relates to a strain of bifidobacteria derived from infant intestine having high ability to promote serotonin secretion, a beverage containing the treated product of the bifidobacteria, and a food.

In recent years, advances in neuroscience and molecular biology have made it possible to form a bidirectional network between central nerves and peripheral organs through signal transduction substances such as nervous system and humoral factors (hormone, cytokines), and receptors. It is clear that For example, various signals generated in the intestine are known to be transmitted to the central nervous system via these pathways and to affect the information processing process, and the intestinal flora is involved in the development of the nervous system. It has been pointed out that they may be deeply involved in

Enteric bacteria produce physiologically active substances such as short-chain fatty acid and GABA, but when butyric acid (BA), which is one of short-chain fatty acids, is administered to mice, brain-derived neural factor BDNF in the hippocampus and frontal lobe (Brain derived The neurotrophic factor) concentration is increased, and antidepressant action is obtained (Non-patent Document 1). On the other hand, it is known that serotonin is essential for the biosynthesis of BDNF (Non-patent Document 2).

Nobuyuki Sudo, Enteric bacteria and brain-gut correlation, Fukuoka Medical Journal 2009; 100 (9): 298-304, 2007.09. Pathophysiology of depression: the concept of synaptic plasticity European Psychiatry Volume 17, Supplement 3, July 2002, Pages 306-310 R.S. Duman, Mol. Psychiatry, 7, Suppl 1, S29 (2002).

An object of the present invention is to provide a strain of bifidobacteria of infant intestinal origin having high ability to promote serotonin secretion and a food and drink containing the treated product of the bifidobacteria.

The present inventors focused on the fact that serotonin is essential for the biosynthesis of BDNF, and as a result of screening a large number of bifidobacteria, they found that there is a bifidobacteria having a high ability to promote serotonin secretion. We came to complete the invention.

That is, the first invention of the present application is a bifidobacteria belonging to the genus Bifidobacterium having the ability to promote serotonin secretion, and in the second invention of the present application, the Bifidobacterium described in the first invention of the present application is a bifidobacteria. It is a longum N61 strain (NITE BP-02214).

Furthermore, the applicant of the present application also intends a food or drink containing the bifidobacteria, or a serotonin secretion promoter containing the bifidobacteria as an active ingredient. That is, the third invention of the present application is a food and drink containing the bifidobacteria described in the second invention of the present application, and the fourth invention of the present application is a serotonin secretion containing the bifidobacteria described in the second invention as an active ingredient It is an accelerator.

The bifidobacteria of the present invention have a serotonin secretion promoting ability.

FIG. 1 is a diagram comparing the serotonin secretion promoting ability of the strain of the present invention and a comparative strain. FIG. 2 is a diagram comparing the skimmed milk medium growth properties of the strain of the present invention and the comparative strain.

Hereinafter, the present invention will be described in detail.
1. Bifidobacterium longum N61 strain (NITE BP-02214)
The Bifidobacterium of the present invention is Bifidobacterium longum. Among bifidobacteria belonging to Bifidobacterium longum, it is Bifidobacterium longum N61 strain (NITE BP-02214). The symbol N61 referred to in the present invention is a number uniquely assigned to the strain by Nisshin Foods Holdings Co., Ltd., and this Bifidobacterium longum N61 strain is isolated for the first time by the present inventor.

The Bifidobacterium longum N61 strain of the present invention has been deposited under the following conditions.
(1) Name of Depositary Organization: Patent Administrative Agency, National Institute of Advanced Industrial Science and Technology Patent Microorganisms Depositary Center (2) Contact information: 〒 292-0818 Kisarazu City, Chiba Prefecture -8 2-5-8 Room 122 (3) Accession No .: NITE BP --02214
(4) Display for identification: N61
(5) Date of original deposit: March 3, 2016 (6) Transfer date to deposit under the Budapest Treaty: January 5, 2017 The mycological properties of Bifidobacterium longum N61 strain of the invention of Japan are as follows: As shown in Tables 1 and 2 of This bacteriological property is according to the method described in Bergey's manual of systematic bacteriology Vol. 2 (1986). Table 1 shows the shape and the like of this strain, and Table 2 shows the results of testing the sugar utilization by Api 50 CH and Api CHL (manufactured by BioMérieux). In Table 2, "+" indicates fermentability and "-" indicates non-fermentability.

2. Serotonin Production Test The Bifidobacterium longum N61 strain of the present invention has a high ability to promote serotonin secretion as shown in the following experimental example. The confirmation of the ability to promote serotonin secretion was carried out by the following test method.

<Preparation of subject (culture fluid) used for evaluation of serotonin secretion promoting ability>
The subject (culture fluid) used for the evaluation of the ability to promote serotonin secretion was obtained by culturing bifidobacteria in GAM medium (GAM Broth "Nissui") shown in Table 3 at 37 ° C for 24 hours.

<Serotonin secretion promotion ability evaluation>
In the evaluation of the ability to promote serotonin secretion, RIN14B cells (ATCC), which are enterochromaffin-like cells derived from rat pancreatic cancer, were used as cells. First, 10% fetal bovine serum (Invitrogen) and 100 U / mL penicillin (Sigma), 100 μg / mL streptomycin, 25 ug / ml amphotericin B (Sigma), 0.2% NaHCO ₃ (gibco), 1 mM L-Glutamine (gibco) , RIN14B cells at 37 ° C, 5% using RPMI 1640 medium (gibco) supplemented with 4.5 g / L D-Glucose (gibco), 2.383 g / L HEPES Buffer (gibco) and 110 mg / L Sodium Pyruvate (gibco) Culture was performed under CO ₂ conditions. At the time of assay, RIN14B cells cultured in a petri dish are detached using Accutase (Innova Cell Technologies) (incubate at 37 ° C for 10 minutes), and then 500 μl of 2 × 10 ⁵ cells / ml of RIN14B cells are seeded on a 24-well plate. Incubate at 37 ° C., 5% CO ₂ for 1 hour. The cell counter Countess (invitrogen) was used for cell count. The above-obtained subject (bifidobacteria culture solution) is adjusted to a constant value of turbidity Optical Density (OD), diluted 20-fold with RIN14B cell culture medium, and RIN14B cell seeded and incubated wells It was added to a final concentration of 10% Te, 37 ° C., and cultured for 24 hours under 5% CO _2. After culture, the culture supernatant of RIN14B cells to which the subject was added was collected, and the serotonin content in the supernatant was measured with a commercially available serotonin immunoassay kit (Enzo).

3. Growth Test with Skim Milk Medium <Skin Milk Medium Growth Test>
The preculture liquid was inoculated into a 10% SM (skimmed milk) medium, and this was cultured at 37 ° C. for 24 hours, and the growth in milk medium was evaluated by the number of bifidobacteria.

4. Food and Beverage The bifidobacteria of the present invention can be used by being contained in food and beverage. The Bifidobacterium of the present invention can be suitably used particularly for dairy products, but, for example, fermented milk containing bifidobacteria and a lactic acid bacteria beverage containing bifidobacteria can be considered. Although the number of bifidobacteria is not particularly defined as a component standard by the Ministerial Ordinance on the current component standards of milk and milk products, fermented milk (with non-fat milk solid content of 8.0% or more) or lactic acid bacteria beverage (non-fat milk) 1.0 × 10 ⁷ cfu / ml or more if it is solid content of 3.0% or more), 1.0 × 10 ⁶ cfu / ml or more is preferable if it is a lactic acid bacteria beverage (less than non-fat milk solid content of 3.0%) The above-mentioned number of bacteria can be realized by growing them in a fermentation broth or growing them in the form of a final product. In addition to fermented milk containing bifidobacteria and lactic acid bacteria containing bifidobacteria, it can be used for dairy products such as butter, processed eggs such as mayonnaise, and confectionery breads such as butter cake. In addition, it can be suitably used for processed foods such as instant noodles and cookies. In addition to the above, the food of the present invention may be formulated (for example, powder, granules, capsules, tablets, etc.) together with the above-mentioned Bifidobacterium, if necessary, with addition of suitable carriers and additives. Good.

The bifidobacteria of the present invention is also useful to be contained in food for specified health use, dietary supplements and the like in addition to general beverages and foods.

In addition to foods, the bifidobacteria of the present invention is used in the cosmetic field such as lotions and the like, the pharmaceutical field such as intestinal conditioners, the daily use field such as tooth powder, silage, animal feed, and animal feed and plant fertilizers such as plant liquid fertilizers. Is also applicable.

The bifidobacteria of the present invention (Bifidobacterium longum strain N61) has a high ability to promote serotonin secretion.

Examples of the present invention will be shown below, but the present invention is not limited to the following examples.
<Test Example 1> Evaluation of Serotonin Secretion-Promoting Ability Bifidobacterium longum N61 strain of the present invention and two Bifidobacterium longum comparative strains (BL1, BL2) owned by the company and Bifidobacterium strain as a standard strain Serotonin secretion promotion ability evaluation was performed about long gum (JCM1217).

The serotonin secretion promoting ability was evaluated by the following procedure. Each of the strain of the present invention and the comparative strain was cultured at 37 ° C. for 24 hours in GAM medium (GAM Broth “Nissui”) shown in Table 3 to obtain a subject (culture fluid).

Separately, 10% fetal bovine serum (Invitrogen) and 100 U / mL penicillin (Sigma), 100 μg / mL streptomycin, 25 ug / ml amphotericin B (Sigma), 0.2% NaHCO ₃ (gibco), 1 mM L-Glutamine (gibco) , RIN14B cells at 37 ° C, 5% using RPMI 1640 medium (gibco) supplemented with 4.5 g / L D-Glucose (gibco), 2.383 g / L HEPES Buffer (gibco) and 110 mg / L Sodium Pyruvate (gibco) Culture was performed under CO ₂ conditions. At the time of assay, RIN14B cells cultured in a petri dish are detached using Accutase (Innova Cell Technologies) (incubate at 37 ° C for 10 minutes), and then 500 μl of 2 × 10 ⁵ cells / ml of RIN14B cells are seeded on a 24-well plate. Incubated at 37 ° C., 5% CO ₂ for 1 hour. The cell counter Countess (invitrogen) was used for cell count. The above-obtained subject (Bifidobacterium culture fluid) is adjusted to a constant value of turbidity Optical Density (OD), diluted 20-fold with RIN14B cell culture medium, and then added to wells after RIN14B cell seeding incubation It was added to a final concentration of 10% and cultured at 37 ° C., 5% CO ₂ for 24 hours. After culture, the culture supernatant of RIN14B cells to which the subject was added was collected, and the serotonin content in the supernatant was measured with a commercially available serotonin immunoassay kit (Enzo).

The results are shown in Table 4 and FIG.

As is clear from Table 4 and FIG. 1, the ability to promote serotonin secretion of the bifidobacteria of the present invention (Bifidobacterium longum N61 strain) is very strong, and compared with other proprietary bifidobacteria and standard strains. It was also confirmed that it has high serotonin secretion promoting ability.

<Test Example 2> Skimmed Milk Medium Proliferation Test The Bifidobacterium longum N61 strain of the present invention and two Bifidobacterium longum comparative strains (BL1, BL2) owned by the company and Bifidobacterium as a standard strain. The skimmed milk medium growth test was carried out for long gum (JCM1217).

The cells were inoculated into a 10% SM (skimmed milk) medium, cultured at 37 ° C. for 24 hours, and the growth of the skimmed milk medium was evaluated by the number of bifidobacteria.

The results are shown in Table 5 and FIG.

The bacterial count of the strain of the present invention (Bifidobacterium longum N61 strain) is 2.1 × 10 ⁸ cfu / ml, which is a level at which there is no problem in producing bifidobacteria-containing fermented milk and bifidobacteria-containing lactic acid bacteria beverage The

Claims (4)

Bifidobacterium belonging to the genus Bifidobacterium which has the ability to promote serotonin secretion. The bifidobacteria of claim 1, wherein the bifidobacteria belonging to the genus Bifidobacterium is Bifidobacterium longum N61 strain (NITE BP-02214). Food-drinks containing the bifidobacteria of Claim 2. The serotonin secretion promoter which uses the bifidobacteria of Claim 2 as an active ingredient.

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