WO2022064839A1 - Composition for regulating expression of gene involved in production or degradation of collagen, elastin, or hyaluronic acid - Google Patents

Abstract

The present invention addresses the problem of providing a technique for regulating expression of a gene involved in the production or degradation of collagen, elastin, or hyaluronic acid. This problem is solved by using a composition, for regulating the expression of a gene involved in the production or degradation of collagen, elastin or hyaluronic acid, which includes Bifidobacterium Breve M-16V (NITE BP-02622) or a culture supernatant thereof as an active ingredient, wherein the gene is selected from the collagen-degrading enzyme 1 gene (MMP1), the type I collagen gene (COL1A1), the elastin gene (ELN), the hyaluronic acid synthase 1 gene (HAS1), and the hyaluronic acid synthase 2 gene (HAS2).

Description

A composition for regulating the expression of genes involved in the production or degradation of collagen, elastin or hyaluronic acid.

The present invention relates to a composition for regulating the expression of genes involved in the production or degradation of collagen, elastin or hyaluronic acid.

Collagen is present in joints and bones. Collagen is known to cause degeneration of articular cartilage when the number of cartilage cells and collagen, which is a cartilage substrate component, decreases (Non-Patent Document 1). In addition, it is known that the collagen content increases after peaking in the 30s to 40s, but then decreases after the middle ages (Non-Patent Document 2). Thus, it is known that collagen is associated with arthritis and arthropathy. It is also known that an increase in collagen can prevent or improve osteoporosis (Patent Document 1). In addition, bullous disease is known as a disease associated with type VII collagen abnormality, specifically, sublamina densa type of acquired epidermolysis bullosa, malnutrition-type congenital epidermolysis bullosa, and linear IgA bullous dermatosis. , Bullous lupus erythematosus bullous SLE is known (Non-Patent Document 3).

Elastin is present in blood vessels and ligaments. In elastic arteries such as the aorta, elastin decreases with age, the arterial wall becomes stiff, loses elasticity, reduces compliance, and as a result, the Windkessel effect disappears, resulting in an increase in systolic blood pressure and a decrease in diastolic blood pressure. It is known to increase blood pressure (Non-Patent Document 4). Thus, it is known that there is a link between elastin and vascular aging. Further, cutis laxa and arteriosclerosis are known as diseases related to elastin abnormality (Non-Patent Document 5).

Hyaluronic acid is present in joints and cartilage. In osteoarthritis (OA) patients or OA model animals, the matrix constituent hyaluronic acid (HA) is fragmented, cartilage tissue migration occurs, and matrix-degrading enzymes from an early stage. Has been shown to be activated, and as a result, it is known that the joint substrate matrix is damaged (Non-Patent Document 6). Thus, it is known that hyaluronic acid is associated with arthritis and arthropathy.

In addition, collagen, elastin, and hyaluronic acid are all present in the skin and are known to decrease with age and to be involved in skin properties such as wrinkles and sagging (Patent Documents 2 and 3). ..

It is known that genes involved in the production or degradation of collagen, elastin or hyaluronic acid exist in the living body. As for collagen, for example, a collagen degrading enzyme 1 gene (MMP1) and a type I collagen gene (COL1A1) are known. For elastin, for example, the elastin gene (ELN) is known. As for hyaluronic acid, for example, one gene for hyaluronic acid synthase (HAS1) and two genes for hyaluronic acid synthase (HAS2) are known.
However, it is not known that Bifidobacterium Breve M-16V (NITE BP-02622) or its culture supernatant regulates the expression of these genes.

Japanese Unexamined Patent Publication No. 2015-96475 Japanese Unexamined Patent Publication No. 2020-090544 JP-A-2017-202990

Physical Therapy-Clinical / Research / Education, 18: 61-66, 2011 The Japan Geriatrics Society Magazine Vol. 50, No. 2, 2013: 3 Connective Tissue, 29: 279-283, 1997 Humans are aging with blood vessels A man is as old as his arteries.-Diagnosis of aging of blood vessels from pulse wave velocity-, Arterial Stiffness, No. 6, 2004 Connective Tissue, 24: 135-140, 1992 Glycative Stress Research, 5 (1): 012-020, 2018

An object of the present invention is to provide a technique for regulating the expression of a gene involved in the production or degradation of collagen, elastin or hyaluronic acid.

The present inventors have found that Bifidobacterium Breve M-16V (NITE BP-02622) or a culture supernatant thereof is a gene involved in the production or degradation of collagen, elastin or hyaluronic acid in a subject ingesting it. We have found that the expression can be regulated, and have completed the present invention.

That is, the present invention
A composition for regulating the expression of genes involved in the production or degradation of collagen, elastin or hyaluronic acid, which comprises Bifidobacterium Breve M-16V (NITE BP-02622) or its culture supernatant as an active ingredient.
The gene is selected from collagen degrading enzyme 1 gene (MMP1), type I collagen gene (COL1A1), elastin gene (ELN), hyaluronan synthase 1 gene (HAS1) and hyaluronan synthase 2 gene (HAS2). The composition is provided.

The composition is
Regarding the collagen degrading enzyme 1 gene (MMP1), the regulation of expression is a decrease in expression.
For the type I collagen gene (COL1A1), elastin gene (ELN), hyaluronan synthase 1 gene (HAS1) and hyaluronan synthase 2 gene (HAS2), it is preferable that the expression regulation is enhanced expression. ..

The present invention can also provide a composition for improving skin properties, which comprises Bifidobacterium Breve M-16V (NITE BP-02622) or a culture supernatant thereof as an active ingredient.

The present invention also contains Bifidobacterium Breve M-16V (NITE BP-02622) or its culture supernatant as an active ingredient for suppressing collagen degradation or promoting the synthesis of collagen, elastin or hyaluronic acid. Can provide things.

The present invention may also provide a composition for suppressing aging of joints, bones or blood vessels, which comprises Bifidobacterium Breve M-16V (NITE BP-02622) or a culture supernatant thereof as an active ingredient. can.

In each of the above compositions, it is preferable that the Bifidobacterium Breve M-16V (NITE BP-02622) is at least one of live and dead bacteria.
It is preferable that all of the compositions are food and drink compositions.
It is preferable that all of the compositions are pharmaceutical compositions.

The composition of the present invention can regulate the expression of genes involved in the production or degradation of collagen, elastin or hyaluronic acid in the subject ingesting it.

The graph which shows the experimental result of Example 1 which is one aspect of this invention. The graph which shows the experimental result of Example 1 which is one aspect of this invention. The graph which shows the experimental result of Example 1 which is one aspect of this invention. The graph which shows the experimental result of Example 2 which is one aspect of this invention. The graph which shows the experimental result of Example 2 which is one aspect of this invention. The graph which shows the experimental result of Example 2 which is one aspect of this invention. The graph which shows the experimental result of Example 2 which is one aspect of this invention.

Next, a preferred embodiment of the present invention will be described in detail. However, the present invention is not limited to the following preferred embodiments, and can be freely changed within the scope of the present invention.

Hereinafter, the present invention will be described in detail.
One aspect of the present invention is the regulation of expression of a gene involved in the production or degradation of collagen, elastin or hyaluronic acid, which comprises Bifidobacterium Breve M-16V (NITE BP-02622) or its culture supernatant as an active ingredient. The above-mentioned genes are a collagen-degrading enzyme 1 gene (MMP1), a type I collagen gene (COL1A1), an elastin gene (ELN), a hyaluronic acid synthase 1 gene (HAS1), and a hyaluronic acid synthase 2 gene. A composition selected from (HAS2).
In the present specification, the composition may be referred to as "the composition of the present invention". The composition of the present invention is a concept containing a mixture, and it does not matter whether the components are uniform or non-uniform.
Further, in the present specification, terms such as "ingestion", "ingestion", and "ingested" are "administered" and "administered", respectively, when used in a pharmaceutical composition or a feed composition. , Can be replaced with words such as "administered".

The composition of the present invention contains a cell of Bifidobacterium Breve M-16V (NITE BP-02622) or a culture supernatant thereof as an active ingredient.

Bifidobacterium Breve M-16V (NITE BP-02622) is a bacterium to which the contract number of NITE BP-02622 is given, and as of January 26, 2018, it is a patented microorganism of the National Institute of Technology and Evaluation. An international deposit was made to the Deposit Center (Room 122, 2-5-8 Kazusakamatari, Kisarazu City, Chiba Prefecture, 292-0818) with the deposit number of NITE BP-02622 based on the Budapest Treaty.

The Bifidobacterium Breve M-16V (NITE BP-02622) of the present invention is the strain itself that has been deposited or registered with a predetermined institution under the name of the bacterium (hereinafter, for convenience of explanation, "deposited strain". (Also referred to as), but substantially equivalent strains (also referred to as “derivative strains” or “derivative strains”) are also included. That is, "Bifidobacterium Breve M-16V (NITE BP-02622)" is not limited to the strain itself deposited with the above depository under the accession number NITE BP-02622, but is substantially equivalent to it. Is also included. The "strain substantially equivalent to the above-mentioned deposit strain" belongs to the same species as the above-mentioned deposit strain, the effect of the present invention is obtained, and the base sequence of the 16SrRNA gene thereof is the base of the 16SrRNA gene of the above-mentioned deposit strain. A strain having preferably 98% or more, more preferably 99% or more, still more preferably 100% identity with respect to the sequence, and preferably having the same mycological properties as the above-mentioned deposited strain. The stock substantially equivalent to the deposited stock may be, for example, a derivative stock having the deposited stock as a parent stock. Derivative strains include strains bred from deposited strains and strains naturally generated from deposited strains. Examples of the breeding method include modification by a genetic engineering method and modification by mutation treatment. Mutation treatment includes irradiation with X-rays, irradiation with ultraviolet rays, and treatment with mutant agents such as N-methyl-N'-nitro-N-nitrosoguanidine, ethylmethanesulfonate, and methylmethanesulfonate. Will be. Examples of naturally occurring strains from the deposited strains include strains naturally occurring during the use of the deposited strains. Examples of such a strain include a mutant strain naturally generated by culturing a deposited strain (for example, subculture). The derivative strain may be constructed by one kind of modification, or may be constructed by two or more kinds of modifications.

As the bacterial cells of Bifidobacterium Breve M-16V (NITE BP-02622) that can be contained in the composition of the present invention, a commercially available product may be used, or a commercially available product may be used. ..
Examples of commercially available products include "Bifidobacterium Breve M-16V" manufactured by Morinaga Milk Industry Co., Ltd. Further, as the composition of the present invention, a composition containing a viable cell of Bifidobacterium Breve M-16V (NITE BP-02622) can also be used, and as a commercially available product, for example, "Baby Bifidobacterium" can be used. (Registered trademark) ”.
In addition, as the cells of Bifidobacterium Breve M-16V (NITE BP-02622) that can be contained in the composition of the present invention, the above-mentioned Bifidobacterium Breve M-16V (NITE BP-02622) is cultured. It can be easily obtained by doing so. The culture method is not particularly limited as long as Bifidobacterium Breve M-16V (NITE BP-02622) can grow. As the culturing method, for example, the method usually used for culturing Bifidobacterium Breve M-16V (NITE BP-02622) can be used as it is or modified as appropriate. The culture temperature may be, for example, 25 to 50 ° C, preferably 35 to 42 ° C. The culture can be preferably carried out under anaerobic conditions, and can be carried out, for example, while aerating an anaerobic gas such as carbon dioxide gas. In addition, the culture can also be carried out under slightly aerobic conditions such as liquid static culture. Culturing can be carried out, for example, until Bifidobacterium Breve M-16V (NITE BP-02622) grows to the desired degree.

The medium used for culturing is not particularly limited as long as Bifidobacterium Breve M-16V (NITE BP-02622) can grow. As the medium, for example, the medium normally used for culturing Bifidobacterium Breve M-16V (NITE BP-02622) can be used as it is or after being appropriately modified. That is, as the carbon source, for example, saccharides such as galactose, glucose, fructose, mannose, cellobiose, maltose, lactose, sucrose, trehalose, starch, starch hydrolysate, and waste sugar honey can be used depending on the assimilation property. .. As the nitrogen source, for example, ammonium salts such as ammonia, ammonium sulfate, ammonium chloride and ammonium nitrate and nitrates can be used. Further, as the inorganic salts, for example, sodium chloride, potassium chloride, potassium phosphate, magnesium sulfate, calcium chloride, calcium nitrate, manganese chloride, ferrous sulfate and the like can be used. Further, organic components such as peptone, soybean flour, defatted soybean meal, meat extract and yeast extract may be used. In addition, as the medium usually used for culturing Bifidobacterium breve, specifically, fortified Clostridia medium (Reinforced Clostridia medium), MRS medium (deMan, Rogosa, and Sharpe medium), mMRS medium (modified MRS medium). ), TOSP medium (TOSpropionatemedium), TOSPMup medium (TOSpropionatemupirocinmedium).

The Bifidobacterium Breve M-16V (NITE BP-02622) that can be contained in the composition of the present invention includes cells of Bifidobacterium Breve M-16V (NITE BP-02622) or a cell thereof. The fraction can be used without particular limitation. That is, as Bifidobacterium Breve M-16V (NITE BP-02622), for example, the culture obtained by culturing may be used as it is, or the culture may be diluted or concentrated and used, or the culture may be used. Bacterial cells recovered from an object may be used. In addition, various additional operations such as heating and freeze-drying can be performed after culturing as long as the effects of the present invention are not impaired. It is preferable that the additional operation is such that the survivability of the bacterial cells is high. That is, as Bifidobacterium Breve M-16V (NITE BP-02622) that can be contained in the composition of the present invention, specifically, culture of Bifidobacterium Breve M-16V (NITE BP-02622). Examples thereof include products, bacterial cells recovered from the culture, and processed products thereof, and examples of the treated products include diluted products, concentrates, dried products, and the like.
The bacterial cell may be a live bacterial cell or a dead bacterial cell, and it is usually preferable to use the bacterial cell in a form containing the live bacterial cell. This is because if it is a viable cell, it can be expected to have an effect as a probiotic. The bacterial cells may be, for example, those consisting of live bacterial cells or a mixture of live bacterial cells and dead bacterial cells. That is, the Bifidobacterium Breve M-16V (NITE BP-02622) that can be contained in the composition of the present invention is preferably at least one of live and dead bacteria. Examples of the dead cells include dead cells sterilized by heating or the like (that is, heat sterilized bodies), and the heat sterilized bodies are, for example, Bifidobacterium Breve M-16V (NITE BP) as described above. After culturing -02622), the obtained culture solution is centrifuged, freeze-dried to obtain viable bacterial powder, suspended in sterile water, and sterilized by heating at 90 ° C. for 30 minutes. .. It can also be obtained as in the examples.

Further, the composition of the present invention may contain a culture supernatant of Bifidobacterium Breve M-16V (NITE BP-02622) as an active ingredient. The culture supernatant of Bifidobacterium Breve M-16V (NITE BP-02622) that can be contained in the composition of the present invention is, for example, as described above, Bifidobacterium Breve M-16V (NITE BP-022-). After culturing 02622), the obtained culture solution can be obtained by centrifugation. It can also be obtained as in the examples.

Targets for ingesting the composition of the present invention include humans and mammals other than humans. Examples of mammals other than humans include cows, goats, sheep, pigs, monkeys, dogs, cats, rats, mice, hamsters, guinea pigs and the like.

The composition of the present invention has an action of regulating the expression of genes involved in the production or degradation of collagen, elastin or hyaluronic acid in the subject ingesting it. The gene is selected from collagen degrading enzyme 1 gene (MMP1), type I collagen gene (COL1A1), elastin gene (ELN), hyaluronan synthase 1 gene (HAS1) and hyaluronan synthase 2 gene (HAS2).

The expression regulation includes expression enhancement and expression decrease. When the expression level when the subject ingests the composition of the present invention is larger than the expression level when the subject does not ingest (or ingests placebo), the composition of the present invention exerts the effect of enhancing the expression of the gene. On the other hand, the composition of the present invention is described above when the expression level when the subject ingests the composition of the present invention is smaller than the expression level when the subject does not ingest (or ingests placebo). It has the effect of reducing gene expression. The expression level can be calculated by a conventional method, for example, by the method described in Examples.

For the collagen degrading enzyme 1 gene (MMP1), it is preferable that the expression regulation is decreased. At this time, the expression level when the subject ingested the composition of the present invention is preferably 0.8 times or less, more preferably 0.6 times or less, still more preferably 0.5 times or less of the expression level when not ingested. On the other hand, it is preferable that the lower limit is small, but for example, it is 0.1 times or more.

Regarding the type I collagen gene (COL1A1), it is preferable that the expression regulation is enhanced expression. At this time, the expression level when the subject ingested the composition of the present invention is preferably 1.2 times or more, more preferably 1.4 times or more, still more preferably 1.6 times or more the expression level when not ingested. On the other hand, it is preferable that the upper limit is large, but for example, it is 2.5 times or less.

For the elastin gene (ELN), it is preferable that the expression regulation is the enhancement of expression. At this time, the expression level when the subject ingested the composition of the present invention is preferably 1.2 times or more, more preferably 1.4 times or more, still more preferably 1.6 times or more the expression level when not ingested. On the other hand, it is preferable that the upper limit is large, but for example, it is 2.5 times or less.

For the hyaluronan synthase 1 gene (HAS1), it is preferable that the expression regulation is the enhancement of expression. At this time, the expression level when the subject ingested the composition of the present invention is preferably 1.2 times or more, more preferably 1.4 times or more, still more preferably 1.6 times or more the expression level when not ingested. On the other hand, it is preferable that the upper limit is large, but for example, it is 2.5 times or less.

For the hyaluronan synthase 2 gene (HAS2), it is preferable that the expression regulation is the enhancement of expression. At this time, the expression level when the subject ingested the composition of the present invention is preferably 1.2 times or more, more preferably 1.4 times or more, still more preferably 1.6 times or more the expression level when not ingested. On the other hand, it is preferable that the upper limit is large, but for example, it is 2.5 times or less.

As described above, the expression level of the collagen degrading enzyme 1 gene (MMP1) is higher when the subject ingests Bifidobacterium Breve M-16V (NITE BP-02622) or its culture supernatant. It is preferably smaller than when not ingested.
In addition, the expression level of the type I collagen gene (COL1A1) was higher when the subject ingested Bifidobacterium Breve M-16V (NITE BP-02622) or its culture supernatant than when it was not ingested. Is also preferable.
In addition, the expression level of the elastin gene (ELN) is higher when the subject ingests Bifidobacterium Breve M-16V (NITE BP-02622) or its culture supernatant than when it does not ingest it. Is preferable.
In addition, the expression level of hyaluronan synthase 1 gene (HAS1) was not ingested when the subject ingested Bifidobacterium Breve M-16V (NITE BP-02622) or its culture supernatant. It is preferable that it is larger than the time.
In addition, the expression level of the hyaluronan synthase 2 gene (HAS2) was not ingested when the subject ingested Bifidobacterium Breve M-16V (NITE BP-02622) or its culture supernatant. It is preferable that it is larger than the time.
Thus, in a preferred embodiment, Bifidobacterium Breve M-16V (NITE BP-02622) or its culture supernatant suppresses the degradation of collagen or of collagen, elastin or hyaluronic acid in the subject ingesting it. It can promote synthesis.

The compositions of the present invention are for diseases (including symptoms herein) that can be prevented or ameliorated by regulating the expression of the gene involved in the production or degradation of collagen, elastin or hyaluronic acid in a subject. It can be used for prevention or improvement. The diseases include, for example, acne vulgaris (also referred to as puff or acne), decreased elasticity, sebum deficiency (also referred to as dry skin disease), bullous pemphigoid (for example, acquired epidermolysis bullosa, dystrophy type). Congenital epidermolysis bullosa, sublamina densa type of linear IgA bullous dermatosis, bullous lupus erythematosus bullous SLE), skin laxity, arthritis, arthritis (eg, osteoarthritis), osteoporosis, arteriosclerosis Can be mentioned.
"Improvement" includes disease improvement, prevention of disease exacerbation, delay in disease progression, maintenance of disease status, reversal of disease progression, and treatment of disease.

Of these, for the prevention or amelioration of acne vulgaris, decreased elasticity, and sebum deficiency, the gene involved in collagen production by reducing the expression of the gene involved in collagen degradation in the subject. One or more by enhancing the expression of the gene involved in the production of elastin, and by enhancing the expression of the gene involved in the production of hyaluronic acid. Is preferable.
Also, in the subject, one or more of by suppressing the decomposition of collagen, by promoting the synthesis of collagen, by promoting the synthesis of elastin, and by promoting the synthesis of hyaluronic acid. Is also preferable.

For the prevention or amelioration of arthritis and arthropathy, in the subject, by reducing the expression of the gene involved in collagen degradation, by enhancing the expression of the gene involved in collagen production, and by hyaluronic acid. It is preferably one or more by enhancing the expression of the gene involved in the production of.
It is also preferable that the subject has one or more of by suppressing the decomposition of collagen, by promoting the synthesis of collagen, and by promoting the synthesis of hyaluronic acid.

For prevention or amelioration of vesicular disease and osteoporosis, by reducing the expression of the gene involved in collagen degradation and by enhancing the expression of the gene involved in collagen production in the subject. It is preferably one or more of them.
It is also preferable that the subject has one or more of by suppressing the decomposition of collagen and by promoting the synthesis of collagen.

For the prevention or amelioration of cutis laxa and arteriosclerosis, it is preferable to enhance the expression of the gene involved in the production of elastin in the subject.
It is also preferable to promote the synthesis of elastin in the subject.

Further, as described above, in a preferred embodiment, Bifidobacterium Breve M-16V (NITE BP-02622) or its culture supernatant suppresses the decomposition of collagen or collagen, elastin in the subject ingesting it. Alternatively, the synthesis of hyaluronic acid can be promoted.
Therefore, in one embodiment, the present invention contains Bifidobacterium Breve M-16V (NITE BP-02622) or a culture supernatant thereof as an active ingredient, suppresses the decomposition of collagen, or synthesizes collagen, elastin or hyaluronic acid. Compositions for facilitation can be provided.

As another aspect, the present invention can provide a composition for improving skin properties, which comprises Bifidobacterium Breve M-16V (NITE BP-02622) or a culture supernatant thereof as an active ingredient.
The skin properties include skin moisture content, dermis collagen content, skin moisturization, luster, firmness, texture, transparency, smoothness after face washing (tightness), wrinkles, makeup paste, dryness / sagging, pimples, and skin condition. Comprehensive evaluation etc. can be mentioned. In addition, skin disorders caused by irradiation with ultraviolet rays (for example, thickening of the skin, etc.) can be mentioned.
The improvement of skin properties is the improvement of the skin properties of the gene involved in the production of elastin by enhancing the expression of the gene involved in the production of collagen by reducing the expression of the gene involved in the degradation of collagen in the subject. It is preferably one or more by enhancing the expression and by enhancing the expression of the gene involved in the production of hyaluronic acid.
In addition, the improvement of skin properties is due to suppressing the decomposition of collagen, promoting the synthesis of collagen, promoting the synthesis of elastin, and promoting the synthesis of hyaluronic acid in the subject. It is also preferable that it is one or more of them.
In the present specification, "improvement of skin properties" means improvement of skin properties, prevention of deterioration of skin properties, delay of deterioration of skin properties, maintenance of deteriorated skin properties, and progress of deterioration of skin properties. Including reversal etc.

The present invention also comprises, as another embodiment, Bifidobacterium Breve M-16V (NITE BP-02622) or a culture supernatant thereof as an active ingredient, a composition for suppressing joint or bone aging. Can be provided.
Suppression of the aging of the joint or bone in the subject is by reducing the expression of the gene involved in collagen degradation, by enhancing the expression of the gene involved in collagen production, and of hyaluronic acid. It is preferably one or more by enhancing the expression of the gene involved in production.
In addition, the suppression of aging of the joint or bone is one of, in the subject, by suppressing the decomposition of collagen, by promoting the synthesis of collagen, and by promoting the synthesis of hyaluronic acid, or one of them. It is also preferable that the above is the case.
Diseases that can be prevented or ameliorated by suppressing joint or bone aging include arthritis, arthropathy, and osteoporosis already described.

The present invention also provides, as another embodiment, a composition for suppressing aging of blood vessels, which comprises Bifidobacterium Breve M-16V (NITE BP-02622) or a culture supernatant thereof as an active ingredient. be able to.
The suppression of aging of the blood vessels is preferably by enhancing the expression of the gene involved in the production of elastin in the subject.
It is also preferable that the suppression of aging of the blood vessels is by promoting the synthesis of elastin in the subject.
Diseases that can be prevented or ameliorated by suppressing aging of blood vessels include arteriosclerosis already described.

When the composition of the present invention contains cells of Bifidobacterium Breve M-16V (NITE BP-02622), the content of the cells is appropriately set depending on the aspect of the composition, but the total amount is 1 ×. It is preferably in the range of 10 ⁴ to 1 × 10 ¹³ cfu / g or 1 × 10 ⁴ to 1 × 10 ¹³ cfu / ml, 1 × 10 ⁵ to 1 × 10 ¹² cfu / g or 1 × 10 ⁵ to. It is more preferably in the range of 1 × 10 ¹² cfu / ml, further preferably in the range of 1 × 10 ⁶ to 1 × 10 ¹⁰ cfu / g or 1 × 10 ⁶ to 1 × 10 ¹⁰ cfu / ml. It is preferably in the range of 1 × 10 ⁷ to 1 × 10 ¹⁰ cfu / g or 1 × 10 ⁷ to 1 × 10 ¹⁰ cfu / ml.
In addition, in this specification, "cfu" represents a colony forming unit (colony forming unit). Further, in the present specification, when the bacterium of the present invention is a dead bacterium, cfu / g or cfu / ml can be replaced with individual cells / g or individual cells / ml.
Further, when the composition of the present invention contains the culture supernatant of the cells of Bifidobacterium Breve M-16V (NITE BP-02622), the content of the culture supernatant is appropriately set depending on the aspect of the composition. However, the amount equivalent to the solid content in the culture supernatant is preferably 0.01% by mass or more, more preferably 0.1% by mass or more, still more preferably 1.0% by mass or more, and particularly preferably 10% by mass or more. be. The upper limit is not particularly limited, but is 40% by mass or less, preferably 30% by mass or less, and more preferably 20% by mass or less.

The intake amount of the composition of the present invention is appropriately set depending on the form, usage, subject, age, sex, and other conditions of the composition, but is not particularly limited as long as the effect of the present invention is exhibited.
When the composition of the present invention contains cells of Bifidobacterium Breve M-16V (NITE BP-02622), the total amount of the cells is 1 × 10 ⁴ to 1 × 10 ¹³ per day and 100 g body weight. It is preferably in the range of cfu, more preferably in the range of 1 × 10 ⁵ to 1 × 10 ¹² cfu, and even more preferably in the range of 1 × 10 ⁶ to 1 × 10 ¹⁰ cfu. It is more preferably in the range of 1 × 10 ⁶ to 1 × 10 ⁹ cfu.
In the present specification, when the bacterium of the present invention is a dead bacterium, cfu can be replaced with an individual cell.
In addition, when the composition of the present invention contains the culture supernatant of the cells of Bifidobacterium Breve M-16V (NITE BP-02622), the amount equivalent to the solid content in the culture supernatant is 0. 001 g / kg body weight or more, preferably 0.01 to 0.1 g / kg body weight can be used as a guide.

The composition of the present invention can be ingested once a day or in multiple divided doses. It may be taken once every few days or weeks, but it is preferably taken every day. In addition, the intake period is preferably 4 weeks or longer.

The ingestion route of the composition of the present invention may be oral or parenteral, but oral is preferable. In addition, parenteral ingestion includes transdermal, intravenous injection, rectal administration, inhalation and the like. Further, for example, it may be ingested nasally, or it may be ingested by a gastrostomy or an intestinal fistula. For example, the subject may be ingested by a nasal gastric feeding tube or the like.

The composition of the present invention can be used as a food or drink composition, a feed composition, or a pharmaceutical composition. for example,
A food and drink composition for regulating the expression of genes involved in the production or degradation of collagen, elastin or hyaluronic acid, which comprises Bifidobacterium Breve M-16V (NITE BP-02622) or its culture supernatant as an active ingredient. hand,
The gene is selected from collagen degrading enzyme 1 gene (MMP1), type I collagen gene (COL1A1), elastin gene (ELN), hyaluronan synthase 1 gene (HAS1) and hyaluronan synthase 2 gene (HAS2). Food and beverage composition;
A feed composition for regulating the expression of genes involved in the production or degradation of collagen, elastin or hyaluronic acid, which comprises Bifidobacterium Breve M-16V (NITE BP-02622) or its culture supernatant as an active ingredient. ,
The gene is selected from collagen degrading enzyme 1 gene (MMP1), type I collagen gene (COL1A1), elastin gene (ELN), hyaluronan synthase 1 gene (HAS1) and hyaluronan synthase 2 gene (HAS2). Feed composition; or
A pharmaceutical composition for regulating the expression of genes involved in the production or degradation of collagen, elastin or hyaluronic acid, which comprises Bifidobacterium Breve M-16V (NITE BP-02622) or its culture supernatant as an active ingredient. ,
The gene is selected from collagen degrading enzyme 1 gene (MMP1), type I collagen gene (COL1A1), elastin gene (ELN), hyaluronan synthase 1 gene (HAS1) and hyaluronan synthase 2 gene (HAS2). Pharmaceutical compositions can be provided.
Hereinafter, they may be described as "food and drink composition of the present invention", "feed composition of the present invention", and "pharmaceutical composition of the present invention", respectively.
A composition for improving skin properties, which comprises the previously described embodiment of Bifidobacterium Breve M-16V (NITE BP-02622) or a culture supernatant thereof as an active ingredient; Bifidobacterium Breve M-. A composition containing 16V (NITE BP-02622) or its culture supernatant as an active ingredient for suppressing collagen degradation or promoting the synthesis of collagen, elastin or hyaluronic acid; Bifidobacterium Breve M-16V (NITE). BP-02622) or a composition for suppressing aging of joints or bones containing a culture supernatant thereof as an active ingredient; and Bifidobacterium Breve M-16V (NITE BP-02622) or a culture supernatant thereof. The same applies to the composition for suppressing aging of blood vessels, which contains the above as an active ingredient, and the same applies to the following items.

The food and drink composition of the present invention is not particularly limited as long as it contains Bifidobacterium Breve M-16V (NITE BP-02622) or a culture supernatant thereof. The food and drink composition may be a food or drink regardless of the form such as liquid, paste, gel-like solid, powder, etc. In addition to beverages, liquid foods, etc., for example, bread, macaroni, spaghetti, noodles, etc. , Cake mix, fried flour, bread flour and other wheat flour products; instant noodles, cup noodles, retort / cooked foods, canned foods, microwave foods, instant soups / stews, instant miso soups / beverages, canned soups, freezes / dry foods, etc. Instant foods such as instant foods; canned agricultural products, canned fruits, jams and marmalades, pickles, boiled beans, dried agricultural products, processed agricultural products such as cereals (processed grain products); canned fishery products, fish hams and sausages, fishery products Processed marine products such as kneaded products, marine delicacies, and boiled Tsukuda; processed livestock products such as canned livestock / pastes, livestock ham / sausage; processed milk, dairy beverages, yogurts, lactic acid bacteria beverages, cheese, ice cream, preparations Milk and dairy products such as powdered milk, cream and other dairy products; Oils and fats such as butter, margarine and vegetable oil; Basic seasonings such as soy sauce, miso, sauces, tomato processing seasonings, mirins and vinegars; Combined seasonings / foods such as cooking mixes, curry bases, sauces, dressings, noodles, spices, and other complex seasonings; Foods; Caramel, candy, gummy, chewing gum, chocolate, cookies, biscuits, cakes, pies, snacks, crackers, Japanese sweets, rice sweets, bean sweets, dessert sweets, jelly, and other sweets; carbonated beverages, natural fruit juice , Fruit juice drinks, soft drinks with fruit juice, fruit drinks, fruit drinks with fruit grains, vegetable drinks, soy milk, soy milk drinks, coffee drinks, tea drinks, powder drinks, concentrated drinks, sports drinks, nutritional drinks, alcoholic drinks, etc. Favorite beverages such as favorite beverages, baby foods, sprinkles, other commercial foods such as Ochazuke paste; preparation powdered milk for childcare; enteral nutritional foods; special purpose foods, health functional foods (specified health foods, nutritional functional foods, etc.) Functionally labeled foods); Examples include nutritional supplements.
Further, the food and drink composition of the present invention may be a supplement, for example, a tablet-shaped supplement. In the case of supplements, Bifidobacterium Breve M-16V (NITE BP-02622) or its culture supernatant can be ingested without being affected by other foods in terms of daily dietary intake and calorie intake.

The food and drink composition of the present invention can be produced by adding Bifidobacterium Breve M-16V (NITE BP-02622) or a culture supernatant thereof to the raw materials of ordinary foods and drinks. It can be produced in the same manner as ordinary foods and drinks except that Bacterium Breve M-16V (NITE BP-02622) or its culture supernatant is added. The addition of Bifidobacterium Breve M-16V (NITE BP-02622) or its culture supernatant may be carried out at any stage of the manufacturing process of the food or drink composition. Further, the food or drink composition may be produced through a fermentation step with the added Bifidobacterium Breve M-16V (NITE BP-02622). Examples of such food and drink compositions include lactic acid bacteria beverages, fermented milk and the like. In addition, there is also a mode of adding to milked breast milk or prepared milk, and it is assumed that the added milk is ingested by infants.

The food and drink composition of the present invention also includes raw materials for manufacturing the food and drink composition, food additives, and the like, which are added to the food and drink composition during or after the manufacturing process of the food and drink composition. For example, Bifidobacterium Breve M-16V (NITE BP-02622) can be used as a starter for fermented milk production. In addition, Bifidobacterium Breve M-16V (NITE BP-02622) or its culture supernatant can be added later to the produced fermented milk.

Further, in the food and drink composition of the present invention, a component having a prebiotic effect known or found in the future or a component assisting the prebiotic effect may be used as long as the effect of the present invention is not impaired. can. For example, the food and drink composition of the present invention comprises various proteins such as whey protein, casein protein, soybean protein, or pea protein (pea protein) or mixtures thereof, decomposition products; amino acids such as leucine, valine, isoleucine or glutamine; Vitamin types such as vitamin B6 or vitamin C; creatine; citric acid; fish oil; or components such as isomaltooligosaccharides, galactooligosaccharides, xylooligosaccharides, soybean oligosaccharides, fructo-oligosaccharides, lactulose, and oligosaccharides such as human milk oligosaccharides. And Bifidobacterium Breve M-16V (NITE BP-02622) or its culture supernatant can be mixed and produced.

The content of Bifidobacterium Breve M-16V (NITE BP-02622) or the culture supernatant thereof in the food and drink composition of the present invention is described above, and the composition of the present invention is Bifidobacterium Breve M-. It is the same as the content of the cells when 16V (NITE BP-02622) is contained, or the content of the culture supernatant when the culture supernatant is contained. Further, the intake amount of the food and drink composition of the present invention is also the same as the intake amount of the composition of the present invention described above. Further, the intake schedule of the food and drink composition of the present invention is also the same as the intake schedule of the composition of the present invention described above.

The food and drink composition of the present invention may be ingested alone or together with other food and drink compositions or foods and drinks, or pharmaceutical compositions or pharmaceuticals. For example, it may be ingested together with other food or drink compositions or foods or drinks or pharmaceutical compositions or pharmaceuticals for regulating the expression of genes involved in the production or degradation of collagen, elastin or hyaluronic acid.

The food and drink composition of the present invention can be sold as a food and drink composition or a food and drink whose use is indicated for the purpose of regulating the expression of genes involved in the production or degradation of collagen, elastin or hyaluronic acid. In addition to this, it goes without saying that any wording that expresses a secondary effect due to the above-mentioned use can be used.

The "display" means all actions for informing the consumer of the above-mentioned use, and if the display is such that the above-mentioned use can be recalled or inferred, the purpose of the display, the content of the display, and the display are used. Regardless of the object, medium, etc., all fall under the "indication" of the present invention. However, it is preferable to display it in an expression that allows the consumer to directly recognize the above-mentioned use.
Specifically, the act of describing the above use in the food or drink composition of the present invention or the product or product packaging related to the food or drink, or the transfer, delivery, transfer or transfer of the product or product packaging in which the above use is described. The act of displaying and importing for delivery, advertisements related to goods, price lists or transaction documents with the above-mentioned uses described or distributed, or the information containing these described with the above-mentioned uses electromagnetically The act of providing by the method (Internet, etc.) can be exemplified, and it is particularly preferable to display it on advertising materials, other documents, etc. at the sales site such as packaging, containers, catalogs, brochures, and POPs.

Further, as the display, it is preferable that the display is permitted by the government or the like (for example, a display obtained based on various systems established by the government and performed in a mode based on such approval). For example, labeling as health functional foods, more specifically health functional foods, functional foods, enteral nutrition foods, special purpose foods, nutritional functional foods, non-medicinal products, etc. may be exemplified. It can exemplify other labeling approved by the Consumer Affairs Agency, such as foods for specified health use, foods with nutritional function, foods with functional claims, and labeling approved by a similar system. Examples of the latter include labeling as a food for specified health use, labeling as a food for specified health use conditionally, labeling to the effect that it affects the structure and function of the body, labeling for reducing disease risk, and functionality based on scientific evidence. Can be exemplified. More specifically, as a food for specified health use stipulated in the Cabinet Office Ordinance (Cabinet Office Ordinance No. 57, August 31, 2001) regarding permission for special use labeling prescribed in the Health Promotion Law. (In particular, indications for health purposes) and similar indications can be exemplified.
To give a specific example, when the food or drink composition of the present invention is used as a food with a functional claim, the "prevention or improvement of a disease" is "improvement of a disease", and the "improvement of a disease" is a disease. Includes improvement, prevention of disease exacerbation, delay in disease progression, maintenance of disease status, reversal of disease progression, but does not include treatment of the disease. In addition, even when it is not possible to use a label intended for a person suffering from a specific disease, for example, the functionality associated with the disease that is useful for maintaining and promoting health can be used as the label.

The food and drink composition for improving skin properties can be sold as a food and drink composition or food and drink for which the purpose of improving skin properties is indicated. It should be noted that the wording used for the above-mentioned display is not limited to the wording such as improvement of skin properties, and other words also indicate the effect of improvement of various diseases related to the improvement of skin properties. Needless to say, the wording is included in the scope of the present invention. In addition, even when it is not possible to use a label intended for a person suffering from a specific disease, for example, the functionality associated with the disease that is useful for maintaining and promoting health can be used as the label.

Such words include, for example, "for those who want moisture", "for those who care about health and beauty", "for those who want to maintain the barrier function of the skin", and "to protect the health of the skin". Words based on various uses such as "toward" can be mentioned to make consumers aware of the improvement of skin properties.

When the improvement of skin properties is improvement of skin moisture content and skin moisturization, for example, "for those who want to increase the moisturizing power of the skin", "for those who want to retain the moisture of the skin", and "for those who want to retain the moisture of the skin". Words based on various uses such as "for those who want to keep their skin moisturized" and "for those who want to relieve dry skin" can be mentioned to make consumers aware of the improvement of skin moisture content and skin moisturization. ..

When the improvement of skin properties is the improvement of the amount of dermal collagen, "for those who want to keep the skin moisturizing power", "for those who want to keep the skin moisturized", "for those who want to relieve the dryness of the skin", To make consumers aware of the improvement in the amount of dermal collagen, such as "for those who want bouncy skin", "for those who want to increase the elasticity of the skin", and "for those who have lost the elasticity of the skin". Wording based on various uses can be mentioned.

When the improvement of skin properties is the improvement of gloss, various methods such as "for those who want the shine of the skin" and "for those who want the pearly feeling of the skin" are made to make the consumer aware of the improvement of the gloss. Wording based on the use of.

When the improvement of skin properties is improvement of firmness, "for those who want bouncy skin", "for those who want to maintain skin elasticity", "for those who have weakened skin elasticity", etc. There are various uses-based language that make consumers aware of the improvement in elasticity.

When the improvement of skin properties is the improvement of texture, consumers such as "for those who want smooth skin", "for those who want smooth skin", "for those who want soft skin", etc. There are various uses-based language that make people aware of the improvement in texture.

When the improvement of the skin property is the improvement of the transparency, the consumer is made to recognize the improvement of the transparency such as "for those who want the transparency of the skin" and "for those who want the transparent skin". Wording based on various uses such as.

If the improvement in skin properties is to improve the feeling of smoothness after washing the face, for consumers such as "because the skin does not become tight after washing the face" and "for those who want to alleviate the stiffness of the skin after washing the face". Words based on various uses such as recognizing the improvement of the feeling of smoothness after washing the face can be mentioned.

When the improvement of skin properties is improvement of wrinkles, for consumers such as "for those who are wrinkled", "for those who want to make wrinkles less noticeable", "for those who want wrinkle-free skin", etc. Words based on various uses that make people aware of the improvement of wrinkles can be mentioned.

If the improvement in skin properties is an improvement in makeup paste, make the consumer aware of the improvement in makeup paste, such as "for those who want to improve the familiarity of makeup" and "for those who want to improve the finish of makeup". Wording based on various uses such as.

When the improvement of skin properties is improvement of dryness / sagging, consumers are aware of the improvement of dryness / sagging, such as "for those who want to relieve dryness / sagging of the skin" and "for those who want to lift up". There are words based on various uses that make it.

When the improvement of skin properties is the improvement of pimples, it is based on various uses such as "for those who have conspicuous pimples" and "for those who want to alleviate pimples" so that consumers can recognize the improvement of pimples. The wording can be mentioned.

When the improvement of skin properties is an improvement of the comprehensive evaluation of skin condition, "for those who want to improve the skin condition as a whole", "for those who want to alleviate skin troubles", and "for those who want to maintain skin health". There are various uses-based words such as "to" that make consumers aware of the improvement in the comprehensive evaluation of skin condition.

When the improvement of skin properties is the improvement of skin disorders caused by irradiation with ultraviolet rays, "for those who are concerned about the effect of ultraviolet rays on the skin", "for those who often go out during the day", etc. Words based on various uses that make consumers aware of the improvement of skin disorders caused by exposure to ultraviolet rays can be mentioned.

When the improvement of skin properties is improvement of skin thickening, improvement of skin thickening for consumers such as "for those who have thickened skin" and "for those who are concerned about skin thickness". There are words based on various uses that make people recognize.

The content of Bifidobacterium Breve M-16V (NITE BP-02622) or its culture supernatant in the feed composition of the present invention is described above, and the composition of the present invention is Bifidobacterium Breve M-16V. It is the same as the content of the bacterial cells when the cells of (NITE BP-02622) are contained, or the content of the culture supernatant when the culture supernatant is contained. Moreover, the dose of the feed composition of the present invention is also the same as the intake amount of the composition of the present invention described above. Further, the administration schedule of the food and drink composition of the present invention is also the same as the ingestion schedule of the composition of the present invention described above.

The feed composition of the present invention may be administered to a subject alone or together with other feed compositions or feeds or pharmaceutical compositions or pharmaceuticals. For example, it may be administered together with other feed compositions or feeds or pharmaceutical compositions or pharmaceuticals for regulating the expression of genes involved in the production or degradation of collagen, elastin or hyaluronic acid.

The pharmaceutical composition of the present invention is not particularly limited as long as it contains Bifidobacterium Breve M-16V (NITE BP-02622) or a culture supernatant thereof. As the pharmaceutical composition of the present invention, Bifidobacterium Breve M-16V (NITE BP-02622) or its culture supernatant may be used as it is, and a physiologically acceptable liquid or solid pharmaceutical carrier may be used. It may be blended, formulated and used.

The dosage form of the pharmaceutical composition of the present invention is not particularly limited, and specifically, tablets, pills, powders, liquids, suspensions, emulsions, granules, capsules, syrups, suppositories, injections, etc. Examples thereof include ointments, patches, eye drops, and nasal drops. In addition, when formulating, the addition of excipients, binders, disintegrants, lubricants, stabilizers, flavoring agents, diluents, surfactants, solvents for injections, etc., which are usually used as pharmaceutical carriers. The agent can be used.

Further, as the pharmaceutical carrier, various organic or inorganic carriers can be used depending on the dosage form. Examples of the carrier in the case of a solid preparation include excipients, binders, disintegrants, lubricants, stabilizers, flavoring agents and the like.

Examples of excipients include sugar derivatives such as lactose, sucrose, glucose, mannit, and sorbit; starch derivatives such as corn starch, horse bell starch, α-starch, dextrin, and carboxymethyl starch; crystalline cellulose, hydroxypropyl cellulose, and the like. Cellulose derivatives such as hydroxypropylmethyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose calcium; Arabic rubber; Dextran; Plulan; Silate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, magnesium aluminometasilicate; Phosphate derivatives such as calcium phosphate; Carbonated Carbonated carbonate derivatives such as calcium; sulfate derivatives such as calcium sulfate can be mentioned.

Examples of the binder include gelatin; polyvinylpyrrolidone; macrogol, etc., in addition to the above-mentioned excipients.

Examples of the disintegrant include, in addition to the above-mentioned excipients, chemically modified starch or cellulose derivatives such as croscarmellose sodium, carboxymethyl starch sodium, and crosslinked polyvinylpyrrolidone.

Examples of the lubricant include talc; stearic acid; metal stearate salts such as calcium stearate and magnesium stearate; colloidal silica; waxes such as pea gum and gay wax; boric acid; glycol; carboxylic acids such as fumaric acid and adipic acid. Carboxylic acid sodium salts such as sodium benzoate; sulfates such as sodium sulfate; leucine; lauryl sulfates such as sodium lauryl sulfate and magnesium lauryl sulfate; silicic acids such as anhydrous silicic acid and silicic acid hydrate; starch derivatives and the like. Be done.

Examples of the stabilizer include paraoxybenzoic acid esters such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol and phenylethyl alcohol; benzalkonium chloride; acetic anhydride; sorbic acid and the like.

Examples of the flavoring agent include sweeteners, acidulants, and fragrances.
Examples of the carrier used in the case of a liquid preparation for oral administration include a solvent such as water, a flavoring and odorant, and the like.

The content of Bifidobacterium Breve M-16V (NITE BP-02622) or its culture supernatant in the pharmaceutical composition of the present invention is described above, and the composition of the present invention is Bifidobacterium Breve M-16V. It is the same as the content of the bacterial cells when the cells of (NITE BP-02622) are contained, or the content of the culture supernatant when the culture supernatant is contained. Further, the dose of the pharmaceutical composition of the present invention is also the same as the ingestion amount of the composition of the present invention described above.

The administration time of the pharmaceutical composition of the present invention is not particularly limited as long as the effects of the present invention are exhibited, and the administration time can be appropriately selected. It may also be administered prophylactically. Further, it is preferable that the administration form is determined according to the pharmaceutical form, the subject, the age of the subject, the sex, the type of the disease, the degree thereof, other conditions and the like. Further, the administration schedule of the pharmaceutical composition of the present invention is also the same as the ingestion schedule of the composition of the present invention described above.

The pharmaceutical composition of the present invention may be administered alone or together with other pharmaceutical compositions or pharmaceuticals, food and drink compositions or foods and drinks, or feed compositions or feeds. For example, it is administered together with other pharmaceutical compositions or pharmaceuticals, food and drink compositions or foods and drinks, or feed compositions or feeds for regulating the expression of genes involved in the production or degradation of collagen, elastin or hyaluronic acid. May be good.

Further, the present invention can also adopt the following configuration.
[1-1] On Bifidobacterium Breve M-16V (NITE BP-02622) or its culture for producing a composition for regulating the expression of genes involved in the production or degradation of collagen, elastin or hyaluronic acid. The use of Qing
The gene is selected from collagen degrading enzyme 1 gene (MMP1), type I collagen gene (COL1A1), elastin gene (ELN), hyaluronan synthase 1 gene (HAS1) and hyaluronan synthase 2 gene (HAS2). use.
[1-2] Bifidobacterium Breve M-16V (NITE) for use in the prevention or amelioration of diseases that can be prevented or ameliorated by regulating the expression of genes involved in the production or degradation of collagen, elastin or hyaluronic acid. BP-02622) or its culture supernatant
The gene is selected from collagen degrading enzyme 1 gene (MMP1), type I collagen gene (COL1A1), elastin gene (ELN), hyaluronan synthase 1 gene (HAS1) and hyaluronan synthase 2 gene (HAS2). Bifidobacterium Breve M-16V (NITE BP-02622) or its culture supernatant.
[1-3] A step of administering a prophylactically or ameliorating effective amount of Bifidobacterium Breve M-16V (NITE BP-02622) or its culture supernatant to a human or patient in need of prevention or improvement. A method for preventing or ameliorating a disease that can be prevented or ameliorated by regulating the expression of genes involved in the production or degradation of collagen, elastin or hyaluronic acid, including.
The gene is selected from collagen degrading enzyme 1 gene (MMP1), type I collagen gene (COL1A1), elastin gene (ELN), hyaluronan synthase 1 gene (HAS1) and hyaluronan synthase 2 gene (HAS2). Prevention or improvement method.
[1-4] The use of Bifidobacterium Breve M-16V (NITE BP-02622) or its culture supernatant for the regulation of expression of genes involved in the production or degradation of collagen, elastin or hyaluronic acid. hand,
The gene is selected from collagen degrading enzyme 1 gene (MMP1), type I collagen gene (COL1A1), elastin gene (ELN), hyaluronan synthase 1 gene (HAS1) and hyaluronan synthase 2 gene (HAS2). use.
[1-5] Bifidobacterium Breve M-16V (NITE BP-02622) or a culture supernatant thereof used for regulating the expression of genes involved in the production or degradation of collagen, elastin or hyaluronic acid.
The gene is selected from collagen degrading enzyme 1 gene (MMP1), type I collagen gene (COL1A1), elastin gene (ELN), hyaluronan synthase 1 gene (HAS1) and hyaluronan synthase 2 gene (HAS2). Bifidobacterium Breve M-16V (NITE BP-02622) or its culture supernatant.
[1-6] (i) Including the step of administering Bifidobacterium Breve M-16V (NITE BP-02622) or its culture supernatant to humans; or (ii) Bifidobacterium Breve M-16V. (NITE BP-02622) or a composition for regulating the expression of a gene involved in the production or degradation of collagen, elastin or hyaluronic acid containing (NITE BP-02622) or its culture supernatant as an active ingredient, which comprises the step of administering collagen, elastin or A method of regulating the expression of genes involved in the production or degradation of hyaluronic acid.
The gene is selected from collagen degrading enzyme 1 gene (MMP1), type I collagen gene (COL1A1), elastin gene (ELN), hyaluronan synthase 1 gene (HAS1) and hyaluronan synthase 2 gene (HAS2). Method.

[2-1] Use of Bifidobacterium Breve M-16V (NITE BP-02622) or a culture supernatant thereof for producing a composition for improving skin properties.
[2-2] Bifidobacterium Breve M-16V (NITE BP-02622) or a culture supernatant thereof for use in the prevention or amelioration of diseases that can be prevented or ameliorated by improving skin properties.
[2-3] A step of administering a prophylactically or ameliorating effective amount of Bifidobacterium Breve M-16V (NITE BP-02622) or its culture supernatant to a human or patient in need of prevention or improvement. A method for preventing or improving a disease that can be prevented or improved by improving skin properties, including.
[2-4] Use of Bifidobacterium Breve M-16V (NITE BP-02622) or its culture supernatant for improving skin properties.
[2-5] Bifidobacterium Breve M-16V (NITE BP-02622) or a culture supernatant thereof used for improving skin properties.
[2-6] (i) Bifidobacterium Breve M-16V (NITE BP-02622) or its culture supernatant comprises the step of administering to humans, or (ii) Bifidobacterium Breve M-16V. (NITE BP-02622) or a method for improving skin properties, which comprises a step of administering a composition for improving skin properties to humans, which comprises the culture supernatant thereof as an active ingredient.

[3-1] On Bifidobacterium Breve M-16V (NITE BP-02622) or its culture for producing a composition for suppressing the decomposition of collagen or promoting the synthesis of collagen, elastin or hyaluronic acid. Use of Qing.
[3-2] Bifidobacterium Breve M-16V (NITE BP-) for use in the prevention or amelioration of diseases that can be prevented or ameliorated by suppressing the decomposition of collagen or promoting the synthesis of collagen, elastin or hyaluronic acid. 02622) or its culture supernatant.
[3-3] A step of administering a prophylactically or ameliorating effective amount of Bifidobacterium Breve M-16V (NITE BP-02622) or its culture supernatant to a human or patient in need of prevention or improvement. A method for preventing or ameliorating a disease that can be prevented or ameliorated by suppressing the decomposition of collagen or promoting the synthesis of collagen, elastin or hyaluronic acid.
[3-4] Use of Bifidobacterium Breve M-16V (NITE BP-02622) or its culture supernatant for suppressing the decomposition of collagen or promoting the synthesis of collagen, elastin or hyaluronic acid.
[3-5] Bifidobacterium Breve M-16V (NITE BP-02622) or a culture supernatant thereof, which is used for suppressing the decomposition of collagen or promoting the synthesis of collagen, elastin or hyaluronic acid.
[3-6] (i) Including the step of administering Bifidobacterium Breve M-16V (NITE BP-02622) or its culture supernatant to humans; or (ii) Bifidobacterium Breve M-16V. (NITE BP-02622) or its culture supernatant as an active ingredient, including the step of injecting a composition for suppressing collagen degradation or promoting the synthesis of collagen, elastin or hyaluronic acid into humans, including collagen degradation. A method of suppressing or promoting the synthesis of collagen, elastin or hyaluronic acid.

[4-1] Use of Bifidobacterium Breve M-16V (NITE BP-02622) or a culture supernatant thereof for producing a composition for suppressing aging of joints, bones or blood vessels.
[4-2] Bifidobacterium Breve M-16V (NITE BP-02622) or its use for the prevention or amelioration of diseases that can be prevented or ameliorated by suppressing the aging of joints, bones or blood vessels. Culture supernatant.
[4-3] A step of administering a prophylactic or ameliorating effective amount of Bifidobacterium Breve M-16V (NITE BP-02622) or a culture supernatant thereof to a human or a patient in need of prophylaxis or amelioration. A method for preventing or ameliorating a disease that can be prevented or ameliorated by suppressing the aging of joints, bones or blood vessels, including.
[4-4] Use of Bifidobacterium Breve M-16V (NITE BP-02622) or its culture supernatant to suppress aging of joints, bones or blood vessels.
[4-5] Bifidobacterium Breve M-16V (NITE BP-02622) or a culture supernatant thereof used for suppressing aging of joints, bones or blood vessels.
[4-6] (i) Including the step of administering Bifidobacterium Breve M-16V (NITE BP-02622) or its culture supernatant to humans; or (ii) Bifidobacterium Breve M-16V. Suppresses joint, bone or blood vessel aging, including the step of administering to humans a composition for suppressing joint, bone or blood vessel aging containing (NITE BP-02622) or its culture supernatant as an active ingredient. Method.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

[Example 1]
This example was carried out using a heat-sterilized body of Bifidobacterium Breve M-16V (NITE BP-02622) as a test substance.

<Cell>
Dermal fibroblast line NB1 RGB cells (cell number: RCB0222, RIKEN BRC, Japan) derived from human neonates were cultured using a CO ₂ incubator (CO ₂ concentration 5%, 37 ° C).

<Medium>
10.0% (v / v) Fetal Bovine Serum (FBS, Cat No. SH30071.03, Hyclone, UK) and 1.0% (v / v) Antifungal Agent (Antibiotic-Antimycotic 100X, Cat No. 15240-062, Invitrogen, Eagle's Minimal Essential Medium (EMEM, Cat No. 051-07615, Wako, Japan) including USA) was used.

<Test substance>
Bifidobacterium Breve M-16V (NITE BP-02622) was anaerobically cultured in MRS broth (Wako Pure Chemical Industries, Ltd.) containing 0.05% L-cysteine (37 ° C, 16h), and a fixed amount of the obtained culture was 24. The mL was separated, and this was newly inoculated into 800 mL of L-cysteine-containing MRS broth, and anaerobic culture was performed under the same conditions.
The bacteria collected from the obtained culture and resuspended in PBS were heated at 90 ° C. for 15 minutes in a commercially available autoclave machine. Subsequently, the supernatant was removed by centrifugation at 8,000 × g for 10 minutes, and then 400 mL of PBS was added for washing. After removing the supernatant, the mixture was suspended in 100 mL of sterile water and lyophilized.
The test substance was diluted with PBS to prepare a total of 3 concentrations (0.1 mg / mL, 1 mg / mL, 10 mg / mL) at the time of use. The prepared test substance was added to the culture medium, and a total of 3 concentrations (1 μg / mL, 10 μg / mL, 100 μg / mL) were used for the test. Medium was added instead of the test substance to the negative control.

<Gene expression analysis>
The expression of the following genes was analyzed using TaqMan Assay (Applied Biosystems, USA).
(a) Human Matrix Metallopeptidase 1 (MMP1, Assay ID. Hs00899658_m1))
(b) Human Hyaluronan Synthase 2 (HAS2, Assay ID. Hs00193435_m1))
(c) Elastin gene (Human Elastin (ELN, Assay ID. Hs00355783_m1))
(d) As an internal standard gene, Human Glyceraldehyde 3 Phosphate Dehydrogenase (GAPDH, Assay ID. Hs02786624_g1)

<Test configuration>
For the analysis of gene expression, the average value of three 35 mm dishes (Cat No. 150318, Thermo Scientific, USA) per treatment group was used. Operations related to the test, including preparation of the test substance, were performed at room temperature unless otherwise stated.

<Test method>
(Cell culture and addition of test substance)
1) 5.0 × 10 ⁵ cells / 2 mL of NB1 RGB cells were seeded on a 35 mm dish and cultured in a CO ₂ incubator for 24 hours.
2) After 24 hours, the medium of the 35 mm dish was removed, the test substance and the control-containing medium were added, and the cells were cultured in a CO ₂ incubator for 24 hours.

(RNA extraction / purification and quantification)
1) The following RNA was extracted and purified using PureLink ™ RNA Mini Kit (Cat No. 12183018A, Invitrogen, USA).
2) After culturing for 24 hours, the 35 mm dish from which the medium had been removed was washed twice with 2 mL of PBS (-) heated to 37 ° C.
3) Lysis Buffer containing 600 μL of 2M Dithiothreitol (CAS No. 27565-41-9, Invitrogen, USA) was added to lyse the cells, and lysate was recovered.
4) Using Homogenizer (Cat No. 12183-026, Invitrogen, USA), the cells in the recovered lysate were disrupted.
5) After adding 600 μL of 70% ethanol (CAS No. 64-17-5, Japan Alcohol, Japan) solution to the cell disruption solution, the cells were transferred to a column with a silica membrane. Centrifuge at 12,000 xg at room temperature for 15 seconds and discard the filtrate.
6) 700 μL of Wash Buffer I containing guanidine isothiocyanate and 500 μL of Wash Buffer II containing ethanol were added to the silica membrane and washed.
7) The silica membrane was washed with ethanol-containing Wash Buffer II, centrifuged at 12,000 × g at room temperature for 15 seconds, and dried.
8) 30 μL of RNase-Free Water was added, and the mixture was allowed to stand at room temperature for 1 minute, and then centrifuged at 12,000 × g at room temperature for 15 seconds. The RNA was eluted from the membrane twice.
9) Part of the eluted RNA was fractionated into UV permeable 96-well plates (Cat No. 8404, Thermo Scientific, USA) and Tris-EDTA Buffer (TE (pH 8.0), Cat No. 310-90023). , NIPPON GENE, Japan) and diluted 25-fold with a microplate reader (SPARK® 10M TECAN, Switzerland) for 230 nm, 260 nm and 280 nm absorbance (OD ₂₃₀ , OD ₂₆₀ and OD ₂₈₀ ). It was measured.
10) Using OD ₂₆₀ , the RNA concentration of the control and test substances was calculated by the following formula, and diluted with TE Buffer to prepare the RNA concentration to 10 μg / mL.
RNA concentration (μg / mL) = A × K × 0.3 × 10 (dilution factor)
A: Control or test substance OD ₂₆₀
K: K = 40, RNA absorption coefficient l: l = 0.3, optical path length (cm)

(Gene expression analysis by real-time PCR method)
1) The following RNA was reverse transcribed using SuperScript ™ IV VILO ™ Master Mix with ezDNase (Cat No. 11766050, Invitrogen, USA).
2) Add 1 μL of 10 × ezDNase Buffer, 1 μL of ezDNase enzyme, 6 μL of Nuclease-free Water and 2 μL of 10 μg / mL RNA to 8 tubes (AB1182, Thermo Scientific, USA). Incubated at 37 ° C for 2 minutes.
3) After 2 minutes, add 4 μL SuperScript ™ IV VILO ™ Master Mix and 6 μL Nuclease-free Water per well to the 8-tube tube and add real-time PCR (QuantStudio® 3, Applied Biosystems, Using USA), the cDNA was synthesized by heating at 25 ° C. for 10 minutes, 50 ° C. for 10 minutes, and 85 ° C. for 5 minutes.
4) 10 μL TaqMan® Fast Advanced Master Mix (Cat No. 4444557, Applied Biosystems, USA), 1 μL TaqMan Gene Expressior, 7 μL per well on PCR plate (Cat No. N8010560, Thermo Scientific, USA) UltraPure ™ Distilled Water (Invitrogen, Cat No. 10977-015, USA) and 2 μL cDNA were added and sealed with a plate seal (Cat No. 4360954, Thermo Scientific, USA).
5) The solution was spun down using a plate centrifuge to remove air bubbles.
6) Real-Time qPCR was performed using a real-time PCR system, and the Threshold Cycle (Ct) value, which is the number of cycles when the fluorescent signal of each gene in the control and the test substance reached an arbitrary threshold, was calculated. The Ct value was corrected by the internal standard gene to obtain the ΔCt value. The ΔCt value was corrected by the average of the ΔCt values of the control, and this was taken as the ΔΔCt value. The gene expression level of the test substance was analyzed when the gene expression level of the control was set to 1 by substituting it into 2 ^-ΔΔCt , assuming that the difference in detection per cycle would be doubled by the ΔΔCt method. A significant difference test was performed on the gene expression levels of the control and the test substance added by the paired t-test. All tests had a significance level of less than 5% on both sides (P <0.05, P <0.01, P <0.001).

<Test results>
The results of gene expression are shown in FIGS. 1 to 3. Specifically, it is as follows.
Figure 1: Results of the collagen degrading enzyme 1 gene (Human Matrix Metallopeptidase 1 (MMP1, Assay ID. Hs00899658_m1)) Figure 2: Results of the hyaluronan synthase 2 gene (Human Hyaluronan Synthase 2 (HAS2, Assay ID. Hs00193435_m1)) 3: Results of the elastin gene (Human Elastin (ELN, Assay ID. Hs00355783_m1))

The heat-sterilized body of Bifidobacterium Breve M-16V (NITE BP-02622) significantly reduced the expression of collagen degrading enzyme 1 gene (MMP1) and significantly reduced the expression of hyaluronan synthase 2 gene (HAS2). It was found that the expression of the elastin gene (ELN) was significantly enhanced.

[Example 2]
This Example was carried out in the same manner as in Example 1 except that the culture supernatant of Bifidobacterium Breve M-16V (NITE BP-02622) was used as a test substance and that the following was performed. ..

<Test substance>
DMEM medium (10% FCS, no antibiotics) was prepared as medium. The medium was inoculated with Bifidobacterium Breve M-16V (NITE BP-02622) and cultured in an anaerobic jar (CO ₂ : 20%, N ₂ : 80%) at 37 ° C. for 48 hours. The culture supernatant after centrifuging the culture medium was used as a test substance.
The test substance was continuously diluted with a medium to prepare a total of 3 concentrations (× 10, × 5, × 1) at the time of use. The prepared test substance was added to the culture medium, and a total of 3 concentrations (× 100, × 50, × 10) were used for the test. Medium was added instead of the test substance to the negative control.

<Gene expression analysis>
The expression of the following genes was analyzed using TaqMan Assay (Applied Biosystems, USA).
(a) Type I collagen gene (Human Collagen Type 1 Alpha 1 (COL1A1, Assay ID. Hs00164004_m1))
(b) Human Hyaluronan Synthase 1 (HAS1, Assay ID. Hs00758053_m1))
(c) Human Hyaluronan Synthase 2 (HAS2, Assay ID. Hs00193435_m1))
(d) Elastin gene (Human Elastin (ELN, Assay ID. Hs00355783_m1))
(e) As an internal standard gene, Human Glyceraldehyde 3 Phosphate Dehydrogenase (GAPDH, Assay ID. Hs02786624_g1)

<Test results>
The results of gene expression are shown in FIGS. 4 to 7. Specifically, it is as follows.
Figure 4: Results of type I collagen gene (Human Collagen Type 1 Alpha 1 (COL1A1, Assay ID. Hs00164004_m1)) Figure 5: Results of Hyaluronan Synthase 1 (HAS1, Assay ID. Hs00758053_m1) Figure 6: Results of the Hyaluronan Synthase 2 gene (HAS2, Assay ID. Hs00193435_m1) Figure 7: Results of the elastin gene (Human Elastin (ELN, Assay ID. Hs00355783_m1))

The culture supernatant of Bifidobacterium Breve M-16V (NITE BP-02622) significantly enhances the expression of type I collagen gene (COL1A1) and significantly enhances the expression of hyaluronan synthase 1 gene (HAS1). It was found that the expression was enhanced, the expression of the hyaluronan synthase 2 gene (HAS2) was significantly enhanced, and the expression of the elastin gene (ELN) was significantly enhanced.

[Example 3]
This example was carried out using a food containing live bacteria of Bifidobacterium Breve M-16V (NITE BP-02622) as a test food.

<Test content>
It was a single group pre- and post-comparative test. The subjects were five employees of Morinaga Milk Industry Co., Ltd. (subjects A to E).
As the test food, a food containing live bacteria of Bifidobacterium Breve M-16V (NITE BP-02622) (trade name: "Baby Bifidobacterium (registered trademark)") was used, and 2 packets per day (number of bacteria: 10 billion / day) was used for ingestion.
The intake period was 4 weeks, and a 2-week washout period was provided before administration.
Objective indicators (skin moisture content, dermis collagen content), subjective indicators (questionnaire: skin moisturizing, gloss, firmness, texture, transparency, smoothness after face washing, wrinkles, makeup paste, dryness / sagging, pimples, and Each item of comprehensive evaluation of skin condition) was evaluated.
In the evaluation by the objective index, the evaluation site was the forearm of the arm opposite to the dominant hand, and the site without skin symptoms such as scratches and dermatitis. After resting for 15 minutes in the anterior room where the temperature and humidity are controlled, the temperature and humidity are constant (temperature: 21 ± 1 ° C, humidity: 50% ± 5) according to the guidelines of the Japan Cosmetic Science Society. %) Was set in the measurement room, and the measurement was carried out after sufficient acclimatization in a resting state. The skin water content was measured using Tewameter TM3008 (courage + manufactured by Khazaka). The collagen score indicating the amount of dermal collagen was measured using DermaLab (registered trademark).
In the evaluation by the subjective index, the subjects were asked to enter 6 levels of subjective symptoms from 1 (poor) to 6 (good) for each of the above items.

<Test results>
The results of evaluation by objective indicators (mean values) are shown in Table 1, and the results of evaluation by subjective indicators (mean values) are shown in Table 2. In addition, Table 3 shows the results of evaluation by the subjective index for each subject before ingestion, and Table 4 shows the results of evaluation by the subjective index for each subject 4 weeks after ingestion. In addition, "-" in Tables 3 and 4 indicates "not applicable".

By ingesting live bacteria of Bifidobacterium Breve M-16V (NITE BP-02622) for 4 weeks, the average value of skin water content increased compared to before ingestion, and the collagen score indicating the amount of dermal collagen. Increased significantly. In addition, the skin condition was subjectively improved.

Claims (8)

A composition for regulating the expression of genes involved in the production or degradation of collagen, elastin or hyaluronic acid, which comprises Bifidobacterium Breve M-16V (NITE BP-02622) or its culture supernatant as an active ingredient.
The gene is selected from collagen degrading enzyme 1 gene (MMP1), type I collagen gene (COL1A1), elastin gene (ELN), hyaluronan synthase 1 gene (HAS1) and hyaluronan synthase 2 gene (HAS2). Composition. Regarding the collagen degrading enzyme 1 gene (MMP1), the regulation of expression is a decrease in expression.
For the type I collagen gene (COL1A1), elastin gene (ELN), hyaluronan synthase 1 gene (HAS1) and hyaluronan synthase 2 gene (HAS2), the expression regulation is enhanced.
The composition according to claim 1. A composition for improving skin properties, which contains Bifidobacterium Breve M-16V (NITE BP-02622) or its culture supernatant as an active ingredient. A composition containing Bifidobacterium Breve M-16V (NITE BP-02622) or its culture supernatant as an active ingredient for suppressing the decomposition of collagen or promoting the synthesis of collagen, elastin or hyaluronic acid. A composition for suppressing aging of joints, bones or blood vessels, which contains Bifidobacterium Breve M-16V (NITE BP-02622) or its culture supernatant as an active ingredient. The composition according to any one of claims 1 to 5, wherein the Bifidobacterium Breve M-16V (NITE BP-02622) is at least one of live and dead bacteria. The composition according to any one of claims 1 to 6, which is a food and drink composition. The composition according to any one of claims 1 to 6, which is a pharmaceutical composition.

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