JP7637474B2 - Composition for promoting the growth of Bifidobacterium breve - Google Patents

Description

The present invention relates to compositions that can be used for specific purposes, such as compositions for promoting the growth of Bifidobacterium breve.

Extracellular vesicles called exosomes exist in milk. It has been reported that milk-derived exosomes promote the growth of Escherichia coli and Lactobacillus plantarum, for example (Non-Patent Document 1).

Yu S et al. Characterization of three different types of extracellular vesicles and their impact on bacterial growth. Food Chem. 2019 Jan 30;272:372-378.

The present invention aims to provide a composition that can be used for specific purposes, such as a composition for promoting the growth of Bifidobacterium breve.

As a result of intensive research, the present inventors discovered that the use of milk-derived exosomes can promote the growth of Bifidobacterium breve, and thus completed the present invention.

That is, the present invention can be exemplified as follows.
[1]
A composition for promoting the proliferation of Bifidobacterium breve, comprising milk-derived exosomes as an active ingredient.
[2]
The composition, wherein the content of milk-derived exosomes at the time of ingestion is 150 ng/mL or more or 150 ng/g or more, calculated as RNA content.
[3]
A composition containing milk-derived exosomes,
A composition having a milk-derived exosome content of 150 ng/mL or more or 150 ng/g or more when ingested, calculated as an RNA content.
[4]
The composition is a nutritional composition.
[5]
The composition is an infant formula.
[6]
A composition for culturing Bifidobacterium breve, comprising milk-derived exosomes as an active ingredient.
[7]
The composition, which is a culture medium.
[8]
The content of milk-derived exosomes during culture was 1 ng/m2 in terms of RNA content.
The composition, wherein the .alpha.-amino acid salt is 1 ng/g or more or 1 L or more.
[9]
The composition, wherein the milk-derived exosomes are milk-derived exosomes.
[10]
1. A method for promoting the growth of Bifidobacterium breve, comprising:
A method comprising a step of administering milk-derived exosomes as an active ingredient to a subject.
[11]
The method, wherein the subject is a human infant or toddler.
[12]
1. A method for culturing Bifidobacterium breve, comprising:
A method comprising a step of culturing Bifidobacterium breve in a medium containing milk-derived exosomes as an active ingredient.
[13]
The method as described above, wherein the content of milk-derived exosomes in the medium is 1 ng/mL or more or 1 ng/g or more, calculated as an RNA content.
[14]
The method, wherein the milk-derived exosomes are milk-derived exosomes.
[15]
Use of milk-derived exosomes to promote the growth of Bifidobacterium breve.
[16]
Use of milk-derived exosomes for the cultivation of Bifidobacterium breve.
[17]
Use of milk-derived exosomes in the manufacture of the composition.
[18]
The above-mentioned use, wherein the milk-derived exosomes are exosomes derived from cow's milk.

The present invention provides a composition that can be used for specific purposes, such as a composition for promoting the growth of Bifidobacterium breve.

The present invention is described in detail below.

<1> Milk-derived exosomes In the present invention, milk-derived exosomes are used. Specifically, in the present invention, milk-derived exosomes may be used as an active ingredient.

"Milk-derived exosomes" refers to exosomes derived from milk. "Exosomes" refers to extracellular vesicles. The type of milk is not particularly limited. Examples of milk include cow's milk, goat's milk, sheep's milk, buffalo milk, reindeer milk, donkey milk, camel milk, and human breast milk. Examples of milk include, in particular, cow's milk. That is, examples of milk-derived exosomes include, in particular, exosomes derived from milk. Milk-derived exosomes may contain RNA. Examples of RNA include mRNA and miRNA.

Milk-derived exosomes can be obtained by collecting exosomes in milk. The method for collecting milk-derived exosomes is not particularly limited. Milk-derived exosomes can be obtained, for example, by
Exosomes can be collected from milk by known methods (Thery C et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J Extracell Vesicles. 2018 Nov 23;7(1):1535750., etc.). The milk used to collect exosomes may be, for example, whole milk or a processed product thereof. The processed product is not particularly limited as long as it contains exosomes. Examples of processed products include skim milk and whey. Specifically, for example, skim milk may be prepared from whole milk, whey may be prepared from skim milk, and exosomes may be collected from the whey. Skim milk can be prepared, for example, by subjecting whole milk to centrifugation. Whey can be prepared, for example, by subjecting skim milk to centrifugation, acid precipitation, or EDTA precipitation. Exosomes can be collected from whey by, for example, ultracentrifugation or size exclusion chromatography. The degree of purification of milk-derived exosomes is not particularly limited. Milk-derived exosomes can be used in any form, such as a dry powder or a suspension.

By using milk-derived exosomes, the proliferation of Bifidobacterium breve may be promoted, i.e., an effect of promoting the proliferation of Bifidobacterium breve may be obtained. This effect is also referred to as a "proliferation-promoting effect." For example, by administering milk-derived exosomes to a subject, a proliferation-promoting effect may be obtained in the subject.

In addition, by using milk-derived exosomes, an effect based on the promotion of the proliferation of Bifidobacterium breve may be obtained. For example, by administering milk-derived exosomes to a subject, an effect based on the promotion of the proliferation of Bifidobacterium breve in the subject may be obtained. An effect based on the promotion of the proliferation of Bifidobacterium breve may include an effect of preventing, improving, and/or treating symptoms for which administration of Bifidobacterium breve is effective for preventing, improving, and/or treating. Such symptoms are also referred to as "Bifidobacterium breve-associated symptoms." Examples of Bifidobacterium breve-associated symptoms include immune diseases such as allergies and necrotizing enterocolitis. The use of milk-derived exosomes to obtain an effect based on the promotion of the proliferation of Bifidobacterium breve may be an example of the use of milk-derived exosomes to promote the proliferation of Bifidobacterium breve.

Milk-derived exosomes may be administered to a subject, for example, in the manner described in the "Method of the Invention" below.

In addition, milk-derived exosomes may be used for culturing Bifidobacterium breve. By culturing Bifidobacterium breve using milk-derived exosomes, a growth-promoting effect may be obtained in the culture. In other words, the use of milk-derived exosomes for culturing Bifidobacterium breve may be an example of the use of milk-derived exosomes for promoting the growth of Bifidobacterium breve.

Milk-derived exosomes may be used, for example, to culture Bifidobacterium breve in the manner described below in the "Method of the Invention."

In addition, milk-derived exosomes may be used as a component contained in the composition of the present invention. That is, milk-derived exosomes may be used in the production of the composition of the present invention so as to be contained in the composition of the present invention.

In addition, milk-derived exosomes may be used in combination with Bifidobacterium breve. For example, milk-derived exosomes and Bifidobacterium breve may be co-administered to a subject. When milk-derived exosomes and Bifidobacterium breve are co-administered to a subject, the proliferation of the administered Bifidobacterium breve may be promoted. That is, "promotion of proliferation of Bifidobacterium breve" is not limited to the case where the proliferation of Bifidobacterium breve inherently possessed by the subject of administration of milk-derived exosomes is promoted, but may also include the case where the proliferation of Bifidobacterium breve administered to the subject of administration of milk-derived exosomes is promoted. "Bifidobacterium breve" used in combination with milk-derived exosomes may mean the bacterial body of Bifidobacterium breve. Bifidobacterium breve may be used in combination with milk-derived exosomes, particularly in a form containing live bacterial bodies.

Specific examples of Bifidobacterium breve include M-16V (NITE BP-02622), MCC1274 (FERM BP-11175), ATCC 15700, B632 (DSM 24706), Bb99 (DSM 13692), ATCC 15698, DSM 24732, UCC2003, YIT4010, YIT4064, BBG-001, BR-03, C50, and R0070.

These strains are available from, for example, the American Type Culture Collection (ATCC, Address: 10801
University Boulevard Manassas, VA 20110, United States of America), Belgian Coordinated Collections of Microorganisms (BCCM, Address: Rue de la Science 8, 1000 Brussels, Belgium), German Collection of Microorganisms and Cell Cultures (DSMZ, Address: Inhoffenstr.7B, D38124 Braunschweig, Germany), Japan Collection of
Microorganisms (JCM, 3-1-1 Takanodai, Tsukuba, Ibaraki, Japan, 305-0074, Japan), or from the depository institutions to which the respective strains have been deposited.

As the Bifidobacterium breve, a commercially available product may be used, or one obtained by appropriate production may be used. An example of a commercially available product of Bifidobacterium breve is Bifidobacterium breve FERM BP-11175 manufactured by Morinaga Milk Industry Co., Ltd. In addition, the bacterial cells of Bifidobacterium breve can be easily obtained by culturing Bifidobacterium breve. The culture method for obtaining the bacterial cells of Bifidobacterium breve can be applied mutatis mutandis to the description of the culture of Bifidobacterium breve in the "Method of the present invention" described below, except that milk-derived exosomes may or may not be used.

The present invention discloses the use of milk-derived exosomes in the above-mentioned applications. That is, the present invention discloses, for example, the use of milk-derived exosomes to promote the growth of Bifidobacterium breve, the use of milk-derived exosomes to prevent, ameliorate, and/or treat Bifidobacterium breve-associated symptoms, the use of milk-derived exosomes to culture Bifidobacterium breve, and the use of milk-derived exosomes in the manufacture of the composition of the present invention.

The present invention also discloses milk-derived exosomes for use in the applications exemplified above. That is, the present invention discloses, for example, milk-derived exosomes for use in promoting the proliferation of Bifidobacterium breve, milk-derived exosomes for use in preventing, ameliorating, and/or treating symptoms associated with Bifidobacterium breve, milk-derived exosomes for use in culturing Bifidobacterium breve, and milk-derived exosomes for use in producing the composition of the present invention.

The present invention also discloses the use of milk-derived exosomes in combination with Bifidobacterium breve.

<2> Composition of the Present Invention The composition of the present invention is a composition containing milk-derived exosomes. Specifically, the composition of the present invention may contain milk-derived exosomes as an active ingredient.

By utilizing the composition of the present invention, the proliferation of Bifidobacterium breve may be promoted, i.e., a proliferation-promoting effect may be obtained. For example, by administering the composition of the present invention to a subject, a proliferation-promoting effect may be obtained in the subject. That is, the composition of the present invention may be a composition for promoting the proliferation of Bifidobacterium breve. Specifically, the composition of the present invention may be a composition for promoting the proliferation of Bifidobacterium breve, which contains milk-derived exosomes as an active ingredient.

By using the composition of the present invention, an effect based on the promotion of the growth of Bifidobacterium breve may be obtained. For example, by administering the composition of the present invention to a subject, an effect based on the promotion of the growth of Bifidobacterium breve may be obtained in the subject. That is, the composition of the present invention may be a composition for obtaining an effect based on the promotion of the growth of Bifidobacterium breve. Specifically, the composition of the present invention may be a composition for obtaining an effect based on the promotion of the growth of Bifidobacterium breve, which contains milk-derived exosomes as an active ingredient. Examples of compositions for obtaining an effect based on the promotion of the growth of Bifidobacterium breve include compositions for preventing, improving, and/or treating symptoms associated with Bifidobacterium breve. The composition for obtaining an effect based on the promotion of the growth of Bifidobacterium breve may be an example of a composition for promoting the growth of Bifidobacterium breve.

The composition of the present invention may be administered to a subject, for example, in the manner described in the "Method of the Present Invention" described below. The description of the subject to which milk-derived exosomes are administered in the "Method of the Present Invention" described below can be applied mutatis mutandis to the subject to which the composition of the present invention is administered. Note that "administering the composition of the present invention to a subject" may be synonymous with "having a subject ingest the composition of the present invention."

The composition of the present invention may also be used for culturing Bifidobacterium breve. That is, the composition of the present invention may be a composition for culturing Bifidobacterium breve. Specifically, the composition of the present invention may be a composition for culturing Bifidobacterium breve, which contains milk-derived exosomes as an active ingredient. By culturing Bifidobacterium breve using the composition of the present invention, a growth-promoting effect may be obtained in the culture. That is, the composition for culturing Bifidobacterium breve may be an example of a composition for promoting the growth of Bifidobacterium breve.

The composition of the present invention may be used, for example, to culture Bifidobacterium breve in the manner described below in the "Method of the present invention."

The composition of the present invention may be, for example, a food and drink composition, a pharmaceutical composition, a feed composition, or a medium composition. The composition of the present invention may be, in particular, a food and drink composition or a medium composition. That is, the present invention may provide, for example, a food and drink composition for the above-exemplified uses such as for promoting the growth of Bifidobacterium breve, a pharmaceutical composition for the above-exemplified uses such as for promoting the growth of Bifidobacterium breve, a feed composition for the above-exemplified uses such as for promoting the growth of Bifidobacterium breve, or a medium composition for the above-exemplified uses such as for culturing Bifidobacterium breve. The composition of the present invention, which is a food and drink composition, a pharmaceutical composition, a feed composition, or a medium composition, is also referred to as the "food and drink composition of the present invention", the "pharmaceutical composition of the present invention", the "feed composition of the present invention", or the "medium composition of the present invention", respectively.

The composition of the present invention may contain, in addition to milk-derived exosomes, components other than milk-derived exosomes, as appropriate.

The components other than milk-derived exosomes are not particularly limited as long as they do not impair the desired effects such as the proliferation-promoting effect. As the components other than milk-derived exosomes, those that are acceptable depending on the mode of use of the composition of the present invention can be used. As the components other than milk-derived exosomes, for example, components that are used by being blended in food and drink, pharmaceuticals, feed, or culture media can be used. Specific examples of the components other than milk-derived exosomes include the components exemplified for the food and drink compositions, pharmaceutical compositions, feed compositions, or culture media compositions described below. Specific examples of the components other than milk-derived exosomes include Bifidobacterium breve. Bifidobacterium breve may be contained in compositions that are administered to subjects, such as food and drink compositions, pharmaceutical compositions, and feed compositions. As the components other than milk-derived exosomes, one type of component may be used, or two or more types of components may be used in combination.

The content and content ratio of each component (i.e., milk-derived exosomes and optionally other components) in the composition of the present invention are not particularly limited as long as the desired effect, such as a proliferation-promoting effect, is obtained. The content and content ratio of each component in the composition of the present invention can be appropriately set depending on various conditions, such as the type of component and the type of composition.

The content of milk-derived exosomes in the composition of the present invention may be, for example, 1 ng/g or more, 2 ng/g or more, 5 ng/g or more, 10 ng/g or more, 20 ng/g or more, 50 ng/g or more, 100 ng/g or more, 150 ng/g or more, 200 ng/g or more, 500 ng/g or more, 1000 ng/g or more, 2000 ng/g or more, 5000 ng/g or more, 1 μg/g or more, 2 μg/g or more, 5 μg/g or more, or 10 μg/g or more, and may be 100 or more. It may be 0 μg/g or less, 500 μg/g or less, 200 μg/g or less, 100 μg/g or less, 50 μg/g or less, 20 μg/g or less, 10 μg/g or less, 5 μg/g or less, 2 μg/g or less, 1 μg/g or less, 5000 ng/g or less, 2000 ng/g or less, 1000 ng/g or less, 500 ng/g or less, 200 ng/g or less, 100 ng/g or less, 50 ng/g or less, 20 ng/g or less, or 10 ng/g or less, or any non-contradictory combination thereof.

The content of milk-derived exosomes in the composition of the present invention may be, for example, 1 ng/mL or more, 2 ng/mL or more, 5 ng/mL or more, 10 ng/mL or more, 20 ng/mL or more, 50 ng/mL or more, 100 ng/mL or more, 150 ng/mL or more, 200 ng/mL or more, 500 ng/mL or more, 1000 ng/mL or more, 2000 ng/mL or more, 5000 ng/mL or more, 1 μg/mL or more, 2 μg/mL or more, 5 μg/mL or more, or 10 μg/mL or more, and may be 1000 ng/mL or more. It may be 1 μg/mL or less, 500 μg/mL or less, 200 μg/mL or less, 100 μg/mL or less, 50 μg/mL or less, 20 μg/mL or less, 10 μg/mL or less, 5 μg/mL or less, 2 μg/mL or less, 1 μg/mL or less, 5000 ng/mL or less, 2000 ng/mL or less, 1000 ng/mL or less, 500 ng/mL or less, 200 ng/mL or less, 100 ng/mL or less, 50 ng/mL or less, 20 ng/mL or less, or 10 ng/mL or less, or any non-contradictory combination thereof.

Specifically, the content of milk-derived exosomes in the composition of the present invention may be, for example, 5 ng/g to 1000 μg/g, 20 ng/g to 500 μg/g, 100 ng/g to 200 μg/g, 150 ng/g to 1000 μg/g, 150 ng/g to 500 μg/g, 150 ng/g to 200 μg/g, or 150 ng/g to 100 μg/g, in terms of the content of RNA. In particular, the composition of the present invention may be within the above ranges when it is a composition to be administered to a subject for use, such as a food or beverage composition, a pharmaceutical composition, or a feed composition.

Specifically, the content of milk-derived exosomes in the composition of the present invention may be, for example, 5 ng/mL to 1000 μg/mL, 20 ng/mL to 500 μg/mL, 100 ng/mL to 200 μg/mL, 150 ng/mL to 1000 μg/mL, 150 ng/mL to 500 μg/mL, 150 ng/mL to 200 μg/mL, or 150 ng/mL to 100 μg/mL, calculated as the content of RNA, and may be within the above ranges particularly when the composition of the present invention is a composition to be administered to a subject, such as a food or beverage composition, a pharmaceutical composition, or a feed composition.

The content of milk-derived exosomes in the composition of the present invention may be, specifically, for example, 1 ng/g to 100 μg/g, 2 ng/g to 20 μg/g, or 4 ng/g to 5 μg/g, calculated as the RNA content, and may be within these ranges particularly when the composition of the present invention is a composition used for culturing Bifidobacterium breve, such as a medium composition.

The content of milk-derived exosomes in the composition of the present invention may be, specifically, for example, 1 ng/mL to 100 μg/mL, 2 ng/mL to 20 μg/mL, or 4 ng/mL to 5 μg/mL, calculated as the RNA content, and may be within these ranges particularly when the composition of the present invention is a composition used for culturing Bifidobacterium breve, such as a medium composition.

When the composition of the present invention is a composition to be administered to a subject, such as a food and drink composition, a pharmaceutical composition, or a feed composition, the content of milk-derived exosomes in the composition of the present invention may be, for example, an amount that allows the content of milk-derived exosomes at the time of ingestion to be within a predetermined range. The content of milk-derived exosomes at the time of ingestion may be, for example, 5 ng/g or more, 10 ng/g or more, 20 ng/g or more, 50 ng/g or more, 100 ng/g or more, 150 ng/g or more, 200 ng/g or more, 500 ng/g or more, or 1000 ng/g or more, or 1 μg/g or less, 5000 ng/g or less, 2000 ng/g or less, 1000 ng/g or less, 500 ng/g or less, 200 ng/g or less, 100 ng/g or less, or 50 ng/g or less, or may be a range of a compatible combination thereof, in terms of the content of RNA. The content of milk-derived exosomes at the time of ingestion may be, in particular, 150 ng/g or more, calculated as the content of RNA. Specifically, the content of milk-derived exosomes at the time of ingestion may be, for example, 5 ng/g to 1 μg/g, 20 ng/g to 5000 ng/g, 100 ng/g to 2000 ng/g, 150 ng/g to 1 μg/g, 150 ng/g to 5000 ng/g, 150 ng/g to 2000 ng/g, or 150 ng/g to 1000 ng/g, calculated as the content of RNA.

When the composition of the present invention is a composition administered to a subject, such as a food and drink composition, a pharmaceutical composition, or a feed composition, the content of milk-derived exosomes in the composition of the present invention may be, for example, an amount such that the content of milk-derived exosomes at the time of ingestion is within a predetermined range. The content of milk-derived exosomes at the time of ingestion may be, for example, 5 ng/mL or more, 10 ng/mL or more, 20 ng/mL or more, 50 ng/mL or more, 100 ng/mL or more, 150 ng/mL or more, 200 ng/mL or more, 500 ng/mL or more, or 1000 ng/mL or more, or 1 μg/mL or less, 5000 ng/mL or less, 2000 ng/mL or less, 1000 ng/mL or less, 500 ng/mL or less, 200 ng/mL or less, 100 ng/mL or less, or 50 ng/mL or less, or may be a range of a compatible combination thereof. The content of milk-derived exosomes at the time of ingestion may be, in particular, 150 ng/mL or more in terms of the content of RNA. Specifically, the content of milk-derived exosomes at the time of ingestion may be, for example, 5 ng/mL to 1 μg/mL, 20 ng/mL to 5000 ng/mL, 100 ng/mL to 2000 ng/mL, 150 ng/mL to 1 μg/mL, 150 ng/mL to 5000 ng/mL, 150 ng/mL to 2000 ng/mL, or 150 ng/mL to 1000 ng/mL in terms of the content of RNA.

Unless otherwise specified, the term "time of ingestion" of the composition of the present invention means the time when the composition of the present invention is in a form suitable for ingestion by a subject (i.e., administration to a subject). In other words, the term "content of milk-derived exosomes at the time of ingestion" of the composition of the present invention means the content of milk-derived exosomes in the composition of the present invention in a form suitable for ingestion by a subject, unless otherwise specified. "Form suitable for ingestion by a subject" means the form of the composition of the present invention itself when the composition of the present invention is a composition to be ingested as is (e.g., a food and beverage composition to be ingested as is). In addition, "form suitable for ingestion by a subject" means the diluted form of the composition of the present invention when the composition of the present invention is a composition to be ingested after dilution (e.g., a food and beverage composition to be ingested after dilution). For example, when the composition of the present invention is a formula, "form suitable for ingestion by a subject" means a form prepared at a standard formula concentration determined by the seller. In addition, when a product is produced using the composition of the present invention (e.g., when another food and beverage composition is produced using the food and beverage composition of the present invention), "time of ingestion" of the composition of the present invention may be read as "time of ingestion" of the product.

When the composition of the present invention is a composition to be administered to a subject, such as a food or drink composition, a pharmaceutical composition, or a feed composition, the content of milk-derived exosomes in the composition of the present invention may be, for example, an amount that results in the administration amount of milk-derived exosomes falling within a predetermined range. The description of the administration amount of milk-derived exosomes in the method of the present invention can be applied mutatis mutandis to the administration amount of milk-derived exosomes by administration of the composition of the present invention.

When the composition of the present invention is a composition used for culturing Bifidobacterium breve, such as a medium composition, the content of milk-derived exosomes in the composition of the present invention may be, for example, an amount in which the content of milk-derived exosomes during culture is within a predetermined range. The content of milk-derived exosomes during culture may be, for example, 1 ng/g or more, 2 ng/g or more, 3 ng/g or more, 4 ng/g or more, 5 ng/g or more, 7 ng/g or more, 10 ng/g or more, 20 ng/g or more, 50 ng/g or more, or 100 ng/g or more, or 1000 ng/g or less, 500 ng/g or less, 200 ng/g or less, 100 ng/g or less, 50 ng/g or less, 20 ng/g or less, or 10 ng/g or less, or may be a range of a compatible combination thereof, in terms of RNA content. Specifically, the content of milk-derived exosomes during culture may be, for example, 1 ng/g to 1000 ng/g, 2 ng/g to 200 ng/g, or 4 ng/g to 50 ng/g, converted into RNA content.

When the composition of the present invention is a composition used for culturing Bifidobacterium breve, such as a medium composition, the content of milk-derived exosomes in the composition of the present invention may be, for example, an amount in which the content of milk-derived exosomes during culture is within a predetermined range. The content of milk-derived exosomes during culture may be, for example, 1 ng/mL or more, 2 ng/mL or more, 3 ng/mL or more, 4 ng/mL or more, 5 ng/mL or more, 7 ng/mL or more, 10 ng/mL or more, 20 ng/mL or more, 50 ng/mL or more, or 100 ng/mL or more, in terms of RNA content, or 1000 ng/mL or less, 500 ng/mL or less, 200 ng/mL or less, 100 ng/mL or less, 50 ng/mL or less, 20 ng/mL or less, or 10 ng/mL or less, or may be a range of a compatible combination thereof. Specifically, the content of milk-derived exosomes during culture may be, for example, 1 ng/mL to 1000 ng/mL, 2 ng/mL to 200 ng/mL, or 4 ng/mL to 50 ng/mL, calculated as the RNA content.

Unless otherwise specified, the term "at the time of culture" for the composition of the present invention means the time point when the composition of the present invention is in a form suitable for culturing Bifidobacterium breve. In other words, the term "content of milk-derived exosomes at the time of culture" for the composition of the present invention means the content of milk-derived exosomes in the composition of the present invention in a form suitable for culturing Bifidobacterium breve, unless otherwise specified. "Form suitable for culturing Bifidobacterium breve" means the form of the composition of the present invention itself when the composition of the present invention is a composition used for culturing as it is (e.g., a liquid medium used for culturing as it is). In addition, "form suitable for culturing Bifidobacterium breve" means the diluted form of the composition of the present invention when the composition of the present invention is a composition used for culturing after dilution (e.g., a powder medium used for culturing after dilution). That is, the "content of milk-derived exosomes during culture" of the composition of the present invention may mean, for example, the content of milk-derived exosomes in a medium when Bifidobacterium breve is cultured using the composition of the present invention as a medium, or when Bifidobacterium breve is cultured in a medium prepared using the composition of the present invention.

When the food and beverage composition of the present invention contains Bifidobacterium breve, the content of Bifidobacterium breve in the composition of the present invention may be, for example, 1 x ¹⁰⁴ cfu/g or more, 1 x ¹⁰⁵ cfu/g or more, 1 x ¹⁰⁶ cfu/g or more, 1 x ¹⁰⁷ cfu/g or more, or 1 x ¹⁰⁸ cfu/g or more, or ¹⁰¹³ cfu/g or less, ¹⁰¹² cfu/g or less, or 1 x ¹⁰¹¹ cfu/g or less, or any combination of these ranges. Furthermore, when the food and beverage composition of the present invention contains Bifidobacterium breve, the content of Bifidobacterium breve in the composition of the present invention may be, for example, 1 x ¹⁰⁴ cfu/mL or more, 1 x ¹⁰⁵ cfu/mL or more, 1 x ¹⁰⁶ cfu/mL or more, 1 x ¹⁰⁷ cfu/mL or more, or 1 x ¹⁰⁸ cfu/mL or more, or ¹⁰¹³ cfu/mL or less, ¹⁰¹² cfu/mL or less, or 1 x ¹⁰¹¹ cfu/mL or less, or a combination of these ranges. The content of Bifidobacterium breve in the composition of the present invention may be, specifically, for example, 1× ¹⁰ to 1× ¹⁰ cfu/g or 1× ¹⁰ to 1× ¹⁰ cfu/mL, preferably 1× ¹⁰ to 1× ¹⁰ cfu/g or 1× ¹⁰ to 1× ¹⁰ cfu/mL, and more preferably 1× ¹⁰ to 1× ¹⁰ cfu/g or 1× ¹⁰ to 1× ¹⁰ cfu/mL. "Cfu" stands for colony forming unit.

The shape of the composition of the present invention is not particularly limited. Any shape that is acceptable depending on the mode of use of the composition of the present invention can be adopted as the shape of the composition of the present invention. Specific examples of the shape of the composition of the present invention include shapes exemplified for food and drink compositions, pharmaceutical compositions, feed compositions, and medium compositions described below.

<Food and drink composition>
The food and drink composition of the present invention is not particularly limited as long as it contains milk-derived exosomes. The food and drink composition of the present invention may be provided in any form, such as a liquid, a paste, a gel-like solid, or a powder. The food and drink composition may be ingested, for example, by humans. Specifically, the food and drink composition may be orally ingested, for example, by humans.

The food and drink composition may be, for example, a food and drink itself, or may be a material used in the manufacture of a food and drink. Examples of such materials include seasonings, food additives, and other food and drink ingredients. A food and drink composition that is a food and drink is also called a "nutritional composition". Specific examples of food and drink compositions include flour products, instant foods, processed agricultural products, processed marine products, processed livestock products, dairy products (fermented milk, cheese, prepared milk powder, etc.), oils and fats, basic seasonings, complex seasonings, frozen foods, confectioneries, beverages, liquid foods, and other commercially available foods and drinks. Examples of food and drink compositions (specifically, nutritional compositions) include, in particular, prepared milk and liquid foods. Examples of food and drink compositions (specifically, nutritional compositions) include, in particular, prepared milk. In addition, specific examples of food and drink compositions include health foods, functional foods, enteral nutritional foods, special purpose foods, health functional foods (specified health foods, nutritional functional foods, functional foods, etc.), nutritional supplements, and pharmaceutical quasi-drugs.

Examples of prepared milk include prepared powdered milk and prepared liquid milk. Prepared powdered milk is defined in the Ministerial Ordinance on the Ingredient Standards of Milk and Dairy Products (Milk Ordinance) as "raw milk, cow's milk, special milk, or food made from these ingredients, processed or used as the main ingredient, with the addition of nutrients necessary for infants, and made into a powder form." Prepared liquid milk is defined in the Ministerial Ordinance on Milk, etc. as "raw milk, cow's milk, special milk, or food made from these ingredients, processed or used as the main ingredient, with the addition of nutrients necessary for infants, and made into a liquid form." Specific examples of prepared milk include those that contain nutritional ingredients such as proteins, fats and oils, carbohydrates, minerals, and vitamins, and are processed into a powder or liquid form. Specific examples of prepared milk include "formulated milk powder for infants," "liquid formulated milk for infants," and "powdered milk for pregnant and lactating women," which are foods for special dietary uses as stipulated in the Health Promotion Act. Specific examples of prepared milk include formulated milk powder for infants, nutritional powder for adults, and nutritional powder for the elderly. The subjects of intake of nutritional compositions such as formula are not particularly limited. The subjects of intake of nutritional compositions such as formula may be people of any age, such as infants, small children, adults, middle-aged and elderly people, and the like. In particular, subjects of intake of nutritional compositions such as formula are infants and small children.

The food and drink composition of the present invention may contain, as appropriate, components other than milk-derived exosomes in addition to milk-derived exosomes. Examples of components other than milk-derived exosomes include long-chain polyunsaturated fatty acids, proteins, fats and oils, carbohydrates, vitamins, and minerals. Any of these components may be contained in, for example, infant formula. For example, breast milk usually contains long-chain polyunsaturated fatty acids necessary for the development of infants, and infant formula is required to contain a certain amount of a specific long-chain polyunsaturated fatty acid (Dietary Table No. 296 (September 9, 2019) Appendix 1
(Standards for approval of labeling of foods for special dietary uses.) As a component other than milk-derived exosomes, one type of component may be used, or two or more types of components may be used in combination.

Examples of long-chain polyunsaturated fatty acids include docosahexaenoic acid (DHA), arachidonic acid (ARA), eicosapentaenoic acid (EPA), linoleic acid, gamma-linolenic acid (GLA), alpha-linolenic acid, dihomo-gamma-linolenic acid (DGLA), stearidonic acid, and docosapentaenoic acid (DPA). These long-chain polyunsaturated fatty acids may be present as free fatty acids when ingested, or may be derivatives that can become free fatty acids after ingestion. Examples of derivatives include oils and fats that contain long-chain polyunsaturated fatty acids as constituent fatty acids. As the long-chain polyunsaturated fatty acids, one type of long-chain polyunsaturated fatty acid may be used, or two or more types of long-chain polyunsaturated fatty acids may be used in combination. As the long-chain polyunsaturated fatty acids, commercially available products may be used, or those obtained by appropriate production may be used. Long-chain polyunsaturated fatty acids can be obtained, for example, by extraction from natural resources such as fish.

Examples of proteins include milk-derived proteins such as whey protein and casein, plant proteins such as soybean protein, and hydrolysates thereof. Examples of whey proteins include β-lactoglobulin, α-lactalbumin, serum albumin, immunoglobulin, lactoferrin, and proteose peptone. For example, a purified high-purity protein may be used, or a low-purity protein containing components other than protein may be used. For example, a milk-derived protein such as whey protein may be a material containing a milk-derived protein such as whole milk, skim milk powder, or whole milk powder. For the protein, a commercially available product may be used, or a product obtained by appropriate production may be used. For example, a milk-derived protein such as whey protein may be obtained from a material containing a milk-derived protein such as whole milk, skim milk powder, or whole milk powder. Methods for purifying whey protein include a method of adding rennet or the like to milk or skim milk powder to remove casein and milk fat, and a method of further purifying the casein and milk fat after the removal by gel filtration, ultrafiltration, ion exchange, or the like. The whey protein concentrate (WPC) and whey protein isolate (WPI) thus obtained can both be used as whey protein. As the protein, one kind of protein may be used, or two or more kinds of proteins may be used in combination. As the protein, for example, at least whey protein may be used, or whey protein may be used in combination with other proteins.

Examples of fats and oils include animal fats and oils, plant fats and oils, fats and oils obtained by culturing microorganisms, and hardened oils thereof. Examples of animal fats and oils include chicken fat, lard, beef tallow, sheep oil, whale oil, fish oil, egg oil, and butter. Examples of fish oils include tuna oil, bonito oil, sardine oil, mackerel oil, salmon oil, and cod oil. Examples of vegetable fats and oils include rapeseed oil, rice oil, safflower oil, sunflower oil, olive oil, peanut oil, palm oil, palm kernel oil, coconut oil, soybean oil, corn oil, cottonseed oil, sesame oil, grape seed oil, and perilla oil. Examples of hardened oils include partially hardened oils and extremely hardened oils. Specific examples of hardened oils include partially hardened oils of the vegetable fats and oils exemplified above, and extremely hardened oils of the vegetable fats and oils exemplified above. As the fats and oils, one type of fat and oil may be used, or two or more types of fats and oils may be used in combination.

Examples of carbohydrates include sugars such as lactose, dextrin, starch, raffinose, and lactulose, and dietary fibers such as resistant dextrin and inulin. As the carbohydrates, one type of carbohydrate may be used, or two or more types of carbohydrates may be used in combination.

Vitamins include water-soluble vitamins such as vitamin B group and vitamin C, and fat-soluble vitamins such as vitamin A, vitamin D, and vitamin E. As vitamins, one type of vitamin may be used, or two or more types of vitamins may be used in combination.

Minerals include salts of sodium, potassium, calcium, iron, zinc, manganese, and copper. Specific examples of minerals include sodium chloride, potassium chloride, calcium carbonate, ferric pyrophosphate, zinc sulfate, manganese sulfate, and copper sulfate. As the minerals, one type of mineral may be used, or two or more types of minerals may be used in combination.

The food and beverage composition of the present invention can be produced, for example, by appropriately combining milk-derived exosomes with other ingredients. The operation of combining milk-derived exosomes with other ingredients is also referred to as "addition of milk-derived exosomes". The method for producing the food and beverage composition of the present invention is not particularly limited. The food and beverage composition of the present invention can be produced, for example, by the same method as that of ordinary foods and beverages using the same raw materials as those of ordinary foods and beverages, except for the addition of milk-derived exosomes. The same applies when the food and beverage composition of the present invention is produced as a material used in the production of foods and beverages such as seasonings, food additives, and other food and beverage ingredients. The addition of milk-derived exosomes may be carried out at any stage of the production process of the food and beverage composition. The addition of milk-derived exosomes may be carried out, for example, during or after the production of the food and beverage composition. That is, for example, the food and beverage composition of the present invention may be produced by adding milk-derived exosomes to a previously prepared food and beverage.

In addition, the food and drink composition of the present invention can be used to produce another food and drink composition. That is, for example, when the food and drink composition of the present invention is provided as a material used in the production of food and drink such as seasonings, food additives, and other food and drink ingredients, another food and drink composition can be produced by adding the food and drink composition of the present invention to the raw materials of the food and drink. Another food and drink composition produced in this way is also one aspect of the food and drink composition of the present invention. For the addition of the food and drink composition of the present invention, the description of the addition of milk-derived exosomes in the production of a food and drink composition can be applied mutatis mutandis.

Below, an example of a method for producing the food and beverage composition of the present invention is described with reference to the case of prepared milk.

A liquid prepared milk (prepared liquid milk) can be prepared by adding a predetermined amount of prepared milk raw materials containing milk-derived exosomes, long-chain polyunsaturated fatty acids, proteins, fats and oils, carbohydrates, vitamins, minerals, etc. to water, raw milk, skim milk, etc., heating appropriately to mix and dissolve, and heat sterilization. The fats and oils may be added after being heated and melted in advance. After the fats and oils are added, homogenization may be performed. Homogenization may be performed, for example, by a homogenizer. For example, fats and oils may be added to a solution in which a part of the raw materials for the prepared milk is dissolved, and the solution may be homogenized once, and then the remaining raw materials for the prepared milk may be added to prepare a liquid prepared milk. The prepared liquid prepared milk may be sterilized. Sterilization may be performed, for example, by heat sterilization. Heat sterilization may be performed, for example, at 75 to 150°C using a plate sterilizer or the like. Homogenization may be performed after heat sterilization. Homogenization may be performed, for example, to adjust the fat globules in the liquid to a uniform size and to bring about a good emulsified state.

The liquid prepared milk (e.g., heat-sterilized liquid prepared milk) may be a final product, or an intermediate product that can be used to manufacture powdered prepared milk, as described below. That is, the liquid prepared milk (e.g., heat-sterilized liquid prepared milk) can be hygienically transferred to a filling machine and directly filled into containers made of paper, plastic, aluminum, etc., to produce the final product.

Liquid prepared milk (e.g., liquid prepared milk that has been heat sterilized) can also be dried to produce powdered prepared milk (prepared powdered milk). Heat sterilization and drying can be carried out in a single step. That is, for example, liquid prepared milk before sterilization can be heat sterilized and dried in a single step to produce powdered prepared milk. Drying can be carried out, for example, by spray drying using hot air or freeze drying. Spray drying using hot air is preferable in that it involves heating and therefore exerts a certain degree of sterilization action. The obtained powdered prepared milk can be filled into containers as it is, or after being mixed with other ingredients as appropriate, to produce the final product.

The food and beverage composition of the present invention can be provided and sold as a food and beverage labeled with uses (including health uses) such as for promoting the growth of Bifidobacterium breve and for preventing, improving, and/or treating symptoms associated with Bifidobacterium breve.

"Representation" includes all acts aimed at informing consumers of the aforementioned uses, and any expression that can recall or infer the aforementioned uses falls under "representation", regardless of the purpose of the representation, the content of the representation, the object and medium on which it is displayed, etc. It is particularly preferable that the representation be made using expressions that enable consumers to directly recognize the aforementioned uses.

Specific examples of labeling include the act of transferring, delivering, displaying for transfer or delivery, or importing a product related to the food and beverage composition of the present invention or a product packaging on which the above-mentioned uses are written, displaying or distributing the product with the above-mentioned uses written in advertisements, price lists, or transaction documents related to the product, or providing the information containing the above-mentioned uses by electromagnetic means (Internet, etc.). Examples of labeling include, in particular, labeling on promotional materials at the point of sale such as packaging, containers, catalogs, pamphlets, and POP, as well as other documents, etc.

In addition, the labeling is preferably a labeling approved by the government or the like (for example, a labeling approved based on various systems established by the government and made in a manner based on such approval). In addition, examples of the labeling include labeling as health food, functional food, enteral nutritional food, special purpose food, health functional food (for example, specified health food, nutrient functional food, functional food), dietary supplement, medical drug, etc. Examples of the labeling approved by the government or the like include labeling approved by the Consumer Affairs Agency. Examples of the labeling approved by the Consumer Affairs Agency include labeling approved under a specified health food or similar system. Examples of the labeling approved by the Consumer Affairs Agency include, specifically, labeling as a specified health food, labeling as a conditional specified health food, labeling that affects the structure or function of the body, labeling for reducing disease risk, labeling of functionality based on scientific evidence, etc., and more specifically, labeling as a specified health food (especially labeling for health purposes) and similar labels as set forth in the Cabinet Office Ordinance on the Permission of Special Purpose Labeling Provided in the Health Promotion Act (Cabinet Office Ordinance No. 57 of August 31, 2009).

The content of milk-derived exosomes in the food and beverage composition of the present invention may be, for example, within the range exemplified above. The content of milk-derived exosomes in the food and beverage composition of the present invention may be, for example, an amount such that the content of milk-derived exosomes at the time of ingestion falls within the range exemplified above. When the food and beverage composition of the present invention is a prepared milk powder, the content of milk-derived exosomes in the prepared milk powder (in a powdered state) may be, for example, 7.7 times the content of milk-derived exosomes at the time of ingestion exemplified above. The content of milk-derived exosomes in the food and beverage composition of the present invention may be, for example, an amount such that the dosage of milk-derived exosomes falls within a predetermined range.

<Pharmaceutical Composition>
The pharmaceutical composition of the present invention is not particularly limited as long as it contains milk-derived exosomes. For example, milk-derived exosomes may be used as the pharmaceutical composition of the present invention as they are, or milk-derived exosomes may be appropriately formulated and used.

The dosage form of the pharmaceutical composition of the present invention is not particularly limited. The dosage form of the pharmaceutical composition of the present invention can be appropriately selected depending on various conditions such as the administration method. The pharmaceutical composition of the present invention may be for oral administration or for parenteral administration. The pharmaceutical composition of the present invention may be for oral administration in particular. In the case of oral administration, the dosage form specifically includes solids such as powders, granules, tablets, and capsules, and liquids such as solutions, syrups, suspensions, and emulsions. In the case of parenteral administration, the dosage form specifically includes suppositories and ointments. In the formulation, physiologically acceptable additives can be used. Examples of additives include various organic and inorganic components. Examples of additives specifically include excipients, binders, disintegrants, lubricants, stabilizers, flavorings, diluents, surfactants, and solvents. These additives can be appropriately selected depending on various conditions such as the dosage form.

Examples of excipients include sugar derivatives such as lactose, sucrose, glucose, mannitol, and sorbitol; starch derivatives such as corn starch, potato starch, α-starch, dextrin, and carboxymethyl starch; cellulose derivatives such as crystalline cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, carboxymethyl cellulose, and calcium carboxymethyl cellulose; gum arabic; dextran; pullulan; silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, and magnesium aluminometasilicate; phosphate derivatives such as calcium phosphate; carbonate derivatives such as calcium carbonate; and sulfate derivatives such as calcium sulfate.

Examples of binders include the above excipients, gelatin, polyvinylpyrrolidone, macrogol, etc.

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

Examples of lubricants include talc; stearic acid; metal stearates such as calcium stearate and magnesium stearate; colloidal silica; waxes such as pea gum and geranium stearate; boric acid; glycol; carboxylic acids such as fumaric acid and adipic acid; sodium carboxylates 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 silicic anhydride and silicic acid hydrate; and starch derivatives.

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

Flavors and flavorings include, for example, sweeteners, acidulants, and fragrances.

The content of milk-derived exosomes in the pharmaceutical composition of the present invention may be, for example, within the range exemplified above. The content of milk-derived exosomes in the pharmaceutical composition of the present invention may be, for example, an amount such that the content of milk-derived exosomes at the time of ingestion falls within the range exemplified above. The content of milk-derived exosomes in the pharmaceutical composition of the present invention may be, for example, an amount such that the administration amount of milk-derived exosomes falls within a predetermined range.

<Feed composition>
The feed composition of the present invention is not particularly limited as long as it contains milk-derived exosomes. The feed composition may be, for example, a composition that can be used as a feed, i.e., a feed. The feed composition (e.g., a feed) may be used as a feed, for example, alone or in combination with other components. Examples of the feed include pet food and livestock feed. The feed composition of the present invention may be provided in any form, such as powder, granules, crumbles, pellets, cubes, paste, liquid, etc. The feed composition may be ingested, for example, by a subject other than a human. Specifically, the feed composition may be orally ingested, for example, by a subject other than a human.

The feed composition of the present invention can be produced, for example, by appropriately combining milk-derived exosomes with other components. The operation of combining milk-derived exosomes with other components is also referred to as "addition of milk-derived exosomes". The method for producing the feed composition of the present invention is not particularly limited. The feed composition of the present invention can be produced, for example, by using the same raw materials as normal feed and by the same method as normal feed, except for the addition of milk-derived exosomes. The addition of milk-derived exosomes may be carried out at any stage of the production process of the feed composition. The addition of milk-derived exosomes may be carried out, for example, during or after the production of the feed composition. That is, for example, the feed composition of the present invention may be prepared by adding milk-derived exosomes to a previously prepared feed.

The content of milk-derived exosomes in the feed composition of the present invention may be, for example, within the range exemplified above. The content of milk-derived exosomes in the feed composition of the present invention may be, for example, an amount such that the content of milk-derived exosomes at the time of ingestion falls within the range exemplified above. The content of milk-derived exosomes in the feed composition of the present invention may be, for example, an amount such that the administration amount of milk-derived exosomes falls within a predetermined range.

<Medium composition>
The medium composition of the present invention is not particularly limited as long as it contains milk-derived exosomes. The medium composition may be, for example, a composition that can be used as a medium, i.e., a medium. The medium composition (e.g., a medium) may be used as a medium, for example, alone or in combination with other components. The medium composition (e.g., a medium) may be used as a medium, for example, by diluting with an aqueous medium such as water or an aqueous buffer solution. The medium composition of the present invention may be provided in any form, such as a powder or liquid. The medium composition may be used, for example, for culturing Bifidobacterium breve.

The medium composition of the present invention may contain, in addition to milk-derived exosomes, components other than milk-derived exosomes as appropriate. Examples of components other than milk-derived exosomes include carbon sources, nitrogen sources, inorganic salts, organic components, and milk components. As components other than milk-derived exosomes, one type of component may be used, or two or more types of components may be used in combination. Examples of carbon sources include sugars such as galactose, glucose, fructose, mannose, cellobiose, maltose, lactose, sucrose, trehalose, starch, starch hydrolysates, and blackstrap molasses. Examples of nitrogen sources include ammonium salts and nitrates such as ammonia, ammonium sulfate, ammonium chloride, and ammonium nitrate. Examples of inorganic salts include sodium chloride, potassium chloride, potassium phosphate, magnesium sulfate, calcium chloride, calcium nitrate, manganese chloride, and ferrous sulfate. Examples of organic components include peptone, soybean flour, defatted soybean meal, meat extract, and yeast extract. Examples of milk components include milk proteins. The milk proteins are as described above.

The medium composition of the present invention can be produced, for example, by appropriately combining milk-derived exosomes with other components. The operation of combining milk-derived exosomes with other components is also referred to as "addition of milk-derived exosomes". The method for producing the medium composition of the present invention is not particularly limited. The medium composition of the present invention can be produced, for example, by using the same raw materials as in a normal medium and by the same method as in a normal medium, except for the addition of milk-derived exosomes. The addition of milk-derived exosomes may be carried out at any stage of the production process of the medium composition. The addition of milk-derived exosomes may be carried out, for example, during or after the production of the medium composition. That is, for example, the medium composition of the present invention may be prepared by adding milk-derived exosomes to a previously prepared medium.

The content of milk-derived exosomes in the medium composition of the present invention may be, for example, within the range exemplified above. The content of milk-derived exosomes in the medium composition of the present invention may be, for example, an amount such that the content of milk-derived exosomes during culture falls within the range exemplified above.

<3> Method of the Present Invention The method of the present invention is a method including a step of using milk-derived exosomes.

By using milk-derived exosomes, the proliferation of Bifidobacterium breve may be promoted, i.e., a proliferation-promoting effect may be obtained. For example, by administering milk-derived exosomes to a subject, a proliferation-promoting effect may be obtained in the subject. That is, the method of the present invention may be a method for promoting the proliferation of Bifidobacterium breve. For example, the method of the present invention may be a method for promoting the proliferation of Bifidobacterium breve, comprising a step of administering milk-derived exosomes to a subject. Specifically, the method of the present invention may be a method for promoting the proliferation of Bifidobacterium breve, comprising a step of administering milk-derived exosomes to a subject. That is, an example of the use of milk-derived exosomes is administering milk-derived exosomes to a subject, and specifically, administering milk-derived exosomes to a subject as an active ingredient.

By using milk-derived exosomes, an effect based on the promotion of the growth of Bifidobacterium breve may be obtained. For example, by administering milk-derived exosomes to a subject, an effect based on the promotion of the growth of Bifidobacterium breve may be obtained in the subject. That is, the method of the present invention may be a method for obtaining an effect based on the promotion of the growth of Bifidobacterium breve. The method of the present invention may be, for example, a method for obtaining an effect based on the promotion of the growth of Bifidobacterium breve, comprising a step of administering milk-derived exosomes to a subject. Specifically, the method of the present invention may be a method for obtaining an effect based on the promotion of the growth of Bifidobacterium breve, comprising a step of administering milk-derived exosomes to a subject. Examples of the method for obtaining an effect based on the promotion of the growth of Bifidobacterium breve include a method for preventing, improving, and/or treating symptoms associated with Bifidobacterium breve. The method for obtaining an effect based on the promotion of the growth of Bifidobacterium breve may be an example of a method for promoting the growth of Bifidobacterium breve.

Note that "administering milk-derived exosomes to a subject" may be synonymous with "allowing a subject to ingest milk-derived exosomes." Ingestion may be voluntary (free ingestion) or forced (forced ingestion). That is, the step of administering milk-derived exosomes to a subject may specifically be, for example, a step of blending milk-derived exosomes into food, drink, or feed and supplying the mixture to a subject, thereby allowing the subject to ingest the milk-derived exosomes freely. Administration may be oral or parenteral. Administration may usually be oral. Examples of parenteral administration include tube administration and rectal administration.

The mode of administration of milk-derived exosomes (e.g., the recipient, the timing of administration, the duration of administration, the number of administrations, the dosage, and other conditions related to administration) is not particularly limited as long as the desired effects, such as a proliferation-promoting effect, are obtained. The mode of administration of milk-derived exosomes can be appropriately set depending on various conditions, such as the type, age, and health condition of the recipient.

The subject to which milk-derived exosomes are administered is not particularly limited as long as the desired effect, such as a proliferation-promoting effect, can be obtained. The subject to which milk-derived exosomes are administered is a mammal. Examples of mammals include humans and pets. Examples of pets include dogs and cats. Examples of mammals include humans, in particular. The mammal may be male or female. The conditions of the human to which milk-derived exosomes are administered are not particularly limited. Milk-derived exosomes may be administered to any human who desires a desired effect, such as a proliferation-promoting effect. The human may be of any age, such as an infant, a toddler, a child, an adult, a middle-aged person, or an elderly person. An "infant" refers to a child less than one year old. An "infant" refers to a child from one year old to the age of entering elementary school. The human may be, for example, a healthy person or an unhealthy person. An example of an unhealthy person is a person who exhibits Bifidobacterium breve-related symptoms.

The dosage of milk-derived exosomes may be, for example, in terms of the dosage of RNA, 1 μg/kg body weight/day or more, 2 μg/kg body weight/day or more, 5 μg/kg body weight/day or more, 10 μg/kg body weight/day or more, 20 μg/kg body weight/day or more, 50 μg/kg body weight/day or more, 100 μg/kg body weight/day or more, or 200 μg/kg body weight/day or more, or 2000 μg/kg body weight/day or less, 1000 μg/kg body weight/day or less, 500 μg/kg body weight/day or less, 200 μg/kg body weight/day or less, 100 μg/kg body weight/day or less, 50 μg/kg body weight/day or less, 20 μg/kg body weight/day or less, or 10 μg/kg body weight/day or less, or a combination of these ranges. Specifically, the dosage of milk-derived exosomes may be, for example, 1 to 2000 μg/kg body weight/day, 5 to 1000 μg/kg body weight/day, 20 to 5000 μg/kg body weight/day, 30 to 2000 μg/kg body weight/day, 30 to 1000 μg/kg body weight/day, 30 to 500 μg/kg body weight/day, or 30 to 200 μg/kg body weight/day, calculated as the dosage of RNA.

The administration period of milk-derived exosomes may be, for example, 4 weeks or more, preferably 8 weeks or more, more preferably 12 weeks or more. Milk-derived exosomes may be, for example, administered once per day, or may be administered in multiple doses per day. Milk-derived exosomes may also be, for example, administered daily, or once every few days. Milk-derived exosomes may be administered daily, in particular. The amount of milk-derived exosomes administered at each administration may be constant, or may not be constant.

For example, milk-derived exosomes may be administered to a subject as is, or may be prepared as a composition containing milk-derived exosomes, such as a food and beverage composition, a pharmaceutical composition, or a feed composition, and then administered to a subject. For compositions containing milk-derived exosomes, the description of the composition of the present invention can be applied mutatis mutandis. Furthermore, milk-derived exosomes may be administered alone or in combination with other components. Examples of other components include pharmaceutical compositions, food and beverage compositions, feed compositions, and components contained therein.

When the composition of the present invention is a composition to be administered to a subject, such as a food and beverage composition, a pharmaceutical composition, or a feed composition, milk-derived exosomes may be administered to a subject, for example, by using the composition of the present invention (i.e., by administering the composition of the present invention). That is, one aspect of the method of the present invention may be a method including a step of administering the composition of the present invention to a subject. That is, "administration of milk-derived exosomes" also includes administration of the composition of the present invention. The administration mode of the composition of the present invention (e.g., the subject to be administered, the administration time, the administration period, the number of administrations, the dosage, and other conditions related to administration) is not particularly limited as long as the desired effect such as a proliferation-promoting effect is obtained. The administration mode of the composition of the present invention can be appropriately set according to various conditions such as the type, age, and health condition of the subject to be administered. The above-mentioned description regarding the administration mode of milk-derived exosomes can be applied mutatis mutandis to the case where the composition of the present invention is administered to a subject. That is, the composition of the present invention can be administered to, for example, a subject as exemplified above. In addition, the dosage of the composition of the present invention can be set so that, for example, the dosage of milk-derived exosomes as exemplified above is obtained. In addition, the composition of the present invention may be administered alone or in combination with other components, such as pharmaceutical compositions, food and drink compositions, feed compositions, and components contained therein.

In addition, milk-derived exosomes may be used in culturing Bifidobacterium breve. That is, the method of the present invention may be a method for culturing Bifidobacterium breve. The method of the present invention may be, for example, a method for culturing Bifidobacterium breve, including a step of culturing Bifidobacterium breve in a medium containing milk-derived exosomes. Specifically, the method of the present invention may be a method for culturing Bifidobacterium breve, including a step of culturing Bifidobacterium breve in a medium containing milk-derived exosomes as an active ingredient. That is, examples of the use of milk-derived exosomes include culturing Bifidobacterium breve in a medium containing milk-derived exosomes, and specifically, culturing Bifidobacterium breve in a medium containing milk-derived exosomes as an active ingredient. By culturing Bifidobacterium breve using milk-derived exosomes, a growth-promoting effect may be obtained in the culture. That is, the method for culturing Bifidobacterium breve may be an example of a method for promoting the growth of Bifidobacterium breve.

Except for using milk-derived exosomes, Bifidobacterium breve can be cultured using, for example, a conventional medium and conditions used for culturing Bifidobacterium breve, either as is or with appropriate modifications.

The medium may contain components other than milk-derived exosomes in addition to milk-derived exosomes. Examples of components other than milk-derived exosomes include the components exemplified for the medium composition of the present invention. As the medium, for example, a medium commonly used for culturing Bifidobacterium bacteria such as Bifidobacterium breve can be used by adding milk-derived exosomes. Examples of media commonly used for culturing Bifidobacterium bacteria include Reinforced Clostridial medium, de Man, Rogosa, and Sharpe medium, modified MRS medium, TOSP medium (TOS propionate medium), and TOSP Mup medium (TOS propionate mupirocin medium).

The culture can be carried out, for example, by batch culture, fed-batch culture, continuous culture, or a combination thereof. The culture medium at the start of the culture is also called the "initial medium". The culture medium supplied to the culture system (e.g., the initial medium) in fed-batch culture or continuous culture is also called the "fed-batch medium". Supplying the fed-batch medium to the culture system in fed-batch culture or continuous culture is also called "fed-batch". 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, for example, while aerating with anaerobic gas such as carbon dioxide. The culture can also be carried out under microaerobic conditions such as liquid stationary culture. The culture can be carried out, for example, until Bifidobacterium breve grows to a desired extent.

The content of milk-derived exosomes in the medium may be, for example, within the range of the content of milk-derived exosomes during culture exemplified above.

The milk-derived exosomes may be contained in the medium throughout the entire culture period, or only during a portion of the culture period. In other words, "culture is carried out in a medium containing milk-derived exosomes" means that it is sufficient that the milk-derived exosomes are contained in the medium during at least a portion of the culture period, and does not require that the milk-derived exosomes be contained in the medium throughout the entire culture period. The milk-derived exosomes may be contained in the medium at the start of culture, or may be supplied to the medium after the start of culture. Furthermore, the milk-derived exosomes may be contained in the medium at the start of culture, and further supplied to the medium after the start of culture, for example.

The milk-derived exosomes may be contained in the medium at the content exemplified above (e.g., the range of the milk-derived exosome content during culture exemplified above) for the entire culture period, or may be contained in the medium at the content exemplified above (e.g., the range of the milk-derived exosome content during culture exemplified above) for only a part of the culture period. In other words, the content "culture is carried out in a medium containing a certain content of milk-derived exosomes" means that the content of milk-derived exosomes in the medium is within the range of the content for at least a part of the culture period, and does not require that the content of milk-derived exosomes in the medium is within the range of the content for the entire culture period. The milk-derived exosomes may be contained in the medium at the content exemplified above at the start of culture, or may be supplied to the medium so as to have the content exemplified above after the start of culture.

The "partial period of the culture" is not particularly limited as long as the desired effect, such as a proliferation-promoting effect, is obtained. The "partial period of the culture" can be set appropriately depending on various conditions, such as the culture conditions and the purpose of the culture. The "partial period" may be, for example, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more of the entire culture period. In addition, the "partial period" may be, for example, 0.5 days or more, 1 day or more, 2 days or more, 3 days or more, 4 days or more, or 5 days or more.

The milk-derived exosome content in the medium may be set to the above-exemplified content as an average value throughout the entire culture period, for example. In other words, "culture is carried out in a medium containing a certain content of milk-derived exosomes" may mean that the average value of the milk-derived exosome content in the medium throughout the entire culture period is within the range of the content. The "average value of the milk-derived exosome content in the medium throughout the entire culture period" is not particularly limited as long as it can grasp the fluctuation of the milk-derived exosome content throughout the entire culture period, and may mean, for example, the average value of the milk-derived exosome content in the medium measured every 60 minutes, every 30 minutes, every 20 minutes, or every 10 minutes throughout the entire culture period.

When the composition of the present invention is a composition used for culturing Bifidobacterium breve, such as a medium composition, milk-derived exosomes may be used for culturing Bifidobacterium breve, for example, by using the composition of the present invention. The composition of the present invention (specifically, the medium composition of the present invention) may be used, for example, as a medium. That is, one aspect of the method of the present invention may be a method including a step of culturing Bifidobacterium breve with the composition of the present invention (specifically, the medium composition of the present invention). That is, "cultivation of Bifidobacterium breve in a medium containing milk-derived exosomes" also includes culturing Bifidobacterium breve with the composition of the present invention (specifically, the medium composition of the present invention). The composition of the present invention (specifically, the medium composition of the present invention) may be used, for example, alone or in combination with other components, as a medium. The composition of the present invention (specifically, the medium composition of the present invention) may be used, for example, as a medium by diluting it with an aqueous medium such as water or an aqueous buffer solution.

The present invention will now be described in more detail with reference to the following non-limiting examples.

Example: Evaluation of the effect of milk-derived exosomes in promoting the proliferation of Bifidobacterium breve Raw milk was centrifuged (1,200 × g, 4 ° C, 10 minutes × 2 times) and the middle layer was collected to obtain skim milk. 1% acetic acid was added to the obtained skim milk, stirred, and centrifuged (4,500 × g, 4 ° C, 30 minutes) to separate whey. The separated whey was subjected to an exosome purification column qEV (Izon) to purify exosomes. The purified exosomes were passed through a 0.22 μm filter to obtain milk-derived exosomes. RNA was extracted from a portion of the milk-derived exosomes using a miRNeasy Serum / Plasma kit (Qiagen), and the RNA concentration was measured using an Agilent 2100 bioanalyzer (Agilent). Milk-derived exosomes were added to the half-diluted MRS medium to obtain a medium containing milk-derived exosomes at an RNA concentration of 5 ng/mL. PBS was added to the half-diluted MRS medium to obtain a control medium. Each medium was inoculated with 1% of the Bifidobacterium bacteria listed in Table 1 and cultured for 16 hours at 37°C under anaerobic conditions. The OD600 values at the start and end of the culture were measured, and the increment in OD600 value over 16 hours of culture was calculated, and the growth promotion rate by milk-derived exosomes was calculated using the following formula.
Proliferation promotion rate by milk-derived exosomes = Increment in OD600 value in medium containing milk-derived exosomes / Increment in OD600 value in control medium

The results are shown in Table 1. No growth-promoting effect of milk-derived exosomes was observed in Bifidobacterium bacteria other than Bifidobacterium breve, whereas a growth-promoting effect was observed in all strains of Bifidobacterium breve. In other words, it was revealed that milk-derived exosomes can promote the growth of Bifidobacterium breve.

Reference Example: Exosome content in commercially available formula A commercially available formula was diluted with water to 13 g/100 mL to obtain a formula. The RNA concentration in the formula was measured and used as an index of exosome content. The results are shown in Table 2.

Claims (7)

A composition for promoting the proliferation of Bifidobacterium breve, comprising raw milk -derived exosomes as an active ingredient ,
A composition that is administered or ingested by a subject, and has a raw milk-derived exosome content, calculated as RNA content, of 150 ng/mL or more or 150 ng/g or more at the time of ingestion . The composition of claim 1 which is a nutritional composition. 3. The composition according to claim 1 or 2 , which is a milk formula. The composition of any one of claims 1 to 3 , which is administered to or ingested by a human infant or child. A composition for culturing Bifidobacterium breve, comprising raw milk -derived exosomes as an active ingredient ,
A composition, wherein the content of raw milk-derived exosomes during culture is 5 ng/mL or more or 5 ng/g or more in terms of RNA content . The composition of claim 5 which is a culture medium. Use of raw milk -derived exosomes in the production of a composition according to any one of claims 1 to 6 .