WO2024005199A1

WO2024005199A1 - Method for producing composition comprising exosome, and composition comprising diacylglycerol-containing exosome

Info

Publication number: WO2024005199A1
Application number: PCT/JP2023/024458
Authority: WO
Inventors: みつる宮戸; 孝広落谷
Original assignee: Ｉｓｍ株式会社; みつる宮戸; 孝広落谷
Priority date: 2022-06-30
Filing date: 2023-06-30
Publication date: 2024-01-04

Abstract

The present invention relates to a method for producing a composition comprising an exosome, the method including a step for treating fish with water at 40 °C or higher. In addition, the present invention relates to a composition comprising a fish-derived diacylglycerol-containing exosome.

Description

Method for producing a composition containing exosomes and composition containing diacylglycerol-containing exosomes

The present invention relates to a method for producing a composition containing exosomes and a composition containing diacylglycerol-containing exosomes.

It has been reported that the consumption of fish in Japan has continued to decline in recent years. On the other hand, by type, it has been reported that the proportion of salmonid fish in fish consumption is on the rise. Behind these trends is a growing demand for fish species that can be purchased in easy-to-cook formats due to a growing preference for simpler cooking (reference materials include the 2021 Fisheries White Paper, Fisheries Agency).

As a result of the trend toward simpler cooking, more and more fish are sold in easy-to-cook formats, and as a result, it is thought that there is an increase in the number of cases in which large parts of fish bodies are discarded as seafood processing residue. However, there is a growing need to reduce food waste, including residues from seafood processing, as it has a negative impact on the environment, including the generation of greenhouse gases, and is an urgent issue for both developed and developing countries. It has become.

It has been reported that fish have excellent nutritional properties and a variety of functionalities, but from the perspective of reducing food waste as mentioned above, there is a need to explore further functionality. There is a need to further explore the functionality of parts that are often discarded. For example, Patent Document 1 discloses a technology related to the effective use of seafood processing residue.

In addition, in recent years, extracellular vesicles called "exosomes" have attracted attention, and research on the functions of exosomes has been increasing at an accelerating pace. In particular, from 2021, in Japan, the ``Special Committee on Therapeutic Preparations Using Extracellular Vesicles (EVs) Containing Exosomes'' will be held, and will Considerations are being made regarding this. In addition, the Japanese Society for Regenerative Medicine has also announced in 2021, "Thoughts on the preparation and treatment of exosomes, etc."

"Exosomes" are extracellular vesicles with a particle size of approximately 20 nm to approximately 300 nm that are released from various cells, and are used for various purposes including intercellular communication, antigen presentation, and transport of proteins and nucleic acids such as mRNA and miRNA. It is a cellular component known to have various functions. Various health effects have also been reported, and it has attracted attention in recent years. For example, Patent Document 2 discloses a cosmetic composition by promoting the secretion of exosomes, and Patent Document 3 discloses a viral infection improving agent containing exosomes.

As mentioned above, exosomes have been attracting attention in recent years, but there are various methods for recovering exosomes from samples, and it is necessary to consider the conditions and methods of recovery depending on the sample and purpose used. Become. Generally, methods such as centrifugation and chromatography are used.

Japanese Patent Application Publication No. 2015-156856 Japanese Patent Application Publication No. 2021-187787 Japanese Patent Application Publication No. 2021-193070

The problem to be solved by the present invention is to provide a new method for producing a composition containing exosomes and a composition containing diacylglycerol-containing exosomes.

As a result of intensive studies, the present inventors have completed the present invention by discovering that a composition containing exosomes can be produced from fish by treating the fish with water at 40°C or higher. The present inventors also discovered that exosomes derived from fish contain diacylglycerol, and completed the present invention.

That is, the present invention includes the following.
(1)
A method for producing a composition containing exosomes, comprising a step of treating fish with water at 40°C or higher.
(2)
The manufacturing method according to (1), wherein the process is performed at a temperature of 50°C or higher and 70°C or lower.
(3)
The manufacturing method according to (1) or (2), wherein the fish is a salmonid fish.
(4)
The production method according to (1) or (2), wherein the fish is King Salmon, Chum Salmon, Coho Salmon, Sockeye Salmon, Rainbow Trout, Sakura Salmon, Biwa Salmon, Kunimasu, Pink Salmon, Char, Yamame, or a hybrid thereof.
(5)
The production method according to (1) or (2), wherein the fish is a hybrid of King Salmon and Rainbow Trout.
(6)
The production method according to any one of (1) to (5), which uses one or more parts selected from the body, head, bone, and skin of the fish.
(7)
The manufacturing method according to any one of (1) to (6), wherein the water is physiological saline.
(8)
The process includes the step of treating one or more parts selected from the body, head, bone, and skin of Fujinosuke, which is a hybrid of King Salmon and Rainbow Trout, with water at 50°C or higher. , a method for producing a composition containing exosomes from Fujinosuke. The water may be physiological saline.
(9)
A composition comprising a fish-derived diacylglycerol-containing exosome.
(10)
The composition according to (9), wherein the content of the diacylglycerol is 4 pmol/10 ¹⁰ particles or more.
(11)
The composition according to (9), wherein the diacylglycerol has a structure derived from a fatty acid selected from the group consisting of fatty acids having 18 or more and 20 or less carbon atoms.

According to the present invention, a method for producing a composition containing exosomes and a composition containing diacylglycerol-containing exosomes can be provided.

The analysis results of diacylglycerol content are shown.

Hereinafter, modes for carrying out the present invention will be described in detail. Note that the present invention is not limited to the following embodiments, and can be implemented with various modifications within the scope of the gist.

The present invention relates to a method for producing a composition containing exosomes, which includes a step of treating fish with water at 40° C. or higher.
The process of treating fish with water is not particularly limited, but for example, water may be added to fish, or fish may be added to water.
Treating with water at 40°C or higher means that the water is at 40°C or higher when treating fish, and the heated water and fish are heated to 40°C or higher. Alternatively, water and fish may be mixed and heated to 40°C or higher.
Furthermore, when treating fish with water at 40° C. or higher, it is preferable to heat the water to an appropriate temperature, preferably 40° C. or higher.
Hereinafter, the process of treating fish with water may be simply referred to as the "main treatment process".

The time for processing fish is not particularly limited, but may be, for example, 5 minutes or more, 10 minutes or more, 15 minutes or more, 20 minutes or more, 30 minutes or more, 40 minutes or more, 50 minutes or more, and 360 minutes or less. , 300 minutes or less, 240 minutes or less, 200 minutes or less, 180 minutes or less, 120 minutes or less, 60 minutes or less, etc.
In this specification, the numerical range may be selected as any combination of an upper limit value (shown below) and a lower limit value (shown above), for example, 20 minutes or more and 240 minutes or less, 20 It may be more than 1 minute and less than 200 minutes.
Further, the time for processing fish may be 20 minutes or more and 120 minutes or less, 20 minutes or more and 60 minutes or less, 15 minutes or more and 120 minutes or less, and 15 minutes or more and 60 minutes or less. The following may be sufficient.
The water temperature during the time of treating fish is preferably 40 ° C. or higher during the treatment time, but this does not mean that the water temperature must always be 40 ° C. or higher during the time of treating fish. In this treatment step, there is no restriction on the water temperature being less than 40°C. Note that the fish processing time and water temperature may be appropriately combined within the ranges described in this specification.

The water used in this treatment step may be pure water, tap water, well water, mineral spring water, mineral water, hot spring water, spring water, fresh water, etc., or water that has been subjected to various treatments. Treatments applied to water include, but are not particularly limited to, purification, heating, sterilization, filtration, ion exchange, osmotic pressure adjustment, buffering, and the like. Therefore, the water used in the process of treating fish may be purified water, heated water, ion exchange water, physiological saline, buffer solution, or the like.

The amount of water used in this treatment step is not particularly limited, but includes, for example, an amount such that all the fish to be treated are submerged in water. For example, the ratio of the volume of water to the weight of fish may be set as appropriate within a range such as weight of fish: volume of water = 1:1 to 1:20. The ratio may be 1:1 to 1:10, 1:1 to 1:5, 1:1 to 1:4, 1:1 to 1:3. In the case of 1:1, the volume of water may be equal to the weight of the fish.

The temperature of the water used in this treatment step is not particularly limited as long as it is 40°C or higher, but may be, for example, 45°C or higher, 50°C or higher, 55°C or higher, 60°C or higher, etc., 90°C or lower, 85°C or higher, etc. It may be below 80°C, below 75°C, below 70°C, below 65°C, etc. For example, 40°C to 90°C, 40°C to 80°C, 40°C to 75°C, 40°C to 70°C, 50°C to 90°C, 50°C to 80°C, 50°C to 75°C , 50°C or higher and 70°C or lower. The temperature of the water may be 55°C or more and 90°C or less, 55°C or more and 80°C or less, 55°C or more and 75°C or less, 55°C or more and 70°C or less, 55°C or more and 65°C or less, and the like.
In this treatment step, any combination of times for treating the fish may be applied within the temperature range of the water.

This treatment step may be performed while standing still, may be performed while stirring, or may be performed while pressurizing. This treatment step may be repeated from the viewpoint of increasing purity.

By treating fish with water and collecting the liquid portion, a liquid (liquid) containing exosomes can be obtained. In this specification, the liquid portion recovered in this treatment step is referred to as "water treatment liquid." Recovery of the liquid portion is not particularly limited, and may be performed, for example, by leaving it still and separating the liquid portion, removing the solid portion, or collecting the supernatant after centrifugation. Conditions for centrifugation treatment may be appropriately set by those skilled in the art.
In the method for producing a composition containing exosomes of the present invention, in order to remove solid portions (solid-liquid separation), for example, filtration treatment may be performed, although not particularly limited. The method of filtration treatment may also be appropriately selected by those skilled in the art.
This treatment step includes at least a step of mixing water at 40° C. or higher and fish.
Exosomes are transferred from fish by treating a mixture of water and fish at a predetermined water temperature. This treatment step can also be understood as a type of exosome extraction step.
Therefore, this treatment step is not particularly limited, but may include, for example, a step of mixing water at 40° C. or higher with fish, and a step of allowing the resulting mixture to elapse for a predetermined period of time. By treating the mixture for a predetermined period of time, exosomes contained in fish are extracted from the fish. In this case, the extracted exosomes will be contained in the liquid. In other words, this treatment step includes a step of mixing water with a temperature of 40° C. or higher and fish, a step of allowing the resulting mixture to elapse at a predetermined water temperature for a predetermined time, and a step of obtaining a water treatment liquid. , may also be included.
The process of obtaining a water treatment liquid can be said to be a process of separating a liquid portion from a mixture from which exosomes are extracted, which is obtained by allowing a predetermined period of time to pass through the resulting mixture.

The method for producing a composition containing exosomes of the present invention may further include a step of concentrating the water treatment liquid. The step of concentrating the water treatment liquid is not particularly limited, but it may be concentrated by, for example, vacuum distillation.
Furthermore, the method for producing a composition containing exosomes of the present invention may further include a step of purifying a water treatment liquid.

The method for producing a composition containing exosomes of the present invention may further include a step of washing the sample to be subjected to this treatment step with water and a step of pre-treating the sample. A person skilled in the art can appropriately perform the step of washing the sample with water or pre-treating it, but for example, washing with water may be carried out by immersing the sample in running water, or the step of pre-treating the sample by removing scales, internal organs, gills, etc. Processing may be performed. Preferably, there is no particular limitation as long as the sample can be separated into meat, head, bones, and skin in the pretreatment step, for example, a method commonly used by those skilled in the art, such as dividing into two pieces or three pieces. Alternatively, high-pressure water treatment in which separation is performed by spraying high-pressure water may be performed.
The method for producing a composition containing exosomes of the present invention includes:
If desired, it may include a step of washing the fish or a step of pre-treating the fish (or a step of washing the fish and/or pre-treating the fish),
The method includes a step of treating fish with water at 40° C. or higher, and preferably includes a step of treating fish with water at 40° C. or higher for a predetermined time (preferably the above-mentioned time).
This processing step is
A step of mixing water with a temperature of 40°C or higher and fish;
A step of allowing the obtained mixture to elapse for a predetermined time at a predetermined water temperature; (Standing, pressurizing, and stirring)
The method may include a step of obtaining a water treatment liquid. The step of obtaining a water treatment liquid may be a step of separating a liquid portion from a mixture from which exosomes are extracted after a predetermined period of time, and preferably a step of obtaining a liquid portion by solid-liquid separation. There may be.

In the method for producing a composition containing exosomes of the present invention, fish are used as the sample to be subjected to this treatment step.

Examples of fish include teleost fishes, cartilaginous fishes, etc., but preferably teleost fishes. In this specification, the term "fish" refers to not only the entire fish body, but also parts of the fish body, such as its tissues and organs. The fish may be natural fish or farmed fish.

Examples of the teleost fishes include Salmonidae, Herringidae, Orientalidae, Acanthidae, Amaranthidae, Salmonidae, Salmonidae, Codidae, Mulletae, Perciformes, Pufferidae, Eelidae, etc., but Salmonidae is preferable. . Among the salmonids, the family Salmonidae is known.

As used herein, "fish of the family Salmonidae" means fish belonging to the order Salmonidae. Known members of the family Salmonidae include the subfamily Salmonidae, the subfamily Salmonidae, and the subfamily Salmonidae.

Examples of the subfamily Salmonidae include the genus Salmon (also referred to as the genus Atlantic Salmon and the genus Oncorhynchus), the genus Atlantic Salmon, the genus Char, the genus Itu, and the genus Smallmouth.
Examples of the salmon genus include Masunosuke (king salmon), chum salmon, coho salmon, sockeye salmon, rainbow trout, cherry salmon, loquat trout, kunimasu, pink salmon, and the like. Examples of the Atlantic salmon genus include Atlantic salmon, brown trout, and the like. Examples of the genus Char include Amemasu trout, Oshorokoma, Miyabeiwana, Brook trout, Char, and the like. Examples of the genus Ito include Ito. Examples of the genus Smallmouth include Smallmouth trout, Gaimamus, Amur trout, and the like.

The genus Shiromasu is known as the subfamily Shiromasu. Examples of the genus Whitefish include whitefish, whitefish, and whitefish.

The genus Kawahimemasu is known as the subfamily Kawahimemasu. Examples of the genus Kawahimemasu include Kitakawahimemasu, Honkawahimemasu, and the like.

Typical species of salmonids include, for example, king salmon, chum salmon, sockeye salmon, coho salmon, rainbow trout, pink trout, cherry salmon, Atlantic salmon, brown trout, itou, Amur itou, char, Examples include Oshorokoma, Arctic char, Omuri, and Shinano snow trout. Among these, preference is given to salmon salmon (king salmon), chum salmon, coho salmon, sockeye salmon, rainbow trout, cherry salmon, loquat trout, kunimasu, pink trout, char, and yamame, and more preferable are salmon salmon and rainbow trout.

In the present invention, a hybrid species of fish may be used. The hybrid species may be a hybrid between the same species or a hybrid between different species. The hybrid is preferably an interspecific hybrid. The hybrid species is preferably an interspecies hybrid of salmonid fish, more preferably a hybrid of Japanese trout and rainbow trout. Crossbreeding of fish can be carried out appropriately by those skilled in the art using known techniques. Commercially available hybrid fish may also be used.

As a hybrid fish species, Fuji no Kai (registered trademark, hereinafter the same) is preferred. Fujinosuke is a hybrid of Masunosuke (king salmon) and rainbow trout developed by the Yamanashi Prefectural Fisheries Technology Center.
Concerning the seed production and aquaculture methods of Fujinosuke, the Fisheries Agency completed confirmation in 2016 based on the ``Guidelines for the Use of Aquatic Products such as Triploid Fish,'' and aquaculture began in 2017.
At the Yamanashi Prefectural Fisheries Technology Center, we manage and raise Fujinosuke by setting standards for production, rearing with prepared feed, and methods for preserving meat color and freshness at the time of shipment. Fujinosuke is a cross-species salmonid species that combines the deliciousness of king salmon with the ease of raising rainbow trout.

In this specification, when a fish head is processed in triplicate by a method commonly used by those skilled in the art, the head of a fish is defined as the length from the tip of the mouth separated from the fish body to the rear end of the bone covering the gills (opercular bone). The bones of fish refer to the middle bones, abdominal bones, blood bones, small bones, etc. that are separated from the fish body, and the skin parts of fish refer to the epidermis and dermis that are separated or stripped from the fish body. It may also include scales.
In this specification, the fish body refers to the fish meat that is left after separating the head, bones, and skin from the fish body.
The body may be attached to the head, bones, and skin of the fish, and the head, bones, and skin may be attached to the body of the fish.

The present invention also relates to a composition containing exosomes produced by the method for producing a composition containing exosomes of the present invention.

A composition containing exosomes produced by the production method of the present invention contains exosomes.
Confirmation that exosomes are included can be performed using, for example, a nanoparticle analysis system (Nanosite LM10, Nippon Quantum Design Co., Ltd.), although there are no particular limitations.

The amount of exosomes contained in the exosome-containing composition produced by the production method of the present invention is not particularly limited, but for example, the number of particles is 2.0 × 10 ⁹ /mL or more, 5.0 × 10 ⁹ 1.0 x 10 ¹⁰ pieces/mL or more, 4.0 x 10 ¹⁰ pieces/mL or more, 5.0 x 10 ¹⁰ pieces/mL or more, 6.0 x 10 ¹⁰ pieces/mL or more, 7 It may be .0×10 ¹⁰ pieces/mL or more, 8.0×10 ¹⁰ pieces/mL or more, etc.
The amount of exosomes contained in the exosome-containing composition produced by the production method of the present invention is not particularly limited, but the number of particles per weight of fish is 8.0 × 10 ⁹ particles / g or more, 9.0 The number may be at least ×10 ⁹ pieces/g, 1.0×10 ¹⁰ pieces/g or more, 1.1×10 ¹⁰ pieces/g or more, 1.2×10 ¹⁰ pieces/g or more, etc.
The particle size of the exosomes contained in the exosome-containing composition produced by the production method of the present invention is not particularly limited, but for example, the average diameter is 20 nm or more, 50 nm or more, 100 nm or more, 120 nm or more, 150 nm or more, 160 nm. The above may be the case. Moreover, the average diameter may be 300 nm or less and 200 nm or less.
The average diameter of exosomes may be 20 nm or more and 300 nm or less, and within this range, the average diameter may be set to the above upper limit or lower limit as appropriate. For example, the average diameter of exosomes may be 100 nm or more and 200 nm or less.
The number of particles as the amount of exosomes and the average diameter of exosomes can be measured as analysis results using a nanoparticle analysis system, as described in Examples.

The composition containing exosomes produced by the production method of the present invention may also contain other water-soluble components eluted by the process of treating fish with water. Such other water-soluble components include, but are not particularly limited to, collagen, gelatin, vitamins, fatty acids, amino acids, polysaccharides, peptides, and the like. These water-soluble components may be derived from fish.

The present invention also relates to compositions comprising diacylglycerol-containing exosomes derived from fish.
The composition contains diacylglycerol-containing exosomes derived from fish, and in diacylglycerol-containing exosomes, the exosomes contain diacylglycerol, and the exosomes and diacylglycerol are, for example, separated into one liquid. It does not mean that it exists inside. Note that as long as the exosome contains diacylglycerol, the diacylglycerol-containing exosome and diacylglycerol may be present separately in one liquid.
Diacylglycerol-containing exosomes derived from fish may contain diacylglycerol as a membrane component of the exosome, or may contain diacylglycerol within the vesicles of the exosome.
BACKGROUND ART Diacylglycerol (DAG) is a molecule in which two fatty acids are bonded to glycerin via an ester bond, and has attracted attention for its various physiological effects.
Therefore, the composition of the present invention containing fish-derived diacylglycerol-containing exosomes is a composition that can be expected to have not only physiological effects due to exosomes but also physiological effects due to diacylglycerol.

The content of diacylglycerol in exosomes may be 3 pmol ^/ ¹⁰ particles or more, 4 pmol/10 particles or more, 4.5 pmol/ ¹⁰ particles or more, or 5 pmol/10 particles or more. The amount may be 10 ¹⁰ particles or more, 5.5 pmol/10 ¹⁰ particles or more, 6 pmol/10 ¹⁰ particles or more, or 6.5 pmol/10 ¹⁰ particles or more.
Further, the content of diacylglycerol in the exosomes may be 9 pmol/ ¹⁰ particles or less, 8 pmol/ ¹⁰ particles or less, 7.5 pmol/ ¹⁰ particles or less, The amount may be 7 pmol/10 ¹⁰ particles or less.
The content of diacylglycerol in exosomes is within any of the above ranges, for example, 4 pmol/10 ¹⁰ particles or more and 8 pmol/10 10 particles or less, 4.5 pmol/ ^{10 10} ^particles or more and 8 pmol/ ^{10 10} particles or less, 5. The amount may be 5 pmol/ ^{10 10} particles or more and 7 pmol/ ^{10 10} particles or less.
The content of diacylglycerol in exosomes can be measured as an analysis result using a DAG (diacylglycerol) assay kit, as described in the Examples. The content of diacylglycerol in exosomes was determined by analysis using an exosome solution obtained by diluting a composition containing exosomes with a solution such as PBS or a solvent (or water used in this treatment step). It may be a value measured as .

Diacylglycerol consists of 1,2-diacyl glycerol (1,2-DAG), in which fatty acids are bound to the 1st and 2nd positions of glycerin, and 1,3-diacylglycerol (1,3-DAG), in which fatty acids are bound to the 1st and 3rd positions of glycerin. DAG) may be any structure.
The fatty acids constituting diacylglycerol may be saturated fatty acids, unsaturated fatty acids, both saturated fatty acids and unsaturated fatty acids, or either one.
The unsaturated fatty acids may be ω-3 (n-3) fatty acids, ω-6 (n-6) fatty acids, ω-9 (n-9) fatty acids, A combination of these may also be used.
The number of double bonds in the unsaturated fatty acid is about 1 to 6 and is not particularly limited.
The number of carbon atoms in the fatty acid is about 2 or more and 22 or less, and may be, for example, 2 or more, 4 or more, 16 or more, 18 or more, or 22 or less, 20 or less, 18 or less. The number of carbon atoms in the fatty acid may be within the above range, for example, 18 or more and 22 or less, 18 or more and 20 or less.
Examples of fatty acids constituting diacylglycerol include acetic acid, butyric acid, palmitic acid, stearic acid, oleic acid, linoleic acid, α-linolenic acid, γ-linolenic acid, dihomo-γ-linolenic acid, arachidonic acid, eicosenoic acid, Examples include eicosapentaenoic acid (EPA), docosapentaenoic acid, docosahexaenoic acid (DHA), and the like.
Among these, fatty acids having 18 or 20 carbon atoms are preferable, and the content of diacylglycerol in exosomes is the content of diacylglycerol to which at least one fatty acid having 18 or 20 carbon atoms is bonded in exosomes. It may be the content of diacylglycerol in which two fatty acids having 18 or 20 carbon atoms are bonded in the exosome.

The composition containing diacylglycerol-containing exosomes derived from fish of the present invention may be produced by isolating exosomes from fish using methods well known to those skilled in the art as methods for isolating exosomes.
Furthermore, a composition containing fish-derived diacylglycerol-containing exosomes may be produced by the production method of the present invention. That is, exosomes contained in a composition containing exosomes produced by a method for producing a composition containing exosomes, which includes a step of treating fish with water at 40° C. or higher, may contain diacylglycerol.

The composition containing exosomes of the present invention may be a composition containing exosomes produced by a method for producing a composition containing exosomes, which includes a step of treating fish with water at 40°C or higher, and may be a composition containing exosomes produced from a fish-derived composition. The composition may include a diacylglycerol-containing exosome.
Such compositions may include any other components in addition to exosomes. Optional other components can be appropriately determined by those skilled in the art depending on the form and purpose of the composition, and may include, for example, collagen, gelatin, vitamins, fatty acids, amino acids, polysaccharides, peptides, etc. may be a component that can be eluted by the process of treating fish with water, that is, a component derived from fish, or may be an added component.
The composition containing exosomes may also contain additives commonly used in the uses described below. In addition, the composition containing exosomes may contain components for promoting the internalization of exosomes and cell targeting effects, buffer components, physiological saline, etc., and other active ingredients used in the following applications. May contain.

The composition containing the exosome of the present invention can be used as an oral preparation or an external preparation.
The composition containing exosomes of the present invention may be used as is, or the exosomes may be concentrated or extracted.
The composition containing exosomes of the present invention can be used in supplements, multivitamin preparations, multivitamin preparations, amino acid preparations, nourishing and tonic altering agents, chondroitin preparations, food fortifying agents, organ preparations, protein and amino acid preparations, cosmetics for anti-wrinkle packs, cold It may be used as a cream, hand cream, shaving cream, foundation cream, medicated cream, hair cosmetic, setting lotion, hair cream, bath oil, bath salt, baby oil, baby powder, etc.
In addition, the composition containing the exosome of the present invention may be used as a food, a cosmetic, or a pharmaceutical. The target is preferably humans, but it may also be used for pets as pet food, supplementary food for pets, and supplements for pets.
Oral preparations, external preparations, and preparations for various uses are not particularly limited, and can be prepared according to conventionally known methods.

The composition containing fish-derived diacylglycerol-containing exosomes of the present invention can be used, for example, in addition to general foods, foods with health functions such as foods for specified health uses, foods with nutritional function claims, and foods with functional claims that claim the physiological effects of diacylglycerol and exosomes. It may be used in foods, health foods (including foods labeled as nutritional supplements, health supplements, nutritional adjustment foods, etc.), supplements, and the like. Specific uses of the composition containing fish-derived diacylglycerol-containing exosomes of the present invention include, for example, reducing fasting neutral fat, postprandial Suppressing the rise in blood triglycerides, lowering LDL cholesterol, reducing body mass index (BMI), suppressing body fat accumulation in obese people, reducing body fat, risk of lifestyle-related diseases It may also be used for the purpose of promoting health, such as reducing.
The usefulness of the composition containing diacylglycerol-containing exosomes derived from fish of the present invention is that the exosomes are absorbed into the body without being degraded in the gastrointestinal tract, are transported to the liver and other organs, and release the diacylglycerol content. It is also believed that diacylglycerol acts in the liver and other organs.

Hereinafter, this embodiment will be described in more detail with reference to Examples and Comparative Examples, but this embodiment is not limited to these Examples.

Three Fujinosuke pieces were processed and separated into a first part (head, bone, and skin) and a second part (body). 200 g of the second portion was blended and 600 mL of water (approximately 3 times the amount) was added. After filtering through a sieve, centrifugation was performed. The floating oil could not be successfully separated, and exosomes were not obtained.

Three Fujinosuke pieces were processed and separated into a first part (head, bone, and skin) and a second part (body). 2.1 kg of the second part was cut into pieces, and 5000 mL of water (approximately 2.5 times the amount) was added. The mixture was heated at 90° C. for 30 minutes, left to stand, and a floating oil was obtained. After suction filtration, the mixture was centrifuged (3500 rpm). Although it was not possible to confirm the presence of exosomes in the floating oil, when PBS was added to the floating oil, the presence of exosomes was confirmed.

Three Fujinosuke pieces were processed and separated into a first part (head, bone, and skin) and a second part (body). 1000 mL (approximately twice the volume) of physiological saline was added to 500 g of each of the first site and the second site. The mixture was heated at 60° C. for 3 hours and subjected to solid-liquid separation by filter paper filtration to obtain each composition containing exosomes.

The obtained exosomes were analyzed using a nanoparticle analysis system (Nanosite LM10, Nippon Quantum Design Co., Ltd., hereinafter the same). The results are shown in Table 1. The analysis conditions are as follows.
Details
NTA Version: NTA 3.4 Build 3.4.4
Script Used: SOP Standard Measurements
Capture Settings
Camera Type: sCMOS
Laser Type: Blue405
Camera level: 13
Slider Shutter: 1232
Slider Gain: 219
DPS: 25.0
Number of frames: 1300
Temperature: 26-27℃
Viscosity: (Water) 0.9 cP
Dilution factor: Dilution not recorded
Analysis Settings
Detect Threshold: 7
Blur Size: Auto
Max Jump Distance: Auto

Three Fujinosuke pieces were processed and separated into a first part (bone part and skin part) and a second part (body part). 600 mL (approximately twice the volume) of physiological saline was added to 300 g of each of the first site and the second site. The mixture was heated at 40° C. or 60° C. for 30 minutes, and subjected to filter paper filtration for solid-liquid separation to obtain exosome-containing compositions.
Although heating at 40° C. for 3 hours was attempted, there was a possibility of spoilage, so production of the composition containing exosomes was discontinued.

The obtained exosomes were analyzed using a nanoparticle analysis system. The results are shown in Table 2. Note that the analysis conditions are the same as above.

When the processing temperature was set to 60°C, the number of general viable bacteria was 0 in the microbial test, and the results were negative for coliform bacteria, E. coli, and Staphylococcus aureus; however, when the processing temperature was set to 40°C In general, the number of viable bacteria was 3.0 to 3.8×10 ² . It is preferable to carry out the treatment at a temperature of 60° C. or higher from the viewpoint of removing bacteria and viruses.
Each microbial test was performed using the following method.
General viable bacteria count Standard agar medium method Coliform group Synthetic enzyme substrate medium method Escherichia coli Synthetic enzyme substrate medium method Staphylococcus aureus Mannitol salt medium method with egg yolk

10 μg of the composition containing exosomes obtained using the first site (bone and skin) was dissolved in 1 mL of PBS(-) to obtain an exosome solution. To the obtained exosome solution, 1.5 mL of ethanol was added, 2.25 mL of 1M NaCl, and 2.5 mL of chloroform were added and stirred, followed by centrifugation at 1,500 g for 10 minutes, and the supernatant was removed. After distilling off the chloroform, it was dissolved in 50 μL of Assay Buffer of DAG (diacylglycerol) assay kit (MET-5028) to obtain a sample solution. The sample solution was operated according to the instructions of the kit, and the content of Diacylglycerol (DAG) was analyzed. Adipose mesenchymal stem cell exosomes (adipose mesenchymal stem cell EVs) were used as a control. The results are shown in Figure 1. In addition, in FIG. 1, "Fujinosuke EVs" means exosomes obtained using the first site (bone and skin).

The following was carried out using wild chum salmon from Hokkaido and farmed rainbow trout from Aomori Prefecture called Aomori Kurenai salmon.
Three pieces of fish were processed and separated into a first part (head, bone, and skin) and a second part (body). 600 mL (approximately twice the volume) of physiological saline was added to 300 g of each of the first site and the second site. The mixture was heated at 60° C. for 30 minutes and subjected to solid-liquid separation by filter paper filtration to obtain each composition containing exosomes. In addition, in the microbiological test, the general viable bacteria count was 0, and the results were negative for coliform bacteria, Escherichia coli, and Staphylococcus aureus.

The obtained exosomes were analyzed using a nanoparticle analysis system. The results are shown in Table 3. Note that the analysis conditions are the same as above.

Claims

A method for producing a composition containing exosomes, which includes the step of treating fish with water at 40°C or higher.
The manufacturing method according to claim 1, wherein the process is performed at a temperature of 50°C or higher and 70°C or lower.
The manufacturing method according to claim 1 or 2, wherein the fish is a salmonid fish.
The production method according to claim 1 or 2, wherein the fish is a salmon salmon (king salmon), a chum salmon, a coho salmon, a sockeye salmon, a rainbow trout, a cherry salmon, a loquat trout, a black trout, a pink trout, or a hybrid thereof.
The production method according to claim 1 or 2, wherein the fish is a hybrid of King Salmon and Rainbow Trout.
The manufacturing method according to claim 1 or 2, wherein one or more parts selected from the head, bone, and skin of the fish are used.
The manufacturing method according to claim 1 or 2, wherein the water is physiological saline.
Fujinosuke, which includes the step of treating one or more parts selected from the head, bone, and skin of Fujinosuke, which is a hybrid of King salmon and rainbow trout, with water at 50°C or higher. A method for producing a composition containing exosomes from a medium.
A composition containing diacylglycerol-containing exosomes derived from fish.
The composition according to claim 9, wherein the diacylglycerol content is 4 pmol/10 10 particles or more.
The composition according to claim 9, wherein the diacylglycerol has a structure derived from a fatty acid selected from the group consisting of fatty acids having 18 or more and 20 or less carbon atoms.