Method for preparing functional components derived from animals and plants or microorganisms

Abstract

To provide a method for measuring and / or concentrating and preparing trace components derived from animals and plants or microorganisms, and a method for screening functional components derived from animals and plants or microorganisms.SOLUTION: Provided is a method for measuring and / or concentrating and preparing trace components derived from animals or plants or microorganisms, including: (1) a step 1 of preparing a clarified liquid containing extracellular vesicles derived from animals and plants or microorganisms; (2) a step 2 of preparing a fraction having extracellular vesicles from the clarified liquid prepared in the step 1; (3) a step 3 of concentrating the extracellular vesicle fraction collected in the step 2 to prepare an extracellular vesicle concentrate; and (4) a step 4 of comparing amounts of components contained in the clarified solution obtained in step 1 and the extracellular vesicle concentrate obtained in step 3 for each component.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for measuring and / or concentrating and preparing a functional component present in trace amounts in animals, plants or microorganisms, and also to a method for screening functional components derived from animals, plants or microorganisms. [Background technology]

[0002] Food functions are said to have three functions: nutritional function (primary function), taste / sensory function (secondary function), and physical condition regulation / physiological activity function (tertiary function). Of these, foods and food ingredients with the tertiary function are generally referred to as functional foods. Among these, "Foods for Specified Health Uses (FOSHU)" and "Foods with Function Claims" are functional foods that have scientific evidence of the effects and efficacy expected from consuming them and are permitted to display such claims. In particular, functional foods, which were introduced in 2015 as a system that allows companies to display health benefits without government review, under their own responsibility, have a lower hurdle compared to FOSHU, which requires huge costs for clinical trials for review. This has attracted many companies, and the market is steadily expanding. More than 6,000 applications for functional foods have already been submitted to the Consumer Affairs Agency.

[0003] Functional food ingredients can be broadly classified by their components into vitamins, minerals, fatty acids, dietary fiber, polysaccharides, proteins, peptides, amino acids, probiologic and prebiologic ingredients (microorganisms such as lactic acid bacteria, oligosaccharides, etc.), and plant extracts (phytochemicals). As this classification demonstrates, functional foods do not contain newly created active ingredients; rather, functional food ingredients are isolated and concentrated from biological raw materials. While elucidating the physiological functions of specific components is important during the development stage of functional foods as commercial products, developing separation and purification processes is key in the search for new functional ingredients (contributing components) and industrial production stages. However, the separation and purification processes for biological materials do not involve particularly novel chemical engineering procedures; rather, they typically involve any combination of techniques, such as extraction, crystallization, membrane processing, centrifugation, and chromatography.

[0004] Extracellular vesicles (EVs) are a collective term for lipid bilayer-bound vesicles released from almost all cells of animals, plants, and microorganisms. Generally, EVs are classified into three categories based on their size and biosynthetic pathway: exosomes, microvesicles, and apoptotic vesicles. Exosomes originate from intracellular multivesicular endosomes and are approximately 20–150 nm in diameter. Microvesicles are approximately 50–1000 nm in diameter and are generated by direct budding from the plasma membrane. Meanwhile, apoptotic vesicles are 50–5000 nm in diameter and are generated from cell fragments during programmed cell death. Extracellular vesicles are known to contain membrane proteins and glycolipids on their surface and various proteins and nucleic acids, including DNA and RNA, inside. When they were first discovered, extracellular vesicles were thought to be a mechanism for disposing of insoluble matter within cells. However, it was later discovered that they also transport their contents from one cell to another, thereby transmitting information between cells. It is now clear that they play an important role in biological phenomena (see Non-Patent Document 1, etc.).

[0005] In recent years, research has progressed aimed at utilizing these extracellular vesicles in disease diagnosis and treatment. Specifically, extracellular vesicles have properties more suitable for drug delivery than synthetic nanoparticles, making them promising candidates for novel DDS nanocarriers. Furthermore, because the composition of proteins and nucleic acids (e.g., mRNA) encapsulated in extracellular vesicles is dependent on the secreting cell, they are expected to be used in liquid biopsies as biomarkers for monitoring disease progression. Furthermore, numerous physiologically active substances within extracellular vesicles have been reported to have therapeutic effects against various diseases, and extracellular vesicles themselves are attracting attention as novel biopharmaceuticals. In particular, extracellular vesicles derived from mesenchymal stem cells (MSCs) have been identified to encapsulate over 150 miRNAs and over 900 proteins related to physiological and pathological processes such as epigenetic regulation, immune regulation, and tumor formation and progression, and have been reported to possess a variety of functions (see, for example, Non-Patent Document 1).

[0006] Plant cells also secrete extracellular vesicles (EPVs) that are biologically and morphologically comparable to animal-derived EPVs. These EPVs (plant-derived EVs; PDVs) have been shown to be essential for regulating physiological processes through the intercellular transport of bioactive substances. Proteomic studies have also shown that PDVs are rich in enzymes essential for cell wall remodeling and proteins with antibacterial properties. Recently, PDVs have been shown to be effective in promoting human health, spurring growing interest. Compared to animal-derived EPVs, PDVs offer advantages such as ease of scale-up and sustainable production. However, research on PDVs is still in its infancy. In particular, there is a lack of standardized protocols for their isolation, and the precise physiological functions of PDVs remain unclear (see, for example, Non-Patent Document 2). [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Patent No. 7160294 [Non-patent literature]

[0008] [Non-Patent Document 1] Ryosuke Mizuta et al., "Extracellular vesicle hybrid engineering and DDS, medical applications," Drug Delivery System 36-2, 2021, pp. 90-99 [Non-patent document 2] Meredith Qikai Lian, et al., “Plant-derived extracellular vesicles: recent advancements and current challenges on their use for biomedical applications”, Journal of Extracellular Vesicles, Vol.11, Issue 12, 2022 Summary of the Invention [Problem to be solved by the invention]

[0009] An object of the present invention is to provide a method for measuring and / or concentrating and preparing trace components contained in animals, plants, or microorganisms, particularly trace components that may be functional components, and a method for screening functional components derived from animals, plants, or microorganisms. [Means for solving the problem]

[0010] The present inventors have extracted and concentrated extracellular vesicles from animals, plants, or microorganisms and found that the extracellular vesicles contain trace amounts of components that could be functional components, and in particular, that the extracellular vesicles contain components whose presence in those animals, plants, or microorganisms was not previously known. Based on these findings, the present inventors have confirmed that by extracting and concentrating extracellular vesicles derived from animals, plants, or microorganisms, it is possible to obtain trace components contained in animals, plants, or microorganisms, including components that have not previously been detected, and that these trace components include many components that correspond to components (functional components) that have previously been known to have various physiological effects. They are convinced that functional components derived from animals, plants, or microorganisms can be searched for and obtained by targeting extracellular vesicles.

[0011] The present invention was completed through further research based on these findings, and includes the following embodiments. (I) Measurement and / or concentration preparation method of trace components (I-1) A method for measuring and / or concentrating and preparing trace components derived from animals, plants, or microorganisms, comprising: (1) Step 1: preparing a clarified solution containing extracellular vesicles derived from animals, plants, or microorganisms; (2) Step 2 of preparing a fraction containing extracellular vesicles from the clarified solution prepared in step 1; (3) Step 3 of concentrating the extracellular vesicle fraction prepared in step 2 to prepare an extracellular vesicle concentrate; and (4) Step 4: Comparing the amounts of components contained in the clarified solution obtained in step 1 and the extracellular vesicle concentrate obtained in step 3. The method, characterized in that it comprises: (I-2) The method according to (I-1), further comprising the steps of: (5) If a component is found in step 4 that is present only in the extracellular vesicle concentrate compared to the clarified solution, or if a component is found that is present in greater amounts in the extracellular vesicle concentrate compared to the clarified solution, step 5 is performed to identify the component. (I-3) The method according to (I-2), further comprising the steps of: (6) In step 4, if a component is found to be present only in the extracellular vesicle concentrate compared to the clarified solution, or if a component is found to be present in greater amounts in the extracellular vesicle concentrate compared to the clarified solution, the extracellular vesicle concentrate containing the component is determined to be an ultratrace component concentrate or a trace component concentrate, respectively, and stored for use or provision. (I-4) The method according to any one of (I-1) to (I-3), wherein the trace component is a functional component. (I-5) A manufacturing method described in any one of (I-1) to (I-4), wherein step 1 is a step of preparing and collecting a clarified liquid containing extracellular vesicles from the juice or culture medium of an animal, plant, or microorganism. (I-6) The production method according to (I-5), wherein the step 1 comprises subjecting a squeezed juice or culture solution of an animal, plant, or microorganism to a 0.22 μm filter to prepare a filtrate. (I-7) A manufacturing method described in any of (I-1) to (I-6), wherein step 2 is a step of extracting a fraction containing extracellular vesicles from the clarified solution prepared in step 1. (I-8) A manufacturing method described in (I-7), in which step 2 is a step of subjecting the clarified liquid prepared in step 1 to an anion exchange membrane to obtain a fraction containing extracellular vesicles. (I-9) A manufacturing method described in any of (I-1) to (I-8), wherein step 3 includes a step of concentrating the extracellular vesicle fraction obtained in step 2 by ultrafiltration. (I-10) The animal or plant is meat, organs, cartilage, milk, or eggs prepared from animals; or the whole or part of a plant, seed, rhizome, or fruit; The microorganism is a yeast, a natto bacterium, a lactic acid bacterium, a koji mold, or a mold. The method described in any one of (I-1) to (I-9). (I-11) The method according to any one of (I-1) to (I-12), wherein the trace component is at least one selected from the group consisting of curcumin, shogaol, stigmasterol, sitosterol, ergosterol, tocopherol, coenzyme Q10, testosterone, vizaclone, palmitoylethanolamide, ursodeoxycholic acid, desmethoxyyangonin, gingerol, nobiletin, piperine, tangeretin, oleamide, tectorigenin, linoleic acid, hydroxyeicosatetraenoic acid, formononetin, glucosylceramide, glycochenodeoxycholic acid, glycocholic acid, glucorithocholic acid, and salts thereof.

[0012] (II) Method for preparing trace element concentrate (II-1) A method for preparing an extracellular vesicle concentrate, comprising the following steps A to C: A preparation method in which the extracellular vesicle concentrate is an ultratrace element concentrate or a trace element concentrate determined by the method described in (I-3): (A) Step A of preparing a clarified solution containing extracellular vesicles derived from the relevant animal, plant, or microorganism; (B) a step B of preparing a fraction containing extracellular vesicles from the clarified solution prepared in the step A; (C) a step C of concentrating the extracellular vesicle fraction prepared in the step B to prepare an extracellular vesicle concentrate; and (D) Step D of recovering the extracellular vesicle concentrate prepared in step C The method, characterized in that it comprises:

[0013] (III) Method for screening functional ingredients (III-1) A method for screening functional components derived from animals, plants, or microorganisms, comprising: (a) Step a) of preparing a clarified solution containing extracellular vesicles derived from the relevant animal, plant, or microorganism (b) Step b of preparing a fraction containing extracellular vesicles from the clarified solution prepared in step a (c) concentrating the extracellular vesicle fraction prepared in step (b) to prepare an extracellular vesicle concentrate; and (d) subjecting the extracellular vesicle concentrate prepared in step c to component analysis; The method, characterized in that it comprises: (III-2) The method according to (III-1), wherein the component analysis is metabolomic analysis. [Effects of the Invention]

[0014] According to the method of the present invention, by preparing an extracellular vesicle concentrate, functional components derived from animals, plants, or microorganisms can be easily and highly concentrated and prepared. Furthermore, according to the method of the present invention, even components whose presence has not been previously known in the target animals, plants, or microorganisms can be confirmed, and their functionality can be evaluated by subjecting them to metabolomic analysis. In other words, according to the method of the present invention, it is possible to search for new functional components from animals, plants, or microorganisms, and to provide functional components derived from animals, plants, or microorganisms. DETAILED DESCRIPTION OF THE INVENTION

[0015] In the present invention, "animals and plants" refers to animals or plants. The types of animals and plants are not particularly limited, as long as they have extracellular vesicles. For example, animals include vertebrates such as mammals (e.g., humans, mice, rats, cats, dogs, pigs, sheep, cows, monkeys, and others), reptiles, birds, amphibians, and fish; as well as organisms such as shellfish, insects, tapeworms, and sponges. Plants are organisms that have cell walls and survive by photosynthesis, obtaining nutrients primarily from the air and water, and include organisms such as spermatophytes, ferns, mosses, green algae, and red algae. These plants include plants that have traditionally been eaten by humans (edible plants), regardless of whether they are cultivated or wild. Specifically, edible plants include, without limitation, vegetables (fruit vegetables, root vegetables, leafy vegetables, stem vegetables, flower vegetables), fruits (mainly the edible fruits of woody plants, including nuts), wild vegetables, algae (green algae, brown algae, red algae, cyanobacteria, etc.), and mushrooms produced by fungi.

[0016] Microorganisms that are the subject of the present invention include, without limitation, bacteria (e.g., lactic acid bacteria, Bacillus subtilis, Escherichia coli, etc.), archaea, fungi (yeast, Aspergillus oryzae, mold, etc.), slime molds, microalgae, protozoa (amoeba, paramecium, etc.), etc., as long as they have extracellular vesicles.

[0017] In the present invention, extracellular vesicles (EVs) are, as described above, microparticles separated by lipid bilayer membranes that are said to be released from almost all cells of animals, plants, and microorganisms. Among these, exosomes with a diameter of approximately 20 to 150 nm are preferred, regardless of whether they are from animals, plants, or microorganisms. Hereinafter, the term "extracellular vesicles" can be interchangeably referred to as "exosomes" as needed.

[0018] (I) Measurement and / or concentration preparation method of trace components The present invention relates to a method for measuring and / or concentrating and preparing trace components derived from animals, plants, or microorganisms. The method is characterized by comprising the following steps 1 to 4 or steps 1 to 5. (1) Step 1: preparing a clarified solution containing extracellular vesicles derived from animals, plants, or microorganisms; (2) Step 2 of preparing a fraction containing extracellular vesicles from the clarified solution prepared in step 1; (3) Step 3 of concentrating the extracellular vesicle fraction collected in step 2 to prepare an extracellular vesicle concentrate; (4) comparing the amounts of components contained in the clarified solution obtained in step 1 and the extracellular vesicle concentrate obtained in step 3; (5) If a component is found in step 4 that is present only in the extracellular vesicle concentrate compared to the clarified solution, or if a component is found that is present in greater amounts in the extracellular vesicle concentrate compared to the clarified solution, step 5 is performed to identify the component.

[0019] In the present invention, "trace components" refer to components contained in the extracellular vesicle concentrate obtained by carrying out steps 1 to 3, and whose presence or absence and amount (quantitative ratio) can be measured by carrying out step 4 in comparison with the clarified solution prepared in step 1. Specifically, these are components that are present only in the extracellular vesicle concentrate compared with the clarified solution, or components that are present in greater amounts in the extracellular vesicle concentrate compared with the clarified solution. Here, "components that are present only in the extracellular vesicle concentrate compared to the clarified solution" refer to components that are not detected in the clarified solution by conventional analytical methods (qualitative or quantitative analytical methods) but are detected only in the extracellular vesicle concentrate, and in order to distinguish them from the trace components that are detected in the clarified solution described below, in the present invention they are also referred to as "ultratrace components." On the other hand, a "component that is present in a greater amount in the extracellular vesicle concentrate than in the clear solution" is a component that is detected in both the clear solution and the extracellular vesicle concentrate by conventional analytical methods, but is determined to be present in a greater amount (content) in the extracellular vesicle concentrate than in the clear solution by conventional quantitative analytical methods. Whether or not a component is present in a greater amount in the extracellular vesicle concentrate (quantitative value 1) can be determined by comparing the content of the component in the extracellular vesicle concentrate (quantitative value 2) with the content in the clear solution (quantitative value 2). For example, if a positive value is obtained by dividing quantitative value 1 by quantitative value 2, the component can be determined to be "present in a greater amount in the extracellular vesicle concentrate than in the clear solution."

[0020] Here, conventional qualitative and quantitative analytical methods refer to analytical methods that can measure the target components qualitatively and quantitatively, respectively. Examples of such analytical methods include high-performance liquid chromatography (HPLC), gas chromatography, electrophoresis, and ion chromatography. Each analytical method may be combined with mass spectrometry (MS, MS / MS, etc.), enzymatic methods, fluorescence methods, and absorbance methods. Liquid chromatography-mass spectrometry (LC / MS) is preferred, and specific examples of the analytical methods described in the Examples section are suitable. For the quantification, for example, an absolute calibration curve method using a standard substance (reference substance) corresponding to the component to be measured, an internal standard method, etc. can be used, although there is no limitation thereto.

[0021] Steps 1 to 5 will be explained below. (Process 1) Step 1 is a step of preparing a clarified solution containing extracellular vesicles derived from animals, plants, or microorganisms. The materials used as the target here are not particularly limited, as long as they contain cells that release extracellular vesicles, whether they are animals, plants, or microorganisms. Examples include, without limitation, meat, organs, cartilage, blood, plasma, serum, saliva, urine, tears, sweat, milk, amniotic fluid, cerebrospinal fluid, bone marrow fluid, pleural effusion, ascites, synovial fluid, aqueous humor, vitreous body, and eggs collected from animals; or the whole or part of a plant (e.g., leaves, petals, seeds, stems, roots, peel, fruit, etc.). Furthermore, these materials may be collected from natural sources or may be grown and produced by artificial cultivation (cultures).

[0022] A clarified liquid containing extracellular vesicles derived from animals, plants, or microorganisms can be prepared by removing solid components from the material using a conventional solid-liquid separation method, although this method is not limited thereto. For example, a method can be used in which a centrifugation supernatant, culture medium, or squeezed juice prepared from the material is further filtered to collect a filtrate. An example of the filtration process is membrane processing using a filter with a diameter of 0.22 μm. Therefore, the clarified liquid containing extracellular vesicles is preferably a liquid that can pass through a membrane with a pore diameter of 0.22 μm.

[0023] (Process 2) Step 2 is a step of preparing a fraction containing extracellular vesicles from the clarified solution prepared in step 1. This step can be carried out by selectively collecting a fraction containing extracellular vesicles from the clarified solution. This step mainly uses "ultracentrifugation," "density gradient centrifugation," "bead immunoprecipitation," "elution using a size exclusion chromatography column," etc. Specifically, as described in the Examples section below, a commercially available exosome purification column kit (product name: EVs Quick Filter TMThis method can also be performed using a centrifuge tube (Midi, manufactured by Human Metabolome Technologies, Inc.). This kit is designed to easily purify extracellular vesicles such as exosomes using anion exchange groups, and includes a column with a built-in anion exchange resin filter, wash buffer, elution buffer, and a manual describing the protocol. One embodiment of the kit and a method for recovering extracellular vesicles using the kit are described in detail in Patent Document 1, the contents of which are incorporated herein by reference.

[0024] (Step 3) Step 3 is a step of concentrating the extracellular vesicle fraction collected in step 2 to prepare an extracellular vesicle concentrate. This step can be conveniently carried out by subjecting the extracellular vesicle fraction collected in step 2 to ultrafiltration. The ultrafiltration membrane used in this ultrafiltration process may be a membrane with a nominal molecular weight cutoff (NMWL) of 30 kDa. For example, an ultrafiltration membrane with a nominal molecular weight cutoff of 30 kDa used in the Amicon Ultra-15 centrifugal filter unit (manufactured by Merck) or an equivalent ultrafiltration membrane may be used, although this is not limited thereto. Specifically, but not limited to, as described in the Examples below, the extracellular vesicle fraction collected in step 2 is placed on the membrane of a container equipped with an ultrafiltration membrane, and centrifuged (at about 2500 x g) at room temperature for 5 to 30 minutes, preferably about 15 minutes, to forcibly pass a solution containing low molecular weight substances through the membrane, and the liquid containing extracellular vesicles remaining on the membrane (extracellular vesicle concentrate) can be collected.

[0025] By carrying out the above steps 1 to 3, extracellular vesicles contained in animals, plants, or microorganisms can be selectively concentrated and prepared. If it is found that the extracellular vesicle concentrate prepared by steps 1 to 3 contains components that fall under "trace components" (including ultratrace components) by carrying out step 4 described below, steps 1 to 3 can also be referred to as steps for concentrating and preparing the trace components. Therefore, the method of the present invention, which includes steps 1 to 3 and steps 1 to 4, can also be referred to as a method for concentrating and preparing "trace components" (including ultratrace components).

[0026] (Step 4) Step 4 is a step of comparing the amounts of each component contained in the clarified solution obtained in step 1 and the extracellular vesicle concentrate obtained in step 3. The components and amounts thereof contained in the clarified solution and the components and amounts thereof contained in the extracellular vesicle concentrate can be measured using conventional qualitative and quantitative analytical methods. Conventional qualitative and quantitative analytical methods are as described above. Liquid chromatography-mass spectrometry (LC / MS) is preferred, and specifically, the analytical methods described in the Examples section can be used as suitable examples.

[0027] (Step 5) Step 5 is a step of identifying a component that is present only in the extracellular vesicle concentrate compared to the clarified solution, or a component that is present in greater amounts in the extracellular vesicle concentrate compared to the clarified solution, if found in step 4. Here, "components present only in the extracellular vesicle concentrate compared to the clear solution" refer to components (ultratrace components) that are not detected in the clear solution but are detected only in the extracellular vesicle concentrate by conventional analytical methods, as described above. Also, "components present in greater amounts in the extracellular vesicle concentrate compared to the clear solution" refer to components that are detected in both the clear solution and the extracellular vesicle concentrate by conventional analytical methods, but whose amount (content) in the extracellular vesicle concentrate is determined to be greater than that in the clear solution by conventional quantitative analytical methods (trace components).

[0028] These trace components (including ultratrace components) can be identified by standard methods, for example, by comparing them with standard substances corresponding to the components. For example, as explained in the Examples below, in the case of liquid chromatography-mass spectrometry (LC / MS), the retention time (Rt) and mass spectrum data (m / z value) of the component obtained by the method can be compared with a database (library) that compiles the results of the same analysis for many known standard substances, and substances with the same or similar values (Rt, m / z value) can be used as indicators for identification. Therefore, in the present invention, the term "measuring" includes detecting, quantifying and / or identifying.

[0029] The trace components (including ultratrace components; the same applies hereinafter) measured or concentrated and prepared by steps 1 to 4 and steps 1 to 5 can preferably be functional components derived from animals, plants, or microorganisms. Here, functional components are components that can have beneficial effects on the biological functions of animals, including humans, and are expected to have various functional effects, such as components that have the effect of maintaining or enhancing human health, or components that have the effect of alleviating the harmful effects of substances (harmful substances) that have the effect of damaging human health. The functional components are preferably those that can be incorporated as ingredients in foods and beverages, cosmetics, pharmaceuticals, and / or quasi-drugs, depending on their effects.

[0030] An example of a functional ingredient is, but is not limited to, a functional ingredient in a food with function claims. Whether or not the trace ingredients measured or concentrated and prepared in steps 1 to 4 and steps 1 to 5 are functional ingredients can be confirmed on the "Food with Function Claims Notification Information Search Website" available on the Consumer Affairs Agency's website. The functionality of the functional ingredients and the evaluation method can also be confirmed on this website.

[0031] The functional ingredients covered by the present invention include those already reported to the Consumer Affairs Agency as functional ingredients for functional foods. Examples include, without limitation, plant secondary metabolic components such as polyphenols, carotenoids, and isothiocyanates; protein-related compounds such as proteins, peptides, and amino acids; carbohydrates such as polysaccharides, oligosaccharides, and monosaccharides; complex lipids and highly unsaturated fatty acids that exhibit bioregulatory functions. Furthermore, although not limited thereto, curcumin, shogaol, stigmasterol, sitosterol, ergosterol, tocopherol, coenzyme Q10, testosterone, vizaclone, palmitoylethanolamide, ursodeoxycholic acid, desmethoxyyangonin, gingerol, nobiletin, piperine, tangeretin, oleamide, tectorigenin, linoleic acid, hydroxyeicosatetraenoic acid, formononetin, glucosylceramide, glycochenodeoxycholic acid, glycocholic acid, glucorithocholic acid, and salts thereof, which were measured and concentrated and prepared as trace components from plants and microorganisms in the Examples described below, are all useful functional ingredients.

[0032] (II) Method for preparing trace element concentrate The present invention provides a method for preparing an extracellular vesicle concentrate. The extracellular vesicle concentrate is a concentrate of trace components (including ultratrace components; the same applies hereinafter) measured by the above-described method. Therefore, the method can also be referred to as a method for preparing a trace component concentrate. The preparation method is characterized by having the following steps A to D. (A) Step A of preparing a clarified solution containing extracellular vesicles derived from the relevant animal, plant, or microorganism; (B) a step B of preparing a fraction containing extracellular vesicles from the clarified solution prepared in the step A; (C) a step C of concentrating the extracellular vesicle fraction prepared in the step B to prepare an extracellular vesicle concentrate; and (D) Step D of recovering the extracellular vesicle concentrate prepared in step C.

[0033] The steps A to C correspond to steps 1 to 3 described in (I) above. Therefore, the explanation of steps 1 to 3 in (I) above can be used to explain these steps.

[0034] Step D is a step of recovering the extracellular vesicle concentrate. By this step, the extracellular vesicle concentrate can be obtained as a trace component concentrate, and the concentrate can be used or provided as a functional ingredient in foods and beverages, cosmetics, pharmaceuticals, and / or quasi-drugs. The explanation of the minor components and functional components is as explained in the above section (I), and the explanation therefor can be used by reference.

[0035] (III) Method for screening functional ingredients The present invention provides a method for screening functional ingredients. The method can be carried out by the following steps a to d. (a) Step a) of preparing a clarified solution containing extracellular vesicles derived from the relevant animal, plant, or microorganism (b) Step b of preparing a fraction containing extracellular vesicles from the clarified solution prepared in step a (c) concentrating the extracellular vesicle fraction prepared in step (b) to prepare an extracellular vesicle concentrate; and (d) A step d of subjecting the extracellular vesicle concentrate prepared in the step c to component analysis.

[0036] The steps a to c correspond to steps 1 to 3 described in (I) above. Therefore, the explanation of steps 1 to 3 in (I) above can be used to explain these steps.

[0037] Although there are no limitations on the component analysis in step d, it can be preferably carried out by metabolome analysis (target analysis).

[0038] As described above, in this specification, the terms "comprise" and "contain" encompass the meanings of "consist of" and "consist essentially of." [Example]

[0039] The present invention will be explained below using experimental examples to aid in understanding the configuration and effects of the present invention. However, the present invention is not limited by these experimental examples. Unless otherwise specified, the following experiments were carried out at room temperature (25±5°C) and atmospheric pressure. Unless otherwise specified, "%" and "parts" in the following descriptions mean "% by mass" and "parts by mass," respectively.

[0040] The materials and equipment used in the following examples and experimental examples are as follows: Tea strainer: 200 mesh Coffee filter: Paper coffee filter (unbleached) manufactured by Daiso Industries Co., Ltd. 0.8μm pore size filter: Product name P PLS Disc Filter (Lot. X0006GC8YT) manufactured by Paperless Daini Seisakusho Co., Ltd. 0.22 μm pore size filter: Model SLGPR33RS (Advantec Toyo) 0.45 μm pore size filter: Model No. 25AS045AS (Advantec Toyo) Amazake base: Product name: Myojin Amazake base (unheated sake lees), made by Amano-ya in front of Kanda Myojin Shrine Exosome purification column kit: Product name: EVs Quick Filter TM Midi, manufactured by Human Metabolome Technologies, Inc. This kit uses anion exchange groups to easily purify extracellular vesicles such as exosomes, and includes a column with an anion exchange resin filter, wash buffer, elution buffer, and a manual with a protocol. Anion exchange resin filter: an accessory of the exosome purification column kit Wash Buffer: Included in the exosome purification column kit Elution Buffer: Included in the exosome purification column kit Ultrafiltration membrane: Product name: Amicon Ultra-15 centrifugal filter unit (nominal molecular weight cutoff 30 kDa), manufactured by Merck, made of regenerated cellulose PBS: Phosphate buffer solution (prepared by dissolving one tablet of Takara Bio's phosphate buffered saline (PBS) tablets, pH 7.4, in 1 L of ultrapure water) Membrane grid:

[0041] Experimental Example 1: Preparation of extracellular vesicle concentrate Extracellular vesicle fractions were extracted and concentrated using mandarin oranges (Example 1), strawberries (Example 2), tomatoes (Example 3), apples (Example 4), and grapefruits (Example 5) as plant materials, as well as amazake (Amazake) containing rice as a plant material and koji as a microbial material (Example 6), to prepare extracellular vesicle concentrates derived from each material.

[0042] (1) Preparation of clear solution containing extracellular vesicles (Step 1) Example 1-1: Preparation of tangerine clarified liquid Tangerines from Wakayama Prefecture were used. The mandarins were cut in half with the skin on and the juice was extracted using a fruit squeezer. The juice was filtered using a tea strainer to remove any large solids present in the juice. The filtered juice was transferred to a sterilized 50 mL centrifuge tube and centrifuged (2000 g) at 4°C for 10 minutes to obtain the supernatant. The obtained supernatant was filtered through a coffee filter and centrifuged again (2900 g) at 4°C for 10 minutes to obtain the supernatant. The obtained supernatant was then filtered through a 0.8 μm filter, and the obtained filtrate was further filtered through a 0.22 μm filter, and the filtrate was collected. This filtrate was used as a tangerine clear liquid containing extracellular vesicles in Example 1-2 described below.

[0043] Example 2-1: Preparation of strawberry clarified liquid Strawberries from Tochigi Prefecture were used. The strawberries were placed in a plastic bag with a seal and crushed from the outside of the bag with a rolling pin. The juice was collected from the plastic bag. The juice was filtered using a tea strainer to remove any large solids present in the juice. The filtered juice was transferred to a sterilized 50 mL centrifuge tube and centrifuged (1500 g) for 15 minutes at 4°C to obtain the supernatant. The supernatant was then centrifuged again (2900 g) for 15 minutes at 4°C to obtain the supernatant. The supernatant was then filtered through a coffee filter and centrifuged again (10000 g) for 30 minutes at 4°C to obtain the supernatant. The supernatant was then filtered through a 0.8 μm filter, and the resulting filtrate was further filtered through a 0.45 μm filter to obtain the filtrate. This filtrate was used as a strawberry clear liquid containing extracellular vesicles in Example 2-2 described below.

[0044] Example 3-1: Preparation of tomato clarified liquid Tomatoes from Kumamoto Prefecture were used. The tomatoes were cut in half with the skin on and the juice was extracted using a fruit squeezer. Because the remaining residue contained water, the juice was extracted using kitchen paper and combined with the juice. The juice was filtered using a tea strainer to remove any large solids present in the juice. The filtered juice was transferred to a sterilized 50 mL centrifuge tube and centrifuged (2000 g) at 4°C for 10 minutes to obtain the supernatant. The supernatant was then centrifuged again (10000 g) at 4°C for 30 minutes to obtain the supernatant. The supernatant was then filtered through a coffee filter to obtain the supernatant. The supernatant was then filtered through a 0.8 μm filter, and the resulting filtrate was further filtered through a 0.22 μm filter to obtain the filtrate. This filtrate was used as a tomato clarified liquid containing extracellular vesicles in Example 3-2 described below.

[0045] Example 4-1: Preparation of apple clarified liquid Apples from Aomori Prefecture were used. The apples were cut into quarters, peeled, and grated. The grated liquid was wrapped in kitchen paper and the juice was squeezed out. The juice was transferred to a sterilized 50 mL centrifuge tube and centrifuged (2000 g) at 4°C for 10 minutes to obtain the supernatant. The supernatant was then centrifuged again (10000 g) at 4°C for 20 minutes to obtain the supernatant. The supernatant was then filtered through a coffee filter to obtain the supernatant. The supernatant was then filtered through a 0.8 μm filter, and the filtrate was further centrifuged (10000 g) at 4°C for 60 minutes to obtain the supernatant. The supernatant was then filtered through a 0.45 μm filter, and the resulting filtrate was further filtered through a 0.22 μm filter to obtain the filtrate. This filtrate was used as apple clarified liquid containing extracellular vesicles in Example 4-2 described below.

[0046] Example 5-1: Preparation of grapefruit clarified liquid Grapefruit from Mexico was used. The grapefruit was cut in half with the skin on and the juice was extracted using a fruit squeezer. The juice was filtered using a tea strainer to remove any large solids present in the juice. The filtered juice was transferred to a sterilized 50 mL centrifuge tube and centrifuged (2000 g) at 4°C for 10 minutes to obtain the supernatant. The obtained supernatant was filtered through a coffee filter and then centrifuged again (10000 g) at 4°C for 40 minutes to obtain the supernatant. The obtained supernatant was then filtered through a 0.8 μm filter, and the obtained filtrate was further filtered through a 0.22 μm filter, and the filtrate was collected. This filtrate was used as a grapefruit clarified liquid containing extracellular vesicles in Example 5-2 described below.

[0047] Example 6-1: Preparation of rice koji clarified liquid Amazake base was used as the rice koji-containing raw material. 100 mL of ultrapure water was added to 600 g of amazake base ("Myojin Amazake Base (Nama)" manufactured by Amano-ya Co., Ltd.), and the mixture was filtered through a tea strainer to collect the filtrate. The filtrate was transferred to a sterilized 50 mL centrifuge tube and centrifuged (1000 g) for 40 minutes at 4°C to collect the supernatant. The supernatant was then filtered through a coffee filter and passed through a 0.8 μm filter to collect the filtrate. The collected filtrate was centrifuged again (10,000 g) for 40 minutes at 4°C to collect the supernatant. The supernatant was then filtered through a 0.22 μm filter to collect the filtrate. This filtrate was used as a rice koji clarified liquid containing extracellular vesicles in Example 6-2 described below.

[0048] (2) Extraction of extracellular vesicle fraction (Step 2): Examples 1-2 to 6-2 Extracellular vesicles (exosomes) were recovered from the clarified solutions prepared in each of the Examples (1) above (Examples 1-1 to 6-1) using an exosome purification column kit (column, wash buffer, elution buffer) according to the protocol described in the instructions included with the kit (Examples 1-2 to 6-2). Specifically, the column included in the kit was placed in a 50 mL tube and centrifuged (2400 g) at room temperature for 1 minute to remove the storage solution from the column. Ultrapure water was then added, and the column was centrifuged again under the same conditions as above to remove the liquid (washing the included column). Next, the washed column was placed in a new 50 mL tube, and each of the clarified solutions prepared in Examples 1-1 to 6-1 in (1) above was added to the column, followed by centrifugation (1400 g) at room temperature for 1 minute, and the entire clarified solution was passed through. The passed-through solution was discarded. The column was placed in a new 50 mL tube, 10 mL of the included wash buffer was added, and the column was centrifuged (1400 g) at room temperature for 1 minute to remove the wash buffer. The column filter was washed with 10 mL of wash buffer three times. The treated column was placed in a new 50 mL tube, and 4 mL of the included Elution buffer was added. The column was then centrifuged (1400 g) at room temperature for 1 minute, and the Elution buffer that had passed through was collected. Another 4 mL of Elution buffer was passed through the column, and the column was centrifuged under the same conditions. The resulting flow-through liquid was combined with the previously collected flow-through liquid. This flow-through liquid contained purified extracellular vesicles. This flow-through liquid was designated the "extracellular vesicle-containing fraction."

[0049] By the above-mentioned treatment, a fraction containing extracellular vesicles derived from mandarin oranges (Example 1-2) was obtained from the mandarin orange clarified liquid (Example 1-1). Similarly, a fraction containing extracellular vesicles derived from strawberry oranges (Example 2-1) was obtained from the strawberry clarified liquid (Example 2-2), a fraction containing extracellular vesicles derived from tomato oranges (Example 3-1) was obtained from the tomato clarified liquid (Example 3-2), a fraction containing extracellular vesicles derived from apple oranges (Example 4-1) was obtained from the apple clarified liquid (Example 4-2), a fraction containing extracellular vesicles derived from grapefruit oranges (Example 5-1) was obtained from the grapefruit clarified liquid (Example 5-2), and a fraction containing extracellular vesicles derived from rice koji oranges (Example 6-1) was obtained from the rice koji clarified liquid (Example 6-2).

[0050] (3) Concentration of extracellular vesicle fraction (Step 3): Examples 1-3 to 6-3 Each of the extracellular vesicle-containing fractions (Examples 1-2 to 6-2) prepared by the method (2) above was subjected to an ultrafiltration membrane to concentrate each fraction. Specifically, 15 mL of PBS was added to the ultrafiltration membrane, which was then centrifuged at 2500 g for 15 minutes at room temperature. The PBS was then discarded (pre-treatment of the ultrafiltration membrane). Next, 8 mL of each extracellular vesicle-containing fraction was added to 7 mL of PBS, which was then centrifuged at 2500 g for 15 minutes at room temperature. 15 mL of PBS was added, and the mixture was centrifuged again under the same conditions as above. The liquid remaining on the ultrafiltration membrane was collected and used as the extracellular vesicle concentrate.

[0051] Through the above-described treatment, 200 μl of extracellular vesicle concentrate derived from mandarin oranges (Example 1) was obtained. Similarly, 200 μl of extracellular vesicle concentrate derived from strawberries (Example 2) was obtained; 200 μl of extracellular vesicle concentrate derived from tomatoes (Example 3) was obtained; 200 μl of extracellular vesicle concentrate derived from apples (Example 4) was obtained; 200 μl of extracellular vesicle concentrate derived from grapefruits (Example 5) was obtained; and 200 μl of extracellular vesicle concentrate derived from rice koji (Example 6) was obtained from amazake (sweet sake) base.

[0052] (4) Identification of extracellular vesicles Each extracellular vesicle concentrate (Examples 1 to 6) prepared in (3) above was mixed with ultrapure water to prepare a sample solution, and a membrane-covered grid was placed on the droplet. A 2% uranium acetate aqueous solution in an amount roughly equal to the sample solution was dripped onto the grid to wash the sample solution. A droplet of 2% uranium acetate aqueous solution (a uranium acetate droplet) was prepared on parafilm, and the sample was placed on top of the droplet so that it was in contact with the droplet. Each was allowed to come into contact with the uranium acetate droplet for approximately 1 second. The excess stain was absorbed with filter paper, air-dried, and observed under an electron microscope.

[0053] Experimental Example 2 Analysis of functional components contained in extracellular vesicle concentrate (Steps 4 and 5) The extracellular vesicle concentrate prepared in Experimental Example 1 was subjected to component analysis, and trace components contained in the extracellular vesicles were analyzed and evaluated. Specifically, each of the clarified solutions prepared in Experimental Example 1(1) (hereinafter collectively referred to as "Sample 1"), each of the extracellular vesicle-containing fractions prepared in Experimental Example 1(2) (hereinafter collectively referred to as "Sample 2"), and each of the extracellular vesicle concentrates prepared in Experimental Example 1(3) (hereinafter collectively referred to as "Sample 3") were subjected to analysis (liquid chromatography-mass spectrometry) using the following equipment under the following conditions. This analysis can be performed by outsourcing to the contract analysis service of the present applicant (Human Metabolome Technology, Inc.) (HMT Metabolome Contract Analysis Plan: https: / / humanmetabolome.com / jpn / service / analysis / ).

[0054] [Device] Vanquish Flex UHPLC system: Thermo Scientific Thermo Scientific Orbitrap Exploris 240: Manufactured by Thermo Scientific

[0055] From the analytical results (peak areas) of Sample 1 and Sample 2, components whose content in Sample 2 was five times or more that in Sample 1 were selected, and the peak retention times (Rt) and m / z values of these components were compared with all substances registered in the applicant's library for identification. The allowable error for identification was ±0.15 minutes for retention time (Rt) and ±5 ppm for m / z values. The components detected in this way can be considered trace components contained in extracellular vesicles. Furthermore, the content of these trace components in Sample 3 was quantified using standard samples by the above analysis.

[0056] Furthermore, from the analytical results (peak areas) of Sample 1, Sample 2, and Sample 3, components that were not detected in Samples 1 and 2 but were detected only in Sample 3 were selected and identified in the same manner as described above. Components detected for the first time in Sample 3 in this way can be positioned as ultratrace components contained in extracellular vesicles. In other words, these can be said to be ultratrace components whose presence cannot be confirmed unless the extracellular vesicles are concentrated. Furthermore, the contents of these components in Sample 3 were quantified using standard samples by the above analysis.

[0057] The results of a summary of the trace and ultratrace components contained in the mandarin orange-derived extracellular vesicle concentrate (sample 3) are shown in Tables 1 and 2. Similarly, the results of a summary of the trace and ultratrace components contained in the strawberry-derived extracellular vesicle concentrate, tomato-derived extracellular vesicle concentrate, apple-derived extracellular vesicle concentrate, grapefruit-derived extracellular vesicle concentrate, and rice koji-derived extracellular vesicle concentrate are shown in Tables 3 to 12, respectively. In each table, components marked with an "*" are components for which a paper has been published regarding their functionality, and components marked with an "**" are components that have been notified as components involved in functional food claims. All of these are known functional components. In the table below, "curcumin 1," "curcumin 2," and "curcumin 3" refer to one of the curcuminoids, such as curcumin, demethoxycurcumin, cyclocurcumin, or bisdemethoxycurcumin.

[0058] [Table 1]

[0059] [Table 2]

[0060] [Table 3]

[0061] [Table 4]

[0062] [Table 5]

[0063] [Table 6]

[0064] [Table 7]

[0065] [Table 8]

[0066] [Table 9]

[0067] [Table 10]

[0068] [Table 11]

[0069] [Table 12]

[0070] As shown in Tables 1 to 12, particularly in odd-numbered Table (1), the preparation of extracellular vesicle concentrates using the method of the present invention allows components contained in the clarified solution to be concentrated to levels higher than expected. Furthermore, as shown in even-numbered Table (2), this allows the detection of components whose presence cannot be confirmed in the clarified solution. In other words, the method of the present invention allows the confirmation of the presence of components (ultratrace components) previously unknown to exist in the target animal, plant, or microorganism, and the evaluation of their functionality by subjecting them to metabolomic analysis. Furthermore, measurement of the extracellular vesicles of animals, plants, or microorganisms allows the screening of functional components contained in the animal, plant, or microorganism. In other words, the method of the present invention allows the discovery of new functional components from animals, plants, or microorganisms, and can provide functional components derived from animals, plants, or microorganisms.

Claims

1. A method for measuring and / or concentrating and preparing trace components derived from animals, plants, or microorganisms, comprising: (1) Step 1: Preparing a clarified solution containing extracellular vesicles derived from animals, plants, or microorganisms; (2) Step 2 of preparing a fraction containing extracellular vesicles from the clarified solution prepared in step 1; (3) Step 3 of concentrating the extracellular vesicle fraction prepared in step 2 to prepare an extracellular vesicle concentrate; and (4) A step 4 in which the amounts of components contained in the clarified solution obtained in the step 1 and the extracellular vesicle concentrate obtained in the step 3 are compared for each component. The method, characterized in that it comprises:

2. 10. The method of claim 1 further comprising the steps of: (5) If a component is found in step 4 that is present only in the extracellular vesicle concentrate compared to the clarified solution, or if a component is found that is present in greater amounts in the extracellular vesicle concentrate compared to the clarified solution, step 5 is to identify the component.

3. 3. The method of claim 2, further comprising the steps of: (6) In step 4, if a component is found to be present only in the extracellular vesicle concentrate compared to the clarified solution, or if a component is found to be present in greater amounts in the extracellular vesicle concentrate compared to the clarified solution, the extracellular vesicle concentrate containing the component is determined to be an ultratrace component concentrate or a trace component concentrate, respectively, and stored for use or provision.

4. The method of claim 1 , wherein the minor ingredient is a functional ingredient.

5. A method for preparing an extracellular vesicle concentrate, comprising the following steps A to D: A method for preparing an extracellular vesicle concentrate, wherein the extracellular vesicle concentrate is an ultratrace element concentrate or a trace element concentrate determined by the method of claim 3: (A) Step A of preparing a clarified liquid containing extracellular vesicles derived from the relevant animal, plant, or microorganism; (B) a step B of preparing a fraction containing extracellular vesicles from the clarified solution prepared in the step A; (C) a step C of concentrating the extracellular vesicle fraction prepared in the step B to prepare an extracellular vesicle concentrate; and (D) Step D of recovering the extracellular vesicle concentrate prepared in step C The method, characterized in that it comprises:

6. A method for screening functional ingredients derived from animals, plants, or microorganisms, comprising: (a) Step a) of preparing a clarified solution containing extracellular vesicles derived from the relevant animal, plant, or microorganism (b) a step b of preparing a fraction containing extracellular vesicles from the clarified solution prepared in the step a (c) concentrating the extracellular vesicle fraction prepared in step (b) to prepare an extracellular vesicle concentrate; and (d) subjecting the extracellular vesicle concentrate prepared in the step (c) to component analysis; The method, characterized in that it comprises:

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