WO2020241562A1 - Nanoparticles and method for producing same - Google Patents

Nanoparticles and method for producing same Download PDF

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WO2020241562A1
WO2020241562A1 PCT/JP2020/020521 JP2020020521W WO2020241562A1 WO 2020241562 A1 WO2020241562 A1 WO 2020241562A1 JP 2020020521 W JP2020020521 W JP 2020020521W WO 2020241562 A1 WO2020241562 A1 WO 2020241562A1
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nanoparticles
electrolyte
animal protein
weight
solid content
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PCT/JP2020/020521
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French (fr)
Japanese (ja)
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鹿島　康浩
小林　進
泰治 松川
健二 長田
泰正 山田
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ユーハ味覚糖株式会社
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/20—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents
    • A23L29/275—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of animal origin, e.g. chitin
    • A23L29/281—Proteins, e.g. gelatin or collagen
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/20—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents
    • A23L29/294—Inorganic additives, e.g. silica
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/115—Fatty acids or derivatives thereof; Fats or oils
    • A23L33/12—Fatty acids or derivatives thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/15—Vitamins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/175—Amino acids
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/05—Phenols
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/52—Purines, e.g. adenine
    • A61K31/522—Purines, e.g. adenine having oxo groups directly attached to the heterocyclic ring, e.g. hypoxanthine, guanine, acyclovir
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/02—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
    • A—HUMAN NECESSITIES
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
    • B01J13/02—Making microcapsules or microballoons
    • B01J13/06—Making microcapsules or microballoons by phase separation
    • B01J13/08—Simple coacervation, i.e. addition of highly hydrophilic material
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2/00—Processes or devices for granulating materials, e.g. fertilisers in general; Rendering particulate materials free flowing in general, e.g. making them hydrophobic
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y40/00—Manufacture or treatment of nanostructures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • the present invention relates to nanoparticles having a reduced amount of catechins as compared with the prior art and a method for producing the same. Further, more specifically, the present invention relates to nanoparticles capable of stably retaining a physiologically active substance by using any of an easily available and inexpensive electrolyte such as NaCl, and a method for producing the same.
  • Natural product-derived gelatin is a protein extracted from cartilage components of pigs, cows, and fish. It can be used as a gelling agent, thickener, stabilizer, etc. for foods, as well as a hemostatic agent, etc. as a base material for capsules, etc. It is also used in the medical field.
  • an emulsified colloid containing potassium bromide and silver nitrate is used as a protective colloid for a photosensitive substance.
  • Nanoparticles using food-derived ingredients such as gelatin are considered to be highly superior from the viewpoint of safety.
  • the present inventors have also found advantages in ease of production and raw material cost, and produce nanoparticles by combining gelatin, collagen, and at least one animal protein selected from decomposition products thereof and gallate-type catechin.
  • Patent Document 1 The method of the present inventors is a method of allowing gallate-type catechin to act as a core cell beta for animal proteins such as gelatin. This is the first method of using a substance that has bioactivity in itself as a nanoparticle forming substance.
  • Epigallocatechin gallate-type catechins, represented by gallate are known to have a wide range of physiological functions such as anti-obesity action, cardiovascular disease preventive action, and anti-cancer action.
  • gallate-type catechin has an action of inhibiting lipase, which is a lipolytic enzyme, a technique used for efficient extraction of vegetable oils and fats has been reported (Patent Document 2).
  • the present inventors have reported a lipase inhibitor in which a gallate-type catechin and a protein having an average molecular weight of 5,000 or more are combined (Patent Document 3).
  • gallate-type catechins are also attracting attention for their antioxidant activity. For example, if a gallate-type catechin can be placed in the vicinity of a physiologically active substance that is not stable to oxidation (so-called “property that is easily altered by oxidation, hereinafter referred to as“ easily oxidized ”), its physiology It is expected that the stability can be improved by preventing the oxidation of the active substance. However, it is very difficult to selectively place a physiologically active substance that easily oxidizes gallate-type catechins in the vicinity.
  • antioxidants containing gallate-type catechins have a unique taste
  • the use of a large amount of antioxidants has the effect of protecting bioactive substances that are easily oxidized. , It may have a great influence on the taste of the obtained product.
  • reduced CoQ10 (ubiquinol), which easily oxidizes in the air, is known as a physiologically active substance that is extremely easily oxidized.
  • Ubiquinol is known to become ubiquinone by being oxidized, and further, it has low solubility in water and easily crystallizes and separates in water, so that it is difficult to stably maintain it in an aqueous solution. It is also known.
  • a method for stabilizing the ubiquinol a method of subsuming it in cyclodextrin (Patent Document 4), a method using citric acid (Patent Document 5), and a method of coexisting with ascorbic acid (Patent Document 6) have been reported.
  • Patent Document 7 a particulate composition in which an oily component containing reduced CoQ10 is dispersed in a water-soluble excipient such as gelatin has been reported. Furthermore, as a technique for stabilizing a bioactive substance that is easily oxidized such as ubiquinol, the present inventors, for example, use 0.01 to 20% by weight of ubiquinol, 0.06 to 2% by weight of gallate-type catechin, and collagen. A liquid composition containing 0.6 to 2.8% by weight and having the ubiquinol dispersed in an aqueous solution containing a complex formed of the gallate-type catechin and the collagen (Patent Document).
  • Patent Document 9 characterized in that the bioactive substances that are easily oxidized are ubiquinol, stilbens, water-soluble vitamins, fat-soluble vitamins or carotenoids, and the average particle size is 10 to 200 nm. There is.
  • Japanese Patent No. 6369207 Japanese Unexamined Patent Publication No. 2014-062192 Japanese Unexamined Patent Publication No. 2013-082673 JP-A-2010-126492 Japanese Patent No. 3790530 Japanese Patent No. 3892881 International Publication No. 2007/148798 Japanese Patent No. 6287123 Japanese Patent No. 6428165
  • the nanoparticles described in Patent Document 9 and the like have improved bitterness and astringency derived from gallate-type catechins, and although the average particle size of the obtained nanoparticles can be measured, the particle size distribution is investigated. As a result, it is broad, and there is room for improvement in terms of improving quality and handling when used as a raw material for products such as foods.
  • Patent Document 1 used gallate-type catechin as an essential component in their production, but in order to carry out industrial production more easily, a cheaper and more easily available raw material component was used. There was room for improvement in terms of manufacturing.
  • the present inventors focused on the solid content ratio of catechin to animal protein, and adjusted the particle size to a smaller amount of catechin to animal protein than before. We have found that we can produce nanoparticles with a uniform amount of proteins. Further research revealed that instead of gallate-type catechins, as electrolytes, for example, organic acid salts such as tripotassium citrate and trisodium citrate, inorganic salts such as potassium chloride and sodium chloride, lactic acid, and succinic acid. , Fumaric acid, gluconic acid and other organic acids have been found to be able to produce nanoparticles of the same or smaller size than conventional nanoparticles, and have completed the present invention.
  • organic acid salts such as tripotassium citrate and trisodium citrate
  • inorganic salts such as potassium chloride and sodium chloride
  • lactic acid, and succinic acid Fumaric acid, gluconic acid and other organic acids have been found to be able to produce nanoparticles of the same or smaller size
  • an object of the present invention is to provide nanoparticles having an average particle size of 10 to 300 nm, having a sharp particle size distribution, and having a good flavor, and a method for producing the same.
  • Another object of the present invention is to provide nanoparticles containing no gallate-type catechin and a method for producing the same.
  • the gist of the present invention is [1] 1% by weight or more of solid content of at least one animal protein selected from the group consisting of whey protein, gelatin, collagen, and decomposition products thereof. Contains 0.001% by weight or more of electrolyte as a solid content and Nanoparticles characterized by a solid content weight ratio (electrolyte / animal protein) of the animal protein to the electrolyte of 0.001 to 1.0 and an average particle size of 10 to 300 nm. , [2] The nanoparticles, wherein the electrolyte is at least one selected from the group consisting of catechins, inorganic salts, organic acid salts, and organic acids.
  • an electrolyte is added at a ratio of a solid content weight of 0.001 to 1.0 to a solid content weight of 1 of the animal protein, and the animal protein-.
  • a method for producing nanoparticles wherein the electrolyte is at least one selected from the group consisting of catechins, inorganic salts, organic acid salts, and organic acids.
  • a method for producing nanoparticles which further comprises a step of adding a physiologically active substance.
  • the present invention relates to a method for producing the nanoparticles, wherein the bioactive substance is at least one selected from the group consisting of ubiquinol, stilbenes, water-soluble vitamins, fat-soluble vitamins, amino acids, caffeine, and carotenoids.
  • the nanoparticles of the present invention have an average particle size of 10 to 300 nm, and have a sharp particle size distribution and a good flavor. Therefore, the nanoparticles can be widely applied to foods. ..
  • the nanoparticles of the present invention have an average particle size of 10 to 300 nm and contain various electrolytes that are often used as food additives, so that they can be absorbed into the body and are safe. It is an excellent nanoparticle that can be widely applied to foods in particular. Further, by supporting the bioactive substance on the nanoparticles of the present invention, it is possible to obtain nanoparticles having more excellent functionality, for example, improved bioavailability of the physiologically active substance.
  • FIG. 1 is a graph showing an outline of the particle size distribution (sharp) of the nanoparticles of the present invention and the particle size distribution (broad) of the conventional nanoparticles.
  • FIG. 2 is a graph of the particle size distribution of nanoparticles prepared in the presence of a high concentration of catechin in Comparative Example a-1. The horizontal axis shows the particle radius, and the particle diameter is calculated by doubling the numerical value.
  • FIG. 3 is a graph of particle size distribution of nanoparticles prepared in the presence of low concentration catechin of Example a-1. The horizontal axis shows the particle radius, and the particle diameter is calculated by doubling the numerical value.
  • Nanoparticles contain at least 1% by weight of an animal protein selected from the group consisting of whey protein, gelatin, collagen, and decomposition products thereof as a solid content, and an electrolyte as a solid content of 0.
  • an animal protein selected from the group consisting of whey protein, gelatin, collagen, and decomposition products thereof as a solid content
  • an electrolyte as a solid content of 0.
  • the weight ratio of the solid content of the animal protein to the electrolyte (electrolyte / animal protein) is 0.001 to 1.0
  • the average particle size is 10 to 300 nm. It is characterized by being.
  • the animal protein used in the present invention may be any protein capable of forming a coacervate with an electrolyte, and specific examples thereof include whey protein, gelatin, collagen, and decomposition products thereof.
  • the whey protein used in the present invention is a general term for proteins contained in whey from which casein and milk fat are removed from raw milk of cows, sheep, humans, etc., for example, whey protein concentrate (whey protein concentrate).
  • Whey Protein Concentrate also called WPC
  • Whey protein isolate also called Whey Protein Isolate, WPI
  • the whey protein may be prepared and used by itself, or a commercially available product may be obtained and used.
  • Examples of commercially available whey proteins include “Enlacto HUS” (Nippon Shinyaku Co., Ltd.), a WPC material derived from cheese whey, “Enlacto ALC” (Nippon Shinyaku Co., Ltd.), and “PROGEL800", a WPC material derived from acid whey. Nippon Shinyaku Co., Ltd.), WPI material “Enlacto SAT” (Nippon Shinyaku Co., Ltd.), “Enlacto YYY” (Nippon Shinyaku Co., Ltd.), “Lact Crystal” (Nippon Shinyaku Co., Ltd.), “GermanProt 9000 (WPI)” ( Saxony Museum) and the like.
  • Gelatin used in the present invention breaks down the triple helix structure consisting of three elongated molecules that make up collagen by heating collagen, which is abundant in animal skin and bone, and changes it into a water-soluble protein. It means what you let.
  • any of cattle, pigs, fish, chickens and the like, and genetically modified organisms can be used. Although some particles of 500 nm or less are formed in animal protein derived from beef bone or pork bone, it is difficult to use in the present invention because aggregation and precipitation are likely to occur. However, even animal proteins containing proteins derived from bovine bone or pork bone can be used without particular limitation as long as nanoparticles having an average particle diameter of 10 to 300 nm can be produced. Further, if necessary, collagen or further decomposed collagen peptide can be used.
  • animal proteins may be used alone or in combination of two or more. Moreover, the animal protein may be emulsified.
  • the solid content of the animal protein in the nanoparticles of the present invention is 1% by weight or more, and from the viewpoint of efficiently producing nanoparticles having an average particle diameter of 10 to 300 nm with little variation in particle size. 1 to 99% by weight is preferable, 1 to 95% by weight is more preferable, and 1 to 90% by weight is further preferable.
  • the "electrolyte” is used to nanoparticle an animal protein such as whey protein in a solvent as described later.
  • the electrolyte used in the present invention include at least one selected from the group consisting of catechins, inorganic salts, organic acid salts and organic acids.
  • catechin is used for nanoparticulating animal proteins such as whey protein in a solvent as described later.
  • the catechins used in the present invention are, for example, epigallocatechin gallate (EGCg), epicatechin gallate (ECg), catechin gallate (Cg), and galocatechin gallate (GCg) having a "gallate group” chemically structurally.
  • Examples thereof include “gallate-type catechins” such as “gallate-type catechins” and “free-type catechins” such as epigallocatechin (EGC), epicatechin (EC), catechins (C), and gallocatechins (GC) without a "gallate group”.
  • the catechin may be a commercially available catechin, preferably a catechin peculiar to green tea.
  • the catechin may be used alone or as a combination of two or more types (catechin mixture).
  • a catechin mixture include a tea extract obtained by drying a tea extract obtained by extracting tea with water.
  • the tea extract may be prepared and used by itself, or a commercially available product may be obtained and used.
  • tea extracts for example, “Sanphenon EGCg-OP” (Taiyo Kagaku Co., Ltd.), “Sanphenon 90S” (Taiyo Kagaku Co., Ltd.), “Sanphenon BG-3” (Taiyo Kagaku Co., Ltd.), “Sanphenon BG” -5 ”(Taiyo Kagaku Co., Ltd.),“ Camellia Extract 30S ”(Taiyo Kagaku Co., Ltd.) and the like can be mentioned.
  • Examples of the electrolyte other than catechin include inorganic salts such as potassium chloride and sodium chloride, organic acid salts such as tripotassium citrate and trisodium citrate, and organic acids such as lactic acid, succinic acid, fumaric acid and citric acid. Can be mentioned.
  • inorganic salts such as potassium chloride and sodium chloride
  • organic acid salts such as tripotassium citrate and trisodium citrate
  • organic acids such as lactic acid, succinic acid, fumaric acid and citric acid.
  • commercially available products may be used, and among them, commercially available products used as food additives are preferably used.
  • Examples of commercially available electrolytes (inorganic salts) include “potassium chloride” (Ako Kasei Co., Ltd.) and “sodium chloride” (Ako Kasei Co., Ltd.).
  • Examples of commercially available electrolytes (organic acid salts) include “purified tripotassium citrate” (Fuso Chemical Industry Co., Ltd.) and “purified trisodium citrate” (Fuso Chemical Industry Co., Ltd.).
  • the electrolyte may be used alone or as a combination of two or more types.
  • the solid content of the electrolyte in the nanoparticles of the present invention is 0.001% by weight or more, and is 0.01 from the viewpoint of efficiently producing nanoparticles having a particle diameter of 10 to 300 nm on average. It is preferably from 6.4% by weight, more preferably 0.08 to 3.2% by weight, still more preferably 0.1 to 1.6% by weight.
  • catechin is used as the electrolyte, nanoparticles having an average particle diameter of 10 to 300 nm and little variation in particle diameter are efficiently produced as the solid content of the catechin in the nanoparticles of the present invention. From the viewpoint, 0.01% by weight or more is preferable, 0.08 to 6.4% by weight is more preferable, 0.1 to 3.2% by weight is more preferable, and 0.8 to 1.6% by weight is further preferable. ..
  • the weight ratio of the solid content (electrolyte / animal protein) of the animal protein and the electrolyte is adjusted to 0.001 to 1.0.
  • the weight ratio of the solid content of the animal protein to the electrolyte within the above range, the particle size of 10 to 300 nm is prevented while preventing the animal protein from agglomerating due to the salting out effect.
  • the lower limit of the weight ratio (electrolyte / animal protein) is preferably 0.001 or more, preferably 0.01 or more, for example, from the viewpoint of efficiently producing nanoparticles having a particle size of 10 to 300 nm with little variation.
  • the upper limit is preferably 1.5 or less, more preferably 1.2 or less, further preferably 0.5 or less, further preferably 0.2 or less, and 0.1 or less. Is even more preferable. More specifically, for example, when catechin is used as the electrolyte, the weight ratio (catechin / animal protein) is adjusted to 0.001 to 0.2, so that the bitterness derived from catechin is expressed. High-quality nanoparticles with a sharp particle size distribution of nanoparticles while suppressing the problem and achieving a good flavor.
  • the weight ratio is 0.001 to 0.15, more preferably 0.02 to 0.125, and even more preferably 0.01 to 0.15, from the viewpoint of efficiently producing nanoparticles having a particle size of 10 to 300 nm. It is 0.03 to 0.1. Further, when an organic acid salt such as tripotassium citrate or trisodium citrate is used as the electrolyte, 0.001 to 0.5 is more preferable, and 0.001 to 0.2 is further preferable. When an inorganic salt such as potassium chloride or sodium chloride is used as the electrolyte, 0.001 to 0.2 is more preferable, and 0.001 to 0.1 is even more preferable. Further, when an organic acid such as lactic acid, succinic acid, fumaric acid, and citric acid is used as the electrolyte, 0.001 to 1.5 is more preferable, and 0.01 to 1.2 is further preferable.
  • an organic acid salt such as tripotassium citrate or trisodium citrate
  • 0.001 to 0.5 is more prefer
  • the gallate-type catechin content can be set to 0, but if necessary, the nanoparticles of the present invention may contain gallate-type catechin. It is possible. In that case, the gallate-type catechin content is preferably 0.001 to 1% by weight.
  • the nanoparticles of the present invention can carry a physiologically active substance, if necessary.
  • the stability can be improved and the bioavailability of the physiologically active substance can be improved.
  • “supporting” refers to a state in which the nanoparticles contain a physiologically active substance and a state in which the nanoparticles are attached to the surface of the nanoparticles. Further, the fact that the physiologically active substance is supported in the nanoparticles can be determined from the fact that the physiologically active substance is not changed and / or decreased in activity and / or the component is not decreased in the abuse test described later. Further, the term “stability” as used in the present invention means that a physiologically active substance is less likely to react with another substance (for example, oxygen, sugar, etc.). Here, examples of the reaction with other substances include an oxidation reaction, a Maillard reaction, and the like. In addition, the "bioavailability” as used in the present invention refers to an index indicating how much of a substance administered to the human body circulates throughout the body.
  • the physiologically active substance may be any substance having physical properties that are easily altered by being oxidized.
  • examples include caffeine and carotenoids.
  • those having relatively high hydrophobicity such as ubiquinol, stilbenes, fat-soluble vitamins, amino acids, particularly hydrophobic amino acids, carotenoids, etc. are preferable from the viewpoint of the support rate on nanoparticles.
  • Ubiquinol used in the present invention is a functional component also called reduced coenzyme Q10, and is an oil-soluble solid substance.
  • ubiquinol a commercially available product may be used, and examples thereof include those manufactured by Kaneka Corporation.
  • Kaneka QH which is a refined product manufactured by Kaneka Corporation
  • Kaneka QH stabilized powder (P30) which is a prepared product can be mentioned, but "Kaneka QH” is desirable in terms of cost and physical properties.
  • stilbenes used in the present invention include resveratrol, pterostilbene, piceatannol and the like. Further, the composition may contain these. Commercially available products may be used as the stilbenes, and examples thereof include Nogrape extract manufactured by Maruzen Pharmaceuticals Co., Ltd. containing piceatannol and Vineatorol manufactured by BNH Co., Ltd. containing resveratrol.
  • Examples of the water-soluble vitamin used in the present invention include B vitamins, vitamin C, and folic acid.
  • the B vitamins include vitamins B1, B2, B3, B5, B6, B7, B9 and B12. Commercially available products may be used for these.
  • Examples of the fat-soluble vitamin used in the present invention include vitamin A, vitamin D, vitamin E, and vitamin K. Commercially available products may be used for these.
  • Amino acids used in the present invention include leucine, isoleucine, valine, histidine, lysine, methionine, phenylalanine, threonine, tryptophan, asparagine, aspartic acid, alanine, arginine, cysteine cystine, glutamine, glutamic acid, glycine, proline, serine, Examples include tyrosine. Commercially available products may be used for these.
  • Examples of the hydrophobic amino acid include leucine, isoleucine, valine, tryptophan, phenylalanine, methionine, proline, alanine, glycine and the like.
  • the carotenoid is not particularly limited, but is ⁇ -cryptoxanthin, ⁇ -cryptoxanthin, isocryptoxanthin, actinoerythrol, astaxanthin, ascin, adonixanthin, phenoxanthin, alloxanthin, anteraxanthin, isoageluxanthin, Isozea saquintin, osylaxanthin, caroxanthin, ⁇ -carotene, ⁇ -carotene, ⁇ -carotenone, ⁇ -carotene, ⁇ -carotene, cantaxanthine, guiloxanthin, gualuxanthin, chryptoxanthin, crocoxanthin, crocetin, chloro Xanthine, ketomixocoxanthin, geliodesanthin, diatoxanthin, dehydrolicopen, citranaxanthin, staphyloxanthine, zeaxanthine, tarax
  • the caffeine may be obtained from coffee tree, tea plant, mate, cacao, guarana, etc., and is not particularly limited.
  • caffeine may be a purified product. Commercially available products may be used for these as needed.
  • catechin is used as the electrolyte, it is known that caffeine easily reacts with catechin to form a complex, but in the present invention, the catechin content is reduced as described above. Therefore, even if caffeine is added, nanoparticles containing caffeine can be effectively produced without forming a cloudy precipitate.
  • the bioactive substance may be a naturally derived substance obtained from the natural world or a chemically synthesized product. Further, the physiologically active substance may be used alone, or two or more kinds may be used in combination.
  • the content of the physiologically active substance is 0.001 to 50% by weight from the viewpoint of efficiently producing nanoparticles having an average particle size of 10 to 300 nm with little variation in particle size. Is preferable, 0.001 to 20% by weight is more preferable, and 0.001 to 10% by weight is further preferable.
  • the average particle size of the nanoparticles of the present invention is 10 to 300 nm. Since the nanoparticles of the present invention are fine particles, for example, when ingested orally, these functions are used when the physiologically active substance is used in addition to the animal protein and electrolyte. Since the sex component is easily absorbed into the body from the intestine or the like, it has the property of improving bioavailability as compared with the case where the physiologically active substance is ingested alone.
  • the average particle size is preferably 10 to 200 nm, more preferably 20 to 150 nm, and further preferably 20 to 120 nm from the viewpoint of dispersion stability of the functional component.
  • the average particle size of the nanoparticles can be measured by a zeta potential / nanoparticle size measurement system (“DelsaMax PRO” manufactured by Beckman Coulter, Inc.) as described in Examples described later.
  • the nanoparticles of the present invention are 70% or more, preferably 75% or more, more preferably 80% or more, still more preferably within a range of 20 nm before and after the average particle size of the nanoparticles. 85% or more will be present.
  • the nanoparticles of the present invention have a sharp particle size distribution, even if the nanoparticles of the present invention are used as raw materials for foods and the like, the physical properties of the obtained foods are expected to be accompanied by variations in the particle size distribution. High-quality products can be efficiently obtained without any changes.
  • the amount of nanoparticles existing in the range of 20 nm before and after the average particle size can be measured by using the zeta potential / nanoparticle size measurement system used for measuring the average particle size.
  • the nanoparticles of the present invention may contain a stabilizer, an emulsifier, a protein binder, etc. for the purpose of enhancing the stability of the carried physiologically active substance.
  • stabilizer examples include xanthan gum, agar, gum arabic, pectin, soybean polysaccharide, CMC (carboxymethyl cellulose), sodium caseinate and the like.
  • sucrose fatty acid ester and the like can be mentioned.
  • a protein binder such as sodium tripolyphosphate or transglutaminase can be mentioned.
  • the animal protein and the electrolyte may both be used in a powder state and mixed with a solvent to form a mixed solution to form nanoparticles.
  • Step (A) of preparing an animal protein solution by dissolving, dispersing or swelling at least one animal protein selected from the group consisting of whey protein, gelatin, collagen, and decomposition products thereof in a solvent is added to the animal protein solution obtained in the step (A).
  • an electrolyte is added at a solid content weight of 0.001 to 1.0 to a solid content weight of 1 of the animal protein, and the animal protein-.
  • Step B Manufactured by going through a step of preparing an electrolyte solution (step B) and a step (C) of heating the animal protein-electrolyte solution obtained in the step (B) at 72 to 95 ° C. for 2 to 60 minutes. can do.
  • Step (A) In this step (A), at least one animal protein selected from the group consisting of whey protein, gelatin, collagen, and decomposition products thereof is dissolved, dispersed, or swollen in a solvent to uniformly contain the animal protein. Prepare an animal protein-containing solution or swelling solution (referred to as animal protein solution).
  • the animal protein used in this step (A) may be whey protein, gelatin, collagen, or a decomposition product thereof that can form a coacervate with the electrolyte.
  • Examples of the solvent used in this step (A) include water, an organic solvent, and a hydrous organic solvent.
  • the hydrous organic solvent is a mixed solvent of water and an organic solvent.
  • the organic solvent is not particularly limited as long as it is compatible with water, but it is preferable to select a solvent suitable for the intended use of the obtained nanoparticles.
  • glycerin is a solvent suitable for food use.
  • Propylene glycol, ethanol and the like examples of the solvent suitable for pharmaceutical use include methanol, acetone, dimethyl sulfoxide and the like in addition to the above.
  • the means for dissolving, dispersing or swelling the animal protein in the solvent is not particularly limited as long as it is a known means.
  • the animal protein can be dissolved, dispersed or swollen by adding and mixing with the solvent.
  • swelling means adding a solvent to an animal protein to form a gel.
  • the state of the animal protein in the mixed solution of the animal protein and the solvent may be completely dissolved or swollen, or a part of the animal protein that is not dissolved or swollen may be dispersed. Good.
  • stirring means that the contents in the container are mixed. Specifically, stirring may be performed manually using a stirring rod or the like, or may be performed using a stirrer such as a magnetic stirrer or a mixer.
  • the solid content value of the animal protein in the animal protein solution is preferably 0.1 to 19% by weight, preferably 0.1 to 19% by weight, from the viewpoint of efficiently producing nanoparticles having an average particle diameter of 10 to 300 nm. It is more preferably about 10% by weight, but it is not particularly limited as long as desired nanoparticles can be produced.
  • the solid content value of whey protein in the animal protein solution is preferably 0.1 to 7% by weight, more preferably 0.2 to 6% by weight, and even more preferably 0. It is 2 to 5% by weight.
  • the solid content value is 20% by weight or more, it becomes difficult to handle due to an increase in the viscosity of the liquid, so 19% by weight or less is preferable.
  • collagen it is preferably 0.1 to 0.3% by weight.
  • Step (B) In this step (B), in the animal protein solution obtained in the step (A), an electrolyte is added at a ratio of 0.001 to 1.0 solid content weight to 1 solid content weight of the animal protein. In addition, an animal protein-electrolyte solution is prepared.
  • the solid content weight of catechin is preferably added at a ratio of 0.001 to 0.2.
  • the particle size distribution that tends to be broad in the conventional method becomes a surprisingly sharp particle size distribution. Nanoparticles with the same particle size can be efficiently obtained (Fig. 1).
  • the curve showing a sharp particle size distribution is the nanoparticles of the present invention
  • the curve showing a broad particle size distribution is the nanoparticles prepared under the conventional conditions with a large amount of catechins.
  • the amount of electrolyte added in this step (B) has a lower limit of 0.001 or more with respect to the solid content weight of 1 of the animal protein from the viewpoint of efficiently producing nanoparticles having a target particle size.
  • the upper limit is preferably 1.5 or less, more preferably 1.2 or less, further preferably 0.5 or less, and 0.2 or less. More preferably, 0.15 or less is further preferable, 0.125 or less is further preferable, and 0.1 or less is further preferable.
  • catechin when catechin is used as the electrolyte, catechin is used as the solid content weight with respect to 1 solid content weight of the animal protein from the viewpoint of efficiently producing nanoparticles having a sharp particle size distribution. , 0.001 to 0.2, preferably 0.001 to 0.15, more preferably 0.02 to 0.125, still more preferably 0.03 to 0.1. Further, when an organic acid salt such as tripotassium citrate or trisodium citrate is used as the electrolyte, 0.001 to 0.5 is more preferable, and 0.001 to 0.2 is further preferable.
  • an inorganic salt such as potassium chloride or sodium chloride
  • 0.001 to 0.2 is more preferable, and 0.001 to 0.1 is even more preferable.
  • an organic acid such as lactic acid, succinic acid, fumaric acid, and citric acid
  • 0.001 to 1.5 is more preferable, and 0.01 to 1.2 is further preferable.
  • the conditions for adding the electrolyte are preferably adjusted to 20 to 50 ° C. Further, when the electrolyte is added, the animal protein solution may be agitated or allowed to stand.
  • the steps (A) and the step (B) are combined. May be carried out at the same time.
  • the animal protein and the electrolyte are added to the solvent to dissolve, disperse or swell the animal protein to prepare an animal protein-electrolyte solution that uniformly contains the animal protein and contains the electrolyte. To do.
  • Step (C) the animal protein-electrolyte solution obtained in the step (B) is heated at 72 to 95 ° C. for 2 to 60 minutes.
  • the temperature is 72 to 95 ° C. for 2 to 60 minutes, preferably 72 to 90 ° C. for 2 to 60 minutes, and more preferably 72 to. It is desirable to treat at 85 ° C. for 2 to 60 minutes, more preferably at 72 to 80 ° C. for 2 to 60 minutes.
  • the heat treatment is preferably carried out while stirring the animal protein-electrolyte solution.
  • the pH on the acidic side is preferably 1.0 to 2.5, more preferably 1.5 to 2.2, and even more preferably 1.7 to 2.0.
  • the pH on the alkaline side is preferably 5.0 to 8.0, more preferably 5.5 to 7.0, and even more preferably 6.0 to 6.8.
  • the pH may be adjusted by using a commercially available pH adjusting agent, and the type of the pH adjusting agent is not particularly limited.
  • Step (D) In the production method of the present invention, in order to support the bioactive substance on the nanoparticles, a physiologically active substance-containing solution in which the bioactive substance is dissolved in water or an organic solvent or a hydrous organic solvent is subjected to the steps (A) to (steps) (A) to (step). It can be added at each preparation stage of C).
  • the timing of carrying out this step (D) is not particularly limited, and for example, between steps (A) and step (B), between steps (B) and step (C), or step (C). It can be carried out at any time after. Further, the step (A), the step (B), and the step (C) may be performed at the same time.
  • the means for dissolving the physiologically active substance in the solvent is not particularly limited as long as it is a known means.
  • it can be dissolved by adding and mixing a physiologically active substance to the solvent.
  • the solid content value of the physiologically active substance in the physiologically active substance-containing solution is preferably 0.001 to 10% by weight, preferably 0.001 to 10% by weight, from the viewpoint of efficiently producing nanoparticles having an average particle diameter of 10 to 300 nm. As long as the nanoparticles of the above can be produced, there is no particular limitation.
  • a stabilizer when carrying out this step (D), a stabilizer, an emulsifier, a protein binder, or the like may be added for the purpose of preventing aggregation or precipitation of the physiologically active substance.
  • the nanoparticles of the present invention prepared by the above production method may be further formulated.
  • the formulation form is not particularly limited, and for example, tablets, coated tablets, powders, fine granules, granules, capsules, liquids, pills, suspensions, emulsions, troches, chewable tablets, syrups and the like. Oral preparations and the like can be mentioned.
  • carriers, excipients, lubricants, binders, disintegrants, diluents, stabilizers, tonicity agents, pH adjusters, buffers and the like are used.
  • carriers and excipients include lactose, sucrose, sodium chloride, glucose, maltose, mannitol, erythritol, xylitol, maltitol, inositol, dextran, sorbitol, albumin, urea, starch, calcium carbonate, kaolin and crystalline cellulose. , Maltitol, methylcellulose, glycerin, sodium alginate, gum arabic and mixtures thereof.
  • Examples of the lubricant include purified talc, stearate, borax, polyethylene glycol and a mixture thereof.
  • Examples of the binder include simple syrup, glucose solution, starch solution, gelatin solution, polyvinyl alcohol, polyvinyl ether, polyvinylpyrrolidone, carboxymethyl cellulose, cellac, methyl cellulose, ethyl cellulose, water, ethanol, potassium phosphate and a mixture thereof. Can be mentioned.
  • disintegrant examples include dried starch, sodium alginate, canten powder, laminarin powder, sodium hydrogen carbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid monoglyceride, starch, lactose and mixtures thereof. Can be mentioned.
  • diluent examples include water, ethyl alcohol, macrogol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters, and mixtures thereof.
  • stabilizer examples include sodium pyrosulfite, ethylenediaminetetraacetic acid, thioglycolic acid, thiolactic acid, and mixtures thereof.
  • Examples of the tonicity agent include sodium chloride, boric acid, glucose, glycerin and a mixture thereof.
  • Examples of the pH adjuster and buffer include sodium citrate, citric acid, sodium acetate, sodium phosphate and a mixture thereof.
  • the nanoparticles of the present invention obtained as described above are produced under conditions that can be used for food (specifically, when a solvent that can be used for food is used), they are blended with food and drink. May be good.
  • Foods and drinks are not particularly limited, and examples thereof include beverages, alcoholic beverages, jellies, confectionery, functional foods, health foods, and health-oriented foods. Considering storage stability, portability, ease of ingestion, etc., confectionery is preferable, and among confectionery, hard candy, soft candy, gummy candy, tablet, chewing gum and the like are preferable.
  • the content of the nanoparticles of the present invention in foods and drinks may be an amount that can be expected to have a bioactive effect. It is preferable to determine the blending amount so that 10 to 10000 mg, more preferably 100 to 3000 mg per day can be ingested. For example, in the case of solid foods, 5 to 50% by weight is preferable, and in the case of liquid foods such as beverages, 0.01 to 10% by weight is preferable.
  • the nanoparticles of the present invention treat non-human animals such as rats, mice, guinea pigs, rabbits, sheep, pigs, cows, horses, cats, dogs, monkeys, chimpanzees and other mammals, birds, amphibians and reptiles. It may be added to an agent or feed.
  • the feed includes, for example, livestock feed used for sheep, pigs, cows, horses, chickens, etc., small animal feed used for rabbits, rats, mice, etc., seafood feed used for eels, Thailand, hamachi, shrimp, etc., dogs, etc. Examples include pet food used for cats, small birds, and squirrels.
  • the nanoparticles of the present invention may be blended in a pharmaceutical product.
  • the pharmaceuticals include solid preparations such as powders, tablets, pills, capsules, fine granules and granules, liquid preparations such as liquid preparations, suspensions and emulsions, gel preparations and the like.
  • the granules of tablets, pills, granules, and capsules containing granules may be coated with sugars such as sucrose and sugar alcohols such as maltitol, or with gelatin, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, etc. It can also be coated. Alternatively, it may be coated with a film of a gastric or enteric substance. Further, in order to improve the solubility of the preparation, a known solubilization treatment can be applied. It may be mixed with an injection or an infusion according to a conventional method.
  • the intake thereof is not particularly limited as long as the desired improvement, therapeutic or preventive effect can be obtained, and usually the embodiment, the age of the patient, and the like. It is appropriately selected according to the sex, constitution and other conditions, the type of disease and the degree thereof. It is preferably about 0.1 mg to 1,000 mg per day, and this can be ingested in 1 to 4 divided doses per day.
  • the nanoparticles of the present invention may be blended in quasi-drugs.
  • the quasi-drugs include quasi-drugs used in the oral cavity, such as toothpaste, mouthwash, and mouth rinse, and nutritional tonic drinks for the purpose of preventing infectious diseases.
  • the nanoparticles of the present invention are added to a quasi-drug, it is usually preferable to add 0.001 to 30% by weight in the quasi-drug.
  • Example a-1 Production of nanoparticles under conditions in which the amount of catechin added is suppressed (production of test specimen) First, add 5 g of whey protein (trade name: Enlacto SAT, protein content 89%, manufactured by Nippon Shinyaku Co., Ltd.) to 95 g of distilled water, and stir well at 20 to 30 ° C. (room temperature) to prepare a 100 g whey protein aqueous solution. did. (Step (A)) Next, 20 mg to 320 mg of green tea extract (trade name: EGCg-OP, EGCg content of 94% or more, manufactured by Taiyo Kagaku) was added to the obtained whey protein aqueous solution at 20 to 30 ° C.
  • EGCg-OP green tea extract
  • Step (B) Next, the obtained mixed solution was adjusted to pH 6 to 7 with a pH adjusting solution, and heated at 80 ° C. for 30 minutes with gentle stirring to form nanoparticles.
  • Step (C) The average particle size of the obtained nanoparticles was measured using a zeta potential / nanoparticle size measurement system (“DelsaMax PRO” manufactured by Beckman Coulter, Inc.).
  • the obtained mixed solution was heated at 80 ° C. for 30 minutes with gentle stirring to form nanoparticles.
  • the average particle size of the obtained nanoparticles was measured using a zeta potential / nanoparticle size measurement system (“DelsaMax PRO” manufactured by Beckman Coulter, Inc.).
  • Example a-2 Evaluation of effect of improving bitterness and astringency
  • the nanoparticles prepared in Example a-1 are compared with the nanoparticles prepared in Comparative Example a-1 by a conventional production method, and the bitterness and astringency are panelized.
  • a sensory evaluation of bitterness and astringency was carried out by 10 people. The evaluation criteria are as shown in Table 1.
  • Table 2 shows the results of measuring the particle size of the nanoparticles prepared in Example a-1 and Comparative Example a-1 multiple times and the results of the sensory test conducted in Example a-2.
  • Example a-1 remarkably suppress the amount of catechin added as compared with the case of Comparative Example a-1 (conventional method). It can be seen that the particles have the same particle size as the conventional method and the bitterness and astringency are remarkably suppressed.
  • Example a-3 Verification of particle size distribution when the amount of catechin added is reduced Zeta potential / nanoparticles of particle size distribution of nanoparticles prepared by the procedure shown in Comparative Example a-1 and Example a-1 A diameter measurement system (“DelsaMax PRO” manufactured by Beckman Coulter Co., Ltd.) was used to measure the particle size in the range of 2,000 nm or less, and typical measurement results are shown in FIGS. 2 and 3, respectively. Since the horizontal axis in the figure represents the radius, the value obtained by doubling the value on the horizontal axis is the particle diameter.
  • FIG. 2 shows the particle size distribution of the nanoparticles prepared under the conventional high-concentration catechin condition (9.1%), and FIG.
  • FIG. 3 shows the particle size distribution of the nanoparticles prepared under the condition of reducing the amount of catechin (0.08%). Shown.
  • the broad range was in the range of 20 to 400 nm in particle diameter.
  • the particle size distribution was such that 99% or more existed in the range of 20 nm before and after the particle diameter centered on 70 nm. Therefore, as is clear from FIGS. 2 and 3, it is clear that by adjusting the amount of catechin with respect to animal protein to a specific range, high-quality nanoparticles having uniform particle size can be produced even if the content of catechin is low. It became.
  • Example a-4 Method for producing nanoparticles carrying a physiologically active substance having high reactivity with other substances
  • whey protein (trade name: Enlacto SAT, protein content 89%, Nippon Shinyaku Co., Ltd.) in 95 g of distilled water
  • a 100 g whey protein aqueous solution was prepared by adding 5 g and stirring well at room temperature.
  • 80 mg of green tea extract (trade name: EGCg-OP, EGCg content of 94% or more, manufactured by Taiyo Kagaku) was added to the obtained whey protein aqueous solution at room temperature (EGCg concentration in the mixed solution: 0.08%). ..
  • a reduced CoQ10 solution prepared by dissolving 0.1 g of astaxanthin (Astabio AP1) or 0.1 g of reduced coenzyme Q10 (manufactured by Kaneka) in 5 g ethanol was added, and whey protein-catechin- A mixture of physiologically active substances was obtained.
  • the obtained mixed solution was adjusted to pH 6 to 7 with a pH adjusting solution, and heated at 80 ° C. for 30 minutes with gentle stirring to form nanoparticles.
  • the average particle size of the obtained nanoparticles was measured using a zeta potential / nanoparticle size measurement system (“DelsaMax PRO” manufactured by Beckman Coulter, Inc.), and it was confirmed that the particles had a predetermined particle size. ..
  • Example a-5 Method for producing nanoparticles carrying caffeine
  • 10 mg of caffeine was further added in the step (C) of adding a physiologically active substance having high reactivity with other substances.
  • Nanoparticles were produced in the same manner as in Example a-4, except that the nanoparticles were produced. Even if caffeine was added, the formation of a cloudy precipitate formed by the complexing of catechin and caffeine was not observed. Therefore, the obtained nanoparticles were made into nanoparticles containing caffeine as well as catechin. It turned out that it became.
  • Example a-6 Stability of bioactive substances By subjecting the nanoparticles obtained in Examples a-4 and a-5 to an abuse test at room temperature and under ultraviolet irradiation, they can be combined with other substances supported. The stability of the highly reactive bioactive substance was measured. As a control, 0.1 g of astaxanthin (Astabio AP1) or 0.1 g of reduced coenzyme Q10 (manufactured by Kaneka Corporation) was dissolved in 5 g of ethanol in 95 g of distilled water, and a liquid was added to obtain an astaxanthin solution or a reduced CoQ10 solution. ..
  • Example a-7 Gummy candy containing nanoparticles Based on a known method, the nanoparticles obtained in Examples a-1, a-4, and a-5 have a solid content of 10% by weight. When it was mixed with the gummy candy solution and solidified, no strong bitterness or astringency was felt regardless of which of the obtained gummy candy was eaten, and it was easy to ingest.
  • Example b-1 Production of nanoparticles using an organic acid salt (production of a test sample) Add 5 g of whey protein (trade name: Enlacto SAT, protein content 89%, manufactured by Nippon Shinyaku Co., Ltd.) to 5 mM (0.162%) tripotassium citrate solution or 5 mM (0.147%) trisodium citrate solution. The mixture was gently stirred at 20 ° C. to dissolve the mixture to 100 ml. Then, the nanoparticles were formed by heating at 80 ° C. for 30 minutes with gentle stirring. The nanoparticles in the obtained solution were measured in particle size using a zeta potential / nanoparticle size measurement system (“Delsa Max PRO” manufactured by Beckman Coulter, Inc.).
  • Example b-2 Production of nanoparticles using an inorganic salt (production of a test sample) Add 5 g of whey protein (Nippon Shinyaku Co., Ltd .: Enlacto SAT) to a 5 mM (0.037%) potassium chloride (KCl) solution or a 5 mM (0.029%) sodium chloride (NaCl) solution and gently stir at 20 ° C. Was dissolved to make 100 ml. Then, the nanoparticles were formed by heating at 80 ° C. for 30 minutes with gentle stirring. The nanoparticles in the obtained solution were measured in particle size using a zeta potential / nanoparticle size measurement system (“Delsa Max PRO” manufactured by Beckman Coulter, Inc.).
  • Example b-3 Production of nanoparticles using an organic acid (production of a test sample) 5 g of whey protein (Nippon Shinyaku Co., Ltd .: Enlacto SAT), 200 mM (1.8%) lactic acid solution, 200 mM (2.36%) succinic acid solution, 200 mM (2.32%) fumaric acid solution, or 200 mM (3. 84%) It was added to a citric acid solution and dissolved by gently stirring at 20 ° C. to make 100 ml. Then, the particles were formed by heating at 80 ° C. for 30 minutes with gentle stirring (where the pH was adjusted to 2.4). The nanoparticles in the obtained solution were measured in particle size using a zeta potential / nanoparticle size measurement system (“Delsa Max PRO” manufactured by Beckman Coulter, Inc.).
  • the obtained mixed solution was heated at 80 ° C. for 30 minutes with gentle stirring to form nanoparticles.
  • the average particle size of the obtained nanoparticles was measured using a zeta potential / nanoparticle size measurement system (“DelsaMax PRO” manufactured by Beckman Coulter, Inc.).
  • Table 3 shows the particle sizes of the nanoparticles prepared in Examples b-1 to b-3 and Comparative Example b-1.
  • the nanoparticles of the present invention prepared using the electrolytes obtained in Examples b-1 to b-3 are equivalent to or better than those in Comparative Example b-1 (conventional method). It turned out to be nanoparticles with a small particle size. As a result, it was found that nanoparticles can be produced by using various electrolytes instead of catechin in the conventional method.
  • Example b-4 Production of nanoparticles carrying a physiologically active substance 5 g of whey protein (Nippon Shinyaku Co., Ltd .: Enlacto SAT), 5 mM (0.162%) It was dissolved by adding to 90 ml of a tripotassium citrate solution and gently stirring at 20 ° C. Next, after adding 5 ml of a 1 mg / mL reduced CoQ10 ethanol solution, add a further 5 mM (0.162%) tripotassium citrate solution while adjusting the pH to 6 to 7 using a pH adjusting solution to adjust the final volume. It was set to 100 mL. Then, while gently stirring, the particles were heated at 80 ° C. for 30 minutes to form nanoparticles.
  • the average particle size was 90.7. It was ⁇ 120.7 nm.
  • Example b-5 Stability of bioactive substance in nanoparticles The stability of the bioactive substance carried on the nanoparticles was examined by an abuse test at room temperature and under UV. The study was carried out using the nanoparticle solution (product of the present invention) obtained in Example b-4 and the coenzyme Q10 solution (comparative product) in which reduced coenzyme Q10 was dispersed with an emulsifier. Each of the prepared samples was placed in a transparent glass container and abused at room temperature and under UV. Samples were collected at weekly intervals, and the concentration of reduced coenzyme Q10 contained in the samples was measured.
  • the product of the present invention can stably retain the reduced coenzyme Q10, which is a physiologically active substance, without being oxidized even when stored for a long period of time, as compared with the comparative product. Therefore, instead of the gallate-type epigalocatechin, which has been conventionally used for the production of nanoparticles, an electrolyte commonly used in the production of foods such as tripotassium citrate was used to stably support the physiologically active substance. It turned out that nanoparticles can be produced.
  • the nanoparticles of the present invention produced as described above stably support a physiologically active substance and have a very small average particle size of 10 to 300 nm. Therefore, for example, oral administration is required.
  • animal proteins, electrolytes, and the physiologically active substance are easily absorbed into the body from the intestine and the like, so that the bioavailability is improved as compared with the case where the physiologically active substance is ingested alone. It can be seen that it has.

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Abstract

The present invention relates to nanoparticles characterized by containing, on a solids basis, 1% by weight or more of at least one animal protein selected from the group consisting of whey protein, gelatin, collagen, and decomposed products thereof and 0.001% by weight or more of an electrolyte, and in that the weight ratio on a solids basis between the animal protein and the electrolyte (electrolyte/animal protein) is 0.001-1.0, and the average particle size is 10-300 nm. The nanoparticles according to the present invention are nanoparticles containing a reduced amount of catechin compared to conventional particles and using a cheaper electrolyte, and can stably retain a biologically active substance.

Description

ナノ粒子及びその製造方法Nanoparticles and their manufacturing methods
　本発明は、従来に比べカテキン量を低減させたナノ粒子及びその製造方法に関する。さらに、詳しくは、ＮａＣｌ等の入手が容易で安価ないずれかの電解質を用いて、生理活性物質を安定的に保持できるナノ粒子及びその製造方法に関する。 The present invention relates to nanoparticles having a reduced amount of catechins as compared with the prior art and a method for producing the same. Further, more specifically, the present invention relates to nanoparticles capable of stably retaining a physiologically active substance by using any of an easily available and inexpensive electrolyte such as NaCl, and a method for producing the same.
　天然物由来のゼラチンは、豚や牛、魚の軟骨成分より抽出したタンパク質であり、食品のゲル化剤、増粘剤、安定剤等としての利用のほかにカプセル等の基材として止血剤等の医療分野でも利用されている。また、臭化カリウムと硝酸銀を加えた乳化コロイドは感光物質の保護コロイドとして用いられている。また、ゼラチンが水溶性であるという性質を利用し、有機溶媒に滴下することでマイクロカプセルを作製する技術も知られている。 Natural product-derived gelatin is a protein extracted from cartilage components of pigs, cows, and fish. It can be used as a gelling agent, thickener, stabilizer, etc. for foods, as well as a hemostatic agent, etc. as a base material for capsules, etc. It is also used in the medical field. In addition, an emulsified colloid containing potassium bromide and silver nitrate is used as a protective colloid for a photosensitive substance. Further, there is also known a technique for producing microcapsules by dropping gelatin into an organic solvent by utilizing the property that gelatin is water-soluble.
　更に、近年ゼラチンをナノ粒子化することにより、医薬品成分を目的の臓器や組織に提供するためのドラッグデリバリーシステム（ＤＤＳ）に利用する技術開発が進んでいる。ゼラチンのような食品由来の成分を用いたナノ粒子は安全性の観点から優位性が高いと考えられる。 Furthermore, in recent years, technological development has been progressing in which gelatin is made into nanoparticles and used in a drug delivery system (DDS) for providing pharmaceutical components to target organs and tissues. Nanoparticles using food-derived ingredients such as gelatin are considered to be highly superior from the viewpoint of safety.
　本発明者らも、製造の簡便性及び原料コストでの優位性を見出したゼラチン、コラーゲン、及びこれらの分解物から選ばれる少なくとも１種の動物性タンパク質とガレート型カテキンを組み合わせたナノ粒子の製造方法を提案してきた（特許文献１）。本発明者らの方法はガレート型カテキンをゼラチン等の動物性タンパク質に対するコアセルベーターとして働かせる方法である。これはそれ自体が生理活性を有する物質をナノ粒子形成物質として用いた初めての方法である。
　エピガロカテキンガレートに代表されるガレート型カテキンは、抗肥満作用や循環器系疾患予防作用、抗癌作用等幅広い生理機能を有していることが知られている。また、ガレート型カテキンには脂肪分解酵素であるリパーゼを阻害する作用を有するため、植物性油脂の効率的な抽出に用いる技術が報告されている（特許文献２）。また、本発明者らはガレート型カテキンと平均分子量５，０００以上のタンパク質とを複合化させたリパーゼ阻害剤を報告している（特許文献３）。 The present inventors have also found advantages in ease of production and raw material cost, and produce nanoparticles by combining gelatin, collagen, and at least one animal protein selected from decomposition products thereof and gallate-type catechin. We have proposed a method (Patent Document 1). The method of the present inventors is a method of allowing gallate-type catechin to act as a core cell beta for animal proteins such as gelatin. This is the first method of using a substance that has bioactivity in itself as a nanoparticle forming substance.
Epigallocatechin gallate-type catechins, represented by gallate, are known to have a wide range of physiological functions such as anti-obesity action, cardiovascular disease preventive action, and anti-cancer action. Further, since gallate-type catechin has an action of inhibiting lipase, which is a lipolytic enzyme, a technique used for efficient extraction of vegetable oils and fats has been reported (Patent Document 2). In addition, the present inventors have reported a lipase inhibitor in which a gallate-type catechin and a protein having an average molecular weight of 5,000 or more are combined (Patent Document 3).
　また、ガレート型カテキンは抗酸化作用についても注目されている。例えば、酸化に対して安定性の悪い（いわゆる、酸化されることで変質しやすい性質、以下、「酸化されやすい」という）生理活性物質の近傍にガレート型カテキンを配置することができれば、その生理活性物質の酸化を防止して、安定性を向上させることができることが予想される。しかしながら、ガレート型カテキンを酸化されやすい生理活性物質を近傍に選択的に配置させることは非常に困難である。また、ガレート型カテキンを含む市販の酸化防止剤は、特有の呈味を有していることから、多量の酸化防止剤を使用すると、酸化されやすい生理活性物質を保護する効果は発揮されるものの、得られる製品の呈味性に大きな影響を及ぼす場合もある。 In addition, gallate-type catechins are also attracting attention for their antioxidant activity. For example, if a gallate-type catechin can be placed in the vicinity of a physiologically active substance that is not stable to oxidation (so-called “property that is easily altered by oxidation, hereinafter referred to as“ easily oxidized ”), its physiology It is expected that the stability can be improved by preventing the oxidation of the active substance. However, it is very difficult to selectively place a physiologically active substance that easily oxidizes gallate-type catechins in the vicinity. In addition, since commercially available antioxidants containing gallate-type catechins have a unique taste, the use of a large amount of antioxidants has the effect of protecting bioactive substances that are easily oxidized. , It may have a great influence on the taste of the obtained product.
　例えば、極めて、酸化されやすい生理活性物質として、空気中で簡単に酸化する還元型ＣｏＱ１０（ユビキノール）が知られている。ユビキノールは、酸化されることでユビキノンになることが知られており、さらに、水への溶解度が低く、水中で結晶化や分離が起こりやすいため、水溶液中に安定に保持させることが困難であることも知られている。前記ユビキノールを安定化する方法としては、シクロデキストリンに包摂させる方法（特許文献４）、クエン酸類を用いる方法（特許文献５）、アスコルビン酸類と共存させる方法（特許文献６）が報告されている。又、ゼラチン等の水溶性賦形剤に還元型ＣｏＱ１０を含有する油性成分を分散させた粒子状組成物（特許文献７）が報告されている。さらに、ユビキノール等の酸化されやすい生理活性物質を安定化させる技術として、本発明者らは、例えば、ユビキノールを０．０１～２０重量％、ガレート型カテキンを０．０６～２重量％、コラーゲンを０．６～２．８重量％含有し、前記ガレート型カテキンと前記コラーゲンとで形成された複合体を含有する水溶液中に前記ユビキノールが分散されていることを特徴とする液状組成物（特許文献８）、ガレート型カテキンを固形分として０．１重量％以上、ゼラチン、コラーゲン、及びこれらの分解物から選ばれる少なくとも１種の動物性タンパク質を固形分として０．１重量％以上含有し、且つ、ガレート型カテキンの固形分と前記動物性タンパク質の固形分の重量比（動物性タンパク質／ガレート型カテキン）が０．０７～８．０であり、且つ、酸化されやすい生理活性物質を担持し、前記酸化されやすい生理活性物質がユビキノール、スチルベン類、水溶性ビタミン、脂溶性ビタミン又はカロテノイドであり、平均粒子径が１０～２００ｎｍであることを特徴とするナノ粒子（特許文献９）を報告している。 For example, reduced CoQ10 (ubiquinol), which easily oxidizes in the air, is known as a physiologically active substance that is extremely easily oxidized. Ubiquinol is known to become ubiquinone by being oxidized, and further, it has low solubility in water and easily crystallizes and separates in water, so that it is difficult to stably maintain it in an aqueous solution. It is also known. As a method for stabilizing the ubiquinol, a method of subsuming it in cyclodextrin (Patent Document 4), a method using citric acid (Patent Document 5), and a method of coexisting with ascorbic acid (Patent Document 6) have been reported. Further, a particulate composition (Patent Document 7) in which an oily component containing reduced CoQ10 is dispersed in a water-soluble excipient such as gelatin has been reported. Furthermore, as a technique for stabilizing a bioactive substance that is easily oxidized such as ubiquinol, the present inventors, for example, use 0.01 to 20% by weight of ubiquinol, 0.06 to 2% by weight of gallate-type catechin, and collagen. A liquid composition containing 0.6 to 2.8% by weight and having the ubiquinol dispersed in an aqueous solution containing a complex formed of the gallate-type catechin and the collagen (Patent Document). 8), containing 0.1% by weight or more of gallate-type catechin as a solid content, 0.1% by weight or more of gelatin, collagen, and at least one animal protein selected from decomposition products thereof as a solid content, and , The weight ratio of the solid content of the gallate-type catechin to the solid content of the animal protein (animal protein / gallate-type catechin) is 0.07 to 8.0, and a physiologically active substance that is easily oxidized is carried. We have reported nanoparticles (Patent Document 9) characterized in that the bioactive substances that are easily oxidized are ubiquinol, stilbens, water-soluble vitamins, fat-soluble vitamins or carotenoids, and the average particle size is 10 to 200 nm. There is.
特許第６３６９２０７号公報Japanese Patent No. 6369207 特開２０１４－０６２１９２号公報Japanese Unexamined Patent Publication No. 2014-062192 特開２０１３－０８２６７３号公報Japanese Unexamined Patent Publication No. 2013-082673 特開２０１０－１２６４９２号公報JP-A-2010-126492 特許第３７９０５３０号公報Japanese Patent No. 3790530 特許第３８９２８８１号公報Japanese Patent No. 3892881 国際公開第２００７／１４８７９８号International Publication No. 2007/148798 特許第６２８７１２３号公報Japanese Patent No. 6287123 特許第６４２８１６５号公報Japanese Patent No. 6428165
　前記特許文献９等に記載のナノ粒子は、ガレート型カテキンに由来する苦渋味については改善されたものとなってはいるが、得られるナノ粒子の平均粒子径は計測できるものの、粒度分布を調べたところ、ブロードなものとなっており、食品等の製品原料として使用するには、品質を向上させる点及び取扱い性を良好にする点で改善の余地があった。 The nanoparticles described in Patent Document 9 and the like have improved bitterness and astringency derived from gallate-type catechins, and although the average particle size of the obtained nanoparticles can be measured, the particle size distribution is investigated. As a result, it is broad, and there is room for improvement in terms of improving quality and handling when used as a raw material for products such as foods.
　また、前記特許文献１等に記載のナノ粒子は、その製造においてガレート型カテキンを必須成分としていたが、工業的な生産をより簡便に実施するにおいては、より安価で入手容易な原料成分を用いて製造する点で改善の余地があった。 Further, the nanoparticles described in Patent Document 1 and the like used gallate-type catechin as an essential component in their production, but in order to carry out industrial production more easily, a cheaper and more easily available raw material component was used. There was room for improvement in terms of manufacturing.
　そこで、本発明者らは、鋭意研究を行ったところ、動物性タンパク質に対するカテキンの固形分比率に着目し、従来より動物性タンパク質に対して少量のカテキンに調整することで、従来に比べ粒子サイズの揃ったナノ粒子を製造することを見出した。
　また、さらに研究を進めたところ、ガレート型カテキンにかえて、電解質として、例えば、クエン酸三カリウム、クエン酸三ナトリウム等の有機酸塩、塩化カリウム、塩化ナトリウム等の無機塩、乳酸、コハク酸、フマル酸、グルコン酸等の有機酸を用いたところ、従来のナノ粒子と同等か、あるいはより小さいサイズのナノ粒子を製造できることを見出し、本発明を完成した。
　すなわち、本発明の目的は、平均粒子径が１０～３００ｎｍであり、さらにその粒径分布がシャープな、風味良好なナノ粒子及びその製造法を提供することである。
　また、本発明の他の目的は、ガレート型カテキンを含有しないナノ粒子及びその製造法を提供することである。 Therefore, as a result of diligent research, the present inventors focused on the solid content ratio of catechin to animal protein, and adjusted the particle size to a smaller amount of catechin to animal protein than before. We have found that we can produce nanoparticles with a uniform amount of proteins.
Further research revealed that instead of gallate-type catechins, as electrolytes, for example, organic acid salts such as tripotassium citrate and trisodium citrate, inorganic salts such as potassium chloride and sodium chloride, lactic acid, and succinic acid. , Fumaric acid, gluconic acid and other organic acids have been found to be able to produce nanoparticles of the same or smaller size than conventional nanoparticles, and have completed the present invention.
That is, an object of the present invention is to provide nanoparticles having an average particle size of 10 to 300 nm, having a sharp particle size distribution, and having a good flavor, and a method for producing the same.
Another object of the present invention is to provide nanoparticles containing no gallate-type catechin and a method for producing the same.
　本発明の要旨は、
　〔１〕ホエイタンパク質、ゼラチン、コラーゲン、及びこれらの分解物からなる群より選ばれる少なくとも１種の動物性タンパク質を固形分として１重量％以上、
　電解質を固形分として０．００１重量％以上含有し、且つ、
　前記動物性タンパク質と前記電解質との固形分の重量比（電解質／動物性タンパク質）が０．００１～１．０であり、且つ
　平均粒子径が１０～３００ｎｍであることを特徴とする、ナノ粒子、
　〔２〕前記電解質が、カテキン、無機塩、有機酸塩、及び有機酸から成る群から選択される少なくとも１種である、前記ナノ粒子、
　〔３〕前記動物性タンパク質と前記電解質との固形分の重量比（電解質／動物性タンパク質）が０．００１～０．２である、前記ナノ粒子、
　〔４〕前記電解質がカテキンであり、前記カテキンを固形分として０．０１重量％以上含有する前記ナノ粒子。
　〔５〕前記ナノ粒子が、その平均粒子径の前後２０ｎｍの範囲内に７０％以上が存在する、前記ナノ粒子。
　〔６〕生理活性物質を担持している、前記ナノ粒子。
　〔７〕前記生理活性物質がユビキノール、スチルベン類、水溶性ビタミン、脂溶性ビタミン、アミノ酸、カフェイン、及びカロテノイドならなる群より選ばれる少なくとも１種である、前記ナノ粒子。
　〔８〕ホエイタンパク質、ゼラチン、コラーゲン、及びこれらの分解物からなる群より選ばれる少なくとも１種の動物性タンパク質を溶媒に溶解、分散又は膨潤させて、動物性タンパク質液を調製する工程（Ａ）、
　前記工程（Ａ）で得られた前記動物性タンパク質液に、前記動物性タンパク質の固形分重量１に対し、電解質を固形分重量０．００１～１．０の割合で加えて、動物性タンパク質－電解質液を調製する工程（工程Ｂ）、及び
　前記工程（Ｂ）で得られた前記動物性タンパク質－電解質液を７２～９５℃で、２～６０分間加熱する工程（Ｃ）、
を有する、ナノ粒子の製造方法、
　〔９〕前記電解質が、カテキン、無機塩、有機酸塩、及び有機酸から成る群から選択される少なくとも１種である、前記ナノ粒子の製造方法、
　〔１０〕さらに、生理活性物質を加える工程を含む、前記ナノ粒子の製造方法、
　〔１１〕前記生理活性物質がユビキノール、スチルベン類、水溶性ビタミン、脂溶性ビタミン、アミノ酸、カフェイン、及びカロテノイドから成る群から選択される少なくとも１種である、前記ナノ粒子の製造方法
に関する。 The gist of the present invention is
[1] 1% by weight or more of solid content of at least one animal protein selected from the group consisting of whey protein, gelatin, collagen, and decomposition products thereof.
Contains 0.001% by weight or more of electrolyte as a solid content and
Nanoparticles characterized by a solid content weight ratio (electrolyte / animal protein) of the animal protein to the electrolyte of 0.001 to 1.0 and an average particle size of 10 to 300 nm. ,
[2] The nanoparticles, wherein the electrolyte is at least one selected from the group consisting of catechins, inorganic salts, organic acid salts, and organic acids.
[3] The nanoparticles having a solid content weight ratio (electrolyte / animal protein) of the animal protein to the electrolyte of 0.001 to 0.2.
[4] The nanoparticles in which the electrolyte is catechin and contains 0.01% by weight or more of the catechin as a solid content.
[5] The nanoparticles in which 70% or more of the nanoparticles are present within a range of 20 nm before and after the average particle diameter.
[6] The nanoparticles carrying a physiologically active substance.
[7] The nanoparticles, which is at least one selected from the group consisting of ubiquinol, stilbenes, water-soluble vitamins, fat-soluble vitamins, amino acids, caffeine, and carotenoids.
[8] A step (A) of preparing an animal protein solution by dissolving, dispersing or swelling at least one animal protein selected from the group consisting of whey protein, gelatin, collagen, and decomposition products thereof in a solvent. ,
To the animal protein solution obtained in the step (A), an electrolyte is added at a ratio of a solid content weight of 0.001 to 1.0 to a solid content weight of 1 of the animal protein, and the animal protein-. A step of preparing the electrolyte solution (step B), and a step of heating the animal protein-electrolyte solution obtained in the step (B) at 72 to 95 ° C. for 2 to 60 minutes (C).
Manufacturing method of nanoparticles,
[9] A method for producing nanoparticles, wherein the electrolyte is at least one selected from the group consisting of catechins, inorganic salts, organic acid salts, and organic acids.
[10] A method for producing nanoparticles, which further comprises a step of adding a physiologically active substance.
[11] The present invention relates to a method for producing the nanoparticles, wherein the bioactive substance is at least one selected from the group consisting of ubiquinol, stilbenes, water-soluble vitamins, fat-soluble vitamins, amino acids, caffeine, and carotenoids.
　本発明のナノ粒子は、平均粒子径が１０～３００ｎｍであり、さらにその粒径分布がシャープな、風味良好なものとなっていることから、食品へ幅広く応用することが可能なナノ粒子である。
　また、本発明のナノ粒子は、平均粒子径が１０～３００ｎｍであり、かつ、食品添加物としてよく用いられている種々の電解質を含有していることで、体内への吸収性及び安全性に優れたものであり、特に食品へ幅広く応用することが可能なナノ粒子である。
　また、本発明のナノ粒子には、生理活性物質を担持させることで、より機能性に優れた、例えば、生理活性物質のバイオアベイラビリティを向上させたナノ粒子とすることができる。 The nanoparticles of the present invention have an average particle size of 10 to 300 nm, and have a sharp particle size distribution and a good flavor. Therefore, the nanoparticles can be widely applied to foods. ..
In addition, the nanoparticles of the present invention have an average particle size of 10 to 300 nm and contain various electrolytes that are often used as food additives, so that they can be absorbed into the body and are safe. It is an excellent nanoparticle that can be widely applied to foods in particular.
Further, by supporting the bioactive substance on the nanoparticles of the present invention, it is possible to obtain nanoparticles having more excellent functionality, for example, improved bioavailability of the physiologically active substance.
図１は、本発明のナノ粒子の粒度分布（シャープ）及び従来のナノ粒子の粒度分布（ブロード）の概略を示すグラフである。FIG. 1 is a graph showing an outline of the particle size distribution (sharp) of the nanoparticles of the present invention and the particle size distribution (broad) of the conventional nanoparticles. 図２は、比較例ａ－１の高濃度のカテキン存在下で調製したナノ粒子の粒子サイズ分布のグラフである。横軸は粒子半径を示しており、数値を２倍することで粒子径が算出される。FIG. 2 is a graph of the particle size distribution of nanoparticles prepared in the presence of a high concentration of catechin in Comparative Example a-1. The horizontal axis shows the particle radius, and the particle diameter is calculated by doubling the numerical value. 図３は、実施例ａ－１の低濃度のカテキン存在下で調製したナノ粒子の粒子サイズ分布のグラフである。横軸は粒子半径を示しており、数値を２倍することで粒子径が算出される。FIG. 3 is a graph of particle size distribution of nanoparticles prepared in the presence of low concentration catechin of Example a-1. The horizontal axis shows the particle radius, and the particle diameter is calculated by doubling the numerical value.
　以下、本発明の具体的な実施形態について詳細に説明するが、本発明は以下の実施形態に何ら限定されるものではなく、本発明の目的の範囲内において、適宜変更を加えて実施することができる。なお、説明が重複する箇所については、適宜説明を省略する場合があるが、本発明を限定するものではない。 Hereinafter, specific embodiments of the present invention will be described in detail, but the present invention is not limited to the following embodiments, and the present invention shall be carried out with appropriate modifications within the scope of the object of the present invention. Can be done. It should be noted that the description may be omitted as appropriate for the parts where the description is duplicated, but the present invention is not limited.
１．ナノ粒子
　本発明のナノ粒子は、ホエイタンパク質、ゼラチン、コラーゲン、及びこれらの分解物からなる群より選ばれる少なくとも１種の動物性タンパク質を固形分として１重量％以上、電解質を固形分として０．００１重量％以上含有し、且つ、前記動物性タンパク質と前記電解質との固形分の重量比（電解質／動物性タンパク質）が０．００１～１．０であり、且つ平均粒子径が１０～３００ｎｍであることを特徴とする。 1. 1. Nanoparticles The nanoparticles of the present invention contain at least 1% by weight of an animal protein selected from the group consisting of whey protein, gelatin, collagen, and decomposition products thereof as a solid content, and an electrolyte as a solid content of 0. When the content is 001% by weight or more, the weight ratio of the solid content of the animal protein to the electrolyte (electrolyte / animal protein) is 0.001 to 1.0, and the average particle size is 10 to 300 nm. It is characterized by being.
　本発明で用いる動物性タンパク質は、電解質とコアセルベートを形成可能なタンパク質であればよく、具体的には、ホエイタンパク質、ゼラチン、コラーゲン、及びこれらの分解物が挙げられる。 The animal protein used in the present invention may be any protein capable of forming a coacervate with an electrolyte, and specific examples thereof include whey protein, gelatin, collagen, and decomposition products thereof.
　本発明において使用されるホエイタンパク質とは、ウシ、ヒツジ、ヒト等の生乳から、カゼインと乳脂肪を取り除いた乳清（ホエイ）に含まれるタンパク質の総称を意味し、例えば、ホエイタンパク質濃縮物（Ｗｈｅｙ Ｐｒｏｔｅｉｎ Ｃｏｎｃｅｎｔｒａｔｅ、ＷＰＣともいう）、ホエイタンパク質分離物（Ｗｈｅｙ Ｐｒｏｔｅｉｎ Ｉｓｏｌａｔｅ、ＷＰＩともいう）、さらにはホエイタンパク質からβ－ラクトグロブリン等の特定のタンパク質を取り出したもの、その他、ホエイの原液（甘性ホエイ、酸ホエイ等）、その乾燥物（ホエイ粉等）、その凍結物等が挙げられる。ホエイタンパク質は、自ら調製して用いてもよいし、市販品を入手して用いてもよい。 The whey protein used in the present invention is a general term for proteins contained in whey from which casein and milk fat are removed from raw milk of cows, sheep, humans, etc., for example, whey protein concentrate (whey protein concentrate). Whey Protein Concentrate (also called WPC), whey protein isolate (also called Whey Protein Isolate, WPI), whey protein extracted from specific proteins such as β-lactoglobulin, and other undiluted whey (sweetness) Whey, acid whey, etc.), its dried product (whey powder, etc.), its frozen product, etc. can be mentioned. The whey protein may be prepared and used by itself, or a commercially available product may be obtained and used.
　市販品のホエイタンパク質としては、例えば、チーズホエイ由来のＷＰＣ素材の「エンラクトＨＵＳ」（日本新薬株式会社）、「エンラクトＡＬＣ」（日本新薬株式会社）、酸ホエイ由来のＷＰＣ素材の「ＰＲＯＧＥＬ８００」（日本新薬株式会社）、ＷＰＩ素材の「エンラクトＳＡＴ」（日本新薬株式会社）、「エンラクトＹＹＹ」（日本新薬株式会社）、「ラクトクリスタル」（日本新薬株式会社）、「ＧｅｒｍａｎＰｒｏｔ　９０００（ＷＰＩ）」（ザクセンミルヒ社）等を挙げることができる。 Examples of commercially available whey proteins include "Enlacto HUS" (Nippon Shinyaku Co., Ltd.), a WPC material derived from cheese whey, "Enlacto ALC" (Nippon Shinyaku Co., Ltd.), and "PROGEL800", a WPC material derived from acid whey. Nippon Shinyaku Co., Ltd.), WPI material "Enlacto SAT" (Nippon Shinyaku Co., Ltd.), "Enlacto YYY" (Nippon Shinyaku Co., Ltd.), "Lact Crystal" (Nippon Shinyaku Co., Ltd.), "GermanProt 9000 (WPI)" ( Saxony Milch) and the like.
　本発明において使用されるゼラチンとは、動物の皮や骨に多く含まれるコラーゲンを加熱することによりコラーゲンを構成する細長い３本の分子からなる三重螺旋構造を崩壊させ、水溶性のタンパク質へと変化させたもの意味する。ゼラチンの由来は、牛、豚、魚、ニワトリ等、及び遺伝子組み換え体のいずれかを用いることができる。なお、牛骨又は豚骨由来の動物性タンパク質は、５００ｎｍ以下の粒子が一部形成されるものの、凝集及び沈殿が起こりやすいため、本発明では使用することが難しい。ただし、牛骨又は豚骨由来のタンパク質が含まれている動物性タンパク質であっても、平均粒子径１０～３００ｎｍのナノ粒子が作製できれば、特に限定はなく使用することができる。
　また、必要に応じて、コラーゲンやさらに分解したコラーゲンペプチドを使用することもできる。 Gelatin used in the present invention breaks down the triple helix structure consisting of three elongated molecules that make up collagen by heating collagen, which is abundant in animal skin and bone, and changes it into a water-soluble protein. It means what you let. As the origin of gelatin, any of cattle, pigs, fish, chickens and the like, and genetically modified organisms can be used. Although some particles of 500 nm or less are formed in animal protein derived from beef bone or pork bone, it is difficult to use in the present invention because aggregation and precipitation are likely to occur. However, even animal proteins containing proteins derived from bovine bone or pork bone can be used without particular limitation as long as nanoparticles having an average particle diameter of 10 to 300 nm can be produced.
Further, if necessary, collagen or further decomposed collagen peptide can be used.
　これらの動物性タンパク質は、単独で使用しても、２種以上を組み合わせて使用してもよい。また、前記動物性タンパク質は乳化されていてもよい。 These animal proteins may be used alone or in combination of two or more. Moreover, the animal protein may be emulsified.
　本発明のナノ粒子中における前記動物性タンパク質の固形分含有量としては、１重量％以上であり、粒子径のばらつきの少ない平均粒子径１０～３００ｎｍのナノ粒子を効率的に作製する観点から、１～９９重量％が好ましく、１～９５重量％がより好ましく、１～９０重量％がさらに好ましい。 The solid content of the animal protein in the nanoparticles of the present invention is 1% by weight or more, and from the viewpoint of efficiently producing nanoparticles having an average particle diameter of 10 to 300 nm with little variation in particle size. 1 to 99% by weight is preferable, 1 to 95% by weight is more preferable, and 1 to 90% by weight is further preferable.
　本発明において「電解質」は、後述のようにホエイタンパク質等の動物性タンパク質を溶媒中でナノ粒子化するために用いられる。
　本発明で使用される電解質としては、例えば、カテキン、無機塩、有機酸塩及び有機酸から成る群から選択される少なくとも１種が挙げられる。 In the present invention, the "electrolyte" is used to nanoparticle an animal protein such as whey protein in a solvent as described later.
Examples of the electrolyte used in the present invention include at least one selected from the group consisting of catechins, inorganic salts, organic acid salts and organic acids.
　本発明において「カテキン」は、後述のようにホエイタンパク質等の動物性タンパク質を溶媒中でナノ粒子化するために用いられる。本発明で使用されるカテキンは、例えば、化学構造的に「ガレート基」が付いているエピガロカテキンガレート（ＥＧＣｇ）、エピカテキンガレート（ＥＣｇ）、カテキンガレート（Ｃｇ）、ガロカテキンガレート（ＧＣｇ）等の「ガレート型カテキン」、及び「ガレート基」が付いていないエピガロカテキン（ＥＧＣ）、エピカテキン（ＥＣ）、カテキン（Ｃ）、ガロカテキン（ＧＣ）等の「遊離型カテキン」が挙げられる。前記カテキンとしては、市販のカテキンであればよく、好ましくは緑茶特有のカテキンであればよい。 In the present invention, "catechin" is used for nanoparticulating animal proteins such as whey protein in a solvent as described later. The catechins used in the present invention are, for example, epigallocatechin gallate (EGCg), epicatechin gallate (ECg), catechin gallate (Cg), and galocatechin gallate (GCg) having a "gallate group" chemically structurally. Examples thereof include "gallate-type catechins" such as "gallate-type catechins" and "free-type catechins" such as epigallocatechin (EGC), epicatechin (EC), catechins (C), and gallocatechins (GC) without a "gallate group". The catechin may be a commercially available catechin, preferably a catechin peculiar to green tea.
　本発明において、前記カテキンを単独で使用してもよいし、二種類以上を組み合わせたもの（カテキン混合物）として使用してもよい。このようなカテキン混合物としては、茶を水で抽出した茶抽出液を乾燥して得られる茶抽出物が挙げられる。茶抽出物は、自ら調製して用いてもよいし、市販品を入手して用いてもよい。市販品の茶抽出物として、例えば、「サンフェノン ＥＧＣｇ－ＯＰ」（太陽化学株式会社）、「サンフェノン９０Ｓ」（太陽化学株式会社）、「サンフェノンＢＧ－３」（太陽化学株式会社）、「サンフェノンＢＧ－５」（太陽化学株式会社）、「カメリアエキス３０Ｓ」（太陽化学株式会社）等を挙げることができる。 In the present invention, the catechin may be used alone or as a combination of two or more types (catechin mixture). Examples of such a catechin mixture include a tea extract obtained by drying a tea extract obtained by extracting tea with water. The tea extract may be prepared and used by itself, or a commercially available product may be obtained and used. As commercially available tea extracts, for example, "Sanphenon EGCg-OP" (Taiyo Kagaku Co., Ltd.), "Sanphenon 90S" (Taiyo Kagaku Co., Ltd.), "Sanphenon BG-3" (Taiyo Kagaku Co., Ltd.), "Sanphenon BG" -5 ”(Taiyo Kagaku Co., Ltd.),“ Camellia Extract 30S ”(Taiyo Kagaku Co., Ltd.) and the like can be mentioned.
　また、カテキン以外の前記電解質としては、塩化カリウム、塩化ナトリウム等の無機塩、クエン酸三カリウム、クエン酸三ナトリウム等の有機酸塩、乳酸、コハク酸、フマル酸、クエン酸等の有機酸等が挙げられる。
　これらの電解質としては、市販品を用いればよく、中でも、食品添加物として使用される市販品を用いることが好ましい。市販品の電解質（無機塩）として、例えば、「塩化カリウム」（赤穂化成株式会社）、「塩化ナトリウム」（赤穂化成株式会社）等を挙げることができる。市販品の電解質（有機酸塩）として、例えば、「精製クエン酸三カリウム」（扶桑化学工業株式会社）、「精製クエン酸三ナトリウム」（扶桑化学工業株式会社）等を挙げることができる。市販品の電解質（有機酸）として、例えば、「発酵乳酸９０」（扶桑化学工業株式会社）、「コハク酸」（扶桑化学工業株式会社）、「フマル酸」（扶桑化学工業株式会社）、「精製クエン酸（結晶）」（扶桑化学工業株式会社）等を挙げることができる。 Examples of the electrolyte other than catechin include inorganic salts such as potassium chloride and sodium chloride, organic acid salts such as tripotassium citrate and trisodium citrate, and organic acids such as lactic acid, succinic acid, fumaric acid and citric acid. Can be mentioned.
As these electrolytes, commercially available products may be used, and among them, commercially available products used as food additives are preferably used. Examples of commercially available electrolytes (inorganic salts) include "potassium chloride" (Ako Kasei Co., Ltd.) and "sodium chloride" (Ako Kasei Co., Ltd.). Examples of commercially available electrolytes (organic acid salts) include "purified tripotassium citrate" (Fuso Chemical Industry Co., Ltd.) and "purified trisodium citrate" (Fuso Chemical Industry Co., Ltd.). As commercially available electrolytes (organic acids), for example, "fermented lactic acid 90" (Fuso Chemical Industry Co., Ltd.), "succinic acid" (Fuso Chemical Industry Co., Ltd.), "fumaric acid" (Fuso Chemical Industry Co., Ltd.), ""Purified citric acid (crystal)" (Fuso Chemical Industry Co., Ltd.) and the like can be mentioned.
　本発明において、前記電解質を単独で使用してもよいし、二種類以上を組み合わせたものとして使用してもよい。 In the present invention, the electrolyte may be used alone or as a combination of two or more types.
　本発明のナノ粒子中における前記電解質の固形分含有量としては、０．００１重量％以上であり、平均粒子径１０～３００ｎｍの粒子径のナノ粒子を効率的に作製する観点から、０．０１～６．４重量％が好ましく、０．０８～３．２重量％がより好ましく、０．１～１．６重量％がさらに好ましい。
　また、前記電解質としてカテキンを用いる場合には、本発明のナノ粒子中における前記カテキンの固形分含有量としては、平均粒子径１０～３００ｎｍの粒子径のばらつきの少ないナノ粒子を効率的に作製する観点から、０．０１重量％以上が好ましく、０．０８～６．４重量％がより好ましく、０．１～３．２重量％がより好ましく、０．８～１．６重量％がさらに好ましい。 The solid content of the electrolyte in the nanoparticles of the present invention is 0.001% by weight or more, and is 0.01 from the viewpoint of efficiently producing nanoparticles having a particle diameter of 10 to 300 nm on average. It is preferably from 6.4% by weight, more preferably 0.08 to 3.2% by weight, still more preferably 0.1 to 1.6% by weight.
When catechin is used as the electrolyte, nanoparticles having an average particle diameter of 10 to 300 nm and little variation in particle diameter are efficiently produced as the solid content of the catechin in the nanoparticles of the present invention. From the viewpoint, 0.01% by weight or more is preferable, 0.08 to 6.4% by weight is more preferable, 0.1 to 3.2% by weight is more preferable, and 0.8 to 1.6% by weight is further preferable. ..
　本発明のナノ粒子は、前記動物性タンパク質と前記電解質との固形分の重量比（電解質／動物性タンパク質）が０．００１～１．０に調整されている。本発明においては、前記動物性タンパク質と前記電解質との固形分の重量比を前記範囲に調整していることで、塩析効果による前記動物性タンパク質の凝集を防ぎながら１０～３００ｎｍの粒子径のナノ粒子を効率的に作製することができるという利点がある。
　前記重量比（電解質／動物性タンパク質）は、例えば、粒子径のばらつきの少ない１０～３００ｎｍのナノ粒子を効率的に作製するための観点から、下限は０．００１以上が好ましく、０．０１以上がより好ましく、０．０２以上がさらに好ましく、また上限は１．５以下が好ましく、１．２以下がより好ましく、０．５以下がさらに好ましく、０．２以下がさらに好ましく、０．１以下がさらに好ましい。
　より詳細には、例えば、電解質としてカテキンを用いる場合には、前記重量比（カテキン／動物性タンパク質）が０．００１～０．２に調整されていることで、カテキンに由来する苦渋味の発現を抑えて良好な風味としながら、ナノ粒子の粒度分布がシャープな高品質のナノ粒子となる。前記重量比は、粒子径のばらつきの少ない１０～３００ｎｍのナノ粒子を効率的に作製するための観点から、０．００１～０．１５、より好ましくは０．０２～０．１２５、さらに好ましくは０．０３～０．１である。
　また、電解質として、クエン酸三カリウム、クエン酸三ナトリウム等の有機酸塩を用いる場合には、０．００１～０．５がより好ましく、０．００１～０．２がさらに好ましい。
　また、電解質として、塩化カリウム、塩化ナトリウム等の無機塩を用いる場合には、０．００１～０．２がより好ましく、０．００１～０．１がさらに好ましい。
　また、電解質として、乳酸、コハク酸、フマル酸、及びクエン酸等の有機酸を用いる場合には、０．００１～１．５がより好ましく、０．０１～１．２がさらに好ましい。 In the nanoparticles of the present invention, the weight ratio of the solid content (electrolyte / animal protein) of the animal protein and the electrolyte is adjusted to 0.001 to 1.0. In the present invention, by adjusting the weight ratio of the solid content of the animal protein to the electrolyte within the above range, the particle size of 10 to 300 nm is prevented while preventing the animal protein from agglomerating due to the salting out effect. There is an advantage that nanoparticles can be efficiently produced.
The lower limit of the weight ratio (electrolyte / animal protein) is preferably 0.001 or more, preferably 0.01 or more, for example, from the viewpoint of efficiently producing nanoparticles having a particle size of 10 to 300 nm with little variation. Is more preferable, 0.02 or more is further preferable, and the upper limit is preferably 1.5 or less, more preferably 1.2 or less, further preferably 0.5 or less, further preferably 0.2 or less, and 0.1 or less. Is even more preferable.
More specifically, for example, when catechin is used as the electrolyte, the weight ratio (catechin / animal protein) is adjusted to 0.001 to 0.2, so that the bitterness derived from catechin is expressed. High-quality nanoparticles with a sharp particle size distribution of nanoparticles while suppressing the problem and achieving a good flavor. The weight ratio is 0.001 to 0.15, more preferably 0.02 to 0.125, and even more preferably 0.01 to 0.15, from the viewpoint of efficiently producing nanoparticles having a particle size of 10 to 300 nm. It is 0.03 to 0.1.
Further, when an organic acid salt such as tripotassium citrate or trisodium citrate is used as the electrolyte, 0.001 to 0.5 is more preferable, and 0.001 to 0.2 is further preferable.
When an inorganic salt such as potassium chloride or sodium chloride is used as the electrolyte, 0.001 to 0.2 is more preferable, and 0.001 to 0.1 is even more preferable.
Further, when an organic acid such as lactic acid, succinic acid, fumaric acid, and citric acid is used as the electrolyte, 0.001 to 1.5 is more preferable, and 0.01 to 1.2 is further preferable.
　なお、本発明のナノ粒子は、カテキン以外の電解質を用いる場合、ガレート型カテキン含量は０とすることもできるが、必要に応じて、本発明のナノ粒子中にガレート型カテキンを含有させることも可能である。その場合、ガレート型カテキン含有量としては、０．００１～１重量％が好ましい。 When an electrolyte other than catechin is used for the nanoparticles of the present invention, the gallate-type catechin content can be set to 0, but if necessary, the nanoparticles of the present invention may contain gallate-type catechin. It is possible. In that case, the gallate-type catechin content is preferably 0.001 to 1% by weight.
　また、本発明のナノ粒子は、必要に応じて、生理活性物質を担持させることができる。本発明のナノ粒子では、ナノ粒子に生理活性物質が担持されることで、安定性がよくなったり、生理活性物質のバイオアベイラビリティを向上させたりすることができる。 Further, the nanoparticles of the present invention can carry a physiologically active substance, if necessary. In the nanoparticles of the present invention, by supporting the physiologically active substance on the nanoparticles, the stability can be improved and the bioavailability of the physiologically active substance can be improved.
　なお、本発明において、「担持」とは、ナノ粒子中に生理活性物質が含まれている状態及びナノ粒子表面に付着している状態をいう。
　また、ナノ粒子中に生理活性物質が担持されていることは、後述の虐待試験においても、生理活性物質が変化及び／又は活性低下及び／又は成分減少していないことから判別することができる。
　また、本発明でいう「安定性」とは、生理活性物質が他物質（例えば、酸素、糖等）と反応しにくくなることをいう。ここで、他物質との反応の例としては、酸化反応、メーラード反応等が挙げられる。
　また、本発明でいう「バイオアベイラビリティ」とは、人体に投与された物質のうち、どれだけの量が全身に循環するのかを示す指標をいう。 In the present invention, "supporting" refers to a state in which the nanoparticles contain a physiologically active substance and a state in which the nanoparticles are attached to the surface of the nanoparticles.
Further, the fact that the physiologically active substance is supported in the nanoparticles can be determined from the fact that the physiologically active substance is not changed and / or decreased in activity and / or the component is not decreased in the abuse test described later.
Further, the term "stability" as used in the present invention means that a physiologically active substance is less likely to react with another substance (for example, oxygen, sugar, etc.). Here, examples of the reaction with other substances include an oxidation reaction, a Maillard reaction, and the like.
In addition, the "bioavailability" as used in the present invention refers to an index indicating how much of a substance administered to the human body circulates throughout the body.
　前記生理活性物質としては、一般的に酸化されることで変質しやすい物性を有する物質であればよく、例えば、有用性の観点から、ユビキノール、スチルベン類、水溶性ビタミン、脂溶性ビタミン、アミノ酸、カフェイン、カロテノイド等が挙げられる。
　中でも、ナノ粒子への担持率の観点から、ユビキノール、スチルベン類、脂溶性ビタミン、アミノ酸、特に疎水性アミノ酸、カロテノイド等のような、比較的疎水性の高いものが好ましい。 The physiologically active substance may be any substance having physical properties that are easily altered by being oxidized. For example, from the viewpoint of usefulness, ubiquinol, stilbenes, water-soluble vitamins, fat-soluble vitamins, amino acids, etc. Examples include caffeine and carotenoids.
Among them, those having relatively high hydrophobicity such as ubiquinol, stilbenes, fat-soluble vitamins, amino acids, particularly hydrophobic amino acids, carotenoids, etc. are preferable from the viewpoint of the support rate on nanoparticles.
　本発明に用いられるユビキノールは、還元型コエンザイムＱ１０とも言われる機能性成分であり、油溶性の固体状物質である。ユビキノールとしては、市販品を用いればよく、株式会社カネカ製のものが挙げられる。例えば、株式会社カネカ製の精製品である「カネカＱＨ」や調製品である「カネカＱＨ安定化粉末（Ｐ３０）」等が挙げられるが、コスト面や物性面で「カネカＱＨ」が望ましい。 Ubiquinol used in the present invention is a functional component also called reduced coenzyme Q10, and is an oil-soluble solid substance. As the ubiquinol, a commercially available product may be used, and examples thereof include those manufactured by Kaneka Corporation. For example, "Kaneka QH" which is a refined product manufactured by Kaneka Corporation and "Kaneka QH stabilized powder (P30)" which is a prepared product can be mentioned, but "Kaneka QH" is desirable in terms of cost and physical properties.
　本発明に用いられるスチルベン類は、レスベラトロール、プテロスチルベン、ピセアタンノール等が挙げられる。またこれらを含有する組成物であってもよい。スチルベン類は市販品を用いればよく、例えば、ピセアタンノールを含有する丸善製薬社製のノブドウエキスやレスベラトロールを含有するビーエイチエヌ社製のビネアトロール等が挙げられる。 Examples of stilbenes used in the present invention include resveratrol, pterostilbene, piceatannol and the like. Further, the composition may contain these. Commercially available products may be used as the stilbenes, and examples thereof include Nogrape extract manufactured by Maruzen Pharmaceuticals Co., Ltd. containing piceatannol and Vineatorol manufactured by BNH Co., Ltd. containing resveratrol.
　本発明に用いられる水溶性ビタミンとしては、ビタミンＢ群、ビタミンＣ、葉酸が挙げられる。ビタミンＢ群にはビタミンＢ１、Ｂ２、Ｂ３、Ｂ５、Ｂ６、Ｂ７、Ｂ９、Ｂ１２がある。これらは市販品を用いればよい。 Examples of the water-soluble vitamin used in the present invention include B vitamins, vitamin C, and folic acid. The B vitamins include vitamins B1, B2, B3, B5, B6, B7, B9 and B12. Commercially available products may be used for these.
　本発明に用いられる脂溶性ビタミンとしては、ビタミンＡ、ビタミンＤ、ビタミンＥ、ビタミンＫ等が挙げられる。これらは市販品を用いればよい。 Examples of the fat-soluble vitamin used in the present invention include vitamin A, vitamin D, vitamin E, and vitamin K. Commercially available products may be used for these.
　本発明に用いられるアミノ酸としては、ロイシン、イソロイシン 、バリン、ヒスチジン、 リジン、メチオニン、フェニルアラニン、スレオニン、トリプトファン、アスパラギン、アスパラギン酸、アラニン、アルギニン、システイン・シスチン、グルタミン、グルタミン酸、 グリシン、プロリン、セリン、チロシン等が挙げられる。これらは市販品を用いればよい。疎水性アミノ酸としては、ロイシン、イソロイシン 、バリン、トリプトファン、フェニルアラニン、メチオニン、プロリン、アラニン、グリシン等が挙げられる。 Amino acids used in the present invention include leucine, isoleucine, valine, histidine, lysine, methionine, phenylalanine, threonine, tryptophan, asparagine, aspartic acid, alanine, arginine, cysteine cystine, glutamine, glutamic acid, glycine, proline, serine, Examples include tyrosine. Commercially available products may be used for these. Examples of the hydrophobic amino acid include leucine, isoleucine, valine, tryptophan, phenylalanine, methionine, proline, alanine, glycine and the like.
　カロテノイドとしては、特に限定されないが、α－クリプトキサンチン、β－クリプトキサンチン、イソクリプトキサンチン、アクチノエリスロール、アスタキサンチン、アスタシン、アドニキサンチン、フェニコキサンチン、アロキサンチン、アンテラキサンチン、イソアゲラキサンチン、イソゼアサキンチン、オシラキサンチン、カロキサンチン、α－カロテン、β－カロテン、β－カロテノン、δ－カロテン、ε－カロテン、カンタキサンチン、ギロキサンチン、グアラキサンチン、クリスタキサンチン、クロコキサンチン、クロセチン、クロロキサンチン、ケトミクソコキサンチン、ゲリオデスキサンチン、ジアトキサンチン、デヒドロリコペン、シトラナキサンチン、スタフィロキサンチン、ゼアキサンチン、タラキサンチン、ツナキサンチン、ネオキサンチン、ノストキサンチン、バクテリオルビキサンチン、バクテリオルベリン、バスタキサンチン、パラシロキサンチン、ハラシンチアキサンチン、ビオラキサンチン、フィリプシアキサンチン、フェニコキサンチン、フコキサンチン、プラシノキサンチン、フラブキサンチン、メソ・ゼアキサンチン、モナドキサンチン、ラクツーカキサンチン、リコペン、ルテイン、ルビキサンチン、ロドキサンチン、ロドピナール、ロドピノール、ロドビブリン等が挙げられる。これらは必要に応じて市販品を用いればよい。 The carotenoid is not particularly limited, but is α-cryptoxanthin, β-cryptoxanthin, isocryptoxanthin, actinoerythrol, astaxanthin, ascin, adonixanthin, phenoxanthin, alloxanthin, anteraxanthin, isoageluxanthin, Isozea saquintin, osylaxanthin, caroxanthin, α-carotene, β-carotene, β-carotenone, δ-carotene, ε-carotene, cantaxanthine, guiloxanthin, gualuxanthin, chryptoxanthin, crocoxanthin, crocetin, chloro Xanthine, ketomixocoxanthin, geliodesanthin, diatoxanthin, dehydrolicopen, citranaxanthin, staphyloxanthine, zeaxanthine, taraxanthin, tunaxanthine, neoxanthine, nostoxanthin, bacterial orbixanthine, bacterial orberin, bastaxanthine , Parasiloxanetin, harascinthiaxanthine, violaxanthin, filipsiaxanthin, phenicoxanthine, fucoxanthine, placenoxanthine, flabxanthine, mesozeaxanthin, monadoxanthin, lactucaxanthin, lycopene, lutein, rubixanthin, rhodoxanthin , Rhodopinal, Rhodopinol, Rhodovivrin and the like. Commercially available products may be used for these as needed.
　カフェインとしては、コーヒーノキ、チャノキ、マテ、カカオ、ガラナ等から得られるものであればよく、特に限定はない。また、カフェインは精製物でもよい。これらは必要に応じて市販品を用いればよい。
　なお、前記電解質としてカテキンを用いる場合、カフェインは、カテキンと反応して複合体を形成し易いことが知られているが、本発明では、前記のように、カテキン含有量が低減されているため、カフェインを添加しても、白濁沈殿物の生成等がせず、カフェインを含有したナノ粒子を効果的に製造することができる。 The caffeine may be obtained from coffee tree, tea plant, mate, cacao, guarana, etc., and is not particularly limited. In addition, caffeine may be a purified product. Commercially available products may be used for these as needed.
When catechin is used as the electrolyte, it is known that caffeine easily reacts with catechin to form a complex, but in the present invention, the catechin content is reduced as described above. Therefore, even if caffeine is added, nanoparticles containing caffeine can be effectively produced without forming a cloudy precipitate.
　本発明において、前記生理活性物質は、自然界から得られる天然由来のものでも、化学合成品でもよい。さらに、前記生理活性物質を単独で使用してもよいし、二種類以上を組み合わせて使用してもよい。 In the present invention, the bioactive substance may be a naturally derived substance obtained from the natural world or a chemically synthesized product. Further, the physiologically active substance may be used alone, or two or more kinds may be used in combination.
　本発明のナノ粒子において、前記生理活性物質の含有量としては、粒子径のばらつきの少ない平均粒子径１０～３００ｎｍのナノ粒子を効率的に作製するための観点から、０．００１～５０重量％が好ましく、０．００１～２０重量％がより好ましく、０．００１～１０重量％がさらに好ましい。 In the nanoparticles of the present invention, the content of the physiologically active substance is 0.001 to 50% by weight from the viewpoint of efficiently producing nanoparticles having an average particle size of 10 to 300 nm with little variation in particle size. Is preferable, 0.001 to 20% by weight is more preferable, and 0.001 to 10% by weight is further preferable.
　本発明のナノ粒子の平均粒子径は、１０～３００ｎｍである。本発明のナノ粒子は、微小な粒子となっていることから、例えば、経口で摂取した場合に、動物性タンパク質、電解質に加えて、前記生理活性物質を用いている場合には、これらの機能性成分を腸等から、体内に吸収し易いため、前記生理活性物質を単で摂取した場合に比べ、バイオアベイラビリティが向上するという性質を有する。
　前記平均粒子径としては、機能性成分の分散安定性の観点から、好ましくは１０～２００ｎｍであり、より好ましくは２０～１５０ｎｍ、さらに好ましくは２０～１２０ｎｍである。
　なお、前記ナノ粒子の平均粒子径は、後述の実施例に記載のように、ゼータ電位・ナノ粒子径測定システム（ベックマン・コールター株式会社製、「ＤｅｌｓａＭａｘ  ＰＲＯ」）にて測定することができる。 The average particle size of the nanoparticles of the present invention is 10 to 300 nm. Since the nanoparticles of the present invention are fine particles, for example, when ingested orally, these functions are used when the physiologically active substance is used in addition to the animal protein and electrolyte. Since the sex component is easily absorbed into the body from the intestine or the like, it has the property of improving bioavailability as compared with the case where the physiologically active substance is ingested alone.
The average particle size is preferably 10 to 200 nm, more preferably 20 to 150 nm, and further preferably 20 to 120 nm from the viewpoint of dispersion stability of the functional component.
The average particle size of the nanoparticles can be measured by a zeta potential / nanoparticle size measurement system (“DelsaMax PRO” manufactured by Beckman Coulter, Inc.) as described in Examples described later.
　また、本発明のナノ粒子は、前記電解質としてカテキンを用いる場合、ナノ粒子の平均粒子径の前後２０ｎｍの範囲内に７０％以上、好ましくは７５％以上、より好ましくは８０％以上、さらに好ましくは８５％以上が存在するものとなる。
　このように、本発明のナノ粒子の粒度分布がシャープなものであることで、本発明のナノ粒子を食品等の原料として使用しても、得られる食品の物性に粒度分布のバラつきにともなう予期しない変化が生じること等がなく、高品質の製品を効率よく得ることができる。
　前記平均粒子径の前後２０ｎｍの範囲内に存在するナノ粒子の量については、前記平均粒子径の測定に使用する、ゼータ電位・ナノ粒子径測定システムを用いて測定することができる。 Further, when catechin is used as the electrolyte, the nanoparticles of the present invention are 70% or more, preferably 75% or more, more preferably 80% or more, still more preferably within a range of 20 nm before and after the average particle size of the nanoparticles. 85% or more will be present.
As described above, since the nanoparticles of the present invention have a sharp particle size distribution, even if the nanoparticles of the present invention are used as raw materials for foods and the like, the physical properties of the obtained foods are expected to be accompanied by variations in the particle size distribution. High-quality products can be efficiently obtained without any changes.
The amount of nanoparticles existing in the range of 20 nm before and after the average particle size can be measured by using the zeta potential / nanoparticle size measurement system used for measuring the average particle size.
　また、本発明のナノ粒子は、担持した生理活性物質の安定性を高める目的で、安定剤、乳化剤、タンパク質結合剤等を含有していてもよい。 Further, the nanoparticles of the present invention may contain a stabilizer, an emulsifier, a protein binder, etc. for the purpose of enhancing the stability of the carried physiologically active substance.
　前記安定剤としては、キサンタンガム、寒天、アラビアガム、ペクチン、大豆多糖類、ＣＭＣ（カルボキシメチルセルロース）、カゼインナトリウム等が挙げられる。 Examples of the stabilizer include xanthan gum, agar, gum arabic, pectin, soybean polysaccharide, CMC (carboxymethyl cellulose), sodium caseinate and the like.
　また、前記乳化剤としては、ショ糖脂肪酸エステル等が挙げられる。
　また、ナノ粒子の形成安定剤としてトリポリリン酸ナトリウムやトランスグルタミナーゼ等のタンパク質結合剤が挙げられる。 Moreover, as said emulsifier, sucrose fatty acid ester and the like can be mentioned.
In addition, as a nanoparticle formation stabilizer, a protein binder such as sodium tripolyphosphate or transglutaminase can be mentioned.
２．ナノ粒子の製造方法
　本発明のナノ粒子は、前記動物性タンパク質と、前記電解質とを、いずれも粉体状態で使用し、溶媒と混合して混合液にしてナノ粒子を形成させてもよいが、効率よくナノ粒子を形成させることができ、また、操作性に優れる観点から、例えば、
　ホエイタンパク質、ゼラチン、コラーゲン、及びこれらの分解物からなる群より選ばれる少なくとも１種の動物性タンパク質を溶媒に溶解、分散又は膨潤させて、動物性タンパク質液を調製する工程（Ａ）、
　前記工程（Ａ）で得られた前記動物性タンパク質液に、前記動物性タンパク質の固形分重量１に対し、電解質を固形分重量０．００１～１．０の割合で加えて、動物性タンパク質－電解質液を調製する工程（工程Ｂ）、及び
　前記工程（Ｂ）で得られた前記動物性タンパク質－電解質液を７２～９５℃で、２～６０分間加熱する工程（Ｃ）を経ることにより製造することができる。 2. 2. Method for Producing Nanoparticles In the nanoparticles of the present invention, the animal protein and the electrolyte may both be used in a powder state and mixed with a solvent to form a mixed solution to form nanoparticles. From the viewpoint of being able to efficiently form nanoparticles and having excellent operability, for example,
Step (A) of preparing an animal protein solution by dissolving, dispersing or swelling at least one animal protein selected from the group consisting of whey protein, gelatin, collagen, and decomposition products thereof in a solvent.
To the animal protein solution obtained in the step (A), an electrolyte is added at a solid content weight of 0.001 to 1.0 to a solid content weight of 1 of the animal protein, and the animal protein-. Manufactured by going through a step of preparing an electrolyte solution (step B) and a step (C) of heating the animal protein-electrolyte solution obtained in the step (B) at 72 to 95 ° C. for 2 to 60 minutes. can do.
　以下、工程（Ａ）～工程（Ｃ）を説明する。なお、前記ナノ粒子と重複する成分については、詳細な説明を省略する。 The steps (A) to (C) will be described below. The detailed description of the component overlapping with the nanoparticles will be omitted.
〔工程（Ａ）〕
　本工程（Ａ）では、ホエイタンパク質、ゼラチン、コラーゲン、及びこれらの分解物からなる群より選ばれる少なくとも１種の動物性タンパク質を溶媒に溶解、分散又は膨潤させて、動物性タンパク質を均一に含む動物性タンパク質含有溶液又は膨潤液（動物性タンパク質液という）を調製する。 [Step (A)]
In this step (A), at least one animal protein selected from the group consisting of whey protein, gelatin, collagen, and decomposition products thereof is dissolved, dispersed, or swollen in a solvent to uniformly contain the animal protein. Prepare an animal protein-containing solution or swelling solution (referred to as animal protein solution).
　本工程（Ａ）で用いる動物性タンパク質については、前記電解質とコアセルベートを形成可能なホエイタンパク質、ゼラチン、コラーゲン、及びこれらの分解物であればよい。 The animal protein used in this step (A) may be whey protein, gelatin, collagen, or a decomposition product thereof that can form a coacervate with the electrolyte.
　本工程（Ａ）において使用される溶媒とは、水、有機溶媒、又は含水有機溶媒が挙げられる。ここで、含水有機溶媒とは、水と有機溶媒の混合溶媒である。
　前記有機溶媒としては、水と混和するものであれば特に限定はされないが、得られたナノ粒子の使用用途に適した溶媒を選択することが好ましく、例えば、食品用途に適した溶媒としてはグリセリン、プロピレングリコール、エタノール等が挙げられ、医薬品用途に適した溶媒としては上記に加えてメタノール、アセトン、ジメチルスルホキシド等が挙げられる。 Examples of the solvent used in this step (A) include water, an organic solvent, and a hydrous organic solvent. Here, the hydrous organic solvent is a mixed solvent of water and an organic solvent.
The organic solvent is not particularly limited as long as it is compatible with water, but it is preferable to select a solvent suitable for the intended use of the obtained nanoparticles. For example, glycerin is a solvent suitable for food use. , Propylene glycol, ethanol and the like, and examples of the solvent suitable for pharmaceutical use include methanol, acetone, dimethyl sulfoxide and the like in addition to the above.
　前記溶媒に前記動物性タンパク質を溶解、分散又は膨潤させる手段としては、公知の手段であれば特に限定はない。例えば、前記動物性タンパク質を、前記溶媒に添加・混合することで、溶解、分散又は膨潤させることができる。
　なお、膨潤とは、動物性タンパク質に溶媒を添加してゲル状にすることをいう。
　また、前記溶解、分散又は膨潤させる際には、効率的に溶解、分散又は膨潤させる観点から、前記溶媒の温度を２０～９０℃に調整しておくことが好ましい。
　なお、前記動物性タンパク質と前記溶媒との混合液中において前記動物性タンパク質の状態としては、完全に溶解又は膨潤していてもよいし、溶解又は膨潤していない一部が分散していてもよい。
　また、前記混合時には、動物性タンパク質液を撹拌することで、効率よく混合することができる。
　なお、本発明において「撹拌」とは、容器内の内容物が混合されることを意味する。具体的には、撹拌は、撹拌棒等を使用して手動で行なってもよいし、マグネチックスターラやミキサーなどの攪拌機を使用して行ってもよい。 The means for dissolving, dispersing or swelling the animal protein in the solvent is not particularly limited as long as it is a known means. For example, the animal protein can be dissolved, dispersed or swollen by adding and mixing with the solvent.
In addition, swelling means adding a solvent to an animal protein to form a gel.
Further, when dissolving, dispersing or swelling, it is preferable to adjust the temperature of the solvent to 20 to 90 ° C. from the viewpoint of efficiently dissolving, dispersing or swelling.
The state of the animal protein in the mixed solution of the animal protein and the solvent may be completely dissolved or swollen, or a part of the animal protein that is not dissolved or swollen may be dispersed. Good.
Further, at the time of the mixing, the animal protein solution can be efficiently mixed by stirring.
In the present invention, "stirring" means that the contents in the container are mixed. Specifically, stirring may be performed manually using a stirring rod or the like, or may be performed using a stirrer such as a magnetic stirrer or a mixer.
　前記動物性タンパク質液中の動物性タンパク質の固形分値は、平均粒子径１０～３００ｎｍのナノ粒子を効率的に作製する観点から、０．１～１９重量％であることが好ましく、０．１～１０重量％であることがより好ましいが、所望のナノ粒子が作製できれば、特に限定されることはない。例えば、ホエイタンパク質を使用する場合、動物性タンパク質液中のホエイタンパク質の固形分値は、好ましくは０．１～７％重量％、より好ましくは０．２～６重量％、さらに好ましくは０．２～５重量％である。また、ゼラチンを使用する場合、前記固形分値が２０重量％以上であれば液の粘度の上昇により扱いにくくなるので、１９重量％以下が好ましい。また、コラーゲンを使用する場合、０．１～０．３重量％が好ましい。 The solid content value of the animal protein in the animal protein solution is preferably 0.1 to 19% by weight, preferably 0.1 to 19% by weight, from the viewpoint of efficiently producing nanoparticles having an average particle diameter of 10 to 300 nm. It is more preferably about 10% by weight, but it is not particularly limited as long as desired nanoparticles can be produced. For example, when whey protein is used, the solid content value of whey protein in the animal protein solution is preferably 0.1 to 7% by weight, more preferably 0.2 to 6% by weight, and even more preferably 0. It is 2 to 5% by weight. Further, when gelatin is used, if the solid content value is 20% by weight or more, it becomes difficult to handle due to an increase in the viscosity of the liquid, so 19% by weight or less is preferable. When collagen is used, it is preferably 0.1 to 0.3% by weight.
〔工程（Ｂ）〕
　本工程（Ｂ）では、前記工程（Ａ）で得られた前記動物性タンパク質液に、前記動物性タンパク質の固形分重量１に対し、電解質を固形分重量０．００１～１.０の割合で加えて、動物性タンパク質－電解質液を調製する。
　また、前記電解質としてカテキンを用いる場合、カテキンの固形分重量は、０．００１～０．２の割合で加えるのが好ましい。
　上記のように、前記動物性タンパク質の固形分重量に対するカテキンを固形分重量を顕著に減らしたことで、従来の方法において、ブロードになりがちな粒度分布が、意外にもシャープな粒度分布となり、粒子サイズの揃ったナノ粒子を効率よく得ることができる（図１）。
　なお、図１において、シャープな粒度分布を示している曲線が本発明のナノ粒子であり、ブロードな粒度分布を示している曲線が従来のカテキン量の多い条件化で調製したナノ粒子である。 [Step (B)]
In this step (B), in the animal protein solution obtained in the step (A), an electrolyte is added at a ratio of 0.001 to 1.0 solid content weight to 1 solid content weight of the animal protein. In addition, an animal protein-electrolyte solution is prepared.
When catechin is used as the electrolyte, the solid content weight of catechin is preferably added at a ratio of 0.001 to 0.2.
As described above, by significantly reducing the solid content weight of catechin relative to the solid content weight of the animal protein, the particle size distribution that tends to be broad in the conventional method becomes a surprisingly sharp particle size distribution. Nanoparticles with the same particle size can be efficiently obtained (Fig. 1).
In FIG. 1, the curve showing a sharp particle size distribution is the nanoparticles of the present invention, and the curve showing a broad particle size distribution is the nanoparticles prepared under the conventional conditions with a large amount of catechins.
　本工程（Ｂ）において添加される電解質の量は、目的の粒子サイズにナノ粒子の作製を効率よく行う観点から、前記動物性タンパク質の固形分重量１に対し、例えば、下限は０．００１以上が好ましく、０．０１以上がより好ましく、０．０２以上がさらに好ましく、また上限は１．５以下が好ましく、１．２以下がより好ましく、０．５以下がさらに好ましく、０．２以下がさらに好ましく、０．１５以下がさらに好ましく、０．１２５以下がさらに好ましく、０．１以下がさらに好ましい。
　より詳細には、前記電解質として、カテキンを用いる場合には、粒度分布のシャープなナノ粒子の作製を効率よく行う観点から、前記動物性タンパク質の固形分重量１に対し、カテキンを固形分重量として、０．００１～０．２、好ましくは０．００１～０．１５、より好ましくは０．０２～０．１２５、さらに好ましくは０．０３～０．１である。
　また、前記電解質として、クエン酸三カリウム、クエン酸三ナトリウム等の有機酸塩を用いる場合には、０．００１～０．５がより好ましく、０．００１～０．２がさらに好ましい。
　また、前記電解質として、塩化カリウム、塩化ナトリウム等の無機塩を用いる場合には、０．００１～０．２がより好ましく、０．００１～０．１がさらに好ましい。
　また、前記電解質として、乳酸、コハク酸、フマル酸、及びクエン酸等の有機酸を用いる場合には、０．００１～１．５がより好ましく、０．０１～１．２がさらに好ましい。 The amount of electrolyte added in this step (B) has a lower limit of 0.001 or more with respect to the solid content weight of 1 of the animal protein from the viewpoint of efficiently producing nanoparticles having a target particle size. Is preferable, 0.01 or more is more preferable, 0.02 or more is further preferable, and the upper limit is preferably 1.5 or less, more preferably 1.2 or less, further preferably 0.5 or less, and 0.2 or less. More preferably, 0.15 or less is further preferable, 0.125 or less is further preferable, and 0.1 or less is further preferable.
More specifically, when catechin is used as the electrolyte, catechin is used as the solid content weight with respect to 1 solid content weight of the animal protein from the viewpoint of efficiently producing nanoparticles having a sharp particle size distribution. , 0.001 to 0.2, preferably 0.001 to 0.15, more preferably 0.02 to 0.125, still more preferably 0.03 to 0.1.
Further, when an organic acid salt such as tripotassium citrate or trisodium citrate is used as the electrolyte, 0.001 to 0.5 is more preferable, and 0.001 to 0.2 is further preferable.
When an inorganic salt such as potassium chloride or sodium chloride is used as the electrolyte, 0.001 to 0.2 is more preferable, and 0.001 to 0.1 is even more preferable.
Further, when an organic acid such as lactic acid, succinic acid, fumaric acid, and citric acid is used as the electrolyte, 0.001 to 1.5 is more preferable, and 0.01 to 1.2 is further preferable.
　前記電解質を添加する際の条件としては、２０～５０℃に調整しておくことが好ましい。
　また、電解質を添加する際には、動物性タンパク質液を撹拌していてもよいし、静置していてもよい。 The conditions for adding the electrolyte are preferably adjusted to 20 to 50 ° C.
Further, when the electrolyte is added, the animal protein solution may be agitated or allowed to stand.
　また、本発明においては、本工程（Ｂ）を実施するかわりに、前記工程（Ａ）で使用する溶媒中に、所定量の電解質を添加することで、工程（Ａ）と工程（Ｂ）とを同時に実施してもよい。この場合、前記動物性タンパク質及び電解質を溶媒に添加して、前記動物性タンパク質を溶解、分散又は膨潤させて、動物性タンパク質を均一に含み、かつ電解質を含有する動物性タンパク質－電解質液を調製する。 Further, in the present invention, instead of carrying out the main step (B), by adding a predetermined amount of electrolyte to the solvent used in the step (A), the steps (A) and the step (B) are combined. May be carried out at the same time. In this case, the animal protein and the electrolyte are added to the solvent to dissolve, disperse or swell the animal protein to prepare an animal protein-electrolyte solution that uniformly contains the animal protein and contains the electrolyte. To do.
〔工程（Ｃ）〕
　本工程（Ｃ）では、前記工程（Ｂ）で得られた動物性タンパク質－電解質液を７２～９５℃で、２～６０分間加熱する。
　本工程（Ｃ）では、平均粒子径１０～３００ｎｍのナノ粒子を効率よく得る観点から、７２～９５℃で２～６０分間、好ましくは７２～９０℃で２～６０分間、より好ましくは７２～８５℃で２～６０分間、さらに好ましくは７２～８０℃で２～６０分間処理することが望ましい。
　なお、前記の加熱処理は、前記動物性タンパク質－電解質液を撹拌しながら行うことが好ましい。 [Step (C)]
In this step (C), the animal protein-electrolyte solution obtained in the step (B) is heated at 72 to 95 ° C. for 2 to 60 minutes.
In this step (C), from the viewpoint of efficiently obtaining nanoparticles having an average particle diameter of 10 to 300 nm, the temperature is 72 to 95 ° C. for 2 to 60 minutes, preferably 72 to 90 ° C. for 2 to 60 minutes, and more preferably 72 to. It is desirable to treat at 85 ° C. for 2 to 60 minutes, more preferably at 72 to 80 ° C. for 2 to 60 minutes.
The heat treatment is preferably carried out while stirring the animal protein-electrolyte solution.
　また、本工程（Ｃ）では、ナノ粒子の粒子径を所望の範囲に調整する観点から、前記動物性タンパク質－電解質液のｐＨは、酸性側又はよりアルカリ性側に調整することが好ましい。
　例えば、酸性側のｐＨとしては、好ましくは１．０～２．５、より好ましくは１．５～２．２、さらに好ましくは１．７～２．０である。
　また、アルカリ性側のｐＨとしては、好ましくは５．０～８．０、より好ましくは５．５～７．０、さらに好ましくは６．０～６．８である。
　なお、ｐＨが１．０未満又は３．９付近では粒子径が大きくなったり、ナノ粒子が溶解して、得られる量が減少したりする傾向がある。
　なお、前記ｐＨの調整については、市販のｐＨ調整剤を用いて行えばよく、ｐＨ調整剤の種類については特に限定はない。 Further, in this step (C), from the viewpoint of adjusting the particle size of the nanoparticles to a desired range, it is preferable to adjust the pH of the animal protein-electrolyte solution to the acidic side or the more alkaline side.
For example, the pH on the acidic side is preferably 1.0 to 2.5, more preferably 1.5 to 2.2, and even more preferably 1.7 to 2.0.
The pH on the alkaline side is preferably 5.0 to 8.0, more preferably 5.5 to 7.0, and even more preferably 6.0 to 6.8.
When the pH is less than 1.0 or around 3.9, the particle size tends to increase, or the nanoparticles tend to dissolve and the amount obtained tends to decrease.
The pH may be adjusted by using a commercially available pH adjusting agent, and the type of the pH adjusting agent is not particularly limited.
〔工程（Ｄ）〕
　本発明の製造方法では、生理活性物質をナノ粒子に担持させるために、水又は有機溶媒又は含水有機溶媒に生理活性物質を溶解させた生理活性物質含有溶液を、前記工程（Ａ）～工程（Ｃ）の各調製段階において、添加することができる。
　本工程（Ｄ）を実施するタイミングとしては、特に限定はなく、例えば、工程（Ａ）と工程（Ｂ）との間、工程（Ｂ）と工程（Ｃ）との間、又は工程（Ｃ）の後のいずれかのタイミングで実施することができる。また、前記工程（Ａ）と工程（Ｂ）と前記工程（Ｃ）とを同時に実施してもよい。 [Step (D)]
In the production method of the present invention, in order to support the bioactive substance on the nanoparticles, a physiologically active substance-containing solution in which the bioactive substance is dissolved in water or an organic solvent or a hydrous organic solvent is subjected to the steps (A) to (steps) (A) to (step). It can be added at each preparation stage of C).
The timing of carrying out this step (D) is not particularly limited, and for example, between steps (A) and step (B), between steps (B) and step (C), or step (C). It can be carried out at any time after. Further, the step (A), the step (B), and the step (C) may be performed at the same time.
　前記溶媒に生理活性物質を溶解させる手段としては、公知の手段であれば特に限定はない。例えば、生理活性物質を、前記溶媒に添加・混合することで、溶解させることができる。 The means for dissolving the physiologically active substance in the solvent is not particularly limited as long as it is a known means. For example, it can be dissolved by adding and mixing a physiologically active substance to the solvent.
　前記生理活性物質含有溶液中の生理活性物質の固形分値は、平均粒子径１０～３００ｎｍのナノ粒子を効率的に作製する観点から、０．００１～１０重量％であることが好ましいが、所望のナノ粒子が作製できれば、特に限定されることはない。 The solid content value of the physiologically active substance in the physiologically active substance-containing solution is preferably 0.001 to 10% by weight, preferably 0.001 to 10% by weight, from the viewpoint of efficiently producing nanoparticles having an average particle diameter of 10 to 300 nm. As long as the nanoparticles of the above can be produced, there is no particular limitation.
　さらに、本工程（Ｄ）を実施する場合、生理活性物質の凝集や沈殿などを防ぐ目的で、安定剤、乳化剤、タンパク質結合剤等を加えてもよい。 Further, when carrying out this step (D), a stabilizer, an emulsifier, a protein binder, or the like may be added for the purpose of preventing aggregation or precipitation of the physiologically active substance.
　以上の製造方法により調製された本発明のナノ粒子は、さらに製剤化してもよい。この製剤形態としては特に限定されず、例えば、錠剤、被覆錠剤、散剤、細粒剤、顆粒剤、カプセル剤、液剤、丸剤、懸濁剤、乳剤、トローチ剤、チュアブル錠、シロップ剤等の経口剤等が挙げられる。製剤化の際には、担体、賦形剤、滑沢剤、結合剤、崩壊剤、希釈剤、安定化剤、等張化剤、ｐＨ調整剤、緩衝剤等が用いられる。 The nanoparticles of the present invention prepared by the above production method may be further formulated. The formulation form is not particularly limited, and for example, tablets, coated tablets, powders, fine granules, granules, capsules, liquids, pills, suspensions, emulsions, troches, chewable tablets, syrups and the like. Oral preparations and the like can be mentioned. At the time of formulation, carriers, excipients, lubricants, binders, disintegrants, diluents, stabilizers, tonicity agents, pH adjusters, buffers and the like are used.
　担体や賦形剤としては、例えば、乳糖、ショ糖、塩化ナトリウム、ブドウ糖、マルトース、マンニトール、エリスリトール、キシリトール、マルチトール、イノシトール、デキストラン、ソルビトール、アルブミン、尿素、デンプン、炭酸カルシウム、カオリン、結晶セルロース、ケイ酸、メチルセルロース、グリセリン、アルギン酸ナトリウム、アラビアゴム及びこれらの混合物等が挙げられる。 Examples of carriers and excipients include lactose, sucrose, sodium chloride, glucose, maltose, mannitol, erythritol, xylitol, maltitol, inositol, dextran, sorbitol, albumin, urea, starch, calcium carbonate, kaolin and crystalline cellulose. , Maltitol, methylcellulose, glycerin, sodium alginate, gum arabic and mixtures thereof.
　滑沢剤としては、例えば、精製タルク、ステアリン酸塩、ホウ砂、ポリエチレングリコール及びこれらの混合物等が挙げられる。
　結合剤としては、例えば、単シロップ、ブドウ糖液、デンプン液、ゼラチン溶液、ポリビニルアルコール、ポリビニルエーテル、ポリビニルピロリドン、カルボキシメチルセルロース、セラック、メチルセルロース、エチルセルロース、水、エタノール、リン酸カリウム及びこれらの混合物等が挙げられる。 Examples of the lubricant include purified talc, stearate, borax, polyethylene glycol and a mixture thereof.
Examples of the binder include simple syrup, glucose solution, starch solution, gelatin solution, polyvinyl alcohol, polyvinyl ether, polyvinylpyrrolidone, carboxymethyl cellulose, cellac, methyl cellulose, ethyl cellulose, water, ethanol, potassium phosphate and a mixture thereof. Can be mentioned.
　崩壊剤としては、例えば、乾燥デンプン、アルギン酸ナトリウム、カンテン末、ラミナラン末、炭酸水素ナトリウム、炭酸カルシウム、ポリオキシエチレンソルビタン脂肪酸エステル類、ラウリル硫酸ナトリウム、ステアリン酸モノグリセリド、デンプン、乳糖及びこれらの混合物等が挙げられる。 Examples of the disintegrant include dried starch, sodium alginate, canten powder, laminarin powder, sodium hydrogen carbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid monoglyceride, starch, lactose and mixtures thereof. Can be mentioned.
　希釈剤としては、例えば、水、エチルアルコール、マクロゴール、プロピレングリコール、エトキシ化イソステアリルアルコール、ポリオキシ化イソステアリルアルコール、ポリオキシエチレンソルビタン脂肪酸エステル類及びこれらの混合物等が挙げられる。
　安定化剤としては、例えば、ピロ亜硫酸ナトリウム、エチレンジアミン四酢酸、チオグリコール酸、チオ乳酸及びこれらの混合物等が挙げられる。 Examples of the diluent include water, ethyl alcohol, macrogol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitan fatty acid esters, and mixtures thereof.
Examples of the stabilizer include sodium pyrosulfite, ethylenediaminetetraacetic acid, thioglycolic acid, thiolactic acid, and mixtures thereof.
　等張化剤としては、例えば、塩化ナトリウム、ホウ酸、ブドウ糖、グリセリン及びこれらの混合物等が挙げられる。ｐＨ調整剤及び緩衝剤としては、例えば、クエン酸ナトリウム、クエン酸、酢酸ナトリウム、リン酸ナトリウム及びこれらの混合物等が挙げられる。 Examples of the tonicity agent include sodium chloride, boric acid, glucose, glycerin and a mixture thereof. Examples of the pH adjuster and buffer include sodium citrate, citric acid, sodium acetate, sodium phosphate and a mixture thereof.
　以上のようにして得られる本発明のナノ粒子は、食品に利用可能な条件（具体的には、食品に利用可能な溶媒等を用いた場合）で作製した場合は、飲食品に配合してもよい。飲食品としては特に限定されず、例えば、飲料、アルコール飲料、ゼリー、菓子、機能性食品、健康食品、健康志向食品等が挙げられる。保存性、携帯性、摂取の容易さ等を考慮すると、菓子類が好ましく、菓子類の中でも、ハードキャンディ、ソフトキャンディ、グミキャンディ、タブレット、チューイングガム等が好ましい。 When the nanoparticles of the present invention obtained as described above are produced under conditions that can be used for food (specifically, when a solvent that can be used for food is used), they are blended with food and drink. May be good. Foods and drinks are not particularly limited, and examples thereof include beverages, alcoholic beverages, jellies, confectionery, functional foods, health foods, and health-oriented foods. Considering storage stability, portability, ease of ingestion, etc., confectionery is preferable, and among confectionery, hard candy, soft candy, gummy candy, tablet, chewing gum and the like are preferable.
　本発明のナノ粒子を飲食品に配合する場合、本発明のナノ粒子の飲食品における含有量は、その生理活性効果が期待できる量であればよい。通常１日あたり１０～１００００ｍｇ、より好ましくは１００～３０００ｍｇ摂取できるように配合量を決定することが好ましい。例えば、固形状食品の場合には５～５０重量％、飲料等の液状食品の場合には０．０１～１０重量％が好ましい。 When the nanoparticles of the present invention are blended in foods and drinks, the content of the nanoparticles of the present invention in foods and drinks may be an amount that can be expected to have a bioactive effect. It is preferable to determine the blending amount so that 10 to 10000 mg, more preferably 100 to 3000 mg per day can be ingested. For example, in the case of solid foods, 5 to 50% by weight is preferable, and in the case of liquid foods such as beverages, 0.01 to 10% by weight is preferable.
　また、本発明のナノ粒子は、非ヒト動物、例えば、ラット、マウス、モルモット、ウサギ、ヒツジ、ブタ、ウシ、ウマ、ネコ、イヌ、サル、チンパンジー等の哺乳類、鳥類、両生類、爬虫類等の治療剤又は飼料に配合してもよい。飼料としては、例えばヒツジ、ブタ、ウシ、ウマ、ニワトリ等に用いる家畜用飼料、ウサギ、ラット、マウス等に用いる小動物用飼料、ウナギ、タイ、ハマチ、エビ等に用いる魚介類用飼料、イヌ、ネコ、小鳥、リス等に用いるペットフードが挙げられる。 In addition, the nanoparticles of the present invention treat non-human animals such as rats, mice, guinea pigs, rabbits, sheep, pigs, cows, horses, cats, dogs, monkeys, chimpanzees and other mammals, birds, amphibians and reptiles. It may be added to an agent or feed. The feed includes, for example, livestock feed used for sheep, pigs, cows, horses, chickens, etc., small animal feed used for rabbits, rats, mice, etc., seafood feed used for eels, Thailand, hamachi, shrimp, etc., dogs, etc. Examples include pet food used for cats, small birds, and squirrels.
　本発明のナノ粒子は医薬品に配合してもよい。前記医薬品としては、散剤、錠剤、丸剤、カプセル剤、細粒剤、顆粒剤等の固形製剤、水剤、懸濁剤、乳剤等の液剤、ゲル剤等が挙げられる。錠剤、丸剤、顆粒剤、顆粒を含有するカプセル剤の顆粒は、必要により、ショ糖等の糖類、マルチトール等の糖アルコールで糖衣を施したり、ゼラチン、ヒドロキシプロピルセルロース、ヒドロキシプロピルメチルセルロース等でコーティングを施したりすることもできる。または胃溶性若しくは腸溶性物質のフィルムで被覆してもよい。また、製剤の溶解性を向上させるために、公知の可溶化処理を施すこともできる。常法に基づいて、注射剤、点滴剤に配合して使用してもよい。 The nanoparticles of the present invention may be blended in a pharmaceutical product. Examples of the pharmaceuticals include solid preparations such as powders, tablets, pills, capsules, fine granules and granules, liquid preparations such as liquid preparations, suspensions and emulsions, gel preparations and the like. If necessary, the granules of tablets, pills, granules, and capsules containing granules may be coated with sugars such as sucrose and sugar alcohols such as maltitol, or with gelatin, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, etc. It can also be coated. Alternatively, it may be coated with a film of a gastric or enteric substance. Further, in order to improve the solubility of the preparation, a known solubilization treatment can be applied. It may be mixed with an injection or an infusion according to a conventional method.
　本発明のナノ粒子を医薬用途で使用する場合、例えば、その摂取量は、所望の改善、治療又は予防効果が得られるような量であれば特に制限されず、通常その態様、患者の年齢、性別、体質その他の条件、疾患の種類並びにその程度等に応じて適宜選択される。１日当たり約０．１ｍｇ～１，０００ｍｇ程度とするのがよく、これを１日に１～４回に分けて摂取することができる。 When the nanoparticles of the present invention are used for pharmaceutical purposes, for example, the intake thereof is not particularly limited as long as the desired improvement, therapeutic or preventive effect can be obtained, and usually the embodiment, the age of the patient, and the like. It is appropriately selected according to the sex, constitution and other conditions, the type of disease and the degree thereof. It is preferably about 0.1 mg to 1,000 mg per day, and this can be ingested in 1 to 4 divided doses per day.
　本発明のナノ粒子は医薬部外品に配合してもよい。前記医薬部外品としては、口腔に用いられる医薬部外品、例えば、歯磨き、マウスウオッシュ、マウスリンスや、感染症予防等を目的とした滋養強壮系ドリンク剤等が挙げられる。
　本発明のナノ粒子を医薬部外品に添加する場合には、該医薬部外品中に、通常０．００１～３０重量％添加するのが好ましい。 The nanoparticles of the present invention may be blended in quasi-drugs. Examples of the quasi-drugs include quasi-drugs used in the oral cavity, such as toothpaste, mouthwash, and mouth rinse, and nutritional tonic drinks for the purpose of preventing infectious diseases.
When the nanoparticles of the present invention are added to a quasi-drug, it is usually preferable to add 0.001 to 30% by weight in the quasi-drug.
　次に、本発明を実施例に基づいて詳細に説明するが、本発明はかかる実施例にのみ限定されるものではない。 Next, the present invention will be described in detail based on examples, but the present invention is not limited to such examples.
〔実施例ａ－１〕カテキン添加量を抑えた条件化でのナノ粒子の製造（試験検体の製造）
　まず、蒸留水９５ｇにホエイタンパク質（商品名：エンラクトＳＡＴ、タンパク質含量８９％、日本新薬株式会社製）５ｇを加え、２０～３０℃（室温）でよく撹拌することで１００ｇのホエイタンパク質水溶液を調製した。（工程（Ａ））
　次いで、得られたホエイタンパク質水溶液に緑茶抽出物（商品名：ＥＧＣｇ－ＯＰ、ＥＧＣｇ含量 ９４％以上、太陽化学製）を２０ｍｇ～３２０ｍｇを２０～３０℃（室温）で添加した（混合溶液中のＥＧＣｇ濃度：０．０２％～０．３２％）。（工程（Ｂ））
　次いで、得られた混合液を、ｐＨ調整液を用いてｐＨ６～７に調整し、緩やかに攪拌しながら、８０℃で３０分間加熱してナノ粒子を形成させた。（工程（Ｃ））
　得られたナノ粒子の平均粒子径をゼータ電位・ナノ粒子径測定システム（ベックマン・コールター株式会社製、「ＤｅｌｓａＭａｘ  ＰＲＯ」）を用いて測定した。 [Example a-1] Production of nanoparticles under conditions in which the amount of catechin added is suppressed (production of test specimen)
First, add 5 g of whey protein (trade name: Enlacto SAT, protein content 89%, manufactured by Nippon Shinyaku Co., Ltd.) to 95 g of distilled water, and stir well at 20 to 30 ° C. (room temperature) to prepare a 100 g whey protein aqueous solution. did. (Step (A))
Next, 20 mg to 320 mg of green tea extract (trade name: EGCg-OP, EGCg content of 94% or more, manufactured by Taiyo Kagaku) was added to the obtained whey protein aqueous solution at 20 to 30 ° C. (room temperature) (in a mixed solution). EGCg concentration: 0.02% to 0.32%). (Step (B))
Next, the obtained mixed solution was adjusted to pH 6 to 7 with a pH adjusting solution, and heated at 80 ° C. for 30 minutes with gentle stirring to form nanoparticles. (Step (C))
The average particle size of the obtained nanoparticles was measured using a zeta potential / nanoparticle size measurement system (“DelsaMax PRO” manufactured by Beckman Coulter, Inc.).
〔比較例ａ－１〕　従来のカテキン添加量でのナノ粒子の製造（対照検体の製造）
　前記と同様に、蒸留水９５ｇにホエイタンパク質（商品名：エンラクトＳＡＴ、タンパク質含量８９％、日本新薬株式会社製）５ｇを加え、よく撹拌することで１００ｇのホエイタンパク質水溶液を調製した。（工程（Ａ））
　次いで、得られたホエイタンパク質水溶液に緑茶抽出物（商品名：ＥＧＣｇ－ＯＰ、ＥＧＣｇ含量 ９４％以上、太陽化学製）を２．５ｇ～１２．５ｇを添加した（溶液中のＥＧＣｇ濃度：２．５％～１２．５％）。
　次いで、得られた混合液を、緩やかに攪拌しながら８０℃で３０分間加熱してナノ粒子を形成させた。
　得られたナノ粒子の平均粒子径をゼータ電位・ナノ粒子径測定システム（ベックマン・コールター株式会社製、「ＤｅｌｓａＭａｘ  ＰＲＯ」）を用いて測定した。 [Comparative Example a-1] Production of nanoparticles with a conventional amount of catechin added (Production of control sample)
In the same manner as described above, 5 g of whey protein (trade name: Enlacto SAT, protein content 89%, manufactured by Nippon Shinyaku Co., Ltd.) was added to 95 g of distilled water, and 100 g of a whey protein aqueous solution was prepared by stirring well. (Step (A))
Next, 2.5 g to 12.5 g of green tea extract (trade name: EGCg-OP, EGCg content of 94% or more, manufactured by Taiyo Kagaku) was added to the obtained whey protein aqueous solution (EGCg concentration in the solution: 2. 5% to 12.5%).
Next, the obtained mixed solution was heated at 80 ° C. for 30 minutes with gentle stirring to form nanoparticles.
The average particle size of the obtained nanoparticles was measured using a zeta potential / nanoparticle size measurement system (“DelsaMax PRO” manufactured by Beckman Coulter, Inc.).
〔実施例ａ－２〕　苦渋味の改善効果の評価
　実施例ａ－１で調製したナノ粒子を、従来の製造方法により比較例ａ－１で調製したナノ粒子を比較対象として、苦渋味をパネラー１０名により、苦渋味の官能評価を実施した。評価基準は表１の通りである。 [Example a-2] Evaluation of effect of improving bitterness and astringency The nanoparticles prepared in Example a-1 are compared with the nanoparticles prepared in Comparative Example a-1 by a conventional production method, and the bitterness and astringency are panelized. A sensory evaluation of bitterness and astringency was carried out by 10 people. The evaluation criteria are as shown in Table 1.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　実施例ａ－１及び比較例ａ－１において調製したナノ粒子の粒子サイズを複数回計測した結果及び実施例ａ－２において実施した官能試験の結果を表２に示した。 Table 2 shows the results of measuring the particle size of the nanoparticles prepared in Example a-1 and Comparative Example a-1 multiple times and the results of the sensory test conducted in Example a-2.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　表２に示した通り、実施例ａ－１で得られた本発明のナノ粒子は、比較例ａ－１（従来法）の場合に比べ、カテキン添加量を顕著に抑えているにもかかわらず、従来法と同様の粒子サイズを備え、かつ苦渋味が顕著に抑えられたものであることがわかる。 As shown in Table 2, the nanoparticles of the present invention obtained in Example a-1 remarkably suppress the amount of catechin added as compared with the case of Comparative Example a-1 (conventional method). It can be seen that the particles have the same particle size as the conventional method and the bitterness and astringency are remarkably suppressed.
〔実施例ａ－３〕　カテキン添加量を減らした場合の粒子サイズ分布の検証
　比較例ａ－１及び実施例ａ－１において示した手順で調製したナノ粒子の粒子サイズ分布のゼータ電位・ナノ粒子径測定システム（ベックマン・コールター株式会社製、「ＤｅｌｓａＭａｘ  ＰＲＯ」）を用いて粒子径２，０００ｎｍ以下の範囲について測定し、代表的な測定結果をそれぞれ図２及び図３に示した。図中の横軸は半径を表しているので、横軸の数値を２倍した数値が粒子径となる。
　図２は従来の高濃度のカテキン条件化（９．１％）で調製したナノ粒子を、図３はカテキン量を減らした条件下（０．０８％）で調製したナノ粒子の粒子サイズ分布を示している。
　なお、図２の比較例ａ－１で得られたナノ粒子では、粒子径が２０～４００ｎｍの範囲にブロードは範囲にわたっていた。
　これに対して、図３の実施例ａ－１で得られたナノ粒子では、粒子径が７０ｎｍを中心にその前後２０ｎｍの範囲内に９９％以上が存在する粒度分布となっていた。
　したがって、図２、３から明らかなように、動物性タンパク質に対するカテキン量を特定の範囲に調整することで、カテキンの含有量が低くても粒度の揃った高品質のナノ粒子を製造できることが明らかとなった。 [Example a-3] Verification of particle size distribution when the amount of catechin added is reduced Zeta potential / nanoparticles of particle size distribution of nanoparticles prepared by the procedure shown in Comparative Example a-1 and Example a-1 A diameter measurement system (“DelsaMax PRO” manufactured by Beckman Coulter Co., Ltd.) was used to measure the particle size in the range of 2,000 nm or less, and typical measurement results are shown in FIGS. 2 and 3, respectively. Since the horizontal axis in the figure represents the radius, the value obtained by doubling the value on the horizontal axis is the particle diameter.
FIG. 2 shows the particle size distribution of the nanoparticles prepared under the conventional high-concentration catechin condition (9.1%), and FIG. 3 shows the particle size distribution of the nanoparticles prepared under the condition of reducing the amount of catechin (0.08%). Shown.
In the nanoparticles obtained in Comparative Example a-1 of FIG. 2, the broad range was in the range of 20 to 400 nm in particle diameter.
On the other hand, in the nanoparticles obtained in Example a-1 of FIG. 3, the particle size distribution was such that 99% or more existed in the range of 20 nm before and after the particle diameter centered on 70 nm.
Therefore, as is clear from FIGS. 2 and 3, it is clear that by adjusting the amount of catechin with respect to animal protein to a specific range, high-quality nanoparticles having uniform particle size can be produced even if the content of catechin is low. It became.
〔実施例ａ－４〕　他物質との反応性が高い生理活性物質を担持したナノ粒子の製造方法
　まず、蒸留水９５ｇにホエイタンパク質（商品名：エンラクトＳＡＴ、タンパク質含量８９％、日本新薬株式会社製）５ｇを加え、室温でよく撹拌することで１００ｇのホエイタンパク質水溶液を調製した。
　次いで、得られたホエイタンパク質水溶液に緑茶抽出物（商品名：ＥＧＣｇ－ＯＰ、ＥＧＣｇ含量 ９４％以上、太陽化学製）を８０ｍｇを室温で添加した（混合溶液中のＥＧＣｇ濃度：０．０８％）。
次いで、得られた混合液に、０．１ｇのアスタキサンチン（Ａｓｔａｂｉｏ　ＡＰ１）或いは０．１ｇの還元型コエンザイムＱ１０（カネカ製）を５ｇエタノールに溶解した還元型ＣｏＱ１０溶液を添加し、ホエイタンパク質－カテキン－生理活性物質混合液を得た。
　次いで、得られた混合液を、ｐＨ調整液を用いてｐＨ６～７に調整し、緩やかに攪拌しながら、８０℃で３０分間加熱してナノ粒子を形成させた。
　得られたナノ粒子の平均粒子径は、ゼータ電位・ナノ粒子径測定システム（ベックマン・コールター株式会社製、「ＤｅｌｓａＭａｘ  ＰＲＯ」）を用いて測定し、所定の粒子径となっていることを確認した。 [Example a-4] Method for producing nanoparticles carrying a physiologically active substance having high reactivity with other substances First, whey protein (trade name: Enlacto SAT, protein content 89%, Nippon Shinyaku Co., Ltd.) in 95 g of distilled water A 100 g whey protein aqueous solution was prepared by adding 5 g and stirring well at room temperature.
Next, 80 mg of green tea extract (trade name: EGCg-OP, EGCg content of 94% or more, manufactured by Taiyo Kagaku) was added to the obtained whey protein aqueous solution at room temperature (EGCg concentration in the mixed solution: 0.08%). ..
Next, to the obtained mixed solution, a reduced CoQ10 solution prepared by dissolving 0.1 g of astaxanthin (Astabio AP1) or 0.1 g of reduced coenzyme Q10 (manufactured by Kaneka) in 5 g ethanol was added, and whey protein-catechin- A mixture of physiologically active substances was obtained.
Next, the obtained mixed solution was adjusted to pH 6 to 7 with a pH adjusting solution, and heated at 80 ° C. for 30 minutes with gentle stirring to form nanoparticles.
The average particle size of the obtained nanoparticles was measured using a zeta potential / nanoparticle size measurement system (“DelsaMax PRO” manufactured by Beckman Coulter, Inc.), and it was confirmed that the particles had a predetermined particle size. ..
〔実施例ａ－５〕　カフェインを担持したナノ粒子の製造方法
　実施例ａ－４において、他物質との反応性が高い生理活性物質を添加する工程（Ｃ）において、カフェイン１０ｍｇをさらに添加している以外は、実施例ａ－４と同様にしてナノ粒子を製造した。
　カフェインを添加しても、カテキンとカフェインとが複合すること形成される白濁沈殿物の生成が見られなかっことから、得られたナノ粒子には、カテキンと共にカフェインも含有したナノ粒子になっていることがわかった。 [Example a-5] Method for producing nanoparticles carrying caffeine In Example a-4, 10 mg of caffeine was further added in the step (C) of adding a physiologically active substance having high reactivity with other substances. Nanoparticles were produced in the same manner as in Example a-4, except that the nanoparticles were produced.
Even if caffeine was added, the formation of a cloudy precipitate formed by the complexing of catechin and caffeine was not observed. Therefore, the obtained nanoparticles were made into nanoparticles containing caffeine as well as catechin. It turned out that it became.
〔実施例ａ－６〕　生理活性物質の安定性
　実施例ａ－４、ａ－５で得られたナノ粒子を常温、紫外線照射下での虐待試験に供することで、担持されている他物質との反応性が高い生理活性物質の安定性を測定した。対照として、蒸留水９５ｇに０．１ｇのアスタキサンチン（Ａｓｔａｂｉｏ　ＡＰ１）又は０．１ｇの還元型コエンザイムＱ１０（カネカ製）を５ｇエタノールに溶解し液体を添加し、アスタキサンチン溶液又は還元型ＣｏＱ１０溶液を得た。次いで、公知の測定方法に基づいて、アスタキサンチン及び特許第６２８７１２３号公報の試験例１に記載の方法に準じて還元型ＣｏＱ１０の含有量をそれぞれ計測した。
　実施例ａ－４、ａ－５で得られたナノ粒子は、対照品と比べてアスタキサンチン及び還元型ＣｏＱ１０の酸化が有意に抑えられ、安定性に優れたものであることが判明した。 [Example a-6] Stability of bioactive substances By subjecting the nanoparticles obtained in Examples a-4 and a-5 to an abuse test at room temperature and under ultraviolet irradiation, they can be combined with other substances supported. The stability of the highly reactive bioactive substance was measured. As a control, 0.1 g of astaxanthin (Astabio AP1) or 0.1 g of reduced coenzyme Q10 (manufactured by Kaneka Corporation) was dissolved in 5 g of ethanol in 95 g of distilled water, and a liquid was added to obtain an astaxanthin solution or a reduced CoQ10 solution. .. Then, based on a known measuring method, the contents of astaxanthin and reduced CoQ10 were measured according to the method described in Test Example 1 of Japanese Patent No. 6287123.
It was found that the nanoparticles obtained in Examples a-4 and a-5 had significantly suppressed oxidation of astaxanthin and reduced CoQ10 as compared with the control product, and were excellent in stability.
〔実施例ａ－７〕　ナノ粒子を配合したグミキャンディ
　公知の手法に基づいて、実施例ａ－１、ａ－４、ａ－５で得られたナノ粒子を固形分含有量が１０重量％となるように、グミキャンディ液に配合し、固化したところ、得られたグミキャンディのいずれを食べても強い苦味・渋味等は感じられず、摂取しやすかった。 [Example a-7] Gummy candy containing nanoparticles Based on a known method, the nanoparticles obtained in Examples a-1, a-4, and a-5 have a solid content of 10% by weight. When it was mixed with the gummy candy solution and solidified, no strong bitterness or astringency was felt regardless of which of the obtained gummy candy was eaten, and it was easy to ingest.
〔実施例ｂ－１〕有機酸塩を用いたナノ粒子の製造（試験検体の製造）
　ホエイタンパク質（商品名：エンラクトＳＡＴ、タンパク質含量８９％、日本新薬株式会社製）５ｇを、５ｍＭ（０．１６２％）クエン酸三カリウム溶液又は５ｍＭ（０．１４７％）クエン酸三ナトリウム溶液に加えて２０℃で緩やかに攪拌して溶解し、１００ｍｌとした。
　次いで、緩やかに攪拌しながら８０℃、３０分間加熱しナノ粒子を形成させた。
　得られた溶液中のナノ粒子を、ゼータ電位・ナノ粒子径測定システム（ ベックマン・コールター株式会社製、「Ｄｅｌｓａ Ｍａｘ ＰＲＯ」）を用いて粒子径を測定した。 [Example b-1] Production of nanoparticles using an organic acid salt (production of a test sample)
Add 5 g of whey protein (trade name: Enlacto SAT, protein content 89%, manufactured by Nippon Shinyaku Co., Ltd.) to 5 mM (0.162%) tripotassium citrate solution or 5 mM (0.147%) trisodium citrate solution. The mixture was gently stirred at 20 ° C. to dissolve the mixture to 100 ml.
Then, the nanoparticles were formed by heating at 80 ° C. for 30 minutes with gentle stirring.
The nanoparticles in the obtained solution were measured in particle size using a zeta potential / nanoparticle size measurement system (“Delsa Max PRO” manufactured by Beckman Coulter, Inc.).
〔実施例ｂ－２〕無機塩を用いたナノ粒子の製造（試験検体の製造）
　ホエイタンパク質（日本新薬製：エンラクトＳＡＴ）５ｇを、５ｍＭ（０．０３７％）塩化カリウム（ＫＣｌ）溶液又は５ｍＭ（０．０２９％）塩化ナトリウム（ＮａＣｌ）溶液に加えて２０℃で緩やかに攪拌して溶解し、１００ｍｌとした。
　次いで、緩やかに攪拌しながら８０℃、３０分間加熱しナノ粒子を形成させた。
　得られた溶液中のナノ粒子を、ゼータ電位・ナノ粒子径測定システム（ベックマン・コールター株式会社製、「Ｄｅｌｓａ Ｍａｘ ＰＲＯ」）を用いて粒子径を測定した。 [Example b-2] Production of nanoparticles using an inorganic salt (production of a test sample)
Add 5 g of whey protein (Nippon Shinyaku Co., Ltd .: Enlacto SAT) to a 5 mM (0.037%) potassium chloride (KCl) solution or a 5 mM (0.029%) sodium chloride (NaCl) solution and gently stir at 20 ° C. Was dissolved to make 100 ml.
Then, the nanoparticles were formed by heating at 80 ° C. for 30 minutes with gentle stirring.
The nanoparticles in the obtained solution were measured in particle size using a zeta potential / nanoparticle size measurement system (“Delsa Max PRO” manufactured by Beckman Coulter, Inc.).
〔実施例ｂ－３〕有機酸を用いたナノ粒子の製造（試験検体の製造）
　ホエイタンパク質（日本新薬製：エンラクトＳＡＴ）５ｇを、２００ｍＭ（１．８％）乳酸溶液、２００ｍＭ（２．３６％）コハク酸溶液、２００ｍＭ（２．３２％）フマル酸溶液、又は２００ｍＭ（３．８４％）クエン酸溶液に加えて２０℃で緩やかに攪拌して溶解し、１００ｍｌとした。
　次いで、緩やかに攪拌しながら８０℃、３０分間加熱しナノ粒子を形成させた（ここで、ｐＨは２．４に調整した）。
　得られた溶液中のナノ粒子を、ゼータ電位・ナノ粒子径測定システム（ベックマン・コールター株式会社製、「Ｄｅｌｓａ Ｍａｘ ＰＲＯ」）を用いて粒子径を測定した。 [Example b-3] Production of nanoparticles using an organic acid (production of a test sample)
5 g of whey protein (Nippon Shinyaku Co., Ltd .: Enlacto SAT), 200 mM (1.8%) lactic acid solution, 200 mM (2.36%) succinic acid solution, 200 mM (2.32%) fumaric acid solution, or 200 mM (3. 84%) It was added to a citric acid solution and dissolved by gently stirring at 20 ° C. to make 100 ml.
Then, the particles were formed by heating at 80 ° C. for 30 minutes with gentle stirring (where the pH was adjusted to 2.4).
The nanoparticles in the obtained solution were measured in particle size using a zeta potential / nanoparticle size measurement system (“Delsa Max PRO” manufactured by Beckman Coulter, Inc.).
〔比較例ｂ－１〕　従来のカテキン添加量でのナノ粒子の製造（対照検体の製造）
　前記と同様に、蒸留水９５ｇにホエイタンパク質（商品名：エンラクトＳＡＴ、タンパク質含量８９％、日本新薬株式会社製）５ｇを加え、２０℃でよく撹拌することで１００ｇのホエイタンパク質水溶液を調製した。
　次いで、得られたホエイタンパク質水溶液に緑茶抽出物（商品名：ＥＧＣｇ－ＯＰ、ＥＧＣｇ含量 ９４％以上、太陽化学製）を２．５ｇ～１２．５ｇを添加した（溶液中のＥＧＣｇ濃度：２．４％～１１．１％）。
　次いで、得られた混合液を、緩やかに攪拌しながら８０℃で３０分間加熱してナノ粒子を形成させた。
　得られたナノ粒子の平均粒子径をゼータ電位・ナノ粒子径測定システム（ベックマン・コールター株式会社製、「ＤｅｌｓａＭａｘ  ＰＲＯ」）を用いて測定した。 [Comparative Example b-1] Production of nanoparticles with conventional catechin addition amount (production of control sample)
In the same manner as described above, 5 g of whey protein (trade name: Enlacto SAT, protein content 89%, manufactured by Nippon Shinyaku Co., Ltd.) was added to 95 g of distilled water, and the mixture was stirred well at 20 ° C. to prepare a 100 g whey protein aqueous solution.
Next, 2.5 g to 12.5 g of green tea extract (trade name: EGCg-OP, EGCg content of 94% or more, manufactured by Taiyo Kagaku) was added to the obtained whey protein aqueous solution (EGCg concentration in the solution: 2. 4% to 11.1%).
Next, the obtained mixed solution was heated at 80 ° C. for 30 minutes with gentle stirring to form nanoparticles.
The average particle size of the obtained nanoparticles was measured using a zeta potential / nanoparticle size measurement system (“DelsaMax PRO” manufactured by Beckman Coulter, Inc.).
　実施例ｂ－１～ｂ－３及び比較例ｂ－１において調製したナノ粒子の粒子サイズを表３に示した。 Table 3 shows the particle sizes of the nanoparticles prepared in Examples b-1 to b-3 and Comparative Example b-1.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
　表３に示した通り、実施例ｂ－１～ｂ－３で得られた電解質を用いて調製した本発明のナノ粒子は、比較例ｂ－１（従来法）の場合と同等か、あるいはより小さな粒度のナノ粒子であることが判明した。これにより、従来法におけるカテキンの代わり種々の電解質を用いてナノ粒子を製造できることが判明した。 As shown in Table 3, the nanoparticles of the present invention prepared using the electrolytes obtained in Examples b-1 to b-3 are equivalent to or better than those in Comparative Example b-1 (conventional method). It turned out to be nanoparticles with a small particle size. As a result, it was found that nanoparticles can be produced by using various electrolytes instead of catechin in the conventional method.
〔実施例ｂ－４〕　生理活性物質を担持したナノ粒子の製造
　ホエイタンパク質（日本新薬製：エンラクトＳＡＴ）５ｇを、５ｍＭ（０．１６２％）
クエン酸三カリウム溶液９０ｍｌに加えて２０℃で緩やかに攪拌して溶解した。次いで、１ｍｇ／ｍＬ還元型ＣｏＱ１０エタノール溶液を５ｍｌ添加後、ｐＨ調整液を用いてｐＨ６～７になるように調整しながら、さらに５ｍＭ（０．１６２％）クエン酸三カリウム溶液を加え最終容量を１００ｍＬとした。その後、緩やかに攪拌しながら、８０℃で３０分間加熱してナノ粒子を形成させた。
　得られた溶液中のナノ粒子を、ゼータ電位・ナノ粒子径測定システム（ ベックマン・コールター株式会社製、「Ｄｅｌｓａ Ｍａｘ ＰＲＯ」）を用いて粒子径を測定したところ、平均粒子径は、９０．７～１２０．７ｎｍであった。 [Example b-4] Production of nanoparticles carrying a physiologically active substance 5 g of whey protein (Nippon Shinyaku Co., Ltd .: Enlacto SAT), 5 mM (0.162%)
It was dissolved by adding to 90 ml of a tripotassium citrate solution and gently stirring at 20 ° C. Next, after adding 5 ml of a 1 mg / mL reduced CoQ10 ethanol solution, add a further 5 mM (0.162%) tripotassium citrate solution while adjusting the pH to 6 to 7 using a pH adjusting solution to adjust the final volume. It was set to 100 mL. Then, while gently stirring, the particles were heated at 80 ° C. for 30 minutes to form nanoparticles.
When the nanoparticles in the obtained solution were measured for particle size using a zeta potential / nanoparticle size measurement system (“Delsa Max PRO” manufactured by Beckman Coulter, Inc.), the average particle size was 90.7. It was ~ 120.7 nm.
〔実施例ｂ－５〕　ナノ粒子における生理活性物質の安定性
　ナノ粒子に担持させた生理活性物質の安定性を常温、ＵＶ下での虐待試験により検討した。検討には、実施例ｂ－４で得られたナノ粒子溶液（本発明品）、還元型コエンザイムＱ１０を乳化剤で分散させたコエンザイムＱ１０溶液（比較品）を用いて実施した。作製した各試料を透明ガラス容器に入れ、常温、ＵＶ下で虐待を行った。１週間間隔にてサンプルを回収し、試料中に含まれる還元型コエンザイムＱ１０濃度を測定した。その結果、本発明品では、比較品と比べると、生理活性物質である還元型コエンザイムＱ１０を長期間保存しても酸化させることなく安定に保持することができることが判明した。
  したがって、従来、ナノ粒子の製造に用いられてきた、ガレート型エピガロカテキンの代わりに、クエン酸三カリウム等の食品製造において汎用される電解質を用いても、生理活性物質を安定的に担持したナノ粒子を製造することができることが判明した。 [Example b-5] Stability of bioactive substance in nanoparticles The stability of the bioactive substance carried on the nanoparticles was examined by an abuse test at room temperature and under UV. The study was carried out using the nanoparticle solution (product of the present invention) obtained in Example b-4 and the coenzyme Q10 solution (comparative product) in which reduced coenzyme Q10 was dispersed with an emulsifier. Each of the prepared samples was placed in a transparent glass container and abused at room temperature and under UV. Samples were collected at weekly intervals, and the concentration of reduced coenzyme Q10 contained in the samples was measured. As a result, it was found that the product of the present invention can stably retain the reduced coenzyme Q10, which is a physiologically active substance, without being oxidized even when stored for a long period of time, as compared with the comparative product.
Therefore, instead of the gallate-type epigalocatechin, which has been conventionally used for the production of nanoparticles, an electrolyte commonly used in the production of foods such as tripotassium citrate was used to stably support the physiologically active substance. It turned out that nanoparticles can be produced.
　以上のように作製された本発明のナノ粒子は、生理活性物質を安定的に担持しているものであり、かつ、平均粒子径が１０～３００ｎｍと非常に小さなものであるため、例えば、経口で摂取した場合に、動物性タンパク質、電解質、さらには前記生理活性物質を腸等から、体内に吸収し易いため、前記生理活性物質を単で摂取した場合に比べ、バイオアベイラビリティが向上するという性質を有することがわかる。 The nanoparticles of the present invention produced as described above stably support a physiologically active substance and have a very small average particle size of 10 to 300 nm. Therefore, for example, oral administration is required. When ingested in, animal proteins, electrolytes, and the physiologically active substance are easily absorbed into the body from the intestine and the like, so that the bioavailability is improved as compared with the case where the physiologically active substance is ingested alone. It can be seen that it has.

Claims (11)

  1. 　ホエイタンパク質、ゼラチン、コラーゲン、及びこれらの分解物からなる群より選ばれる少なくとも１種の動物性タンパク質を固形分として１重量％以上、
    　電解質を固形分として０．００１重量％以上含有し、且つ、
    　前記動物性タンパク質と前記電解質との固形分の重量比（電解質／動物性タンパク質）が０．００１～１．０であり、且つ
    　平均粒子径が１０～３００ｎｍであることを特徴とする、ナノ粒子。     1% by weight or more of solid content of at least one animal protein selected from the group consisting of whey protein, gelatin, collagen, and decomposition products thereof.
    Contains 0.001% by weight or more of electrolyte as a solid content and
    Nanoparticles characterized by a solid content weight ratio (electrolyte / animal protein) of the animal protein to the electrolyte of 0.001 to 1.0 and an average particle size of 10 to 300 nm. ..
  2. 　前記電解質が、カテキン、無機塩、有機酸塩、及び有機酸から成る群から選択される少なくとも１種である、請求項１に記載のナノ粒子。 The nanoparticles according to claim 1, wherein the electrolyte is at least one selected from the group consisting of catechins, inorganic salts, organic acid salts, and organic acids.
  3. 　前記動物性タンパク質と前記電解質との固形分の重量比（電解質／動物性タンパク質）が０．００１～０．２である、請求項１に記載のナノ粒子。 The nanoparticles according to claim 1, wherein the weight ratio of the solid content (electrolyte / animal protein) of the animal protein to the electrolyte is 0.001 to 0.2.
  4. 　前記電解質がカテキンであり、前記カテキンを固形分として０．０１重量％以上含有する請求項３に記載のナノ粒子。 The nanoparticles according to claim 3, wherein the electrolyte is catechin, and the catechin is contained in an amount of 0.01% by weight or more as a solid content.
  5. 　前記ナノ粒子が、その平均粒子径の前後２０ｎｍの範囲内に７０％以上が存在する、請求項３又は４に記載のナノ粒子。 The nanoparticles according to claim 3 or 4, wherein 70% or more of the nanoparticles are present within a range of 20 nm before and after the average particle diameter.
  6. 　生理活性物質を担持している、請求項１～５のいずれかに記載のナノ粒子。 The nanoparticles according to any one of claims 1 to 5, which carry a physiologically active substance.
  7. 　前記生理活性物質がユビキノール、スチルベン類、水溶性ビタミン、脂溶性ビタミン、アミノ酸、カフェイン、及びカロテノイドならなる群より選ばれる少なくとも１種である、請求項１～６のいずれかに記載のナノ粒子。 The nanoparticles according to any one of claims 1 to 6, wherein the physiologically active substance is at least one selected from the group consisting of ubiquinol, stilbenes, water-soluble vitamins, fat-soluble vitamins, amino acids, caffeine, and carotenoids. ..
  8. 　ホエイタンパク質、ゼラチン、コラーゲン、及びこれらの分解物からなる群より選ばれる少なくとも１種の動物性タンパク質を溶媒に溶解、分散又は膨潤させて、動物性タンパク質液を調製する工程（Ａ）、
    　前記工程（Ａ）で得られた前記動物性タンパク質液に、前記動物性タンパク質の固形分重量１に対し、電解質を固形分重量０．００１～１．０の割合で加えて、動物性タンパク質－電解質液を調製する工程（工程Ｂ）、及び
    　前記工程（Ｂ）で得られた前記動物性タンパク質－電解質液を７２～９５℃で、２～６０分間加熱する工程（Ｃ）、
    を有する、ナノ粒子の製造方法。     Step (A) of preparing an animal protein solution by dissolving, dispersing or swelling at least one animal protein selected from the group consisting of whey protein, gelatin, collagen, and decomposition products thereof in a solvent.
    To the animal protein solution obtained in the step (A), an electrolyte is added at a ratio of a solid content weight of 0.001 to 1.0 to a solid content weight of 1 of the animal protein, and the animal protein-. A step of preparing the electrolyte solution (step B), and a step of heating the animal protein-electrolyte solution obtained in the step (B) at 72 to 95 ° C. for 2 to 60 minutes (C).
    A method for producing nanoparticles.
  9. 　前記電解質が、カテキン、無機塩、有機酸塩、及び有機酸から成る群から選択される少なくとも１種である、請求項８に記載のナノ粒子の製造方法。 The method for producing nanoparticles according to claim 8, wherein the electrolyte is at least one selected from the group consisting of catechins, inorganic salts, organic acid salts, and organic acids.
  10. 　さらに、生理活性物質を加える工程を含む、請求項８又は９に記載のナノ粒子の製造方法。 The method for producing nanoparticles according to claim 8 or 9, further comprising a step of adding a physiologically active substance.
  11. 　前記生理活性物質がユビキノール、スチルベン類、水溶性ビタミン、脂溶性ビタミン、アミノ酸、カフェイン、及びカロテノイドから成る群から選択される少なくとも１種である、請求項１０に記載のナノ粒子の製造方法。
     
     
          The method for producing nanoparticles according to claim 10, wherein the bioactive substance is at least one selected from the group consisting of ubiquinol, stilbenes, water-soluble vitamins, fat-soluble vitamins, amino acids, caffeine, and carotenoids.
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