WO2020241562A1 - Nanoparticules et leur procédé de production - Google Patents

Nanoparticules et leur procédé de production 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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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
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    • 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
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    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
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    • A61K31/12—Ketones
    • A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
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    • 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]
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    • 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
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    • 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
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    • 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
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    • 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
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    • 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
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    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
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    • 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
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
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    • 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

La présente invention concerne des nanoparticules caractérisées en ce qu'elles contiennent, sur la base des solides, 1 % en poids ou plus d'au moins une protéine animale sélectionnée dans le groupe constitué par une protéine du lactosérum, la gélatine, le collagène, et des produits décomposés de ces derniers, et de 0,001 % en poids ou plus d'un électrolyte, et en ce que le rapport pondéral, sur la base des solides, entre la protéine animale et l'électrolyte (électrolyte / protéine animale) est compris entre 0,001 et 1,0, et la taille moyenne des particules est compris entre 10 et 300 nm. Les nanoparticules selon la présente invention sont des nanoparticules contenant une moindre quantité de catéchine par rapport aux particules classiques et utilisant un électrolyte moins onéreux, et peuvent retenir de manière stable une substance biologiquement active.
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