JP6443009B2 - Method for producing nanoparticles having improved loading of bioactive substances - Google Patents

Description

  The present invention relates to a method for producing nanoparticles with improved carrying ability of a physiologically active substance.

  Gelatin derived from natural products is a protein extracted from cartilage components of pigs, cattle, and fish. In addition to its use as a food gelling agent, thickener, stabilizer, etc., it can also be used as a base material for capsules, etc. It is also used in the medical field. An emulsion colloid obtained by adding potassium bromide and silver nitrate to gelatin is used as a protective colloid for photosensitive materials. In addition, a technique for producing microcapsules by using a property that gelatin is water-soluble and dropping it into an organic solvent is also known.

  Furthermore, in recent years, the development of technology for use in drug delivery systems (DDS) for providing pharmaceutical ingredients to target organs and tissues by making gelatin into nanoparticles has been progressing. Nanoparticles using food-derived ingredients such as gelatin are considered to be highly advantageous from the viewpoint of safety. As an example, a method for producing nanoparticles combined with chitosan can be mentioned (Patent Documents 1, 2, and 3).

  The present inventors have also proposed a method for producing nanoparticles in which gelatin and gallate-type catechin, which have been found to be advantageous in production and superior in raw material cost, are combined (Patent Document 4). Our method is a method in which gallate-type catechin is used as a coacervator for animal proteins such as gelatin. This is the first method using a substance having its own physiological activity as a nanoparticle-forming substance. In other words, gallate-type catechins typified by epigallocatechin gallate, the most widely studied in terms of functionality, are known to have a wide range of physiological functions such as anti-obesity action, cardiovascular disease prevention action, and anti-cancer action. It has been. Further, since gallate 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 5). In addition, the present inventors have reported a lipase inhibitor by complexing gallate catechin and gelatin (Patent Document 6), and not only by the use of gallate catechin as a nanoparticle-forming substance but also by combination with protein. An advantage of improving physiological activity has also been found.

  As described above, the present inventor has reported nanoparticles in which a bioactive substance is supported in Patent Document 4. However, as will be shown in this Example, which will be described later, a phenomenon has been found in which the physiologically active substance once supported on the nanoparticles is detached from the nanoparticles, although the cause is unknown. If the amount of the physiologically active substance to be detached is increased, the amount of the physiologically active substance carried in the nanoparticles is reduced, so that the effect of carrying the physiologically active substance on the nanoparticles may be greatly reduced. is there.

  Since nanoparticles can be expected to improve bioavailability, their significance is great. Accordingly, there is a demand for the development of a new technique for producing nanoparticles having a further improved ability to support physiologically active substances.

Japanese Patent No. 5564200 JP 2009-090160 A US Pat. No. 8,642,088 Japanese Patent Application No. 2014-160745 JP 2014-062192 A JP 2013-082673 A

  An object of this invention is to provide the manufacturing method of the nanoparticle which improved the carrying power of the bioactive substance.

  The present inventors paid attention to the phenomenon that the physiologically active substance carried by the nanoparticles is detached, and as a result of earnestly examining the technology for suppressing this phenomenon, the physiologically active substance, gallate catechin, gelatin, collagen , And these degradation products, whey proteins and at least two animal proteins selected from these degradation products are mixed under appropriate conditions to prepare a solution containing nanoparticles (nanoparticle-containing solution). The nanoparticle-containing solution is dried by a very simple method, and the average particle diameter is 10 to 200 nm. It came to do.

The gist of the present invention is as follows:
[1] 0.1% to 20% by weight of gallate catechin as a solid content, and two animal proteins consisting of gelatin, collagen or a degradation product thereof and whey protein or a degradation product as a solid content The above three components are mixed in water, a water-containing organic solvent or an organic solvent so that the total amount is 0.1 to 20% by weight and the acid is 0.1 to 20% by weight as a solid content. Obtaining a protein-containing solution (A),
A step of dissolving a physiologically active substance in water, a water-containing organic solvent or an organic solvent to obtain a physiologically active substance-containing liquid (B),
A step (C) of mixing the gallate-type catechin obtained in the step (A) and the animal protein-containing solution with the physiologically active substance-containing solution obtained in the step (B);
Step (D) of obtaining nanoparticles by drying the liquid mixture obtained in Step (C)
A method for producing nanoparticles having improved carrying ability of a physiologically active substance, characterized by comprising:
[2] The method for producing nanoparticles with improved supporting ability of the physiologically active substance according to [1], wherein the liquid mixture obtained in the step (C) is diluted with water, a water-containing organic solvent or an organic solvent. ,
[3] A method for producing nanoparticles with improved loading of the physiologically active substance according to [1] or [2], wherein the drying is heating under reduced pressure, freeze drying, or spray drying,
[4] Production of nanoparticles with improved carrying ability of the physiologically active substance according to any one of [1] to [3], wherein the physiologically active substance is ubiquinol, stilbenes, water-soluble vitamins, fat-soluble vitamins or carotenoids Method,
[5] The nanoparticle production method according to any one of [1] to [4], wherein the nanoparticle has an average particle size of 10 to 200 nm, and has an improved ability to support a physiologically active substance.

  Since the nanoparticles obtained in the present invention have significantly improved supporting ability of physiologically active substances than the nanoparticles described in Patent Document 4, gallate catechins, animal proteins, the physiologically active substances, etc. Nanoparticles with improved bioavailability of a plurality of functional components.

  Hereinafter, the present invention will be described in detail.

The method for producing nanoparticles with improved carrying ability of the physiologically active substance of the present invention,
Two animal proteins consisting of 0.1 to 20% by weight of gallate catechin as a solid content, gelatin, collagen or a degradation product thereof, and whey protein or a degradation product thereof as a solid content in a total of 0. The above three components are mixed in water, a water-containing organic solvent or an organic solvent so as to be 1 to 20% by weight and 0.1 to 20% by weight of acid as a solid content, and a gallate catechin and animal protein-containing liquid (A) to obtain
A step of dissolving a physiologically active substance in water, a water-containing organic solvent or an organic solvent to obtain a physiologically active substance-containing liquid (B),
A step (C) of mixing the gallate-type catechin obtained in the step (A) and the animal protein-containing solution with the physiologically active substance-containing solution obtained in the step (B);
Step (D) of obtaining nanoparticles by drying the liquid mixture obtained in Step (C)
It is characterized by having.

In the present invention, “supporting” refers to a state in which a physiologically active substance is contained in nanoparticles.
In the present invention, “supporting force” refers to a force for maintaining a state in which a bioactive substance is supported in nanoparticles.
The amount of the physiologically active substance carried on the nanoparticles can be determined in the test described below.

The average particle diameter of the nanoparticles obtained in the present invention is preferably 10 to 200 nm, more preferably 10 to 100 nm, from the viewpoint of dispersion stability of the functional component and absorbability into the body.
The average particle diameter of the nanoparticles can be measured with a zeta potential / nanoparticle diameter measurement system (“DelsaMax PRO” manufactured by Beckman Coulter, Inc.) as described in the Examples below.

  Hereinafter, process (A)-process (D) are demonstrated.

(Process (A))
In this step, gallate-type catechin as a solid content is 0.1 wt% to 20 wt%, and two animal proteins consisting of gelatin, collagen, or a degradation product thereof, and whey protein or the degradation product as a solid content. Gallate-type catechin / animality by mixing the above three components in water, a water-containing organic solvent or an organic solvent so that the total amount is 0.1 to 20% by weight, and the acid content is 0.1 to 20% by weight. A protein-containing solution is obtained.

  Specifically, a solution or dispersion containing gallate catechin and animal protein is prepared by dissolving or dispersing gallate catechin, two types of animal protein and acid in water or a water-containing organic solvent or organic solvent. To do.

  Examples of the gallate catechin used in the present invention include EGCg, ECg, GCg, and Cg. The gallate catechin may be a non-polymer or a polymer, and they may be mixed or used alone. From the viewpoint of efficient particle formation, it is preferable to contain EGCg and / or ECg. Moreover, the composition containing a gallate type catechin can also be used, for example, the tea extract, coffee extract, etc. containing the said gallate type catechin are mentioned. Further, from the viewpoint of efficiently producing nanoparticles, the amount of gallate catechin in the composition containing gallate catechin is preferably 20% by weight or more, more preferably 30% by weight or more, and more preferably 60% by weight or more. Are more preferred.

One animal protein used in the present invention includes gelatin, collagen, or a degradation product thereof, which can form gallate catechin and coacervate.
As for the origin of gelatin, any of pigs, fish, chickens, etc., and genetically modified organisms can be used. In addition, although animal protein derived from cow bone or pork bone is partially formed with particles of 500 nm or less, it is difficult to use in the present invention because aggregation and precipitation are likely to occur. However, even if it is animal protein containing the protein derived from a cow bone or a pork bone, if a nanoparticle with an average particle diameter of 10-200 nm can be produced, it can use without limitation.
Examples of the collagen include those obtained by decomposing the gelatin with an enzyme or the like, and include collagen peptides.
Examples of the degradation product of gelatin or collagen include degradation products other than collagen and collagen peptides.
In the present invention, any one of the gelatin, collagen, and degradation products thereof may be used alone, or two or more kinds may be mixed and used.

The other animal protein used in the present invention includes whey protein or a degradation product thereof.
Whey protein is a soluble protein obtained when milk coagulates, preferably whey protein isolate (WPI) or hydrolyzed whey peptide (WPH), more preferably albumin and casein purified from milk It is.
The whey protein degradation product includes a product obtained by degrading whey protein using an enzyme or the like.

  The animal protein may be emulsified. When emulsifying animal protein, a known emulsifier may be used.

  What is necessary is just to select the acid which can be used for the acid used for this invention according to the use application of a nanoparticle. For example, citric acid, ascorbic acid, gluconic acid, carboxylic acid, tartaric acid, succinic acid, acetic acid or phthalic acid, organic acids such as trifluoroacetic acid, inorganic acids such as hydrochloric acid, perchloric acid, carbonic acid, or buffers However, it is not limited to these.

Examples of the water used as the solvent include pure water, distilled water, tap water, and commercial drinking water, but are not particularly limited.
The organic solvent used as the solvent is not particularly limited as long as it is miscible with water. In addition, it is preferable to select a solvent suitable for the intended use of the obtained nanoparticles, and examples of the solvent suitable for food use include glycerin, propylene glycol, ethanol, etc. In addition to the above, methanol, acetone, dimethyl sulfoxide and the like can be mentioned.
The hydrous organic solvent used as the solvent refers to a mixed solvent of the organic solvent and water.

  Examples of means for mixing the gallate catechin, the two types of animal proteins, and the acid into the solvent include, for example, the gallate type catechin, the two types of animal proteins, and an acid. It can be dissolved or dispersed in.

  When preparing the gallate-type catechin / animal protein-containing liquid as described above, the raw materials may be mixed in a powder state and then dissolved or dispersed, or a solution or dispersion of each raw material may be prepared. May be mixed. Moreover, when dissolving or dispersing, it is preferable to adjust the temperature of the solvent to 20 to 90 ° C. from the viewpoint of solubility of gallate catechin, two kinds of animal proteins and acid, If it melt | dissolves or disperses, there will be no limitation in particular.

  The solid content value of the gallate catechin in the gallate catechin / animal protein-containing liquid is 0.1 to 20% by weight from the viewpoint of efficiently producing nanoparticles having an average particle size of 10 to 200 nm. It is preferably -20% by weight, more preferably 10-20% by weight.

  The solid content values of the two animal proteins in the gallate catechin / animal protein-containing solution are 0.1 to 20% by weight in total from the viewpoint of efficiently producing nanoparticles having an average particle size of 10 to 200 nm. The total content is preferably 1 to 10% by weight, but is not particularly limited as long as desired nanoparticles can be produced. From the viewpoint of efficient nanoparticle formation, the mixing ratio of the two animal proteins is (gelatin, collagen, or a degradation product thereof) :( whey protein or a degradation product thereof) = 0.1: 9.9-5 : 5 is preferred. Even outside this range, nanoparticles with an average particle diameter of 10 to 200 nm are formed, but this is not preferable because the formed nanoparticles tend to aggregate.

  The solid content value of the acid in the gallate-type catechin / animal protein-containing liquid is 1 to 20% by weight and 10 to 20% by weight from the viewpoint of efficiently producing nanoparticles having an average particle size of 10 to 200 nm. Preferably there is.

In this step, the weight ratio of gallate catechin to animal protein in the gallate catechin / animal protein-containing solution (animal protein / gallate catechin) is 0.07-8. Adjust to zero. By adjusting the ratio within the above range, nanoparticles having an average particle diameter of 10 to 200 nm can be efficiently obtained.
When the weight ratio of gallate-type catechin to animal protein (animal protein / gallate-type catechin) exceeds 8.0, a part of nanoparticles of 200 nm or less are produced, but the average particle diameter exceeds 200 nm. The same is true even if the weight ratio of the gallate catechin to the animal protein is less than 0.07.

  Moreover, it is preferable to adjust pH of the said gallate type catechin and animal protein containing liquid to 1.0-8.0. If the pH is too lower than 1.0, the nanoparticles are dissolved or the particle size is increased. There are few reports of adjusting the particle size of the nanoparticles at such a low pH. On the other hand, if the pH is higher than 8.0, particles are temporarily formed, but aggregation and precipitation are likely to occur. On the other hand, if the pH is more than 8.0, the stability of the gallate catechin decreases, so that efficient nanoparticles cannot be formed. The pH of the mixed solution is more preferably 1.5 to 6.0, and more preferably 1.5 to 4.0.

  In order to adjust the pH of the gallate-type catechin and animal protein-containing liquid, for example, the pH of the gallate-type catechin-containing solution or dispersion and the animal protein-containing solution or swelling liquid may be adjusted in advance. By adjusting the pH in advance in this way, the pH of the mixed solution can be adjusted to a range of 1.0 to 8.0 even by mixing the gallate-type catechin-containing solution or dispersion and the animal protein-containing solution or swelling solution. Can be adjusted. In addition, when powder mixing gallate catechin and animal protein, the acid for pH adjustment or the like may be mixed in the powder mixture, and then the solvent may be mixed.

  In the gallate catechin and animal protein-containing liquid adjusted to a pH in the range of 1.0 to 8.0 as described above, the gallate catechin and the animal protein form a coacervate.

(Process (B))
In this step, the physiologically active substance is dissolved in water or a water-containing organic solvent or an organic solvent to prepare a physiologically active substance-containing liquid.

  Examples of the physiologically active substance include ubiquinol, stilbenes, water-soluble vitamins, fat-soluble vitamins, and carotenoids.

  Ubiquinol used in the present invention is a functional component also called reduced coenzyme Q10, and is an oil-soluble solid substance. As 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 preparation, can be mentioned. “Kaneka QH” is preferable in terms of cost and physical properties.

  Examples of the stilbenes used in the present invention include resveratrol, pterostilbene, piceatannol and the like. Moreover, the composition containing these may be sufficient. A commercially available product may be used as the stilbene, and examples thereof include no grape extract manufactured by Maruzen Pharmaceutical Co., Ltd., which contains piceatannol, and vine atrol, manufactured by BN Co., which contains resveratrol.

  Examples of water-soluble vitamins used in the present invention include vitamin B group. The vitamin B group includes vitamins B1, B2, B3, B5, B6, B7, B9, and B12. These may be commercial products.

  Examples of fat-soluble vitamins include vitamin A, vitamin D, vitamin E, vitamin K, and the like. These may be commercial products.

  Examples of carotenoids include lutein, astaxanthin, lycopene, and carotene. These may be commercial products.

Examples of the water used as the solvent include pure water, distilled water, tap water, and commercial drinking water, but are not particularly limited.
The organic solvent used as the solvent is not particularly limited as long as it is miscible with water. In addition, it is preferable to select a solvent suitable for the intended use of the obtained nanoparticles, and examples of the solvent suitable for food use include glycerin, propylene glycol, ethanol, etc. In addition to the above, methanol, acetone, dimethyl sulfoxide and the like can be mentioned.
The hydrous organic solvent used as the solvent refers to a mixed solvent of the organic solvent and water.

  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, the physiologically active substance can be dissolved by adding and mixing with the solvent. Moreover, about mixing conditions, such as temperature conditions, what is necessary is just normal temperature, and there is no limitation in particular.

  In addition, a stabilizer, an emulsifier and the like may be added for the purpose of preventing aggregation and precipitation of the physiologically active substance. Examples of the stabilizer include gum arabic, pectin, soybean polysaccharide, CMC (carboxymethyl cellulose), sodium caseinate and the like. Examples of emulsifiers include sucrose fatty acid esters.

  The solid content value of the physiologically active substance in the physiologically active substance-containing liquid is preferably 0.001 to 50% by weight from the viewpoint of efficiently producing nanoparticles having an average particle size of 10 to 200 nm. As long as the nanoparticles can be produced, there is no particular limitation.

(Process (C))
In this step, the gallate-type catechin and animal protein-containing liquid prepared in the step (A) and the physiologically active substance-containing liquid prepared in the step (B) are mixed, and the resulting mixture is nano-sized. Form particles.

The mixing method of the gallate-type catechin, the animal protein-containing liquid and the physiologically active substance-containing liquid in this step is not limited as long as it can be uniformly mixed. As the mixing method, a method of adding while stirring, homogenizing, or the like. However, there is no particular limitation, although a method of adding while using can be used.
Moreover, you may add solvents, such as water, when producing a liquid mixture as needed.

  In this step, conditions such as the temperature at the time of mixing are not particularly limited as long as the components can be uniformly mixed without causing a significant change in the components, and may be any temperature suitable for the components to be used. For example, in the case of gelatin, when the temperature is low, the viscosity of the solution increases, and when the concentration is as high as several percent or more, it becomes difficult to mix uniformly. Furthermore, in the case of high temperature, since the change of a component tends to occur, 50-100 degreeC is more preferable, More preferably, 50-90 degreeC is good.

  The nanoparticle-containing liquid obtained as described above may be subjected to ultrafiltration, dialysis or the like in order to concentrate or purify the nanoparticles. If dialysis is performed, it is easy to separate non-particulate components. Examples of the ultrafiltration membrane include a pencil-type UF membrane (manufactured by Asahi Kasei), and examples of the dialysis membrane include SnakeSkin (manufactured by Pierce). There is no particular limitation as long as ultrafiltration and dialysis can be performed without losing nanoparticles.

  Moreover, you may dilute the nanoparticle containing solution obtained at the said process (C) with water, a water-containing organic solvent, and an organic solvent. By further diluting the nanoparticle-containing solution as described above, there is an advantage that aggregation of the obtained nanoparticles is suppressed, and that it is easy to dry while maintaining a desired average particle size even at the time of drying described later.

  As the degree of dilution, for example, a solvent of 5 to 100 times the amount of the obtained nanoparticle-containing solution may be mixed.

  The diluted solution may be mixed with gum arabic, pectin, soybean polysaccharide, CMC (carboxymethylcellulose), sodium caseinate and the like as a stabilizer. Moreover, you may mix sucrose fatty acid ester etc. as an emulsifier. In addition, as a nanoparticle formation stabilizer, phosphates such as monosodium phosphate, disodium phosphate, trisodium phosphate, sodium pyrophosphate, sodium tripolyphosphate, sodium tetrapolyphosphate, sodium metaphosphate, potassium tripolyphosphate Enzyme-based protein binders such as transglutaminase can be mixed. The content of the stabilizer, the emulsifier, the formation stabilizer and the like is not particularly limited as long as the nanoparticles in the diluted solution are 10 to 200 nm.

  In this step, the conditions such as the temperature when diluting may be any conditions that do not cause a significant change in the components and can be mixed uniformly, for example, room temperature.

(Process (D))
In this step, the diluted solution is dried to obtain nanoparticles. In this step, the nanoparticle formed in the mixed solution obtained in the step (C) is further subjected to a drying treatment, whereby the supporting ability of the physiologically active substance in the nanoparticle can be remarkably improved. .

Examples of the drying method include heating under reduced pressure, freeze drying, and spray drying.
The heating and drying under reduced pressure means a method in which the inside of the heating apparatus is decompressed to lower the boiling point, thereby promoting drying and evaporating / drying with less energy. Freeze-drying refers to a method of first freezing, then lowering the boiling point of the frozen dried product in a vacuum, sublimating the moisture of the dried product, and drying. Spray drying refers to a method of producing a dry powder by spraying a liquid into a gas and drying it rapidly.
Any of the drying methods may be performed using a known drying apparatus.

  The temperature condition at the time of drying may be set to an appropriate temperature range according to each drying method. For example, it is 20 to 100 ° C. for heating under reduced pressure, 20 to 60 ° C. for freeze drying, and the outlet temperature for spray drying. Although adjusting to 50-100 degreeC is mentioned, there is no limitation in particular.

(Nanoparticles)
The content of the physiologically active substance in the nanoparticles obtained in the steps (A) to (D) is preferably 0.01 to 20% by weight.

  In addition, since the nanoparticle has improved ability to carry physiologically active substances, it can be added to foods, pharmaceuticals, quasi drugs, cosmetics, etc. in addition to gallate catechins and animal proteins. The bioavailability of a plurality of physiologically active substances such as physiologically active substances can be improved.

  For example, when the nanoparticles are produced under conditions that can be used for foods (specifically, when a solvent that can be used for foods is used), the nanoparticles may be blended in food or drink. It does not specifically limit as food-drinks, For example, a drink, alcoholic beverage, jelly, confectionery, functional food, health food, health-oriented food, etc. are mentioned. In consideration of preservability, portability, ease of ingestion and the like, confectionery is preferable, and among confectionery, hard candy, soft candy, gummy candy, tablet, chewing gum and the like are preferable.

  When the nanoparticles are blended in a food or drink, the content of the nanoparticles in the food or drink may be an amount that can be expected to have a physiological activity effect. Usually, it is preferable to determine the blending amount so that 10 to 10000 mg, more preferably 100 to 3000 mg can be taken per day. For example, 5 to 50% by weight is preferable for solid foods, and 0.01 to 10% by weight for liquid foods such as beverages.

  The nanoparticles may be used as therapeutic agents for non-human animals such as mammals such as rats, mice, guinea pigs, rabbits, sheep, pigs, cows, horses, cats, dogs, monkeys, chimpanzees, birds, amphibians, reptiles, etc. You may mix | blend with feed. As feed, for example, livestock feed used for sheep, pigs, cattle, horses, chickens, etc., feed for small animals used for rabbits, rats, mice, etc., feed for seafood used for eel, Thailand, yellowtail, shrimp, etc., dogs, Pet foods used for cats, small birds, squirrels, etc.

You may mix | blend the said nanoparticle with a pharmaceutical. Examples of the pharmaceutical include solid preparations such as powders, tablets, pills, capsules, fine granules and granules, liquids such as liquids, suspensions and emulsions, gels and the like. If necessary, the granules of capsules containing tablets, pills, granules, granules can be sugar-coated with sugars such as sucrose, sugar alcohols such as maltitol, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose, etc. It can also be coated. Alternatively, it may be covered with a film of gastric or enteric material. Moreover, in order to improve the solubility of a formulation, a well-known solubilization process can also be performed. Based on a conventional method, it may be used in an injection or a drip.
When the nanoparticles are used for pharmaceutical purposes, for example, the amount of intake thereof is not particularly limited as long as the desired improvement, therapeutic or preventive effect is obtained. Usually, the mode, patient age, sex, It is appropriately selected according to the constitution and other conditions, the type of disease, its degree, and the like. About 0.1 mg to about 1,000 mg per day is preferable, and this can be taken in 1 to 4 times a day.

You may mix | blend the said nanoparticle with a quasi-drug. Examples of the quasi-drug include quasi-drugs used in the oral cavity, such as toothpaste, mouthwash, mouth rinse, and nourishing tonic drinks for the purpose of infectious disease prevention.
When adding the said nanoparticle to a quasi drug, it is preferable to add normally 0.001-30 weight% in this quasi drug.

You may mix | blend the said nanoparticle with cosmetics. Examples of the cosmetics include lotions, emulsions, creams, packs, finished cosmetics, hair cosmetics, facial cleansers, bath agents, and antiperspirants. These cosmetics are expected to have a cosmetic effect due to the antioxidant effect, and can be used for antibacterial purposes due to the antibacterial effect.
Moreover, when using the said nanoparticle as cosmetics, it is preferable to make it become a density | concentration of 0.1 ppm-2000 ppm in cosmetics.

  EXAMPLES Next, although this invention is demonstrated in detail based on an Example, this invention is not limited only to this Example.

(Example 1: Preparation of nanoparticle powder)
Gelatin (trade name: G fine powder, manufactured by Nitta Gelatin Co.) 0.1 g, whey protein (trade name: Enlacto SAT, manufactured by Nippon Shinyaku Co., Ltd.) 0.9 g, green tea extract (gallate catechin 64%) 1.8 g Then, 2 g of citric acid was mixed with the powder, and 5.2 g of distilled water was added thereto, and the mixture was homogenized while heating at 80 ° C. to obtain 10 g of a gelatin / gallate catechin-containing solution (pH 2.0). Obtained.
Subsequently, 9 g of ethanol and 1 g of resveratrol (manufactured by Kanto Chemical Co., Inc.) were added and mixed to obtain 10 g of a physiologically active substance solution.
A gelatin / gallate catechin-containing solution 10 g and a physiologically active substance solution 10 g were uniformly mixed, and this was diluted with 5 times amount of 10% ethanol solution. Further, this was diluted with a 10-fold amount of 0.4 wt% soybean polysaccharide (trade name: SM-1200, manufactured by Saneigen Co., Ltd.) solution, diluted with an equal volume of 0.2 wt% phosphate, and nano-sized. A particle-containing liquid was obtained.
The nanoparticle-containing liquid was spray-dried using a spray dryer (manufactured by Tokyo Science) to obtain 10 g of nanoparticle powder.
The spray drying conditions were an inlet temperature of 180 ° C., an outlet temperature of 90 ° C., a dry air amount of 0.4 m ³ / min, a spray pressure of 50 kPa, and a flow rate of 400 mL / h.
The obtained nanoparticle powder was dispersed again in water, and the average particle size was measured using a zeta potential / nanoparticle size measurement system (“DelsaMax PRO” manufactured by Beckman Coulter, Inc.). As a result, the average particle size was 86. .6 nm.

(Example 2: Measurement of bioactive substance loading)
Measurement of the loading capacity of the physiologically active substance in the nanoparticles was performed using a dialysis method.

  The nanoparticle-containing liquid before drying produced in Example 1 (comparative product, the same as the nanoparticle produced in Patent Document 4) and the dried nanoparticle powder (product of the present invention) are redispersed in water again. Each of the nanoparticle-containing liquids was put into a dialysis membrane (trade name: Snake skin, manufactured by Pierce, fractional molecular weight: 3500 Da), 20 mL each, and dialyzed against 5 L of water for 6 hours. Thereafter, the water was changed and dialyzed for 16 hours. 200 μL of ethanol was added to 200 μL of the solution remaining in the dialysis membrane and 200 μL of the nanoparticle-containing solution before dialysis, and 1 mL of ethyl acetate was further added. After mixing for 5 minutes, the mixture was centrifuged and the supernatant was collected. The obtained supernatant was completely dried with a vacuum drier, redissolved in 200 μL of methanol, and resveratrol was quantified by HPLC. The results are shown in Table 1.

As shown in Table 1, in the product of the present invention, it can be seen that the amount of resveratrol supported on the nanoparticles after dialysis is remarkably improved as compared with the comparative product.
From the above, by the production method of the present invention, the detachment of the physiologically active substance supported on the nanoparticles can be suppressed, and by producing nanoparticles containing a desired amount of the physiologically active substance, the gallate catechin is produced. It can be seen that the bioavailability of a plurality of functional components such as animal proteins and physiologically active substances can be further improved.

Claims (4)

0.1 wt% to 20 wt% gallate catechins as solids, gelatin and a total 0.1 wt% to 20 wt% of two or animal protein consisting of whey protein as a solid content, acid (A) to obtain a gallate-type catechin and animal protein-containing liquid by mixing the above three components in water, a water-containing organic solvent or an organic solvent so that the solid content is 0.1 to 20% by weight.
A step of dissolving a physiologically active substance in water, a water-containing organic solvent or an organic solvent to obtain a physiologically active substance-containing liquid (B),
A step (C) of mixing the gallate-type catechin obtained in the step (A) and the animal protein-containing solution with the physiologically active substance-containing solution obtained in the step (B);
Step (D) of obtaining nanoparticles by drying the liquid mixture obtained in Step (C)
I have a,
The whey protein is whey protein isolate (WPI);
Production method of the biologically active substance is resveratrol, pterostilbene or Pisea tan, characterized in Oh Rukoto in Nord bioactive nanoparticles supported force to improve the material.   The method for producing nanoparticles with improved bioactive substance supporting ability according to claim 1, wherein the mixed liquid obtained in the step (C) is further diluted with water, a water-containing organic solvent or an organic solvent. The method for producing nanoparticles with improved loading of a physiologically active substance according to claim 1 or 2, wherein the drying is heating under reduced pressure, freeze drying or spray drying . The average particle diameter of the nanoparticles is 10 to 200 nm, The method for producing nanoparticles with improved carrying ability of the physiologically active substance according to any one of claims 1 to 3 .