JP2016101135A - Manufacturing method of small nanoparticle - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a manufacturing method for safety making small nanoparticle derived from a natural product using a functional raw material such as gallate-type catechin and animal proteins and having average particle diameter of 1 to 50 nm.SOLUTION: A gallate-type catechin-containing liquid is dissolved in a pure water so that the gallate-type catechin is 0.1 wt.% as a solid content, an animal protein-containing liquid is obtained by dissolving at least one kind of animal protein selected from gelatin, collagen and a decomposition product thereof in a pure water so that the animal protein is 0.1 wt.% as a solid content and the gallate-type catechin-containing liquid and the animal protein-containing liquid are mixed with the pure water of 2 to 100 times amount based on the total weight of these liquids to manufacture nanoparticles having average particle diameter of 10 to 50 nm.SELECTED DRAWING: None

Description

  The present invention relates to a method for producing fine nanoparticles comprising a component derived from a natural product and having an average particle size of 10 to 50 nm.

  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 also serves as a base material for capsules, hemostatic agents, 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 using chitosan (Patent Documents 1, 2, and 3) can be mentioned.

  The present inventors have also reported a method for producing nanoparticles (patent document 4) in which gelatin and gallate-type catechin are combined, which have been found to be advantageous in production and superior in raw material cost. Our method is to make gallate catechins act as a coacervator for gelatin. This is the first method using a physiologically active substance as a nanoparticle forming substance. In other words, gallate-type catechins represented by epigallocatechin gallate, the most widely studied in terms of functionality, have a wide range of physiological functions such as anti-obesity action, cardiovascular disease prevention action, and anti-cancer action. It has been. In addition, 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), not only by the use of gallate catechin as a nanoparticle-forming substance but also by a combination of proteins. Significance in improving bioactivity has also been found.

  Here, with regard to the average particle diameter of known nanoparticles, for example, chitosan fine particles described in Patent Document 1 are 0.1 to 50 μm, and it is said that handling is difficult if the particle diameter is less than 0.1 μm (paragraph [0017]). . Moreover, although the volume average particle diameter of the oil droplet particles in the emulsion described in Patent Document 2 is 1 nm to 100 nm (Claim 2), it is produced using a water-soluble organic solvent in the production stage, and is pure water. It is not a technology that uses.

  From the above, a method for safely producing nanoparticles having an average particle diameter of 1 to 50 nm using pure water has not been known.

Japanese Patent No. 0556200 JP 2009-090160 A US Pat. No. 8,642,088 Japanese Patent Application No. 2014-160745 JP 2014-062192 A JP 2013-082673 A

  Therefore, the present invention is a natural product-derived nanoparticle using functional materials such as gallate-type catechin and animal protein, and even a nanoparticle having an average particle diameter of 1 to 50 nm can be produced safely. An object of the present invention is to provide a manufacturing method.

  The present inventors diligently investigated nanoparticles that can be used in foods. Among them, the present inventors have found that the particle size of the nanoparticles is increased by sodium, calcium and other ionic mineral components. As a result of further intensive studies based on this knowledge, a coacervate of gallate catechin and animal protein was formed by a very simple method of mixing gallate catechin and animal protein using pure water, The present inventors have succeeded in producing nanoparticles having an average particle diameter of 1 to 50 nm including a natural product-derived raw material, and have completed the present invention.

The gist of the present invention is as follows:
[1] A step (A) of obtaining a gallate-type catechin-containing liquid by dissolving gallate-type catechin in pure water so that the solid content is 0.1% by weight or more;
Step (B) of obtaining an animal protein-containing solution by dissolving at least one animal protein selected from gelatin, collagen, and degradation products thereof in pure water so that the solid content is 0.1% by weight or more. When,
The gallate catechin-containing liquid obtained in step (A) and the animal protein-containing solution obtained in step (B) are mixed with 2 to 100 times the amount of pure water based on the total weight of these liquids. Process (C)
A method for producing nanoparticles having an average particle size of 10 to 50 nm,
[2] The method for producing nanoparticles having an average particle diameter of 10 to 50 nm according to [1], wherein pure water has a conductivity of 10.0 μS / cm or less.

  Nanoparticles obtained in the present invention are made of raw materials derived from natural products such as gallate catechins and animal proteins, and are expected to have excellent health functions derived from gallate catechins and animal proteins. For example, the present inventors have reported a lipase-inhibiting composition using collagen and gallate-type catechins in Patent Document 6, but the nanoparticles obtained by the present invention also retain their functions. Be expected.

  Hereinafter, the present invention will be described in detail.

The method for producing nanoparticles according to the present invention is a method for producing nanoparticles having an average particle diameter of 10 to 50 nm, and based on natural product-derived components such as gallate catechins and animal proteins,
A step (A) of obtaining a gallate-type catechin-containing liquid by dissolving gallate-type catechin in pure water so that the solid content is 0.1% by weight or more;
Step (B) of obtaining an animal protein-containing solution by dissolving at least one animal protein selected from gelatin, collagen, and degradation products thereof in pure water so that the solid content is 0.1% by weight or more. When,
The gallate catechin-containing liquid obtained in step (A) and the animal protein-containing solution obtained in step (B) are mixed with 2 to 100 times the amount of pure water based on the total weight of these liquids. Process (C)
It is characterized by having.

The average particle size of the nanoparticles prepared in the present invention is 10 to 50 nm, and is preferably 10 to 30 nm, more preferably 10 to 20 nm from the viewpoint of good absorbability into the body and manufacturability. is there.
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.

  The natural product-derived component as used in the present invention means that at least one animal protein selected from gallate catechin, gelatin, collagen, and degradation products thereof, which are raw materials, is derived from a natural product. In addition, when using a reagent etc. as said raw material, the reagent should just be derived from a natural product.

  Hereinafter, each step will be described.

(Process (A))
In the method for producing nanoparticles of the present invention, the gallate catechin is dissolved in pure water to prepare a gallate catechin-containing liquid.

  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, you may use the composition containing a gallate type catechin as a gallate type catechin. Examples of the composition containing gallate catechin include tea extract and coffee extract containing the gallate catechin. From the viewpoint of particle production efficiency, the gallate catechin content in the composition is preferably 20% by weight or more, more preferably 30% by weight or more, and more preferably 60% by weight or more. .

  The pure water used as a solvent in the present invention refers to water from which impurities such as salts and residual chlorine contained in general tap water are removed, for example, water through a reverse osmosis membrane, deionized water, and distillation. Water, purified water, etc. are mentioned.

Among these, pure water having a conductivity of 10.0 μS / cm or less is preferable from the viewpoint of efficiently obtaining nanoparticles having an average particle diameter of 1 to 50 nm.
The conductivity can be measured by a conductivity meter. Examples of the conductivity meter include a compact electrical conductivity meter (manufactured by HORIBA). It can also be measured by specific electrical resistance (MΩ · cm) which is the reciprocal of conductivity.

  The means for dissolving the gallate catechin in the pure water is not particularly limited as long as it is a known means. For example, gallate catechin can be dissolved by adding and mixing with the pure water. Moreover, when making it melt | dissolve, it is preferable to adjust the temperature of the said pure water to 20-90 degreeC from a soluble viewpoint of gallate type catechin, but if it melt | dissolves, there will be no limitation in particular. Note that the gallate catechin may be partially dissolved in pure water, and the gallate catechin-containing liquid may be in a dispersion state.

  The solid content value of the gallate catechin in the gallate catechin-containing liquid is preferably 0.01 to 24% by weight from the viewpoint of efficiently producing nanoparticles having an average particle size of 10 to 50 nm. More preferably, it is 0.1 to 20 weight%.

(Process (B))
In this step, an animal protein-containing solution is prepared by dissolving at least one animal protein selected from gelatin, collagen, and degradation products thereof in pure water to a solid content of 0.1% by weight or more. To do.

The animal protein used in the present invention may be gelatin, collagen, or a degradation product thereof, which can form coacervate with gallate catechin. Animal protein can be derived from pigs, fish, chickens, etc., and genetically modified organisms. These natural products can be used alone or in combination of two or more. May be. Preferably, the animal protein solution having a pH of 4.5 or less is preferable. Even if the pH of the animal protein solution is 4.5 or more, it can be used by adding an acid to 4.5 or less. The acid in this case is not particularly limited. In addition, although animal protein derived from bovine bone or pork bone is partially formed with particles of 50 nm or less, its average particle diameter exceeds 50 nm, so it is difficult to use in the present invention.
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 1-50 nm can be produced, it can use without limitation.

  The pure water used as the solvent may be the same as the pure water that can be used for the gallate catechin-containing solution.

The means for dissolving the animal protein in the pure water is not particularly limited as long as it is a known means. For example, the animal protein can be dissolved by adding and mixing with the solvent. In the present invention, the animal protein may be swollen as one of the states dissolved in pure water.
In addition, swelling means adding a pure water to animal protein and making it gelatinous.
Moreover, when making it melt | dissolve or swell, it is preferable to adjust the temperature of the said solvent to 20-90 degreeC from a viewpoint of making it melt | dissolve or swell efficiently.

The solid content value of the animal protein in the animal protein-containing liquid is preferably 0.1 to 19% by weight, more preferably from the viewpoint of efficiently producing nanoparticles having an average particle diameter of 1 to 50 nm. Is 0.1 to 10% by weight.
When gelatin is used, if the solid content is 20% by weight or more, it becomes difficult to handle due to an increase in the viscosity of the animal protein-containing solution.

(Process (C))
In this step, the gallate-type catechin-containing liquid obtained in the step (A) and the animal protein-containing solution obtained in the step (B) and 2 to 100 times the total weight of these liquids Mix with pure water.

  For example, a method of mixing the gallate-type catechin-containing liquid and the animal protein-containing liquid in 2 to 100 times the amount of pure water with respect to the total weight of these two kinds of liquids can be mentioned.

  As described above, there is an advantage that the particle diameter is easily reduced by mixing the gallate-type catechin-containing liquid and the animal protein-containing liquid in a large amount of pure water.

  In this step, the gallate type catechin-containing liquid, the animal protein-containing liquid, and the pure water may be mixed as long as these components can be mixed uniformly. A method of adding the animal protein-containing solution and pure water to the catechin-containing solution, a method of adding the gallate-type catechin-containing solution and pure water to the standing animal protein-containing solution, and a standing water In addition, a method of adding the gallate-type catechin-containing solution and the animal protein-containing solution, a method of adding while stirring, a method of adding while homogenizing, etc. can be used, but there is no particular limitation. .

  In this step, the conditions such as the temperature at the time of mixing may be any conditions that do not cause a significant change in the components and can be uniformly mixed, 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 becomes easy to occur, 20-80 degreeC is more preferable, More preferably, 50-60 degreeC is good.

In addition, from the viewpoint of efficiently obtaining nanoparticles having an average particle diameter of 1 to 50 nm, as the amount of the gallate catechin-containing liquid and the animal protein-containing liquid,
(A) Solid content of gallate type catechin (b) Weight of animal protein solid content is preferably adjusted and mixed so that 0.07 ≦ (b) / (a) ≦ 8.0. As for the weight ratio, the upper limit value is preferably 7.0 or less.

  In addition, the pH of the mixed solution of the gallate-type catechin-containing liquid, the animal protein-containing liquid, and pure water is 1.0 to from the viewpoint that it can be efficiently formed without dissolving, aggregating, and precipitating the nanoparticles. It is preferable to adjust to 4.0. The pH is more preferably 1.5 to 3.5, and further preferably 1.5 to 3.1.

  The pH is not particularly limited as long as it is a usable acid depending on the intended use of the nanoparticles. 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. When the obtained nanoparticles are used for pharmaceuticals, cosmetics, foods, etc., it is preferable to select an acid suitable for each use application.

  In order to adjust the pH of the mixed solution, the pH of the gallate catechin-containing solution and the animal protein-containing solution may be adjusted in advance. By adjusting the pH in advance in this way, the pH of the resulting mixed solution can be adjusted to a range of 1.0 to 4.0 simply by mixing the gallate-type catechin-containing solution and the animal protein-containing solution. it can.

  The gallate catechin and the animal protein form a coacervate in the mixed solution whose pH is adjusted to the range of 1.0 to 4.0 as described above, and nanoparticles having an average particle diameter of 10 to 50 nm are formed in the coacervate. Occurs.

In the mixed solution, from the viewpoint of efficiently producing nanoparticles, the solid content derived from gallate catechin or a composition containing gallate catechin is 0.01% by weight or more, gelatin, collagen, and degradation products thereof. It is preferable to contain 0.01% by weight or more of a solid content derived from at least one animal protein selected from Further, the total amount of the solid content derived from the gallate catechin and the solid content derived from the animal protein is preferably 0.02 to 0.4% by weight, more preferably 0.1 to 0.3% by weight, and 2 to 0.3% by weight is most preferred.
In addition, you may adjust so that it may become a desired density | concentration at the time of mixing the said gallate type catechin containing liquid and the said animal protein containing liquid, and you may concentrate after producing a nanoparticle.

  The nanoparticle-containing liquid obtained as described above may be subjected to ultrafiltration, dialysis and the like. 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.

  The nanoparticles obtained in the present invention are excellent in stability. A method for measuring the zeta potential on the nanoparticle surface is known as an index indicating the stability of the nanoparticle, and it can be said that the larger the absolute value of this zeta potential, the better the stability. For example, as the nanoparticles obtained in the present invention, a nanoparticle-containing liquid adjusted to a solid content value of 0.2 to 1.0% by weight is used as a zeta potential / nanoparticle diameter measurement system (manufactured by Beckman Coulter, Inc. It is preferable that the absolute value of the zeta potential obtained using DelsaMax PRO ") and the analysis setting as water is 10 mV or more.

  Note that the solvent of the nanoparticle-containing liquid at the time of measurement is pure water, but water other than pure water, a hydrous solvent, and an organic solvent may be added thereto. A water-containing solvent is preferred.

  The organic solvent used as the solvent is not particularly limited as long as it is miscible with water, and examples thereof include glycerin, propylene glycol, ethanol, methanol, acetone, and dimethyl sulfoxide. The hydrous solvent used as the solvent refers to a mixed solvent of the organic solvent and water.

  When the nanoparticles obtained through the above steps (A) to (C) are produced under conditions that can be used for food, they may be blended in food and 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.

  Further, since the nanoparticles obtained in the present invention are considered to be excellent in safety, not only for humans, but also for non-human animals such as rats, mice, guinea pigs, rabbits, sheep, pigs, You may mix | blend with mammals, such as a cow, a horse, a cat, a dog, a monkey, a chimpanzee, birds, amphibians, a reptile, etc., or a 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, The pet food used for a cat, a small bird, a squirrel, etc. is mentioned.

  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 nanoparticles using pure water)
The study of nanoparticles using pure water was performed according to the following procedure.
1 g of gelatin (trade name: G fine powder, Nitta gelatin) was dissolved in 99 g of pure water (conductivity: 0.1 μS / cm) at 50 ° C. to prepare a gelatin solution.
1.8 g of green tea extract (gallate type catechin 64%) was dissolved in 98.2 g of pure water at 50 ° C. to prepare a gallate type catechin solution.
After 1 g of gelatin solution was added to 8 g of pure water and mixed, 1 g of gallate catechin solution was added and mixed at 50 ° C. to prepare 10 g of a solution (pH 4.3) in which nanoparticles were formed.
In addition, the said conductivity was measured with the compact electric conductivity meter (made by HORIBA).

(Comparative example)
As a comparative product, nanoparticles were prepared in the same manner as in Example 1 except that tap water (conductivity 120 μS / cm) was used as a solvent. The results are shown in Table 1.

As can be seen from Table 1, nanoparticles with an average particle size of 10 to 50 nm can be formed safely by using pure water with low conductivity. Further, the nanoparticles obtained in Example 1 are expected to be excellent in bioavailability of gallate catechins and animal proteins since the particle diameter is very small.

Claims (2)

A step (A) of obtaining a gallate-type catechin-containing liquid by dissolving gallate-type catechin in pure water so that the solid content is 0.1% by weight or more;
Step (B) of obtaining an animal protein-containing solution by dissolving at least one animal protein selected from gelatin, collagen, and degradation products thereof in pure water so that the solid content is 0.1% by weight or more. When,
The gallate catechin-containing liquid obtained in step (A) and the animal protein-containing solution obtained in step (B) are mixed with 2 to 100 times the amount of pure water based on the total weight of these liquids. Process (C)
A method for producing nanoparticles having an average particle diameter of 10 to 50 nm.   The method for producing nanoparticles having an average particle diameter of 10 to 50 nm according to claim 1, wherein pure water has a conductivity of 10.0 μS / cm or less.