JP6866060B2 - Manufacturing method of refined tea extract - Google Patents

Description

The present invention relates to a method for producing a purified tea extract.

Tea leaves are rich in non-polymer catechins, and non-polymer catechins can be obtained by extracting from tea leaves, but at the time of extraction, impurities such as caffeine are also extracted at the same time. It ends up.

As a technique for reducing such impurities, for example, tea leaves are extracted with boiling water or an aqueous solution of an organic solvent, the solution containing the extracted components is washed with chloroform, then the extracted components are transsolved in an organic solvent, and then the organic solvent is used. Distillation method (Patent Document 1), method of contacting tea extract with active white clay or acidic white clay (Patent Document 2), tea extract dissolved or suspended in water or a hydrous organic solvent, and synthesized under alkaline conditions. A method of contacting with an adsorbent (Patent Document 3) and the like are known.

On the other hand, it has been reported that the astringency of tea extract can be reduced by coexisting the tea extract with phospholipid (Patent Document 4). Is not known.

Japanese Unexamined Patent Publication No. 59-21938 Japanese Unexamined Patent Publication No. 6-142405 Japanese Unexamined Patent Publication No. 8-109178 Japanese Patent Application Laid-Open No. 2012-503479

An object of the present invention is to provide a method for producing a purified tea extract in which caffeine in the tea extract is reduced.

The present inventor mixes a tea extract and a phospholipid and causes the non-polymer catechins to be incorporated into the phospholipid membrane or its inner aqueous phase, or the non-polymer catechins are adsorbed on the phospholipid membrane. It was found that the non-polymeric catechins and caffeine were separated, and the phospholipid phase was recovered to obtain a purified tea extract with reduced caffeine.

That is, the present invention provides a method for producing a purified tea extract, which comprises a step of mixing a tea extract and a phospholipid and recovering a phospholipid phase from the mixed solution.

According to the present invention, a purified tea extract in which caffeine in the tea extract is reduced can be produced by a simple operation.

Hereinafter, a method for producing the purified tea extract of the present invention will be described.
The method for producing a purified tea extract of the present invention includes a step of mixing a tea extract and a phospholipid and recovering a phospholipid phase from the mixed solution.

Examples of the tea extract include a tea extract, a concentrate thereof, and a purified product thereof, and there are various forms thereof such as a solid, a liquid, a solution, and a slurry. These can be used alone or in combination of two or more. Here, the term "tea extract" as used herein refers to a tea extract extracted from tea leaves using hot water or a hydrophilic organic solvent, which has not been concentrated or purified. As the extraction method and extraction conditions, known methods and conditions can be adopted and are not particularly limited. Examples of tea leaves include the genus Camellia, for example, C. sinensis var. Sinensis (including Camellia species), C. sinensis var. Assamica and tea plants (Camellia sinensis) selected from hybrids thereof. Tea plants can be classified into non-fermented tea, semi-fermented tea, and fermented tea according to the processing method.
Examples of the non-fermented tea include green tea such as sencha, bancha, tencha, potted tea, kukicha, bar tea, and mecha. Examples of semi-fermented tea include oolong tea such as Tieguanyin, color species, golden katsura, and Wuyi tea. Further, examples of fermented tea include black teas such as Darjeeling, Assam and Sri Lanka. These can be used alone or in combination of two or more. Among them, non-fermented tea is preferable, and green tea is more preferable, from the viewpoint of the content of non-polymer catechins.

The "tea extract concentrate" is obtained by removing a part of the solvent from the tea extract to increase the concentration of non-polymer catechins. For example, as a concentration method, normal pressure concentration or vacuum concentration is used. , Membrane concentration and the like. Commercially available products may be used as the concentrate of the tea extract. For example, green tea such as "Polyphenone" by Mitsui Norin Co., Ltd., "Theafran" by ITO EN Co., Ltd., and "Sanphenon" by Taiyo Kagaku Co., Ltd. Examples include concentrates of extracts. Further, the "purified product of the tea extract" is a product obtained by purifying the tea extract or its concentrate to increase the purity of the non-polymer catechins. For example, JP-A-2004-147508, JP. The methods described in JP-A-2004-149416, JP-A-2006-160656, JP-A-2007-282568, JP-A-2008-079609, and the like can be adopted.

The tea extract to be mixed is preferably in the form of an aqueous solution. As the aqueous solution in which the tea extract is dissolved, for example, even if the tea extract extracted from the tea leaves with water is diluted or concentrated with water as necessary, the concentrate of the tea extract or the purified product thereof is used as water. It may be diluted and used, or the tea extract, its concentrate, or a dried product of those purified products may be dissolved again in water and used.

The content of the non-polymer catechins in the aqueous solution in which the tea extract is dissolved can be appropriately selected, but from the viewpoint of purification efficiency, 0.02% by mass or more is preferable, and 0.05% by mass or more is more preferable. , 0.06% by mass or more, more preferably 10.0% by mass or less, more preferably 7.0% by mass or less, still more preferably 5.0% by mass or less. The content of the non-polymer catechins is preferably 0.02 to 10.0% by mass, more preferably 0.05 to 7.0% by mass, and further preferably 0.06 to 5.0% by mass. %. Here, in the present specification, the "non-polymeric catechins" are composed of epigallocatechin gallate, galocatechin gallate, epicatechin gallate and catechin gallate, and epigallocatechin, galocatechin, epicatechin and catechin. It is a general term for non-gallate bodies. The content of the non-polymer catechins is defined based on the total amount of the above eight types, and in the present invention, at least one of the above eight types of non-polymer catechins may be contained.

Further, in the tea extract used in the present invention, the proportion of gallate in the non-polymer catechins (hereinafter, also referred to as “gallate content”) is preferably 30% by mass or more, preferably 35% by mass, from the viewpoint of physiological effects. % Or more is more preferable, 40% by mass or more is further preferable, and from the viewpoint of flavor, 70% by mass or less is more preferable, 65% by mass or less is more preferable, and 60% by mass or less is further preferable. The range of the gallate body ratio is preferably 30 to 70% by mass, more preferably 35 to 65% by mass, and further preferably 40 to 60% by mass. Here, in the present specification, the "gallate form ratio" is the mass ratio of the above four types of gallate form to eight kinds of non-polymer catechins.

Further, the tea extract used in the present invention preferably has a mass ratio [(C) / (A)] of (A) non-polymer catechins and (C) caffeine of 0.01 or more, preferably 0.03. The above is more preferable, 0.05 or more is further preferable, 3.0 or less is preferable, 2.0 or less is more preferable, and 1.5 or less is further preferable. The range of the mass ratio [(C) / (A)] is preferably 0.01 to 3.0, more preferably 0.03 to 2.0, and further preferably 0.05 to 1.5. ..

As the phospholipid, those derived from egg yolk, soybean and other animal and plant materials can be used without particular limitation, and semi-synthetic phospholipids such as hydrogenated compounds and derivatives of hydroxides thereof, synthetic products and the like can be used. You may. The constituent fatty acids of the phospholipid are not particularly limited, and may be either saturated fatty acids or unsaturated fatty acids. Further, the phospholipid may include a charged phospholipid such as an anionic phospholipid and a cationic phospholipid, and a polymerizable phospholipid in addition to the neutral phospholipid.
Phospholipids can be used alone or in combination of two or more.

Examples of phospholipids include glycerophospholipids, lysoglycerophospholipids, and sphingolipids. Of these, glycerophospholipids and sphingolipids are preferable. Examples of the glycerophospholipid include phosphatidylcholine, phosphatidylglycerol, phosphatidylate, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine, and among them, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine are preferable. Examples of sphingolipids include sphingomyelin and ceramide myelin. As a raw material containing these phospholipids, natural lecithin or a refined product obtained by hydrogenating the natural lecithin can also be used. Examples of natural lecithin include egg yolk lecithin, soybean lecithin, squid lecithin and the like, and examples of hydrogenated phospholipids include hydrogenated soybean phosphatidylcholine and hydrogenated egg yolk lecithin.

The phospholipid when mixed with the tea extract preferably forms a phospholipid film, more preferably in the form of liposomes. By making the phospholipid in the form of a phospholipid membrane or a liposome, it is easy to take up the non-polymer catechins into the phospholipid membrane or its inner aqueous phase and to adsorb the non-polymer catechins to the phospholipid membrane. Become. Here, the term "liposome" as used herein refers to a closed vesicle having an inner aqueous phase portion surrounded by a phospholipid bilayer membrane, and depending on its size and the number of lipid bimolecules, a multiphase liposome (Multimulellar Vesicle: It is classified into three types: MLV), large single-membrane liposome (LUV), and small single-membrane liposome (Small Universal Vesicle: SUV). Any type of liposome can be used in the present invention.

When adsorbing non-polymer catechins on a phospholipid membrane, for example, a tea extract and a phospholipid are mixed and stressed by shaking or the like to form a phospholipid membrane and non-polymer catechins on the phospholipid membrane. May be adsorbed substantially at the same time. It is also possible to apply stress to the phospholipid by shaking or the like to form a phospholipid film in advance, and then mix the tea extract with the phospholipid film.

When phospholipids are made into liposomes, phospholipids and one or two selected from glycolipids and dialkyl-type synthetic surfactants may be appropriately combined as the membrane constituents thereof. Further, cholesterol or the like as a membrane stabilizer, stearylamine, phosphatidic acid or the like as a charged substance, and tocopherol or the like as an antioxidant may be added as appropriate in one or a combination of two or more.

The method for forming liposomes is not particularly limited, and methods such as the Bangham method, the lipid dissolution method, the mechanochemical method, and the freeze-dried liposome method can be adopted. For example, in the Bangham method, a phospholipid is dissolved in an organic solvent such as chloroform, ether, or ethanol, the solvent is distilled off under reduced pressure, the phospholipid is further dried under reduced pressure, and then suspended at a temperature equal to or higher than the phase transition temperature of the phospholipid. It can be formed by allowing it to form. Further, the particle size of the liposome can be appropriately adjusted by utilizing the membrane permeation or the shearing force due to the mechanical force.

The phospholipid, phospholipid membrane, and liposome used in the present invention preferably have a phase transition temperature. Here, the "phase transition temperature" as used herein means a temperature at which a phase transition occurs between the gel and liquid crystal states that phospholipids can take, and when a sufficient amount of water is present for phospholipids. Is the value of. The phase transition temperature can be measured by differential thermal analysis using a differential scanning calorimetry (DSC). As the differential scanning calorimeter, for example, DSC7020 (manufactured by Hitachi High-Tech Science) can be used. The phase transition temperature in the present specification indicates a measured value based on the method described in Examples described later.

The phase transition temperature of the phospholipid is preferably −25 ° C. or higher, more preferably −20 ° C. or higher from the viewpoint of handling, and preferably 60 ° C. or lower from the viewpoint of the uptake rate and adsorption rate of non-polymer catechins. 50 ° C. or lower is more preferable, 40 ° C. or lower is further preferable, and 30 ° C. or lower is even more preferable. The range of the phase transition temperature is preferably −25 ° C. to 60 ° C., more preferably −20 ° C. to 50 ° C., further preferably −20 ° C. to 40 ° C., and even more preferably −20 ° C. to 30 ° C. ..

Examples of the phospholipid having a phase transition temperature include soybean phosphatidylcholine (phase transition temperature of about -10 ° C. measured by the method described in Examples described later), hydrogenated soybean phosphatidylcholine (manufacturer's nominal phase transition temperature of about 53 ° C.), and di. Millistylphosphatidylcholine (phase transition temperature of reference 1 below), dipalmitoylphosphatidylcholine (phase transition temperature of reference 1 below), distearoylphosphatidylcholine (phase transition of reference 1 below) Temperature about 54 ° C), dihexadecylphosphatidylcholine (phase transition temperature of about 45 ° C described in Reference 2 below), stearoyl paritemilphosphatidylcholine (phase transition temperature of reference 3 below), dipalmitoylphosphatidyl Ethanolamine (phase transition temperature of about 63 ° C. described in Reference 2 below) and the like can be mentioned. Of these, phosphatidylcholine having a choline group is preferable. As the phospholipid used in the present invention, a commercially available product or a synthetic product can be appropriately selected and used, but the phospholipid content is preferably 60% by mass or more, preferably 80% by mass or more. More preferred.

Reference 1: Kinshin et al., Phosphorlipid bilayer membrane under high pressure. Science and technology of high pressure, vol9, No3, p.213-220 (1999)
Reference 2: Matsuki et al. Membrane state of biological membrane lipids-Structural-function correlation revealed from pressure studies-, Science and technology of high pressure, vol23, No1, p.30-38 (2013)
Reference 3: Abstracts of the 32nd Physical Chemistry Study Group

The median diameter of the liposome is preferably 10 nm or more, more preferably 30 nm or more, further preferably 50 nm or more, further preferably 80 nm or more, and particularly preferably 100 nm or more, from the viewpoint of the separation operation between the phospholipid phase and the liquid phase. From the viewpoint of the uptake rate of non-polymer catechins, 20,000 nm or less is preferable, 15,000 nm or less is more preferable, 10,000 nm or less is further preferable, 5,000 nm or less is further preferable, and 3,000 nm or less. Is more preferable, 1,000 nm or less is further preferable, and 500 nm or less is particularly preferable. The range of the median diameter is preferably 10 to 20,000 nm, more preferably 30 to 15,000 nm, still more preferably 50 to 10,000 nm, still more preferably 50 to 5,000 nm, still more preferably 50 to 50 to It is 3,000 nm, more preferably 80 to 1,000 nm, and even more preferably 100 to 500 nm. The "median diameter" here means a particle size corresponding to _{a cumulative value of 50% (d 50} ) in a volume-based cumulative particle size distribution measured by a laser diffraction / scattering method.

The mixing ratio of the tea extract and the phospholipid is the mass ratio of (A) non-polymer catechins and (B) phospholipid in the tea extract [(A) / (B)] from the viewpoint of production efficiency. Therefore, 0.001 or more is preferable, 0.01 or more is more preferable, 0.02 or more is further preferable, and from the viewpoint of the recovery rate of non-polymer catechins, 0.20 or less is preferable, and 0.18 or less is preferable. More preferably, 0.11 or less is further preferable, and 0.09 or less is particularly preferable. The range of the mass ratio [(A) / (B)] is preferably 0.001 to 0.20, more preferably 0.01 to 0.18, still more preferably 0.01 to 0.11, particularly. It is preferably 0.02 to 0.09.

The mixing temperature of the tea extract and the phospholipid is preferably a temperature higher than the phase transition temperature of the phospholipid used, and more preferably 5 ° C. or more higher than the phase transition temperature of the phospholipid used. Preferably, the temperature is 10 ° C. or higher, more preferably 20 ° C. or higher, and even more preferably 20 ° C. or higher.
The mixing time of the tea extract and the phospholipid is not uniform depending on the production scale and the like, but from the viewpoint of the recovery rate of non-polymer catechins, 10 minutes or more is preferable, 15 minutes or more is more preferable, and 20 minutes or more. Is more preferable, and from the viewpoint of purification efficiency, 360 minutes or less is preferable, 120 minutes or less is more preferable, and 60 minutes or less is further preferable. The range of such mixing time is preferably 10 to 360 minutes, more preferably 15 to 120 minutes, and even more preferably 20 to 60 minutes. A liquid temperature control means can be provided so that the temperature of the mixed liquid is kept constant for a predetermined time.

The pH (25 ° C.) when the tea extract and the phospholipid are mixed is preferably 2.5 or more, more preferably 4.0 or more, further preferably 6.0 or more, and further preferably 6.0 or more, from the viewpoint of preventing equipment corrosion. From the viewpoint of the stability of the non-polymer catechins, 9.0 or less is preferable, 8.0 or less is more preferable, and 7.0 or less is further preferable. The pH range is preferably 2.5 to 9.0, more preferably 4.0 to 8.0, and even more preferably 6.0 to 7.0.

The mixing order of the tea extract and the phospholipid is not particularly limited, and the tea extract and the phospholipid may be added at the same time and mixed, or one may be added to the other and mixed. Further, when the tea extract and the phospholipid are mixed, treatments such as stirring, shaking, and ultrasonic irradiation may be performed.

By mixing the tea extract and phospholipids, non-polymer catechins are incorporated into the phospholipid membrane or into liposomes, or non-polymer catechins are adsorbed on the phospholipid membrane, while impurities such as caffeine are adsorbed. Is present as it is outside the phospholipid membrane or in the outer aqueous phase of liposomes, so that non-polymer catechins and impurities such as caffeine can be separated.

After mixing, the phospholipid phase is recovered from the mixed solution, and examples of the recovery method include solid-liquid separation such as ultrafiltration, centrifugation, gel chromatography, and dialysis. Solid-liquid separation can be performed by one type or a combination of two or more types. Of these, centrifugation is preferred. By centrifuging the mixed solution, the phospholipid phase can be easily recovered as a precipitate layer.

As the centrifuge, general equipment such as a separation plate type, a cylindrical type, and a decanter type can be used, and an ultracentrifuge may be used if necessary.
From the viewpoint of the recovery rate of the phospholipid phase, the temperature at the time of centrifugation is preferably 1 ° C. or higher, more preferably 2 ° C. or higher, further preferably 3 ° C. or higher, and preferably 60 ° C. or lower, more preferably 50 ° C. or lower. It is preferable, and more preferably 40 ° C. or lower. The temperature range is preferably 1 to 60 ° C, more preferably 2 to 50 ° C, and even more preferably 3 to 40 ° C.
From the viewpoint of the recovery rate of the phospholipid phase, the centrifugation time is preferably 3 to 300 minutes, more preferably 5 to 200 minutes, still more preferably 10 to 100 minutes, and even more preferably 15 to 60 minutes.
Further, the conditions for centrifugation are such that the relative centrifugal acceleration is preferably 300 G or more, more preferably 500 G or more, still more preferably 800 G or more, from the viewpoint of caffeine removal and recovery rate of non-polymer catechins. Further, from the viewpoint of shortening the processing time, 10,000 G or more is preferable, 60,000 G or more is more preferable, and 120,000 G or more is further preferable. The upper limit is not limited, but is preferably 170,000 G or less, for example. The rotation speed and radius of rotation of the centrifuge can be appropriately selected so that the relative centrifugal acceleration is within the above range. Here, in the present specification, the "relative centrifugal acceleration" means a value calculated by the following equation (1).

Relative centrifugal acceleration (G) = 1188 × r × N ² × ^10-8 (1)

[In the formula (1), r indicates the maximum turning radius (cm) of the centrifuge, and N indicates the number of revolutions per minute (rpm). ]

The phospholipid phase obtained by solid-liquid separation can be separated from phospholipids by performing separation operations such as membrane separation after extraction with a solvent such as ethanol, if necessary. It is possible, and it can also be used as it is as a purified tea extract.

The amount of lipid in the obtained phospholipid phase can be quantified by using, for example, a phospholipid quantification kit such as phospholipid C Test Wako (manufactured by Wako Junyaku Co., Ltd.) or HPLC.

In this way, a refined tea extract with reduced caffeine can be produced. In addition, the production method of the present invention can also recover non-polymer catechins in a yield of preferably 50% or more, more preferably 55% or more, still more preferably 60% or more.

The purified tea extract obtained by the production method of the present invention has a mass ratio [(C) / (A)] of (A) non-polymer catechins and (C) caffeine, preferably 1.0 or less. , More preferably 0.5 or less, still more preferably 0.1 or less, even more preferably 0.01 or less, and particularly even more preferably 0.009 or less. The mass ratio [(C) / (A)] may be 0, but from the viewpoint of production efficiency, it is preferably 0.00001 or more, and more preferably 0.0001 or more.

1. 1. Measurement of non-polymer catechins and caffeine The purified tea extract was appropriately diluted with a 0.1 mol / L acetate-dimethylsulfooxide solution and filtered through a 0.2 μm filter to prepare a sample. For the measurement of non-polymer catechins and caffeine, a high performance liquid chromatograph (model SCL-10AVP, manufactured by Shimadzu Corporation) was used, and a packed column for an octadecyl group-introduced liquid chromatograph (L-column TM ODS, 4.6 mmφ ×). 250 mm: (manufactured by Chemical Substance Evaluation and Research Organization) was attached, and the column temperature was 35 ° C. by the gradient method. As a standard product of non-polymer catechins, those manufactured by Mitsui Norin Co., Ltd. were used and quantified by the calibration curve method. The mobile phase A solution was a distilled aqueous solution containing 0.1 mol / L of acetic acid, and the B solution was an acetonitrile solution containing 0.1 mol / L of acetic acid. The sample injection amount was 20 μL and the UV detector wavelength was 280 nm. .. The conditions for the gradient are as follows.

Hours (minutes) Liquid A concentration (volume%) Liquid B concentration (volume%)
0.0 97 3
5.0 97 3
37.0 80 20
43.0 80 20
43.5 0 100
48.5 0 100
49.0 97 3
60.0 97 3

2. pH measurement The pH was measured at 25 ° C. using a pH meter (HORIBA compact pH meter, manufactured by HORIBA, Ltd.).

3. 3. Measurement of median diameter A volume-based cumulative particle size distribution measurement was performed using a laser diffraction / scattering particle size distribution measuring device (HORIBA LA-920, manufactured by HORIBA, Ltd.) to determine the median diameter.

4. Measurement of Phase Transition Temperature of Phospholipids (1) For phospholipids having a phase transition temperature at a temperature exceeding 0 ° C., the phase transition temperature is measured by the following procedure. First, a sample in which phospholipids are sufficiently dispersed in water is prepared so that the phospholipid concentration is 1 to 10 g / 100 g so that a sufficient amount of water is present with respect to the phospholipids. Next, 0.005 g of the sample is weighed in an aluminum sample container, covered with an aluminum cover, and then sealed using an electric sample sealer. Place the sealed sample container on the holder unit in the electric furnace. Further, a blank container in which air is sealed is placed on the blank side of the holder unit. Cover the electric furnace and keep it in a nitrogen atmosphere at 1 ° C. for 5 minutes. Next, heating is performed from 1 ° C. to 98 ° C. at a heating rate of 0.5 ° C./min (heating step). In this temperature raising step, the DSC curve is measured using a differential scanning calorimeter DSC7020 (manufactured by Hitachi High-Tech Science). At this time, the temperature at which the endothermic peak is observed in the temperature raising step is defined as the phase transition temperature of the phospholipid.
(2) For phospholipids whose phase transition temperature cannot be confirmed due to the above measurement conditions, the phase transition temperature is measured by the following procedure. First, a sample in which 50% by mass of an ethylene glycol aqueous solution with respect to phospholipid is sufficiently dispersed in water so that the phospholipid concentration is 1 to 10 g / 100 g is prepared. Next, a sample container and a blank container are prepared and placed on the holder unit by the same operation as described above. Cover the electric furnace and keep it in a nitrogen atmosphere at 25 ° C. for 5 minutes. Then, the mixture is cooled from 25 ° C. to −40 ° C. at a temperature lowering rate of 0.5 ° C./min (cooling step). In this cooling step, the DSC curve is measured using a differential scanning calorimeter DSC7020 (manufactured by Hitachi High-Tech Science). At this time, the temperature at which the exothermic peak is observed in the cooling step is defined as the phase transition temperature of the phospholipid.

Preparation Example 1
Preparation of Liposome Suspension A Soybean phosphatidylcholine (Coatsome NC20, manufactured by Nichiyu Co., Ltd., measured phase transition temperature: −10 ° C.) was used as a phospholipid, and 0.312 g of this was collected in a 100 mL eggplant flask. Then, 10 mL of chloroform was added dropwise, and the mixture was completely dissolved while being heated to 25 ° C. in a water bath. Then, chloroform was distilled off using a rotary evaporator. Further, in order to completely remove the organic solvent, after drying under reduced pressure for 30 to 60 minutes _{, 6 mL of phosphate buffer (17 mM-Na 2} HPO ₄ + 49 mM-KH ₂ PO ₄ , pH 6.4) was added dropwise. Then, the mixture was stirred while heating at 25 ° C. to hydrate soybean phosphatidylcholine to form liposomes. Then, in a sealed state, the container was cooled in a cooling bath at −40 ° C. for 30 minutes or more and frozen. Then, the sample was heated in a water bath at 25 ° C. until the sample was completely dissolved. This freezing and thawing operation was repeated 3 times. Next, the particle size was adjusted by passing the suspension through a polycarbonate film having a pore size of 100 nm 11 times or more in a state of being heated to 25 ° C. By the above operation, liposome suspension A (phospholipid concentration 4943 mg / 100 mL, median diameter of liposomes 168 nm) was obtained.

Preparation Example 2
Preparation of Liposome Suspension B As the phospholipid, hydrogenated soybean phosphatidylcholine (Coatsome NC21, manufactured by Nichiyu Co., Ltd., measured value of phase transition temperature: 51 ° C.) having a manufacturer's nominal phase transition temperature of 53 ° C. was used, and the heating temperature was adjusted. The same operation as in Preparation Example 1 was carried out except that the temperature was changed to 60 ° C. to obtain a liposome suspension B (phospholipid concentration 4943 mg / 100 mL, liposome median diameter 215 nm).

Preparation Example 3
Preparation of Liposome Suspension C The same procedure as in Preparation Example 1 was carried out except that a polycarbonate membrane having a pore size of 1000 nm was used instead of passing through the polycarbonate membrane having a pore size of 100 nm, and liposome suspension C (phospholipid concentration: 4943 mg) was performed. / 100 mL, liposome median diameter 733 nm) was obtained.

Preparation Example 4
Preparation of Liposome Suspension D The same operation as in Preparation Example 1 was carried out except that the shearing treatment with a small sealed high-pressure homogenizer was performed at 4000 rpm for 20 minutes instead of the polycarbonate membrane having a pore size of 100 nm. (Phospholipid concentration 4943 mg / 100 mL, liposome median diameter 2461 nm) was obtained.

Preparation Example 5
Preparation of Liposome Suspension E The same procedure as in Preparation Example 1 was carried out except that the polycarbonate membrane having a pore size of 100 nm was not passed through, and the liposome suspension E (phospholipid concentration 4943 mg / 100 mL, liposome median diameter 6378 nm) was performed. Got

Adjustment example 6
Preparation of Liposome Suspension F 0.124 g of soybean phosphatidylcholine (Coatsome NC20, manufactured by NOF Corporation) and 0.124 g of hydrogenated soybean phosphatidylcholine (Coatsome NC21, manufactured by NOF Corporation) were used as phospholipids. Further, the same operation as in Preparation Example 1 was carried out except that the heating temperature was 60 ° C. and the dropping phosphoric acid buffer was 10 mL, and the liposome suspension F (phospholipid 2471 mg / 100 mL, liposome median diameter 141 nm, phase) was carried out. Transition temperature measurement value: 36 ° C.) was obtained.

Preparation Example 7
Preparation of Lipid Suspension A 100 parts by mass of mixed fatty acid of soybean oil fatty acid: rapeseed oil fatty acid = 7: 3 (mass ratio) using deodorized fat as a raw material and 15 parts by mass of glycerin are mixed and esterified by an enzyme. It was. Fatty acids and monoacylglycerol were removed from the obtained esterified product by top-cut distillation, and then acid treatment (0.5% addition of 50% aqueous citric acid solution) was performed at 80 ° C. Next, the oil and fat was washed with 10% distilled water three times to obtain diacylglycerol water-washed oil (diacylglycerol 88%). Next, deodorization was performed at 235 ° C. for 60 minutes to obtain a deodorizing oil. 0.312 g of this deodorizing oil (86% by mass of diacylglycerol) was dispersed in 6 mL of a phosphate buffer to prepare a lipid suspension A.

Preparation Example 8
Preparation of Lipid Suspension B Lipid suspension B was prepared by dispersing 0.312 g of commercially available rapeseed oil (99% by mass of triacylglycerol) in 6 mL of phosphate buffer.

Preparation Example 9
Preparation of Green Tea Extract A Green tea extract (non-polymer catechin concentration 32% by mass) and acid clay (Mizuka Ace # 600, manufactured by Mizusawa Industrial Chemicals Co., Ltd.) were dispersed in 800 g of a 68 mass% ethanol aqueous solution and heated to 40 ° C. Stirring was continued for 6 hours in this state. Then, the generated precipitate and acid clay were filtered through No. 2 filter paper. The obtained filtrate was brought into contact with 30 g of activated carbon (Kuraraycol GLC, manufactured by Kuraray Chemical Co., Ltd.), and subsequently filtered through a 0.2 μm membrane filter. Finally, 200 g of ion-exchanged water was added and ethanol was distilled off under reduced pressure, and then the water content was adjusted to obtain "green tea extract A". The concentration of non-polymer catechins in green tea extract A is 0.2% by mass, the concentration of caffeine is 0.016% by mass, the percentage of gallate is 50% by mass, and the mass ratio of caffeine / non-polymer catechins is 0. It was .08.

Preparation Example 10
Preparation of Green Tea Extract B 100 g of acid clay (Mizuka Ace # 600, manufactured by Mizusawa Industrial Chemicals, Inc.) was dispersed in 800 g of a 92% by mass ethanol aqueous solution at room temperature, and after stirring for about 10 minutes, green tea extract (non-polymer catechins). 200 g (concentration 32% by mass) was added, and stirring was continued for 6 hours. Then, the generated precipitate and acid clay were filtered through No. 2 filter paper. 417 g of ion-exchanged water was added to the obtained filtrate, and the mixture was stirred at 15 ° C. for about 5 minutes. The mixed solution was separated from the turbid component precipitated at an operating temperature of 15 ° C. using a small cooling centrifuge (manufactured by Hitachi Koki Co., Ltd.) (6000 rpm, 5 minutes). The separated solution was brought into contact with 30 g of activated carbon (Kuraraycol GLC, manufactured by Kuraray Chemical Co., Ltd.), followed by filtration through a 0.2 μm membrane filter. Finally, 200 g of ion-exchanged water was added and ethanol was distilled off under reduced pressure, and then the water content was adjusted to obtain "green tea extract B". The concentration of non-polymer catechins in green tea extract B was 0.13% by mass, the concentration of caffeine was 0.0034% by mass, the percentage of gallate was 46.4% by mass, and the mass with caffeine / non-polymer catechins. The ratio was 0.026.

Example 1
1.0 mL of the liposome suspension A obtained in Preparation Example 1 and 1.0 mL of green tea extract A (non-polymer catechin concentration 197 mg / 100 mL) obtained in Preparation Example 9 were mixed at 25 ° C. The mixture was incubated at 25 ° C. for 30 minutes with shaking. Then, as a separation operation, centrifugation was performed under the conditions of 4 ° C. and 166000 G (rotation speed: 50,000 rpm, 30 minutes) to precipitate the liposome phase, and the liquid phase of the supernatant was removed. The purified green tea extract of the precipitated phase was recovered. The obtained purified green tea extract was analyzed. The uptake rates of non-polymer catechins and caffeine in the precipitated phase were converted based on the blended mass assuming that 100% of the phospholipids had precipitated by centrifugation. The results are shown in Table 1.

Example 2
Instead of the liposome suspension A, 0.5 mL of the liposome suspension B obtained in Preparation Example 2 and 1.0 mL of green tea extract B instead of the green tea extract A, and a phosphate buffer (17 mM-). A purified green tea extract was obtained by the same operation as in Example 1 except that 0.5 mL of _{Na 2} HPO ₄ + 49 mM-KH ₂ PO _{4, pH 6.4) was used and these were mixed at 60 ° C. for 300 minutes.} .. The obtained purified green tea extract was analyzed. The results are shown in Table 1.

Comparative Examples 1 and 2
Instead of the liposome suspension A, 0.4 mL of the lipid suspension A obtained in Preparation Example 7 was used and mixed with 1.0 mL of the green tea extract A and 0.6 mL of the phosphate buffer at 25 ° C. or 60 ° C. Except for the above, the same operation as in Example 1 was carried out, and the lipid phase floating on the upper part of the aqueous phase was recovered to obtain a purified green tea extract. The obtained purified green tea extract was analyzed. The results are shown in Table 1.

Comparative Examples 3 and 4
Instead of the liposome suspension A, 0.4 mL of the lipid suspension B obtained in Preparation Example 8 was used and mixed with 1.0 mL of the green tea extract A and 0.6 mL of the phosphate buffer at 25 ° C. or 60 ° C. Except for this, the same operation as in Example 1 was carried out, and the lipid phase floating on the upper part of the aqueous phase was recovered to obtain a purified green tea extract. The obtained purified green tea extract was analyzed. The results are shown in Table 1.

Examples 3 to 6
Except that the mixing ratio of the liposome suspension A, the green tea extract A, and the phosphate buffer was changed to the amount shown in Table 2, and the mixing time was changed to 60 minutes in Examples 4 and 5. A purified green tea extract was obtained by the same operation as in Example 1. The obtained purified green tea extract was analyzed. The results are shown in Table 2 together with the results of Example 1.

Examples 7-9
Example 1 except that the liposome suspensions C to E obtained in Preparation Examples 3 to 5 were used instead of the liposome suspension A, and the mixing time was changed to 60 minutes in Example 8. A purified green tea extract was obtained by the same operation as in the above. The obtained purified green tea extract was analyzed. The results are shown in Table 3 together with the results of Example 1.

Example 10
A purified green tea extract was obtained by the same operation as in Example 1 except that the green tea extract B was used instead of the green tea extract A and the mixing time was changed to 300 minutes. The obtained purified green tea extract was analyzed. The results are shown in Table 4 together with the results of Example 2.

Example 11
A purified green tea extract was obtained by the same operation as in Example 10 except that the mixing temperature was set to 60 ° C. The obtained purified green tea extract was analyzed. The results are shown in Table 4 together with the results of Example 2.

Example 12
A purified green tea extract was obtained by the same operation as in Example 2 except that the mixing temperature was changed to 25 ° C. The obtained purified green tea extract was analyzed. The results are shown in Table 4 together with the results of Example 2.

Examples 13 and 14
Instead of the liposome suspension A, 1.0 mL of the liposome suspension F obtained in Preparation Example 6 and 1.0 mL of the green tea extract B instead of the green tea extract A were added at 5 ° C. or 60 ° C. A purified green tea extract was obtained by the same operation as in Example 1 except that the mixture was mixed for 30 minutes. The obtained purified green tea extract was analyzed. The results are shown in Table 4 together with the results of Example 2.

Example 15
As phospholipids, 0.806 g of soybean phosphatidylcholine (Coatsome NC20, measured value of phase transition temperature manufactured by Nichiyu Co., Ltd .: -10 ° C) and 0 of green tea extract powder (non-polymer catechin concentration 32% by mass). .247 g was mixed with 10.104 g of ion-exchanged water. The mixture was incubated at 25 ° C. for 30 minutes with shaking. 1.0 mL of the mixture after shaking was collected, and the suspension composition was analyzed after mixing. Next, as a separation operation, centrifugation was performed under the conditions of 25 ° C. and 1000 G (rotation speed 3000 rpm) for 30 minutes to precipitate the phospholipid phase and remove the liquid phase of the supernatant. The purified green tea extract of the precipitated phase was recovered. The obtained purified green tea extract was analyzed. The results are shown in Table 5.

Example 16
As phospholipids, 0.203 g of soybean phosphatidylcholine and 0.124 g of green tea extract powder were mixed with 10.09 g of ion-exchanged water, and the purified green tea extract was prepared by the same operation as in Example 15. Obtained. The obtained purified green tea extract was analyzed. The results are shown in Table 5.

From Tables 1 to 5, it can be seen that a purified tea extract with reduced caffeine can be obtained by mixing the tea extract and the phospholipid and recovering the phospholipid phase from the mixed solution.

Claims (8)

A tea extract having a mass ratio of (A) non-polymeric catechins and (C) caffeine [(C) / (A)] of 0.01 or more and a phospholipid are mixed and mixed in the presence of water. A method for producing a purified tea extract, which comprises a step of recovering the phospholipid phase from the liquid. The method for producing a purified tea extract according to claim 1, wherein the tea extract and the phospholipid are mixed at a temperature higher than the phase transition temperature of the phospholipid. The method for producing a purified tea extract according to claim 1 or 2, wherein the phase transition temperature of the phospholipid is 25 to 60 ° C. The method for producing a purified tea extract according to any one of claims 1 to 3, wherein the phospholipid is in the form of liposomes. The method for producing a purified tea extract according to claim 4, wherein the liposome has a median diameter of 10 to 20,000 nm in a volume-based cumulative particle size distribution. The mixing ratio of the tea extract and the phospholipid is 0.001 to the mass ratio of (A) non-polymeric catechins and (B) phospholipid in the tea extract [(A) / (B)]. The method for producing a purified tea extract according to any one of claims 1 to 5, which is 0.20. The method for producing a purified tea extract according to any one of claims 1 to 6, wherein the mixing time of the tea extract and the phospholipid is 10 to 360 minutes. The method for producing a purified tea extract according to any one of claims 1 to 7, wherein the phospholipid phase is recovered from the mixed solution by centrifugation.