JP7307757B2 - Bilberry fruit extract containing reduced amounts of organic acids and extraction method therefor - Google Patents

Description

The present invention relates to an extract of bilberry fruit containing reduced amounts of organic acids and an extraction method therefor.

Anthocyanins are water-soluble pigments responsible for the attractive colors of many flowers, fruits and leaves. Generally, they can be extracted from plants with acidified alcoholic solvents and many are commercially available as food colorings. They are often supplied with maltodextrin as a diluent of suitable concentration for incorporation into beverages or other foods such as cereals.

Anthocyanidins, which are the aglycon components of anthocyanins, have the basic structure shown in formula (I).

Due to their particular chemical structure, anthocyanins and anthocyanidins are characterized by electron deficiency, which makes them highly reactive towards reactive oxygen species (ROS), also known as free radicals, As a result, they are considered to be powerful natural antioxidants.

More specifically, the bioavailability of anthocyanins is low due to their sensitivity to changes in pH. Anthocyanins are generally stable at pH values below 3.5 and are therefore stable under gastric conditions.

Brief description of the invention

The present invention provides an extract of bilberry fruit containing reduced amounts of organic acids and an extraction method therefor.

The present inventors have found that by pressing bilberry fruit, the water content including organic acids is partially or completely removed to produce fruit with a reduced water content (e.g., pressed product, particularly presscake). It was found that by further treatment with acid, large amounts of anthocyanins can be recovered from bilberry fruit, and the recovered anthocyanins can further reduce the organic acid content (particularly the chlorogenic acid content). The present invention is based on such findings. In addition, anthocyanins separated from organic acids have improved intestinal absorbability and are suitable as oral preparations.

According to the present invention, the following inventions are provided.
[1] A method for extracting a bilberry extract,
pressing bilberry fruit to obtain fruit with reduced moisture content;
treating the fruit with reduced water content with an acid to obtain a bilberry extract as the water-soluble fraction;
Method.
[2] The method of [1] above, wherein the acid treatment is hydrochloric acid treatment.
[3] The method according to [1] or [2] above, further comprising obtaining a water-soluble fraction from the acid-treated product.
[4] The method according to any one of [1] to [3] above, further comprising concentrating polyphenols in the obtained water-soluble fraction.
[5] The method according to any one of [1] to [4] above, wherein the obtained bilberry extract has an anthocyanin purity of 50% or more and a contaminant content of 15% by weight or less relative to the total anthocyanins. .
[6] The method according to any one of [1] to [4] above, wherein the obtained bilberry extract has an anthocyanin purity of 50% or more and a quinic acid content of 2% by weight or less relative to the total anthocyanins. .
[7] The method according to any one of the above [1] to [6], wherein in the pressing step, the fruit is pressed at 10°C or lower and pectinase is not added to the fruit.
[8] A method for producing an anthocyanin composition,
A method comprising the extraction method according to any one of [1] to [7] above.
[9] A bilberry extract containing anthocyanins, having an anthocyanin purity of 50% by weight or more and a contaminant content of 15% by weight or less relative to total anthocyanins.
[10] The bilberry extract according to [9] above, wherein the obtained bilberry extract has an anthocyanin purity of 50% or more and a quinic acid content of 2% by weight or less relative to the total anthocyanins.
[11] The bilberry extract according to [9] or [10] above, wherein the bilberry extract contains depolymerized anthocyanins.
[12] A supplement containing the bilberry extract or dried product thereof according to any one of [9] to [11] above.

FIG. 1 shows the time course of the total blood anthocyanin concentration in rats after oral administration of the indicated bilberry extracts. FIG. 2 is a flow chart showing the steps of extracting an anthocyanin extract from bilberry fruit in the present invention. In the flowchart, step S1 is compression, step S2 is acid treatment of the residue, and step S3 is filtration as an example of removing the water-soluble fraction. After that, the obtained water-soluble fraction is subjected to purification as step S4.

Specific description of the invention

As used herein, "bilberry" is a low shrub of the genus Vaccinium of the Ericaceae family. Bilberries produce edible fruits rich in anthocyanins (referred to as "bilberry fruits"). Bilberry includes Vaccinium myrtillus L. and several related species.

As used herein, "bilberry extract" means an extract from bilberry fruit containing anthocyanins. The bilberry extract can be in liquid form. The bilberry extract can be in solid form (eg, dried solid form). The bilberry extract contains anthocyanins (Mw: about 550) and can also be called an anthocyanin composition.

As used herein, "anthocyanin" includes both glycosylated anthocyanins (anthocyanosides) and aglycones of anthocyanosides (anthocyanidins). Anthocyanosides are compounds in which a sugar is covalently attached to an unsugar aglycone, anthocyanidin. Anthocyanidin, which is an aglycon component of anthocyanin, has a basic structure represented by formula (II) below. Anthocyanins can exist in polymeric form with other molecules such as organic acids. As used herein, "depolymerized anthocyanins" refers to anthocyanins that are not polymerized (monomers of anthocyanins), ie, anthocyanins other than anthocyanins that have been polymerized with molecules other than organic acids or organic acids.

R ₁ -R ₇ in formula (II) in representative anthocyanidins are summarized in the table below.

Glycosylated forms of anthocyanins are more water soluble and stable than anthocyanidins. Anthocyanosides are classified by the number of glycosyl units they contain. Monoglycosides contain one sugar moiety predominantly attached to the 3-hydroxyl group of the aglycone. Diglycosides generally contain two monosaccharides at the 3- and 5-hydroxyl positions, and occasionally at the 3- and 7-hydroxyl positions. A triglycoside generally has a bond when there are two units at the 3-position and one at the C-5 or C-7 position. Glycosylation at the 3', 4' and/or 5' positions is also possible.

The most common sugars of anthocyanins include the monosaccharides glucose, rhamnose, galactose, arabinose and xylose. The di- and trisaccharides most frequently found in anthocyanins are rutinose, sophorose, sambubiose and glucortinose.

The terms "squeeze" or "squeeze" mean to squeeze under pressure. According to the invention, pressing can be carried out on the bilberry fruit, whereby the juice can be partially or completely separated from the fruit.

The terms "purified" and "isolated" are used to refer to the purification and/or isolation of one or more anthocyanins from the anthocyanin extracts described above. Again, using routine methods known in the art, the various components of the anthocyanin extract can be separated into purified materials. In one aspect of the invention, the anthocyanins of the extract are substantially purified and isolated by techniques known in the art.

According to the present invention, a bilberry extract containing a low chlorogenic acid concentration and a high polyphenol content can be obtained by treating a pressed product obtained by pressing bilberry fruit (especially, presscake) with an acid.

According to the invention,
A method for extracting a bilberry extract,
Pressing bilberry fruit to obtain fruit with reduced water content (pressed product);
treating the press with acid to obtain a bilberry extract as a water-soluble fraction;
A method is provided.

(A) Pressing bilberry fruit to obtain fruit with reduced water content According to the present invention, bilberry fruit can be pressed to reduce the water content of the fruit. Pressing may partially or completely remove the juice from the bilberry fruit. In (A) above, it is possible to obtain a fruit whose moisture content has been reduced by pressing (hereinafter sometimes referred to as "pressed product", but for example, the pressed product may be "press cake"). . In (A), compression can be performed according to a conventional method. The pressing can be carried out at a low temperature, preferably below 10°C, more preferably below 4°C {provided that the temperature does not freeze the fruit}. The pressing may be performed after thawing the frozen fruit. Native bilberry fruit, on average, contains solids (8% by weight) and water (92% by weight) including organic acids. In (A), the more the water content is reduced by pressing, the more the organic acid content can be reduced, which can improve the purity of anthocyanins. This is probably because the water-soluble fraction of bilberry fruit contains a large amount of organic acids as impurities and has a relatively low anthocyanin content. In some embodiments, in preparing the press, pressing reduces the water content of the fruit to 1/2 or less, 1/3 or less, 1/4 or less, 1/5 or less, 1/7 or less, or 1/10. can be reduced to As a result, the organic acid is removed together with the removed water, and the organic acid content in the pressed product is reduced (e.g., 1/2 or less, 1/3 or less, 1/4 or less, 1/5 or less, 1/7 or less, or reduced to 1/10 or less). In one aspect of the present invention, the step of obtaining a pressed product from bilberry fruit may or may not use a pectin-degrading enzyme, preferably not using a pectin-degrading enzyme. Also, the fruit need not be heated to 60°C or higher, 50°C or higher, 40°C or higher, or 30°C or higher during the pressing step. Pressing allows the fruit to be separated into a press and a fresh juice. Since the fresh juice contains water-soluble impurities, it is desirable that the fresh juice is sufficiently dehydrated by pressing in the pressing step of the present invention. For step (A), see pressing and dewatering (S1) in FIG.

The pressed product obtained by pressing the bilberry fruit contains abundant anthocyanin, and water-soluble impurities (organic acids, etc.) are removed from the pressed product, so the extraction process of the bilberry extract It is considered suitable as the first step of In particular, organic acids can associate with anthocyanins to form intermolecular and intramolecular copigmentations. Organic acids are the main cause of anthocyanin polymerization, and removal of the organic acids is thought to suppress the formation or reformation of aggregates of the organic acids and anthocyanins in subsequent steps. Therefore, in (A), it would be possible to obtain fruit (pressed product) with a reduced moisture content containing organic acids.

(B) Treating fruit (pressed product) with reduced water content with acid to obtain bilberry extract as a water-soluble fraction. According to the present invention, the obtained pressed product can be treated with acid. . An aqueous solution containing a pharmaceutically acceptable acid (eg, hydrochloric acid) is added to the compressed product, and the water-soluble fraction contains bilberry extract. The acid can be, for example, a pH of 3.5 or less, a pH of 3 or less, a pH of 2.5 or less, or a pH of 2 or less. In some preferred embodiments, the acid can be a pH 1-2 (eg, pH 1.5) acid. Treatment of the presses with acid is believed to promote the release of anthocyanins from the presses.
Anthocyanin is contained in the pressed product in the form of a covalent bond with an organic acid, and when the acid treatment breaks the bond between the anthocyanin and the organic acid, the anthocyanin is released from the organic acid. Therefore, the acid treatment has one aspect of being advantageous in the extraction of anthocyanins and in the separation of organic acids and anthocyanins. See acid treatment of residue (squeezed product) in FIG. Anthocyanins are abundantly contained in the extract obtained from the pressed product. Moreover, the anthocyanin contained is depolymerized anthocyanin, and may be, for example, an anthocyanin having a molecular weight of about 550.

(C) Obtaining a water-soluble fraction from the obtained acid-treated product The extraction method of the present invention may further comprise obtaining a water-soluble fraction from the obtained acid-treated product. The water-soluble fraction can be obtained by various methods well known to those skilled in the art, such as centrifugation and filtration. For example, filtration can be performed by those skilled in the art according to routine methods. Filtration can be performed, for example, by a filter machine (eg, filter press). The filter may be driven with a filter aid. For example, filtration can be carried out by a filter using a filter aid containing calcined diatomaceous earth produced by calcining diatomaceous earth (eg, Celite ^™ , Radiolite ^™ ) and volcanic rock-derived perlite (Topco ^™ ). This allows insoluble components to be removed from the extract.

According to the present invention, the obtained bilberry extract can be used as it is or after further purification. Purification may be performed for the purpose of increasing the purity of anthocyanins and/or for the purpose of reducing the organic acid content in the extract.
Purification can be, for example, purification using a column. The column can be a gel filtration column, or an anthocyanin adsorption column. As the anthocyanin adsorption column, many columns having affinity for anthocyanin are known, and these can be used. As the anthocyanin adsorption column, for example, a column (C18 column) packed with silica gel whose surface is modified with octadecylsilyl groups, HP-20 column, SP207 column, and other columns used for purifying anthocyanins are used to purify anthocyanins. be able to. Elution of anthocyanins from the column can be carried out as appropriate by those skilled in the art. Since anthocyanins are known to be stable at pH 3.5 or lower, organic solvents at pH 3.5 or lower (e.g., 50%-70% ethanol solution, e.g., 50%-70% ethanol-HCl (pH 3.5)) can be extracted with

It is believed that by increasing the purity of the anthocyanin and/or further reducing the organic acid content, the depolymerized anthocyanin can be prevented from polymerizing again via the organic acid. Therefore, increasing the purity of the anthocyanin and/or further reducing the organic acid content by purification may be beneficial in maintaining the depolymerized anthocyanin in a depolymerized state. Whether the anthocyanin is depolymerized anthocyanin can be determined by methods well known to those skilled in the art, such as mass spectrometry. The concentration of depolymerized anthocyanins can ideally be determined, for example, by subtracting the organic acid concentration from the total anthocyanin concentration after removing the free organic acids. The resulting bilberry extract may be acidic (eg, pH 2-5, such as pH 3-4, eg about pH 3.5).

According to the present invention, the obtained bilberry extract can be dried and processed into dried solid or powder form and used in such processed form. According to the present invention, the bilberry extract in dry or powder form can be formulated as supplements, pharmaceuticals, and the like. Supplements and medicaments can be tablets, pills, capsules, granules, granules, or powders.

The bilberry extract of the present invention may have a lower chlorogenic acid content than conventional bilberry extracts. In one aspect, the bilberry extract of the present invention comprises 1.5% or less by weight of chlorogenic acid relative to total anthocyanins. In one aspect, the obtained bilberry extract is 1.4 wt% or less, 1.3 wt% or less, 1.2 wt% or less, 1.1 wt% or less, 1.0 wt% or less relative to total anthocyanins. , 0.9 wt% or less, 0.8 wt% or less, 0.7 wt% or less, 0.6 wt% or less, 0.5 wt% or less, 0.4 wt% or less, 0.3 wt% or less, 0.2% by weight or less, or 0.15% by weight or less of chlorogenic acid.

The bilberry extract of the present invention may contain less quinic acid than conventional bilberry extracts. In one aspect, the bilberry extract of the present invention comprises 4.0% or less by weight of quinic acid relative to total anthocyanins. In one aspect, the obtained bilberry extract is 3.5% by weight or less, 3.0% by weight or less, 2.5% by weight or less, 2.0% by weight or less, 1.5% by weight or less based on total anthocyanins , 1.4% by weight or less, 1.3% by weight or less, 1.2% by weight or less, 1.1% by weight or less, 1.0% by weight or less, 0.9% by weight or less, 0.8% by weight or less, 0.7 wt% or less, 0.6 wt% or less, or 0.5 wt% or less of quinic acid. In one aspect, the resulting bilberry extract contains, for example, 2.0% or less by weight of quinic acid relative to total anthocyanins. The resulting bilberry extract contains, for example, 1.5% by weight or less of quinic acid relative to total anthocyanins. The resulting bilberry extract contains, for example, 1.0% by weight or less of quinic acid relative to total anthocyanins.

The bilberry extract of the present invention may contain less malic acid than conventional bilberry extracts. In one aspect, the bilberry extract of the present invention comprises 3.0% or less by weight of malic acid relative to total anthocyanins. In one aspect, the obtained bilberry extract is 2.5% by weight or less, 2.0% by weight or less, 1.5% by weight or less, 1.4% by weight or less, 1.3% by weight or less based on total anthocyanins. , 1.2 wt% or less, 1.1 wt% or less, 1.0 wt% or less, 0.9 wt% or less, 0.8 wt% or less, 0.7 wt% or less, 0.6 wt% or less, Alternatively, it contains 0.5% by weight or less of malic acid. In one aspect, the resulting bilberry extract contains, for example, 2.0% or less by weight of malic acid relative to total anthocyanins. In one aspect, the resulting bilberry extract contains, for example, 1.5% or less by weight of malic acid relative to total anthocyanins. In one aspect, the resulting bilberry extract contains, for example, 1.0% or less by weight of malic acid relative to total anthocyanins.

The bilberry extract of the present invention may contain less citric acid than conventional bilberry extracts. In one aspect, the bilberry extract of the present invention comprises 7% or less by weight of total anthocyanins of citric acid. In one embodiment, the resulting bilberry extract contains 6 wt% or less, 5.5 wt% or less, 5 wt% or less, 4.5 wt% or less, 4 wt% or less, 3.5 wt% of total anthocyanins. or less, or 3% by weight or less of citric acid. In one aspect, the resulting bilberry extract contains, for example, 6% or less by weight of citric acid relative to total anthocyanins. The resulting bilberry extract contains, for example, 5.5% by weight or less of citric acid relative to total anthocyanins. The obtained bilberry extract contains, for example, 4% by weight or less of citric acid relative to total anthocyanins.

The bilberry extract of the present invention may have less succinic acid content than conventional bilberry extracts. In one embodiment, the bilberry extract of the present invention contains 0.15% or less succinic acid relative to total anthocyanins. In one aspect, the obtained bilberry extract is 0.14% by weight or less, 0.13% by weight or less, 0.12% by weight or less, 0.11% by weight or less, 0.10% by weight or less based on total anthocyanins. , 0.09 wt.% or less, 0.08 wt.% or less, 0.07 wt.% or less, 0.06 wt.% or less, or 0.05 wt.% or less of succinic acid. In one aspect, the resulting bilberry extract contains, for example, 0.13% or less by weight of succinic acid relative to total anthocyanins. In one aspect, the resulting bilberry extract contains, for example, 0.10% or less by weight of succinic acid relative to total anthocyanins. In one aspect, the resulting bilberry extract contains, for example, 0.08% or less by weight of succinic acid relative to total anthocyanins.

The bilberry extract of the present invention may contain less contaminants including organic acids than conventional bilberry extracts. In one aspect, the bilberry extract of the present invention is 20 wt% or less, 19 wt% or less, 18 wt% or less, 7 wt% or less, 16 wt% or less, 15 wt% or less, 14 wt% of total anthocyanins. 13% by weight or less, 12% by weight or less, 11% by weight or less, 10% by weight or less, 9% by weight or less, 8% by weight or less, 7% by weight or less, 6% by weight or less, or 5% by weight or less of contaminants including. In one aspect, the contaminant content is, for example, 20% or less by weight relative to the total anthocyanin. In some embodiments, for example, 15% by weight or less of contaminants is included relative to total anthocyanins. In one aspect, the contaminant content is, for example, 10% by weight or less relative to the total anthocyanin. Contaminants include, for example, one or more selected from the group consisting of tartaric acid, quinic acid, malic acid, citric acid, succinic acid, protocatechuic acid, gallic acid, caffeic acid, chlorogenic acid, and caffeine. sell.

The bilberry extract of the present invention contains 50% by weight or more, 51% by weight or more, 52% by weight or more, 53% by weight or more, 54% by weight or more, 55% by weight or more, 56% by weight or more, 57% by weight or more, 58% by weight or more. It may be included at a concentration of weight percent or more, 59 weight percent or more, or 60 weight percent or more. In one aspect, the bilberry extract of the present invention contains 50% by weight or more of anthocyanins and 1.5% by weight or less of chlorogenic acid. In one aspect, the bilberry extract of the present invention contains 50% by weight or more of anthocyanins and 1% by weight or less of chlorogenic acid. In one aspect, the bilberry extract of the present invention contains 50% by weight or more of anthocyanins and 0.5% by weight or less of chlorogenic acid. In one aspect, the bilberry extract of the present invention contains 55% by weight or more of anthocyanins and 0.5% by weight or less of chlorogenic acid. Thus, the bilberry extract of the present invention may contain highly pure anthocyanins, especially depolymerized anthocyanins. Bilberry extracts of the present invention may also contain reduced amounts of organic acids such as chlorogenic acid (eg, aromatic organic acids).

As is well known to those skilled in the art, the anthocyanin content in the bilberry extract can be determined by liquid chromatography, and the organic acid content can be determined by liquid chromatography and mass spectrometry.
Specifically, one type of cyanidin chloride 3-glucoside is used as a standard product, and the peak area of each substance of the standard product is determined when developed with a high-performance liquid chromatography device. A calibration curve is prepared from the peak area and the solution concentration of the standard, and the anthocyanin content can be calculated from the obtained calibration curve by the following equation.

｛式中、標準溶液濃度（μｇ／ｍＬ）＝標準品秤量値（ｍｇ）×１０００×標準品純度（％）／１００×１／１００×希釈率であり、試験溶液濃度（μｇ／ｍＬ）＝試料秤量値（ｍｇ）×１０００×１／５０×５／２５である。｝

{Wherein, standard solution concentration (μg/mL) = standard product weighed value (mg) × 1000 × standard product purity (%) / 100 × 1/100 × dilution rate, test solution concentration (μg/mL) = Sample weighed value (mg) x 1000 x 1/50 x 5/25. }

Organic acid content can be measured using a liquid chromatography device and a mass spectrometer. Specifically, each standard solution is prepared from standard products of each organic acid such as citric acid. Quantitative values of components can be calculated.

The bilberry extract of the present invention is highly absorbable of anthocyanins when orally administered. Therefore, supplements or medicines containing the bilberry extract of the present invention can be oral preparations. A supplement or medicament containing the bilberry extract of the present invention may further contain anthocyanins and excipients. The excipients added to the supplement are food science acceptable excipients. The excipients added to the medicament are pharmaceutically acceptable excipients.

The bilberry extract of the present invention, or a supplement or medicine containing the same, can have higher absorption of anthocyanins than the ethanol extraction method.

Anthocyanins can be quantified by methods well known to those skilled in the art. For example, anthocyanins can be quantified by the method described in the Examples. For example, it can be measured by liquid chromatography-mass spectrometry (LC-MS). If necessary, a standard bilberry extract can be used.

[material]
The bilberry extracts used in the test were an extract (conventional method) and an extract obtained by pressing. The extracted extract (conventional method) and the pressed product extracted extract were prepared as follows.
The extract obtained by the conventional method (ethanol extraction method) is an extract obtained by purifying an acid water-containing ethanol extract of bilberry fruit. Specifically, pectinase is allowed to act on the fruit under heating (60 ° C.) conditions to decompose the fiber of the fruit, and then the decomposition product is mixed with a hydrous ethanol solution under acidic (about pH 3.5) conditions. bilberry fruit anthocyanins were leached into aqueous ethanol. This operation allowed the anthocyanin concentration in ethanol to reach saturation (2000 ppm to 2500 ppm). The numerical value of pH 3.5 is a pH value selected from the condition of suppressing corrosion of stainless steel equipment in contact with the extract while maintaining the chemical stability of anthocyanins. Thereafter, centrifugation was performed according to a conventional method, and the supernatant was removed to collect the precipitate.
Anthocyanin concentration was measured as follows. A sample of 0.100 g to 0.110 g was weighed in a large weighing dish, transferred into a 50 mL volumetric flask equipped with a funnel, 10 mL of 2% hydrochloric acid methanol was added four times, and mixed for 5 minutes with an ultrasonic cleaner. After that, 2% hydrochloric acid methanol was added to adjust the volume. 5 mL of this solution was accurately weighed, added to a 25 mL volumetric flask, and 10% phosphoric acid was added to adjust the volume. A portion of this solution was passed through a 0.45 μm membrane filter and then transferred to a vial to obtain a test solution. Anthocyanin concentration was measured using a high-performance liquid chromatography device (ELITE LaChrom, Chromaster, manufactured by Hitachi High-Tech Science Co., Ltd. or Prominence LC-20AB, manufactured by Shimadzu Corporation). The column used was Kinetex C18 (4.6×250 mm, 5 μm, manufactured by Phenomenex), and the column temperature was 30°C. The column flow rate was 1.0 ml/min, and the gradient conditions of mobile phase A (10% formic acid aqueous solution) and mobile phase B (methanol, acetonitrile, formic acid, pure water (225:225:100:400) mixture) were applied ( 0.00 minutes (A: 93%, B: 7%), 35 minutes (A: 75%, B: 25%), 45 minutes (A: 35%, B: 65%), 46 minutes (A: 0 %, B: 100%), 50 minutes (A: 90%, B: 10%), and 60 minutes (A: 90%, B: 10%)). The sample cooler was set at 4° C. and a 40 μL injection was measured. Anthocyanin was detected by measuring absorbance at 535 nm, and the peak area value of each substance was used to calculate the total anthocyanin concentration. Using one type of cyanidin chloride 3-glucoside as a standard, it was calculated from the calibration curve and the molecular weight ratio by the following formula.

｛式中、標準溶液濃度（μｇ／ｍＬ）＝標準品秤量値（ｍｇ）×１０００×標準品純度（％）／１００×１／１００×希釈率であり、試験溶液濃度（μｇ／ｍＬ）＝試料秤量値（ｍｇ）×１０００×１／５０×５／２５である。｝

{Wherein, standard solution concentration (μg/mL) = standard product weighed value (mg) × 1000 × standard product purity (%) / 100 × 1/100 × dilution rate, test solution concentration (μg/mL) = Sample weighed value (mg) x 1000 x 1/50 x 5/25. }

As the extract extracted from the pressed product, an extract extracted from the press cake, that is, a press cake extract (press cake extract) was used. The presscake extract was prepared as follows. Frozen and thawed bilberry fruits (10,000 kg; 9,200 kg of liquid component and 800 kg of solid content) were pressed at 2° C. to obtain 2,500 kg of pressed material. In this way the water content was reduced to about 1/5 or less. The presscake was then acid treated at 45°C. Specifically, 4800 mL of pH 1.5 HCl was added to 600 g of press cake, and the mixture was stirred at 150 rpm for 15 minutes, allowed to stand for 45 minutes, and then stirred twice for 15 minutes to obtain an extract. Next, the extract was filtered to obtain a filtrate. Specifically, Radiolite #600 (5 g) (manufactured by Showa Chemical Industry Co., Ltd.) was added to 500 mL of the extract obtained above, and suction filtration was performed to obtain a filtrate. An extract was extracted from bilberry in this way. The extracted extract was purified on a resin column suitable for polyphenol purification by the following procedure. Specifically, the resulting filtrate was passed through Zepabeads ^™ SP207 (80 mL) (manufactured by Mitsubishi Chemical Corporation) to adsorb anthocyanin to the resin, washed with pH 3.5 HCl (60 mL), and washed with 70% ethanol-pH 3. Eluted with .5 HCl (240 mL). The resulting eluate was concentrated using a thin-film concentrator and dried by dry spraying to obtain 867.6 mg of bilberry extract. The obtained bilberry extract (press cake extract) contained about 61% by weight of anthocyanins.
In the conventional method, ethanol is removed and water is removed and concentrated by using a vacuum concentrator in combination. While more association or intramolecular or intermolecular copigment occurs, thin-film concentration can remove ethanol and water even in a short period of time (for example, about 30 seconds), thereby anthocyanin association or This is because it has been clarified that intramolecular or intermolecular copigment is suppressed.

[animal]
Wistar rats (Slc:wistar, body weight 189-203 g, male, 9 weeks old) were used as experimental animals. After the animals were brought in, they were preliminarily bred for one week and used for experiments. Food and water were given ad libitum, and the animals were fasted overnight from 17:00 on the previous day before the experiment.

[Bilberry extract administration and sample collection]
A preparation table for each bilberry extract administration solution is shown in Table 2-1. The amount of each bilberry extract was calculated so that the amount of anthocyanin to be orally administered was 37 mg/kg, and each bilberry extract was weighed in a 5 mL centrifuge tube. Water was added to each of the weighed bilberry extracts, dissolved by vortexing, and diluted to a volume to obtain an administration solution. The oral dose was 4 mL/kg.
After exposing the jugular vein of the rat under inhalation anesthesia (isoflurane) and securing a blood sampling site, each bilberry extract solution was orally administered using a sonde. After oral administration, blood was collected over time at 0.25, 0.5, 1, 1.5, 2, 4, and 6 hours, a total of 7 times. A heparinized syringe was used for blood collection, and the volume of blood collected was 500 μL/time. The collected blood was immediately ice-cooled and centrifuged (4° C., 10,000×g, 3 minutes) within 30 minutes to collect plasma. During the experiment, warming was performed to maintain the body temperature of the rats under anesthesia.

[Measurement of anthocyanin concentration]
Sample preparation for anthocyanin extraction was performed by collecting 200 μL of plasma in a 1.5 mL microtube and adding 200 μL of 5% formic acid aqueous solution. The solid phase extraction column used was Oasis HLB 1 cm ³ (Waters). In the solid-phase extraction operation, 1 mL of 10% formic acid acetone is placed on the column, and then 1 mL of 10% formic acid aqueous solution is loaded twice. A column is loaded with 400 μL of a sample for anthocyanin extraction and loaded with 1 mL of 1% formic acid aqueous solution (washing). This operation was performed twice. Finally, a 1.5 mL microtube for collecting the eluate was set and loaded with 400 μL of 10% formic acid acetone. The eluate in the microtube was dried with nitrogen (N ₂ ) gas. After drying with N ₂ gas, 100 μL of 10% formic acid aqueous solution was added to the microtube, dissolved by vortexing, and transferred to a 300 μL capacity PP vial to obtain a measurement solution.
Anthocyanin concentration was measured using a liquid chromatography system (Nexera X2, Shimadzu Corporation) and a mass spectrometer (LCMS-8050, Shimadzu Corporation). A Shim-Pack FC-ODS (150×2 mm) (Shimadzu Corporation) was used as a column, and the column oven was set at 30°C. The mobile phase flow rate was 0.25 ml/min, and the gradient conditions of mobile phase A (5% formic acid aqueous solution) and mobile phase B (acetonitrile containing 5% formic acid) (0.00 → 4.00 min (A: 90%, B: 10%), 4.00 → 10.00 min (A: 90 → 75%, B: 10 → 25%), 10.00 → 12.00 min (A: 75 → 62%, B: 25 → 38%), 12.01→14.00 min (A: 0%, B: 100%), 14.01→20.00 min (A: 90%, B: 10%)). The sample cooler was set at 4° C. and a 2 μL injection was measured. The mass spectrometer (electrospray method: ESI) had a nebulizer flow rate of 3 L/min, a heating gas flow rate of 5 L/min, an interface temperature of 250°C, a DL temperature of 250°C, a heat block temperature of 250°C, and a drying gas flow rate of Set to 15 L / min, Del-3-Gal, Del-3-Glu 464.9>303.0, Del-3-Ala 435.1>303.0, Cya-3-Gal, Cya-3 -Glu is 449.0>287.0, Cya-3-Ala is 419.0>287.0, Pet-3-Gal, Pet-3-Glu is 479.2>317.0, Pet-3-Ala is 449.2>317.0, Peo-3-Gal, Peo-3-Glu is 463.2>301.0, Peo-3-Ala is 433.2>301.0, Mal-3-Gal, Mal MRM (positive) of 493.1>331.0 for -3-Glu and 463.2>331.0 for Mal-3-Ala was measured as quantitative ions.
For quantification of anthocyanins, the bilberry extract (extract by conventional method) was quantified by C3G with LC-UV, and the quantitative values of 15 kinds of anthocyanins in each of the bilberry extract solutions were determined, and the bilberry extract solution was used as a standard solution.

[Statistical analysis]
All data are presented as mean ± standard deviation (Mean ± SD). Each data was compared using Student's t-test. The level of significance was p<0.05.

[result]
Table 2-2 shows the body weight of rats in each administration group immediately before administration. No significant difference was observed between the two groups in comparison with the extract administration group by the conventional method.

Changes over time in plasma anthocyanin concentration in the group administered with the extract by the conventional method were confirmed (Fig. 1, Tables 3-1 and 3-2). In the extract administration group by the conventional method, the maximum concentration (C _max ) was reached about 2 hours after oral administration. The time-dependent change in plasma anthocyanin concentration was compared between the conventional extract administration group and the compressed product extract administration group (Fig. 1, Tables 3-1 and 3-3). In the pressed product extract administration group, the plasma anthocyanin concentration continued to increase even 6 hours after oral administration. In addition, AUC _0-6 was statistically significantly higher in the group administered with the compressed extract than in the group administered with the extract obtained by the conventional method (Table 3-1).

In the compressed product extract administration group, the C _max of each individual was higher than in the extraction extract administration group by the conventional method. In addition, the AUC _0-6 of the compressed product extract administration group showed a value more than twice as high as that of the extraction extract administration group by the conventional method, and this difference was statistically significant (see Table 3-1). ), and the C _max increased about twice (maximum six times among individuals), it is possible that the compressed product extract has better absorption of anthocyanins after administration than the extract obtained by the conventional method. was suggested. In addition, in the extract administration group by the conventional method, an individual with particularly low C _max (No. 3) was observed, but in the compressed extract, a stable C _max with little variation among individuals was achieved. As described above, the extract of the pressed product, which is considered to contain a large amount of depolymerized anthocyanin, exhibits the characteristic that it is easy to increase the concentration of anthocyanin in the blood by oral administration.

Organic acid content of various bilberry extracts was analyzed. Specifically, 0.050 g to 0.060 g of the sample was weighed with a weighing dish, transferred to a 50 mL volumetric flask with a funnel, 10 mL of a 10% methanol aqueous solution was added four times, and mixed for 5 minutes with an ultrasonic cleaner. . After that, a 10% methanol aqueous solution was added to adjust the volume. A portion of this was diluted appropriately, passed through a 0.45 μm membrane filter, and then transferred to a vial to obtain a test solution. Next, the organic acid content was measured using a liquid chromatograph (Nexera X2, Shimadzu Corporation) and a mass spectrometer (LCMS-8050, Shimadzu Corporation). Sigma F5 (250×2.1 mm, 5 μm) (Shimadzu Corporation) was used as a column, and the column oven was set at 30°C. The column flow rate was 0.2 mL/min, and the gradient conditions of mobile phase A (0.2% formic acid aqueous solution) and mobile phase B (acetonitrile containing 0.2% formic acid) were set for 0.00 min (A100%, B: 0% ), 2.00 minutes (A100%, B: 0%), 5.00 minutes (A75%, B: 25%), 11.00 minutes (A65%, B: 35%), 15.00 minutes (A5 %, B: 95%), 18.00 minutes (A5%, B: 95%), 18.01 minutes (A100%, B: 0%), 30.00 minutes (A100%, B: 0%)) measured in The sample cooler was set at 4° C. and 2 μL of sample was injected. The mass spectrometer (electrospray method: ESI) had a nebulizer flow rate of 3 L/min, a heating gas flow rate of 10 L/min, a DL temperature of 200°C, a heat block temperature of 300°C, and a drying gas flow rate of 10 L/min. . Each organic acid was quantified using an organic acid mixed standard solution. A standard undiluted solution was prepared from each organic acid standard product, each standard undiluted solution was mixed to obtain an organic acid mixed standard solution, and a quantitative value was calculated from the calibration curve. The results were as shown in Tables 4-1 and 4-2.

As shown in Table 4-1, the pressed product obtained by pressing the frozen bilberry fruit has a significantly reduced organic acid content. This means that the organic acids have been removed by pressing. In addition, as shown in Table 4-2, in the pressed product extract, the content of tartaric acid, quinic acid, malic acid, citric acid, succinic acid, gallic acid, and chlorogenic acid is higher than that of the extract obtained by the conventional method. was reduced. Organic acids were removed in the process of preparing the pressed product by pressing, and the acid treatment of the pressed product destroyed the bond between the anthocyanin and the organic acid, thereby enabling effective removal of the organic acid as a contaminant. It is thought that On the other hand, the pressed product extract contained a high concentration of anthocyanins, so it should be noted that the fact that anthocyanins are less likely to decrease by pressing or acid treatment is an advantage of the pressed product extract. is.

Anthocyanin increases color by being combined with an organic acid (for example, chlorogenic acid, etc.). This phenomenon is due to intramolecular copigmentation. In the pressed extract, the organic acid content is reduced, and the formation of copigments is dramatically reduced to less than 10-50% compared to extracts extracted by conventional methods. In particular, the anthocyanin weight:total organic acid weight ratio was reduced to about 1/3 in the pressed extract as compared with the extract obtained by the conventional method.

Conventionally, the purity of anthocyanin in the substance adsorbed to the ion-exchange resin was about 45% at maximum, but the purity of the extract obtained by pressing was found to be about 60%. As a result, the organic acid in the solution can cause the formation of intramolecularly or intermolecularly polymerized anthocyanins such as associations of anthocyanins and organic acids (intermolecular copigmentation or intramolecular copigmentation). It is considered that the removal of water containing organic acids by the pressing step leads to the reduction of re-formation of inter-molecular co-pigmentation and intra-molecular co-pigmentation in subsequent steps. In addition, the subsequent acid treatment promotes dissociation from anthocyanin for the organic acid that was not removed by water removal, thereby reducing the aggregate of the organic acid and anthocyanin (depolymerized). there). In addition, anthocyanins with low molecular weights (depolymerized anthocyanins) preferentially bind to ion exchange resins, and anthocyanins containing intramolecular or intermolecular bonds with organic acids have low binding properties. This is considered to be the reason for the increased content of depolymerized anthocyanins in the pressed product extract. In this way, the extract of the pressed product has a reduced organic acid concentration, a remarkably low copigment formation rate by anthocyanin, and a content of depolymerized monomolecular anthocyanin extracted by a conventional method. It is thought that it is higher than the extract. From the results of the in vivo administration test, _Cmax and AUC were high in the group that was administered the extract of the compressed product. This result suggests that the increase in the yield of depolymerized anthocyanin contributed to the enhancement of anthocyanin absorption in the intestinal tract. In the anthocyanin solution used in the experiment, anthocyanin is darkened, and it is considered that self-associated anthocyanin is formed. It was also found that increasing the content of self-associated anthocyanins (or decreasing intramolecular or intermolecular copigmentation) can improve the intestinal absorption and blood levels of anthocyanins.

According to the method of the present invention, not only can a large amount of anthocyanin be extracted from bilberry fruit, but the ratio of depolymerized anthocyanin in the extracted anthocyanin is high, and bioavailability is improved.

Claims (8)

A method for extracting a bilberry extract,
Pressing bilberry fruit under a temperature condition of 10 degrees Celsius or less to obtain a pressed product of the fruit having a reduced moisture content and a reduced organic acid content relative to anthocyanin ;
treating the fruit press with reduced water content and organic acid content relative to anthocyanins with an acid to obtain a bilberry extract as a water-soluble fraction;
Method. 2. The method of claim 1, wherein the acid treatment is hydrochloric acid treatment. 3. The method of claim 1 or 2, further comprising obtaining a water-soluble fraction from the acid-treated product. The method of any one of claims 1-3 , further comprising concentrating the polyphenols in the water-soluble fraction obtained. The method according to any one of claims 1 to 4, wherein the obtained bilberry extract has an anthocyanin purity of 50% or more and a contaminant content of 15% by weight or less relative to total anthocyanins. The method according to any one of claims 1 to 4, wherein the obtained bilberry extract has an anthocyanin purity of 50% or more and a quinic acid content of 2% by weight or less relative to total anthocyanins. A method according to any one of claims 1 to 6, wherein no pectinase is added to the fruit in the pressing step. A method for producing an anthocyanin composition,
A method comprising an extraction method according to any one of claims 1-7.

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