SG188097A1

SG188097A1 - Process and apparatus for producing a high polyphenol-containing composition

Info

Publication number: SG188097A1
Application number: SG2013007604A
Authority: SG
Inventors: Satoshi Hanamura; Ken Chikauchi; Shigeru Kanazawa; Jinichiro Koga; Minoru Kanegae
Original assignee: Meiji Co Ltd
Priority date: 2008-02-07
Filing date: 2009-02-06
Publication date: 2013-03-28
Also published as: MY159608A; JPWO2009099220A1; JPWO2009099222A1; JP5448852B2; WO2009099222A1; WO2009099220A1

Abstract

PROCESS AND APPARATUSFOR PRODUCING A HIGH POLYPHENOL-CONTAINING COMPOSITIONAn object is to provide a process for producing, simply and effectively, a high polyphenol-containing composition, in5 particular, a composition the cacao polyphenol content is high while the theobromine content is low.The polyphenol-containing composition producing process comprises the following steps: (a) performing an extraction of a polyphenol-containing plant or a processed product thereof10 with a solvent, to obtain a crude polyphenol extract; (b) applying the crude polyphenol extract to a cation exchange resin subjected beforehand to hydrogen ion substitution, andsubsequently passing a first solvent containing no ionic substance through the cation exchange resin, to obtain a first15 polyphenoleluate; (c) passing the first polyphenol eluate again through a cation exchange resin subjected beforehand to hydrogen ion substitution, and then passing a second solvent containing no ionic substance through the cation exchange resin, to obtain a second polyphenol eluate; and (d) concentrating or drying the20 second polyphenol eluate. The process makes it possible to yield a polyphenol-containing composition that the polyphenol content. in solid content is 33% or more by weight, and the theobromine content is 1% or less by weight.FIG,

Description

DESCRIPTION

PROCESS AND APPARATUS

FOR PRODUCING A HIGH POLYPHENOL~CONTAINING COMPOSITION

Technical Field

[0001]

The present invention relates to a high polyphenol-containing composition, and a producing process thereof. More specifically, the invention relates to a process comprising extraction of the polyphenol, with a high selectivity, from a plant in which a thecbromine and a polyphenol coexist or a processed product thereof, and thereby producing a composition that a theobromine content is low and a polyphenol content is high.

Background Art

[0002]

It is known that the intake of plant polyphenols contained in various fruits and vegetables is generally useful for keeping health. Among the polyphenols, it is known that, cacao polyphencl contained in cacao has various physiological effects such as an antioxidative effect, an effect of restraining the formation of dental calculus, an anti-tumor effect, an anti-stress effect, and an effect of preventing a cancer (see,

Z5 for example, Fabent Documents 1 fo Hi.

[0003]

Cacao polyphenol can be obtained as an extract from cacao.

In recent years, attention has been paid to cocoa and chocolate and other similar cacao food and drink using such an extract.

In general, however, the cacao polyphenol contained in commercially available cocoa, chocolate or the like is low.

Therefore, in order that the above-mentioned physiological effects can be exhibited by cacao food or drink, it is necessary to take a considerably large amount of the cacao food or drink.

However, such an intake of a large amount thereof may induce various side effects. Accordingly, if a process for producing a composition that the polyphenol content is high, in particular a high cacao polyphenol-containing composition, can be established, it becomes possible to take effortlessly polyphenols in an amount sufficient for exhibiting the physioclogical effects without taking a large amount of cocoa or chocolate.

[6004]

From such viewpoints, investigations have been made on various methods for extracting cacao polyphenol from cacac. For example, suggested are methods of using hot water or ethanol when the polyphenol is extracted (see, for example, Patent

Documents 1, 6 and 7). As seen in the cacao polyphenol extracting methods given as the examples, conventional polyphenol extracting methods require complicated operations, such as extraction, adsorption onto a synthetic adsorbent, washing, and elution with a solvent. As a result, in many cases, it is difficult to extract polyphenols effectively and inexpensively.

Thus, 1t has been awaited and desired to develop a process of using fruits or vegetables as raw materials to produce a high wolyphenol-containing composition effectively and inexpensively. [0C05]

Patent Document 1: JP-A-7-213251

Patent Document 2: JP-A-7-238028 24 Patent Document 3: JP-A-7-2748%4

Patent Document 4: JpP-A-9-206026

Patent Document 5: JP-A-9-234018

Patent Document 6: JP-A-9-224606

Patent Document 7: JP-R-20G00-256345

Non—-Patent Document 1: Martin Li. Price and Larry G. Butler,

L. Agric Food Chem., Vel. 25 No. 6, 1268-1273, 1977

Non-Patent Document 2: AOAC Official Methods of Analysis (1880) 980. 14, “Theobromine and Caffeine in Cacao Products

Liquid Chromatographic Method”

Non~Patent Document 3: Food Chemistry 68 (2000) Yinrong

Lu. L. Yeap Foo “Anticxidant and radical scavenging activities of pelyphenols from apple pomace”

Non~-Patent Document 4: Journal of Nutrition, 199%9, 129; 1725-1730, Satoshi Nagaoka, Kenji Miwa, Michiko Eto, Yasuo

Kuzuya, Goro Hori and Kazuhiro Yamamoto “Soy Protein Peptic

Hydrolysate with Bound Phospholipids Decreases Micellar

Solubility and Cholesterol Absorption in Rats and Caco-2 Cells”

Disclosure of the Invention

[0006]

In many cases, a crude extract obtained from cacao originally contains, besides cacao polyphenol, theobromine, amino acids, proteins and other components. Thus, it is difficult to say that the cacao polyphenol content in the crude extract Ls high, Even when Uhe crude exUraci is further subjected to separating purification {hereinafier called “purification”}, cacao polyphenol and thecbromine are not easily separated from each other. Thus, in many cases, a fraction of cacao polyphenol contains thecobromine. IU is known that when a person takes a large amount of theohromine, the person comes to feel sick although theeobromine has an excellent physiclogical effect. Accordingly, when a cacao polyphenol extract is used for food or drink, it is desired that the cacao polyphenol content in the extract is high while the thecbromine content is low.

[0007]

However, conventional extracting methods require complicated operations; besides cacao polyphenol and theobromine are not easily separated from each other, and the theobromine content in the extract tends to be low. Accordingly, an object of the invention is to provide a high polyphenol-containing composition simply and effectively, in particular, to provide a process for producing a composition that the cacac polyphenol content is high while the theobromine content is low.

[0008]

In light of the above-mentioned situation, the inventors have made eager investigations to find out that when a crude extract solution wherein a polyphenol and theobromine coexist is applied to a cation exchange resin subjected beforehand to hydrogen ion substitution, theobromine is effectively adsorbed onto the resin and the polyphenol is effectively eluted oul.

The invention is based on this finding, and has, as features thereof, subject matters described in the following:

[0009] (1) A process for producing a polyphenol~containing composition, comprising the following steps: pe! (a) performing an extraction of polvphenoi-ocontaining plant or a processed product thereof with a solvent, to obtain a crude polyphenol extract: (bh) applying the crude polyphenol extract to a cation exchange resin subjected beforehand to hydrogen lon substitution, and subsequently passing a First solvent containing no lonic substance through the cation exchange resin, to obtain a first polyphenol eluate; {c) passing the first polyphenol eluate again through a cation exchange resin subjected beforehand to hydrogen ion substitution, and then passing a second solvent containing no lonic substance through the cation exchange resin, to obtain a second polyphencl eluate; and (d} concentrating or drying the second polyphenol eluate. 0010) (2) The producing process according to item (1), wherein the plant is cacao.

[0011] {3) The producing process according to item (1) or (2), wherein the sclvent used for the extraction in the step (a) is a 40-70% by weight aqueous ethanol solution.

[0012] (4) The producing process according to any one of items (1) to (3), wherein in the steps (b} and {(¢}), the first and second solvents are each passed at a flow rate SV of 2 to 10.

[0013] (5) The producing process according to any one of items (1) to (4), wherein before the step (¢), a precipitate of theobromine is obtained from the first polyphenol eluate, the precipitate is next removed to vield a solution, and the solution is used in the step (oo). 2h [00147 (6) The producing process according to any one of items (1) to (5), wherein the first and second solvents each containing no ionic substance are each delionizied water or an aqueous ethanol solution.

A [0015] (7) The producing process according to any one of items

{1) to (6), wherein the second solvent containing no ionic substance is the deionizied water having a temperature of 35°C or lower, or an agueous ethanol scoluticen having an ethanol concentration of 30% or less by welght.

[0016] {8) A polyphenol-containing composition yielded by the producing process according to any one of items (1) to {7}. reoi7) (9) A polyphenol-containing composition that the 16 polyphenol content in a solid content is 33% or more by weight, and the theobromine content therein is 13% or less by weight.

[0018] (10} An apparatus for producing a polyphenol-containing composition from a polyphencol-containing plant or a processed product thereof, comprising: an extraction section having a means for obtaining a crude polyphenol extract from the polyphenol-containing plant or the processed product thereof; a purification section providing a first purification and a second purification, wherein the first purification has a means for obtaining a first polyphenol eluate by applying the crude polyphenol extract transferred through a means for transferring from the extraction section to a cation exchange resin subjected beforehand to

Zh hydrogen ion substitution, and subsequently naseing a First solvent containing no ionic substance through the cation exchange resin, and the second purification has a means for obiaining a second prelyphencl eluate by passing the first polyphenol eluate transferred through the means for transferring from the first purification section again through a cation exchange resin subjected beforehand to hydrogen ion substitution, and subsequently passing a second solvent containing no ionic substance through the cation exchange resin; and a recovery section having a means flor concentrating or drying the second polyphenol eluate transferred through the means for transferring from the purification section.

[0019]

The present application claims the priorify based on

Japanese Patent Application No. 2008-27946 filed on February 7, 2008, the contents of which are incorporated into this specification by reference to the specification of the earlier filed application.

[0020]

According te the producing process of the invention, a high polyphencl~containing composition can be effectively produced in a simple way. The invention has advantages that the steps are simple, the recovery of polyphenols is high and a required amount of the solvent is small since it is unnecessary to subject the crude extract sclution beforehand to pre-treatment with a porous resin or the like in the invention. The composition obtained by the producing process of the invention has high polyphencl content in the solid content thereof than conventional ordinary cacao extracts. Thus, the composition is useful for various use purposes. In oa high cacao polyphenol-containing composition obtained by the invention, the theobromine content is lower than in conventional cacao extracts; therefore, the composition is easily blended with food or drink, medical drugs, and others.

Brief Description of the Drawings

L0021 ”

[FIG. 1] FIG. 1 is a fiowchart describing an embodiment of the producing apparatus of The invention. [FIG. 2] FIG. 2 1s a graph showing a relationship between the ethanol concentration and extraction effect in the case of using an aqueous ethanol solution as an extracting solvent. [FIG. 3) FIG. 3 1s a graph showing measurement results of micelle insolubilizing capabllity examined in Example 6 and

Comparative Example 1.

Description of Reference Numerals

[0022] 100: Extraction section 100a, and 100b: Pipes 110: Extracting tank 120: Raw material tank 130: Solvent tank 1490; Centrifuge 200: Purification section 200a, 200b and 200c: Pipes 210: Column 212: Waste tank 220: Concentrator 230: Column 2372: Waste tank 40a, 240Gb, 2400 and Z40d: Solvent tanks 300: Recovery section 300a: Pipe 310: Concentrator 320: Spray drier

J22: Collecting tank

Best Mode for Carrying Out the Invention

[0023]

Hereinafter, the invention will be described in detail.

A first of the invention relates to a process for producing polyphenol-containing composition. This producing process is characterized by having the step of performing an extraction of a polyphenol-containing plant or a processed product thereof with a solvent, to obtain a crude polyphenol extract; and the step of applying the crude polyphenol extract to a cation exchange resin subjected beforehand to hydrogen ion substitution, and subsequently eluting out the polyphenol, to obtain a polyphenol eluate; and the step of concentrating or drying the polyphenol eluate as the need arises. According to this process, unnecessary components in the crude polyphenol extract can be adsorbed to the cation exchange resin so as to be removed. As a result, fractions rich in the polyphenol can be selectively eluted cut. In order to increase the polyphenol content in the solid content of the composition, it is preferred to applying the eluate to the cation resin repeatedly. In an embodiment of the producing process of the invention, it is preferred to carry oul the purification step, using a cation exchange resin subjected beforehand to hydrogen ion substitution, at least two

Limes.

[0024]

A preferred embodiment of the producing orocess of the invention has a step (a) of performing an extraction of a polyphenol-containing plant or a processed product thereof with a solvent, to obtain a crude polyphencl extract, a step (bh) of applying the crude polyphenol extract to a cation exchange resin 20 subjected beforehand to hydrogen ion substitution, and subsequently passing a first solvent containing no ionic substance through the cation exchange resin, to obtain a first polyphenol eluate, and a step (¢) of passing the first polyphenol eluate again through a cation exchange resin subjected beforehand to hydrogen ion substitution, and then passing a second solvent containing no ionic substance through the cation exchange resin, to obtain a second polyphenol eluate, and a step (d) of concentrating or drying the second polyphenol eluate as the need arises. In the cation exchange resin used in the step (¢), the cation exchange resin used in the precedent step (b) may be regenerated so as to be reused, or the same cation exchange resin that has been described above and is separately prepared may be used.

[0025]

The term “polyphenol-containing plant or a processed product thereof” used in the present specification denotes any plant or any processed product thereof that contains, as a compenent therecf, a polyphenol, and means that no especial limitation is imposed onto one or more components coexisting therewith. In other words, the producing process according to

Che invention may be applied to various polyphenol-containing plants or processed products thereof. The invention is useful for extracting a polyphenol component selectively from a plant in which a polyphenol and theobromine coexist, or a processed product thereot, suchas cacao, lex paraguariensis {Yerba mate),

AH Camellia sinengiaitens Tree), koala, guarana, and Coffealonffns tree), although the plant or product to which the invention is to be applied is not particularly limited.

[0026]

The following will describe the process for producing a 20 poiyphenol-containing composition according to the invention in detail, giving, as an example, the case of using a cacac plant

Or a processed product thereof as a raw material. It is however needless to say that the producing process according to the invention can be carried out in the same way even when plants other than cacao, and processed products thereof are used as 2 a raw material. The term “high polyphencl-containing composition” used in the specification means that the content cf polyphenol components in the solid content is 33% or more by weight, more preferably 40% or more by weight. In the case of using, in particular, a cacao plant or a processed product thereof as a raw material, the cacao polyphenol content in the solid content is 33% or more, more preferably 40% or more by weight. By contrast, it is intended that the theobromine content in the sclid content is 1% or less by weight, more preferably 0.9% or less by weight.

[0027]

A “cacao plant or a processed product” usable as a raw material in the invention may be, for example, cacao barks, cacao leaves, cacao beans, cacao shells or some other cacao plant; or cacao nib, cacao liquor, defatted cacao liquor, cocoa powder or some other cacao bean processed product. Cacao liquor is obtained by grinding cacao beans, and defatted cacao liquor is obtained by removing fat from cacao liquor. The method for removing fat from cacao liquor is not particularly limited, and

LU dis possible to apply a known method such as squeezing. Cocoa powder can he obtained hy pulverizing defatted cacao | iouor,

Of these raw materials given as the examples, cacao liquor and cocoa powder are preferred since these are subjected, in a processing process therefor, to finely-granulating such as grinding, and pulverizing, so that polyphenol canbe effectively exlracted. fo02e)

In the embodiment of Che producing process according to the invention, the step (a) is concerned with the step of yielding a crude polyphenol extract from a polyphenol-containing raw material. No especial limitation is imposed to this step as far as extraction with a solvent is conducted, a well-known extracting technique may be applied thereto. The technique may be appropriately selected from well-known solvent extraclion methods, for example, a method of putting the raw material into an extracting kettle, and soaking the material in a predetermined amount of an extracting solvent for a specified period to yield an extract solution, and a method of sending an extracting solvent to the raw material filled into a column to yield a predetermined amount of an extract solution. Components insoluble in the extracting solvent, that is residues, are removed by centrifugaticn, filtration, or scme other method.

The thus cbtained crude polyphenol extract may be a solution obtained by the extracting and not subjected to any subsequent treatment wherein the solvent is contained in a large amount, or a solution wherein the extracting solvent is partially distilled off. When the extracting solvent is partially distilled off, in this case, a precipitate may be generated, the precipitate is removed by a known method such as centrifugation and filtration.

[0029]

When a cacao plant or a nrocessed product thereof ig need as a raw material, an extract resulting from the solvent extraction {abbreviated to a “crude cacao extract” hereinafter) is prepared in the step (a). The method for the preparation is not particularly limited as far as the solvent extraction is applied. The solvent used for the extraction may be water, an organic solvent such as ethanol, methanol and acetonitrile, or an aqueous soluticn thereof. Although the extracting solvent is not particularly limited, preferred therefor are water, ethancl, and a solution thereof in water since the solvents are each a solvent widely used in the production of food. These solvents are preferred also since the solvents are harmless to human. About the solvents, ethanol may be used alone; more preferably, water, or a mixture of water and ethanol, that is, an aqueous ethanol solution is used. The ethanol concentration in the aqueous ethancl sclution is preferably from 30 to 95% by weight, more preferably from 40 to 70% by weight, even more preferably 50% by weight.

[0030]

It is preferred that the temperature at the time of the extraction ranges theoretically from 0 to 100°C, preferably from 50 to 90°C when water is used as the extracting solvent. When the aqueous ethanol solution is used as the solvent, it is preferred that the temperature ranges theoretically from 0 to 80°C, preferably from 40 tec 70°C. It is advisable to decide the extracting period appropriately in accordance with a desired targel to be extracted under the consideration of the used raw material, and other extracting parameters. The extracting period, which is not particularly limited, is usually from about 1G to 60 minutes. The crude cacao extract obtained as described above contains theobronine, amino acids, saccharldes, colorants, fats, and other unnecessary components in a large amount together with cacao polyphenol.

[0031]

In the embodiment of the producing process according to the invention, the steps (b) and (¢) are each concerned with purification using a cation exchange resin subjected beforehand to hydrogen ion substitution. In the embodiment, an object of the step (b) is to remove mainly proteins, amino acids, and sone other unnecessary components from the crude polyphenol extract vielded in the step (a) to yield a first polyphenol eluate. An object of the step (c) is to remove theobromine and some of the other unnecessary components to yield a second polyphenol eluate.

The removal of the unnecessary components is attainedby applying each of the crude polyphenol extract and the first polyphenol eluate to the cation exchange resin, and then passing an appropriate solvent therethrough.

[0032]

The term “cation exchange resin” used in the specification means a strongly acidic or weakly acidic cation exchange resin, which is well known and is not particularly limited. The “cation exchange resin beforehand subjected to hydrogen ion substitution” denotes that the cations in the resin, which are to be reactive groups, are beforehand subjected to substitution with hydrogen ions. In the invention, specific examples of the usable cation exchange resin include DIAION (registered trade name) series SKI1RB, SKL10, SKilé, P206 and WK40, manufactured by Mitsubishi Chemical Corp., AMBERLITEs (registered trade name)

TR-1208B, IR-200{T, IRCHO and IR-124, manufactured by Rohm and

Haas Co., and DOWEX (registered trade name} 50W-X8 manufactured by the Dow Chemical Co.

LOOE3]

The substitution treatment of the cation exchange resin with hydrogen ions can be conducted, for example, by applying an acid having an appropriate concentration to the resin. In order to conduct the substitution, for example, 1 N hydrochloric acid may be used. In the invention, it appears that theobromine 50 is more easily adsorbed onto the cation exchange resin by subjecting the cation exchange resin beforehand to the hydrogen ion substitution. Accordingly, even when any resin is used, it is essential to subject the resin beforehand to the hydrogen ton substitution although the cation exchange resin used in the invention is not particularly limited.

[0034]

In general, for the elution of a target material from an ion exchange chromatograph wherein a cation exchange resin or the like is used as a filler, an ionic solution is used which contains a material capable of attaining ion exchange for the ion exchange resin, examples of the solution including respective ionic solutions of hydrochloric acid, sodium hydroxide, calcium chloride, and sodium chloride. However, in the producing process according to the invention, the elution of the polyphenol from the cation exchange resin is carried out by passing a solvent containing no ionic substance, such as deionized water, through the resin. The term “solvent containing no ionic substance” used in the specification means a solvent which does not contain any material that is adsorbed onto or desorbed from a cation exchange resin. Specific examples of the solvent containing no ionic substance in the invention include deionized water and an aqueous solution of an organic solvent. Examples of the organic solvent solution in water are as given previously as the examples in the step {a}. The solvent is preferably an aqueous ethanol solution. In the invention, deionized water is most preferred from tha viewpoint of costs.

[0035]

In the invention, by a combination of cation exchange resins that 1s subjected beforehand to substitution with

SU hydrogen ions, with a solvent containing no ionic substance, theobromine and any unnecessary component in the crude cacao extract are selectively adsorbed onto the cation exchange resin.

In this way, cacao polyphenol can be preferentially eluted out.

In the embodiment of the invention, it is preferred that appropriate solvents are selected and used in accordance with the objects of the steps (b} and {(c¢), respectively. Hereinafter, the step (b) and (¢) will be described in more detail. [003el

In the step (b), any method for the applying crude polyphenol extract, more specifically the crude cacao extract, to the cation exchange resin may be used as far as the method is capable of adsorbing unnecessary component other than cacao polyphenol in the crude cacao extract onto the cation exchange resin, and subsequently eluting out a cacao polyphenol fraction selectively. Conditions for the applying the crude cacao i5 extract to the cation exchange resin may be appropriately selected in accordance with the concentration in the crude cacao extract, and others. [0C37]

The step (b) in the invention may be carried out, for example, by filling a cation exchange resin subjected beforehand to substitution with hydrogen icons into a column, and next passing the crude cacao extract through the cation exchange resin in the column. In another method, the step (b) can be carried cul by putting a catlon exchange resin subjected beforehand to hydrogen ion substitution directly into the arude cacao extract and then stirring the mixture. When such a method is used, it is necessary to adsorb the unnecessary components in the crude cacao extract onto the cation exchange resin and subsequently collect the cation exchange resin by filtration or some other separating method. In other words, the latter method Ls a bateh treatment, and thus the column-used method is more preferred from the viewpoint of work efficiency.

[0038]

When the step (b) is carried out by the column~used method, a solvent for equilibrating the cation exchange resin is substantially required. In the invention, as the equilibrating solvent, an aqueous ethanol solution having an ethanol content of 0 to 50% by weight, more preferably from 0 fo 10% by weight is used. After the cation exchange resin is equilibrated, the crude cacao extract is passed therethrough, and subsequently a solvent containing no ionic substance is passed therethrough to yield a first cacao polyphenol eluate.

[0039]

The solvent used for the elution in the step (bh) is not particularly limited as far as the solvent is a solvent containing no ionic substance. In the step (bl), from the viewpoint of separating the unnecessary components contained in the crude polyphenol extract, such as proteins, amino acids and others, it is preferred to select appropriately a solvent making the separation easy, and use the selected solvent appropriately. Examples thereof include deionized water, and an aqueous solution of an organic solvent, such as an agueous ethanol sclution. In general, the ethanol concentration of the solvent used for the elution is made equal to that of the extract.

Accordingly, for example, an agueous ethanol sciutlon having

Zh an ethanol content of 30 to 95% hy weight may be used. However, even when deionized water is used, the unnecessary components can be adsorbed onto the resin and separated in the same way in the invention.

[0040]

The temperature of the solvent used for the elution is not particularly limited. In the case of using, for example,

deionized water, the temperature is theoretically from 0 to 100°C, preferably from 5 to 50°C. In the case of using an aqueous ethanol solution, the temperature is theoretically from 0 to 80°C, preferably from 5 to 50°C, It is preferred to pass the solvent containing no ionic substance into the column at a flow rate

SV of about 1 to 10, preferably about 2 to 10. When the solvent is passed at such a flowrate, a composition (in a solution stare) that the cacao polyphencl content is high in solid content is easily obtained. About the flow rate, for example, an SV of 1 means that a liquid is caused to flow in a volume eguivalent to (as much as) the resin volume per hour.

[0041]

In the step (b), the amount of the cation exchange resin filled into the column can be appropriately selected. The amount of the filled resin is preferably, for example, 1/50 times or more, more preferably 1/25 times or more the liquid volume of the crude cacao extract. When a column is separately prepared in the step {c¢), the amount of the filled resin may be made small.

When the amount of the resin used in the step (b) is small, only proteins, amino acids, and some of the unnecessary components are adsorbed and thecbromine is easily eluted out without being adsorbed. Accordingly, the eluate contains a large amount of theobromine together with the polyphenol. fond zy ah in the embodiment according to the invention, the ston (rm) is a step of removing some of the unnecessary components, mainly thecbromine, from the first polyphenol eluate vielded in the precedent step (b). As the cation exchange resin used in the step (cc), the cation exchange resin used in the precedent step (b} may be used regenerated resin, or the same cation exchange resin that has been described above and is separately prepared may be used. Thecbromine is more easily adsorbed onto this cation exchange resin than cacao polyphenol. For this reason, by applying the first polyphenol eluate to the cation exchange resin, theobromine can be removed so that a fraction rich in the polyphenol is yielded. The method for applying the eluate to the cation exchange resin is not particularly limited, and the applying can be effectively conducted by a column-used method in the same manner as in the step (b). Specifically, the cation exchange resin is equilibrated, the first polyphenol eluate is then passed therethrough, and subsequently a solvent containing no ionic substance is passed therethrough.

[0043]

In an embodiment of the step (¢), the first polyphenol eluate yielded in the step {(b) may be used as it is. In another embodiment, it is allowable to precipitate some of the unnecessary components from the eluate, remove the precipitate to yield a solution, and use the solution. Such an embodiment is particularly preferred in a case where the use amount of the cation exchange resin is small in the step {(b}. The reason is as follows: when the use amount of the cation exchange resin is small in the step (b), the eluate contains a large amount of theobromine; accordingly, before the step (¢), theobromine contained in the first polyphenol eluate becomes able to be remcved as a precipitate. Therefore, such an embodiment is as! useful for vielding effectively a hich polyphenol -ocontaind no composition in which the theobromine content is small. [0C44]

The precipitation of theobromine may be carried out by a known method such as distillation-off of the solvent in the eluate. Separated theobromine way be appropriately purified by a known method, and used as a by-product. In the case of carrying oul a theobromine-removing step as described above, in the step (c} the solution obtained by removing the thecbromine precipitate is brought, as it is, into apply to the cation exchange resin, and further a sclvent containing no ionic substance is passed therethrough. In a different embodiment, ethanol may be appropriately added to the above-mentioned solution in order to make the ethanol concentration in the solution equal or similar to that in the eluate. In such an embodiment, the sclution to which ethanol is added is applied to the cation exchange resin and further a solvent containing ne ionic substance is passed therethrough.

[0045]

In order to remove thecbromine effectively in the step (¢), it is preferred in the invention to select the solvent used for the elution appropriately. The solvent used for the elution in the step (c) may be any solvent that contains no ionic substance, can cause theobromine to be effectively adsorbed onto the cation exchange resin and further can cause the polyphenol to be selectively eluted out. Examples thereof include deionized water, and an aqueous solution of an organic solvent, such as an aqueous ethanol solution. [code]

When deionized water is used as the eluting solvent, it la desired thal the temperature of the deionized water ia 35°C or lower, move preferably 25% ar lower, Tf deilsnized water the temperature of which is higher than 35°C is used, theobromine tends not to be easily adsorbed onto the resin so that theobromine 1s easily eluted out together with the polyphencl. When an organic solvent solution in water, such as an aqueous ethanol solution, 1s used as the eluting solvent, it is preferred to use a solvent which is lower in the organic solvent concentration than the solvent used in the precedent step. For example, an aqueous ethanol solution which is lower in concentration than the aqueous ethanol solution used at the time of the extraction is used. More specifically, when an aqueous ethanol sclution

Dis used in the step (¢), it is preferred to use an aqueous ethanol solution having an ethanol content of 0 to 30% by weight, more preferably 0 to 10% by weight. If the ethanol concentration in the aqueous ethanol sclution is more than 30% by weight, theobromine tends not to be easily adsorbed so that theobromine is easily eluted out together with the polyphenol.

[0047]

The flow rate of the eluting solvent in the step (c¢) may be the same as in the step (b) descried above. It is preferred that the amount of the filled resin in the step (cc) is equal to or more than the liguid amount of the first polyphenol eluate or the solution yielded by subjecting the eluate to further treatment, preferably 1.5 times or more the liquid amount. In another method, the cation exchange resin used in the step (b) may be used regenerated to be reused.

[0048]

As described above, in the producing process according to the invention, purification by use of a cation exchange resin subjected beforehand to hydrogen ion substitution is carried cut in plural divided steps, thereby making it possible to make 2h small the amount of the resin filled into the column or each of the columns and the amount of the solvent passed therethrough.

Thus, the process is good in working efficiency. The plural purification operations are preferred alsc from the viewpoint of an increase in the polyphenol content in the finally cbtained 20 composition. In the producing process according Lo the invention, the purification with the cation exchange resin (s)

isnot limited to purification carried out two times in the steps (b} and (¢). As the need arises, the same purification may be repeatedly carried out. In addition to the extraction in the step (a), and the purifications in the steps (b) and {(c¢}, a technique known about purification may be used.

[0049]

It is preferred that as the need arises, the producing process according to the invention has the step (dY of concentrating or drying the polyphenol extract yielded through the purification step comprising the steps (a) to (¢). By the concentration or drying, a high polyphenol-containing composition, which is a target, can easily be cbtained in a solid state. For the concentration or the drying, a known method may be used. An example of the method for the concentration is concentration under reduced pressure or heating concentration.

An example of the method for the drying is spray drying or freeze drying. When a precipitate of theobromine is generated in the concentration or drying in the step (d}, the theobromine content in the finally obtained composition can be made lower by removing the precipitate. The removal of the precipitate of thecbromine may be carried out by a known method such as filtration or centrifugation. {0050

A second of the invention relates Lo a high polyohenol-containing composition vielded bv the producing process according to the first of the invention. Since the composition is high in the polyphenol content in the solid content thereof, the composition may be used for various articles for which a polyphenol is effective. The cacao polyphenol compesition yielded by the invention is particularly characterized in that the cacao polyphenol content is high while the thecobromine content is remarkably lower than that of compositions in the prior art. Thus, the composition can be appropriately added to food or drink, or a medical supply. [G051]

For example, when the composition according to the invention is blended with various. types of an exciplent, a stabilizer, a flavoring agent, and others and may be used in ordinary drugs, a medical supply can be provided, examples of

Lhe supply comprising a tablet, a capsule agent, a granule, and a powder that each has pharmaceutical effects. When the composition according to the invention is incorporated into a food or drink, such as cocoa, coffee, chocolate, biscuit, snack, candy, tablet sweets, gum, gummy candy, jelly, sweetened and jellied bean paste (youkan), ice cream, sherbet, beverage, dairy product, bread, sausage and ham, pharmaceutical effects can be given thereto. When the use of the composition in food, drink, or a medical supply 1s considered, the composition according to the invention is preferably a cacao polyphenol-containing composition that the polyphenol content in solid content is 33% or more by weight and the theobromine content therein is 1% or less by weight. The invention makes it possible to supply such a desired composition, that the cacao polyphenol content is high and the theobromine content is low. [oon

A third of the invention relates to an apparatus for producing a polyphencl-containing composition from a polyphenol~containing plant or a processed product thereof.

The producing apparatus of the invention has: an extraction section having a means for chtaining a crude polyphenol extract from the polyphenol-containing plant or the processed product thereof;

a purification section providing a first purification and a second purification, wherein the first purification has a means for obtaining a first polyphenol eluate by applying the crude polyphenol extract transferred through a means for transferring from the extraction section to a cation exchange resin subjected beforehand to hydrogen ion substitution, and subsequently passing a first solvent containing no ionic substance through the cation exchange resin, and the second purification has a means for obtaining a second polyphenol eluate by passing the first polyphenol eluate transferred through the means for transferring from the first purification section again through a cation exchange resin subjected beforehand to hydrogen ion substitution, and subsequently passing a second solvent containing no ionic substance through the cation exchange resin; and a recovery section having a means for concentrating or drying the second polyphenol eluate transferred through the means for transferring from the purification section,

[0053]

The extraction section is equipped with at least a container that can hold the above-mentioned plant or the processed product, which may be referred to as the raw material hereinafter, and a solvent used in an extraction therefrom. The 2h extraction ls conducted by causing the vaw material and the solvent to contact each other in the container, and the insoluble components (residues) are removed by a removing means, to obtain a crude polyphenol extract. The container preferably has at least one opening to pour the raw material and the extracting solvent, and an opening to discharge the crude polyphenol extract.

The container may be, for example, a column, an extracting kettle,

or a tank. In order to make the efficiency of the extraction high, a stirring means such as a propeller and a screw may be arranged in the container. A means for pulverizing the raw material into fine pieces, such as a rotating blade, may also be arranged in the container. The removing means of insoluble component may be, for example, a centrifuge, or a filltrating device set up oulside the container. By adding a concentrating means such as an evaporator and a reduced-pressure concentrator to the extraction section as the need arises, the solvent in the crude extract can be distilled off before the extract is transferred to the purification sections, so thal the liquid amount can be decreased.

[0054]

The crude polyphenol extract vielded in the extraction section is transferred through a means for transferring to a purification section. The means for transferring may be, for example, a liguid-sending pump and a pipe. As the need arises, a control valve may be set to the pipe. By the liquid-sending pump and the control valve, the flow quantity and the flow rate of the crude polyphencl extract can be adjusted when the extract solution is transferred to the purification section.

[0055]

The purification section has a layout capable of attaining al least two purifications including First and second

Zh purifications. In other words, the First and second purifications may be arranged as sections independent of each other, or may be arranged to have a means for transferring which can attain a clrculation in the same sectlon, such as pipes. [C056] 30 When the f[irst and second purifications are arranged in sections independent of each other, the first purificaticn section is equipped with a first column holding a cation exchange resin subjected beforehand to hydrogen ion substitution, and a first solvent tank holding a first solvent containing no ionic substance. The crude polyphenol extract transferred from the extraction section is passed through the resin held in the column, and then the first solvent, which contains no ionic substance, shifted from the first solvent tank is passed therethrough, to obtain a first polyphencl eluate.

[0057]

In the same manner, the second purification section is equipped with a second column holding a cation exchange resin subjected beforehand to hydrogen ion substitution, and a second solvent tank holding a second solvent containing no ionic substance. The crude polyphenol extract transferred from the extraction section is passed through the resin held in the column, and then the second solvent, which contains no ionic substance, transferred from the second solvent tank is passed therethrough, to obtain a second polyphenol eluate. The first purification section and the second purification section are connected to each other through a means for transferring, such as a pipe, and the first polyphenol eluate yielded in the first purification section is transferred through the shifting means to the second purification section.

Loans

Whan the amount of The resin held in ihe First column ia made small in the first purification section, theobromine comes to be contained in a large amount in the first polyphenol eluate. it may be, therefore, arranged a removal section for removing theobromine before the first polyphenol eluate is transferred 20 to the second purification section. The removal section is located between the first and second purification sections, and is equippedwith a container holding the first polyphenol eluate, a means for precipitating and removing theobromine from the eluate, and a means for transferring, such as a pipe, for transferring the solution yielded after the removal of thecbromine to the second purification section. The removing means may be, for example, a filtrating device or a centrifuge. inorder to promote the precipitation of theobromine, the removal section may have a temperature controller such as a cooler, and a concentrator such as an evaporator and a reduced-pressure concentrator. [C059]

In the meantime, when the first and second purifications are arranged to make a circulation in the same section, the purification section is equipped with a column holding a cation exchange resin subjected beforehand to hydrogen ion substitution, a first solvent tank holding a first solvent containing no iconic substance, a second solvent tank holding a second solvent containing no ionic substance, and a means for transferring, such as a pipe, for shifting a first polyphenol eluate yielded by passing the first solvent through the resin to the column again, In the purification section, the crude polyphenol extract transferred from the extraction section is passed through the column, and further the first solvent supplied from

Che first solvent tank is passed through the resin, to abltain a first nolyphenol eluate. Next, this first nolvphennl eluate is again transferred to the column, which has been regenerated, through the means for transferring, and passed through the column.

Thereafter, the second solvent supplied from the second solvent tank is passed therethrough to yield a second polyphenol eluate.

L0060]

The purification section may have third and fourth solvent tanks holding hydrochloric acid for subjecting the cation exchange resin to the hydrogen ion substitution, a solution of sodium hydroxide that is used to wash the resin, and some other treating solution. Furthermore, a sclvent tank holding a solvent tobe supplied to the column or one or each of the columns may be added thereto as the need arises. After the polyphenol eluate is yielded, by passing the treating solutions from the third and fourth solvent tanks into the column, the resin in the column is regenerated, so that the purification can be continuously conducted. The supply of the solvent from each of the solvent tanks to the column is attained by a means for transferring, such as a liquid-sending punp and a pipe. The means for transferring may contain a control valve as the need arises. By the liquid-sending pump and the control valve, the flow quantity and the flow rate of the solvent supplied to the column may be adjusted.

[0061]

The polyphenol eluate yielded in the purification section is transferred through a means for transferring to a recovery section. The means for transferring may be, for example, a

Liguid-sending pump, and a pipe. As the need arises, a control valve may be sel to the pipe. By the liquid-sending pump and the control valve, the flow quantity and the flow rate of the

Crude polyphenol extract can be adjusted when the exiract Ls transferred to the recovery soct ion,

L0062]

The polyphenol eluate is transferred through the means for transferring to the recovery section. The recovery section has a container holding the transferred polyphencl eluate, and a means for removing the solvent in the eluate from the container.

The means for removing solvent may be, for example, a device used for ordinary concentration such as a reduced-pressure concentrator, or adevice used for ordinary drier such as a freeze drier and spray drier. 10063]

An embodiment of the producing apparatus of the invention is illustrated in FIG. 1. As illustrated inFIG. 1, the producing apparatus of the invention has an extraction section 100, a purification section 200, and a recovery section 300. The extraction section 100 has an extracting tank 110 for conducting i0 an extraction from the raw material, a raw material tank 120 for holding the raw material, a solvent tank 130 holding an extracting solvent, and a centrifuge 140 for removing insoluble components after the extraction. The extracting tank 110 and the centrifuge 140 are connected to each other through a pipe 1004. [C064]

The purification section 200 has a column 210 for conducting a first purification, a concentrator 220 for concentrating an eluate, a column 230 for conducting a second purification, a solvent tank 240a for holding a solvent for conducting any columnn-washing, a solvent tank 240b for holding a solvent for conducting any column~equilibrating, and solvent tanks 240c and 240d each for holding a solvent used for elution.

The coluw 210, the concentrator 220 and the column 234 are connected to each other through a pips 200a and a pipe 200h.

The purification section 200 is connected to the extraction section 100 through a pipe 100bh. Reference numbers 212 and 232 each represent a waste tank for holding a waste.

[0065]

A The recovery section 300 has a concentrator 210 for concenlbrating an eluate transferred through a pipe 200c¢ from the purification section 200, and a spray drier 320 for drying the concentrated liquid. The concentrator 310 and the spray drier 320 are connected to each other through a pipe 300a.

Reference number 322 represents a collecting tank holding a composition yielded by the drving,

[0066] hoccording to the producing apparatus of the invention having such a structure, a polyphencl-containing plant or a processed product thereof is used as a raw material to make it possible to vield a high polyphenol-containing composition effectively. The producing apparatus of the invention is intended to be used to carry out the producing process of the invention described above. Accordingly, the solvent used in each of the sections, the resin{s) held in the columni(s) and the other various conditions are equivalent to the various conditions in the producing process.

Examples

[0067]

Hereinafter, the invention will be specifically described by way of working examples; however, the invention is not limited to these working examples. [Goo8]

The cacao polyphenol content and the theobromine content described in the individual working examples and individual comparative examples are values obtained by measurements according to the following methods,

[0069] (Polyphenol content) 34 The polyphenol content are measured by the Prussian blue method. More specifically, the content are each a value obtained by using commercially available epicatechin as a standard product to quantitative the solid content of a composition vielded in the individual working examples and the individual comparative examples with reference to a method described in Martin L. Price and Larry G. Butler, J. Agric Food

Chem., Vol. 25 No. 6, 1268-1273, 1977.

[0070]

The method for the analysis is specifically as follows:

Each of the samples was defatted by n-hexane, and then a 50% by weight aqueous methanol solution was used to extraction.

The extract solution was used as a “test solution”. Next, 100

UL each of the test solution was added to 50mL of distilled water,

While the solutionwas stirred, 3mLofa0.1Miron (III) ammonium sulfate solution in 0.1 NW hydrochloric acid was added thereto.

After 20 minutes from the addition, thereto was added 3 mil of an 8 mM hexacyanoiron (111) potassium solution. After 20 minutes, the absorbance was measured at 720 nm. Furthermore, the same processing was performed, using a solution in which 100 uL of the solvent (50% by weight methanol) of each of the sample liquids was added to 50 mL of distilled water, and a (~Jepicatechin {manufactured by Sigma Aldrich Corp.) solution in methanol, so as to prepare a calibration curve. The polyphenol content in each of the test solutions was calculated

Out. jucilg {(Thechromine content)

The theobromine content are each a value obtained by using commercially available theobromine as a standard product, and quantitating the solid content of the composition vielded in the individual working examples and the individual comparative examples with reference to a method described in ACAC Official

Methods of Analysis (1990) 980. 14, “Theobromine and Caffeine in Cacao Products Liguid Chromatographic Method”.

[0072]

The method for the analysis is specifically as follows:

First, each of the samples was precisely weighed into a centrifugal tube, and 30 mL of petroleum ether was added thereto.

The mixture was sufficiently stirred, and then centrifuged and the supernatant was thrown away. The defatted sample was transferred into a conical flask, and water was added to fill up to about 100 mL. This solution was heated in 100°C boiled water for 25 minutes. After the heating, the solution was immediately cooled, and thereto were added 10 nl. of a 2% by weight zinc sulfate solution and 10 wml of a 1.8% by welght barium hydroxide solution. The components were mixed and then the mixture was allowed to stand still. Water was added to this sclution to make the solution into a fixed volume of 200 ni, and the sclution was again heated in 100°C boiled water for 10 minutes. After the heating, the solution was filtrated to prepare a sample solution. The thus obtained sample solution was used to make a measurement by high-performance liquid chromatography. Conditions for the high-performance liquid chromatographic measurement are as follows:

Column for the analysis: Waters p-Bondapk C18 10 um 4 nmlD 2 300 mm {or an eguivalent bherelo)

Mobile phase: water and acetonitrile (85:15)

Mobile phase flow rate: 1.0 mL/minute, and Detection: UV 273 mm

[0073] {Investigation on extracting sclvent)

An aqueous ethanol solution was used as an extracting solvent, and an investigation was made on the composition of the solvent onto the polyphenol recovery and the polyphenol content in the solid content. Steps therefor are as follows:

First, 10 g of cacao beans (dried in the production area thereof; the cacac polyphenol content: 7.1% by weight, and the theobromine content: 1.3% by weight) was ground. The ground cacao beans were added to 200 mL of each of 0-100% by weight solutions of ethanol in water. The mixture was stirred at 50°C for 30 minutes. Next, the insoluble components were removed by centrifugation to yield a crude cacao extract. The crude cacao axtract was freeze-dried, and the polyphenol content in this solid content was measured. The results are shown in FIG. 2.

When the polyphenol recovery and the polyphenol content in the solid content are considered, it is understood that the solvent having an ethanol concentration of about 50% by weight is preferred as the extracting solvent.

[0074] (Example 1)

Cocoa powder (the fat content: 12% by weight, the cacao polyphencl content: 10.4% by weight, and the theobromine content: 2.0% by weight) was used to prepare a polyphencl~containing composition in accordance with the following process:

FPirst, 100 g of the cocoa powder was dispersed in 1000 ml of water, and The wixture was stirred at 50°C for 30 minutes.

Thereafter, the insoluble components were removed by centrifugation to yield a crude cacao extract.

Next, the crude cacao extract was passed at a flow rate sV oof © through a column into which 1000 mL of a cation exchange resin (AMBERLITE IR-120B) subjected beforehand to hydrogen ion substitutionwas filled. Subsequently, 25% deionizedwabter was passed through the column. By the passing of the crude cacao extract and the deionized walter, 2000 ml of an eluate was fractionated.

Next, the 2000 mL eluate was freeze-dried to yield a polyphencol-containing composition. Components in the solid content of this composition were analyzed. As a result, the polyphenol content was 40.2% by weight, and the theobromine content was 0.6% by welght.

A crude cacao extract prepared separately in the same way was freeze-dried. Components in the solid content were analyzed.

As a result, the polyphencl content was 8.2% by weight, and the theobromine content was 3.3% by weight.

[0075] (Example 2)

Cocoa powder (the fat content: 12% by weight, the cacao polyphenol content: 10.4% by weight, and the theobromine content: 2.0% by weight) was used to prepare a polyphenol-containing composition in accordance with the following process:

First, 2 g of the cocoa powder was dispersed in 20 ml of a 50% by weight aqueous ethanol solution, and the mixture was stirred at 50°C for 30 minutes. Thereafter, the insoluble components were removed by centrifugation to yield a crude cacao extract.

Next, the crude cacao extracl was passed av a flow rate £25 8V of 5 through a column into which 20 nh of a cation exchange resin {(DOWEX 50W-X8) subjected beforehand to hydrogen ion substitutionwas filled. Subsequently, 25°Cdeionizedwater was passed through the column. By the passing of the crude cacao extract and the deionized water, 50 ml of an eluate was fractionated.

Next, the 50 ml eluate was freeze-dried to vield a polyphencl-containing composition. Components in the solid content of this composition were analyzed. As a result, the polyphencl content was 53.4% by weight, and the theobromine content was 0.7% by weight.

As a result, the polyphenol content was 12.9% by weight, and the theobromine content was 5.3% by welghtl.

[0076] (Example 3)

Ten grams of cacao beans (dried in the production area thereof; the cacao polyphencl content: 7.3% by weight, and the theobromine content: 1.3% by weight) were used to prepare a polyphenol~-containing composition in accordance with the following process:

First, 10 g of the cacao beans were ground, and then thereto was added 200 ml of a 50% by weight aqueous ethanol sclution.

The mixture was stirred at 50°C for 30 minutes. Next, the inscluble components were removed by centrifugation to yield a crude cacac extract.

Next, the crude cacac extract was passed at a flow rate

SV of 5 through a column into which 200 mi of a cation exchange resin (AMBERLITE IR-120B) subjected beforehand to hydrogen ion substitution was f1)1ed, Subsequently, 20°C deionized walter was 25% passed through the column. By the passing of the crude cacao extract and Lhe deionized water, 600 ml. of an eluate was fractionated.

Next, the 600 ml, eluate was freeze-dried to vield a polyphenol-containing composition. Components in the solid content of this composition were analyzed. As a result, the polyphenol content was 62.3% by weight, and Lhe Cheobromine content was 0.6% by weight.

As a result, the polyphenol content was 28.4% by weight, and the theobromine content was 8.5% by weight.

[0077] (kxample 4)

One hundred grams of cacao shells (the cacao polyphenol content: 3.5% by weight, and the theobromine content: 2.0% by weight) were used to prepare a polyphenol-containing composition in accordance with the following process:

First, 100 g of the cacac shells were ground, and then thereto was added 500 mL of a 50% by weight aqueous ethanol solution. The mixture was stirredat 50°C for 30 minutes. Next, the insoluble components were removed by centrifugation to yield a crude cacao shell extract.

Next, the crude cacao shell extract was passed at a flow rate SV of 5 through a column into which 500 mL of a cation exchange resin (AMBERLITE IR~120B) subjected beforehand to hydrogen ion substitutionwas filled. Subsequently, 25°C deionizedwater was passed through the column, By the passing of the crude cacao shell extract and the deionized water, 1500 ml of an eluate was fractionated.

Next, the 1500 ml eluate was freeze-dried to vield a polyphenol-containing composition. Components in the solid content of this composition were analyzed. As a result, the polyphenol content was 20.5% by weight, and the thecbromine content was 0.4% by weight.

As a result, the polyphenol content was 7.8% by weight, and the theobromine content was 0.9% by weight.

[0072] (Example 5)

One hundred grams of cacao beans (dried in the production area thereof; the cacao polyphenol content: 7.1% by weight, and the theobromine content: 1.3% by weight) were used to prepare a polyphencol-containing composition in accordance with the following process:

First, 100 g of the cacao beans were ground, and then thereto was added 1000 ml of a 50% by weight aqueous ethanol solution. The mixture was stirred at 50°C for 10 minutes. Next, the inscluble components were removed by centrifugation to yield a crude cacao extract.

Next, Lhe crude cacao extract was passed at a flow rate 38V of 5 through a column into which 1000 mL of a cation exchange resin (AMBERLITE TR-120B} subjected beforehand to hydrogen ion substitutionwas filled. Subsequently, 25°Cdeionizedwater was passed through the column. By the passing of the crude cacac extract and the deionized water, 3000 mL of an eluate was fractionated.

Next, the 3000 ml eluate was spray-dried under a condition that the drying temperature (inlet temperature} was 160°C to yield a polyphenol-~containing composition. Components in the solid content of this composition were analyzed. As a result, 25h the polyphenol content was 59.0% by weight, and the Uheobromine content: was 0.6% by weight,

A crude cacao extract prepared separately in the same way was [reere-dried. Components in the solid content were analyzed.

As a resull, the polyphenol content was 28.4% by weight, and the thecobhroming content was #2. 5% hy weight,

[0079]

(Example ©)

The polyphenol-containing composition yielded in Example was used to investigate physiclogical effects produced by the composition from evaluation l1tems described below. The results 5 are shown in Table 1 and FIG. 3.

[0080] (DPPH radical scavenging capability)

The DPPH (1, l-diphenyl-2-picrylhydrazyl: DPPH) radical scavenging capability was measured, using commercially avallable epicatechin as an index in accordance with a method described in Food Chemistry 68 (2000) Yinrong Lu. L. Yeap Foo “Antioxidant and radical scavenging activities of polyphenols from apple pomace”. A summary of the method for the measurement is as follows:

Epicatechin as the index and individual samples were each dissolved into a 50% by weight aqueous ethanol solution. To 100 nL of this solution was added 2 ml of a 0.1 mM DPPH solution, and the mixture was allowed to stand still at room temperature for 30 minutes. Thereafter, the absorbance was measured at 520 nm, The inhibitor ratio of each of the samples was calculated under the condition that the value of the absorbance of a blank solution (DPPH solution) was regarded as an inhibition ratio of 100%. As the numerlcal value obtained in the measurement is lower, the DPPH radical scavenging capability ls higher. 20 [0081] (Micelle insolubilizing capability)

The micelle inscolublilizing capability was measured, using commercially avallable epicatechin as an index in accordance with a method described in Journal of Nutrition, 1989, 129: 34 L725=-1730, Satoshi Nagaoka, Kenji Miwa, Michiko Ete, Yasuo

Kuzuva, Goro Hori and Kazuhiro Yamamoto “Soy Protein Peptic

Hydrolysate with Bound Phospholipids Decreases Micellar

Solubility and Cholestercl Absorption in Rats and Caco-2 Cells”,

A summary of the method for the measurement is as follows:

Into a phosphoric acid buffer (pH: 7.4) were dispersed 7.44 mmol/L of sodium taurocholate, 1.94 mmol/L of cholesterol, 1 mmol/L of oleic acid, 0.5 mmol/L of monooleic acid, 0.6 mmol/l of phosphatidylicholine, and 132 mmol/L of sodium chloride, and then the mixture was subjected to ultrascnic treatment for 10 minutes to prepare a micelle solution. Into this solution were added epicatechin and the sawple each, and the solution was sufficientlymixedwith a 50% by weight aqueous ethanol solution.

The mixture was heated at 37°C for 60 minutes. Thereafter, the precipitate was removed by centrifugation, and then the cholestercl amount in the supernatant was measured, using a commercially available kit, CHOLESTEROL E - TEST WAKO (manufactured by Wako Pure Chemical Industries, Ltd.). As the numerical value obtained by the measurement is higher, the micelle insolubilizing capability is higher.

[0682] (Comparative Example 1)

A cacao polyphencl material (trade name: “CACAO

POLYPHENOL”, manufactured by Melll Food Materia Co., Ltd.) as a commercially available product was used to make the same investigation as in Dxample ¢. The results are shown in Table 2h oand Fla. 3. 0083] [Table 1]

Tabie 1 [Es [EEE (composition of Example 5) | (commercially available product) {n

DPPH radical scavenging capability 0.227 0.382

ERT ee | em me |e (% by weight)

[0084]

Notes (1): The content by percentage were each a content in the total weight of the solid content.

[0085]

As 1s evident from Table 1, it is understood that the polyphenol-containing composition of the invention (Example 6) had a higher polyphenol content and a lower theobromine content compared to the commercially avallable product (Comparative

Example 1). It is also understood that the composition had a higher radical scavenging capability compared to the commercially available product. Furthermore, it is also understood from FIG. 3 that the composition had a higher micelle insolubilizing capability compared to the commercially

Lh avallable product. Thematter that “the DPPH radical scavenging capability is higher” suogests thal the composition has a higher anti-oxidative activity. The matter that “the micelle insolubilizing capability is higher” suggests that the composition is higher in the capability of insclubilizing 26 cholesterol micelle and has a capability of lowering the cholesterol concentration in blood. From such viewpoints, it is evident that according to the invention, a more useful polyphenol-containing composition can be supplied than according to commercially available products in the prior art.

[0086] {Example 7)

The present example is concerned with an investigation on cation exchange resins used in the purification step. Cacao beans (dried in the production area thereof; the cacao polyphenol content: 7.0% by weight, and the thecobromine content: 1.2% by weight) were used to prepare a polyphenol-containing composition in accordance with the following process:

First, 10 g of the cacao beans were ground, and then thereto was added 200 mL of a 50% by weight aqueous ethanol solution.

The mixture was stirred at 50°C for 30 minutes. Next, the insoluble components were removed by centrifugation to yield a crude cacao extract.

Next, the crude cacao extract was passed at a flow rate

SV of 5 through a column into which 200 mL of each of various cation exchange resins (see Table 2) subjected beforehand to hydrogen ion substitution was filled. Subsequently, 25°C deionized water was passed through the column. By the passing of the crude cacao extract and the deionized water, 600 ml of an eluate was fractionated.

Next, the 600 ml, eluate was freeze-dried to vield a polyphenol-vontaining composition. Components in Lhe solid content of this composition were ansivzed. Ry comparing tha components between the individual compositions, an effect produced by the resin species was investigated. The results are shown in Table 2.

[0087] 20 {Comparative Example 2)

An eluate was yielded by making the same operations as in

Example 7 except that instead of the various cation exchange resing used in Example 7, an anion exchange resin (see Table 2) subjected to hydroxide ion substitution was used. Next, in the same way as in the Example, the eluate was freeze-dried to wield a polyphenol-containing composition. Components in the solid content of the composition were analyzed in the same way as in Example 7. The results are shown in Table 2.

[0088] [Table 2]

Table 2

Resin name Polyphenol content™ | Theobromine content!" (% by weight) (% by weight)

DIAION 2 WK40 (weak acidity) 53.4 0.9 (Example 7) ~~ FpiAION SK116 TT

Cation exchange | (strong acidity) 50.2 0.9

DIAION PK206 51 3 (strong acidity)

AMBERLITE IRA-96SB 28 3 4 (weak basicity) {Comparative res :

Example 2) AMBERLITE XE583 5.2 3.4

Anion exchange | (weak basicity) IR BE resins DIAION WA10 (weak basicity) 8.9 | 3.4

DIAION HPAZ5 5 Te {strong basicity)

[0089]

Notes: {1) The content by percentage were each a content in the total weight of the solld content. {2) “DILATION” (registered trade name) series are manufactured by Mitsubishi Chemical Corp. {3) “AMBERLITE” (registered trade name) series are manufactured by Rohm and Haas Co. [ooan]

As shown in Table 2, in the case of using each of the cation exchange resins (Example 7) in the purification, the polyphenol content in the solid content was higher while the theobromine content therein was lower than in the case of using the anion exchange resin (Comparative Example 2). From this matter, it has been understood that even when either a strongly acid or a weakly acid cation exchange resin is used, theobromine can be satisfactorily adsorbed and thecbromine and the polyphenol are effectively separated from each other. By contrast, when the anion exchange resin (Comparative Example 2) was used, the polyphenol content was remarkably lower and the theobromine content was higher than when the cation exchange resins were used (Example 7). From such results, it has been understood that when an anion exchange resin is used as the filler, the resin tends to adsorb not only thecbremine but also the polyphenol.

[0091] {Example 8)

The present example 1s concerned with an investigation on solvents used in a purification step using a cation exchange resin. Specifically, various solvents were each used as an ean Liberating solulion Lo a column into which a cabion exchange resin was filled, as well as an eluent from the column.

Cacao beans (dried in the production area thereof; the cacao polyphenol content: 7.4% by weight, and the theobromine content: 1.3% by weight) were used to prepare a polyphencl-containing composition in accordance with the following process:

First, 2 g of Lhe cacao beans were ground, and then thereto was added 20 mL of a 50% by weight aqueous ethanol solution.

Next, 100 mi, of each of various equilibrating solutions shown in Table 3 described below was passed through a cation exchange resin (AMBERLITE IR~120B, strongly acid cation exchange resin} subjected beforehand te hydrogen ion substitution.

Thereafter, Lhe crude cacao extract was passed at an 3V of 5 therethrough. Subsequently, the corresponding one out of column eluents shown in Table 3 was passed therethrough.

Next, by the passing of the crude cacao extract and the column eluent, an eluate was fractionated, and the eluate was freeze-dried. As a result, each polyphenol-containing composition was yielded. Individual components in the solid content of each of the compositions were analyzed. By comparing the components therebetween, an effect of the solvents was investigated. The results are shown in Table 3.

[0092] [Table 3]

Table 3 rest division | Equiirating | Column | Polyphenol | Theobrormine joe Cl ; if i 0% Deionized water | Deionized water 53.4 0.7 0% {10 che Em] 75 50% Ethan

[0093]

Notes: {1) The temperature of each of the equilibrating solutions was 25°C. (2) The temperature of each of the column eluents was 25°C, (3) The content by percentage were each a content in the total weight of the solid content.

[0094]

As 1s evident from Table 3, when deionized water was used as the equilibrating solution and the column eluent, a composition wherein the polyphenol content was the highest and the theobrecmine content was the lowest was yielded. When the ethanol sclutions in water were used, it was observed that theobromine tended Lo elute oul more easily as the ethanol concentration was higher. It has been understood from this matter that when deionized water is used, theocbromine is most easily adsorbed onto the cation exchange resin. [0CG95] {Example 9)

Cacao beans (dried in the production area thereof; the cacao polyphenol content: 7.3% by weight, and the thecbromine content: 1.3% by welght) were used to prepare a polyphenol-containing composition in accordance with the following process:

First, 100 g of the cacao beans were ground, and then 2h thereto was added 1000 ml of 2 H0% by welghl agueons ethanol solution. Themixture was stirred at 50°C for 30 minutes. Next, the insoluble components were removed by centrifugation te yield a crude cacao extract.

Next, the crude cacac extract was passed at a flow rate

SV of bh through a column into which 20 ml of a catlon exchange resin (AMBERLITE IR-120B, strongly acid cation exchange resin)

subjected beforehand to hydrogen ion substitution was filled.

Subsequently, 25° deionized water was passed through the column.

By the passing of the crude cacao extract and the deionized water, 600 mL of an eluate was fractionated.

Next, the 600 ml, eluate was concentrated under reduced pressure to yield 40 ml of a concentrated liguid. This concentrated liquid was allowed fo stand still for 18 hours to obtain a precipitate. And then the precipitate was removed by centrifugation.

Next, the centrifugal supernatant vielded by the above-mentioned operation was again passed at a flow rate SV of 5 through a column into which 80 mL of a cation exchange resin (AMBERLITE IR-1208) subjected beforehand to hydrogen ion substitutionwas filled. Subsequently, 25°C deionized water was passed therethrough. By the passing of the centrifugal supernatant and the deionized water, 200 mL of an eluate was fractionated.

Next, the 200 ml. eluate was freeze-dried to vield a polyphenol-containing composition. Components in the solid content of this composition were analyzed. As a result, the polyphenol content was 52.9% by welght, and the thecobromine content was (0.9% by weight.

[0096] (Example 10]

Cacao beans (dried in the production area thereof; the cacao polyphenol content: 7.3% by weight, and the theobromine content: 1.3% by weight) were used Lo prepare a polyphenol-containing composition in accordance with the following process:

First, 100 g of the cacao beans were ground, and then thereto was added 1000 mL of a 50% by weight aqueous ethanol solution. The mixture was stirred at 50°C for 30 minutes. Next, the insoluble components were removed by centrifugation to yield a crude cacao extract.

Next, the crude cacao extract was passed at a flow rate 8V of 5 through a column into which 20 mL of a cation exchange resin (AMBERLITE IR-120B, strongly acid cation exchange resin) subjected beforehand to hydrogen icon substitution was filled.

Subsequently, 25°C deionized water was passed through the column.

By the passing of the crude cacao extract and the deionized water, ic 600 mL of an eluate was fractionated. Subsequently, the 600 mL eluate was concentrated under reduced pressure to yield 40 ml of a condensed liguid.

Next, the concentrated liquid yielded by the above-mentioned operation was again passed at a flow rate gv of 5 through a column into which 160 nL of a cation exchange resin (AMBERLITE IR-120B) subiected beforehand to hydrogen ion substitutionwas filled. Subsequently, 25°C deionizedwater was passed therethrough. By the passing of the concentrated liquid and the deionized water, 360 mL of an eluate was fractionated.

Next, the 360 ml eluate was freeze~dried to vield a polyphenol-containing composition. Components in the solid content of this composition were analyzed. As a result, the polyphenol content was 51.9% by weight, and the theobromine content was 0.9% by welght.

Pa FO0S7]

From the above description, it is evident that the invention can be made into various and different embodiments without disobeying neither the scope nor the sprit of the invention. The invention is not limited by any specified one of the embodiments except that the invention ls limited by the claims,

Claims

1. A process for producing a polyphenol-containing compositien, comprising the following steps: (a) performing an extraction of polyphencl-containing plant or a processed product thereof with a solvent, to obtain a crude polyphenol extract, (b) applying the crude polyphenol extract to a gation exchange resin subjected beforehand to hydrogen ion substitution, and subsequently passing a first solvent containing no ionic substance through the cation exchange resin, to obtain a first polyphenol eluate, {c} passing the first polyphenol eluate again through a cation exchange resin subjected beforehand to hydrogen ion substitution, and then passing a second solvent containing no iconic substance through the cation exchange resin, to obtain a second polyphencl eluate, and (d) concentrating or drying the second polyphenol eluate.

2. The producing process according to claim 1, wherein the plant is cacao.

3. The producing process according to claim 1 or 2, wherein the solvent used {or the extraction in the step (a) is 2h a 40-70% by weight aqueous ethanol solution.

4, The producing process according Lo any one of claims 1 to 3, wherein in the steps (b) and {c¢}), the first and second solvents are each passed att a {low rate 3V of 2 to 10. 20 a. The producing process according fo any one of claims

1 to 4, wherein before the step {(¢), a precipitate of theobromine is obtained from the first polyphenol eluate, the precipitate is next removed to yield a solution, and the solution is used in the step (¢).

6. The producing process according to any one of claims I to 5, wherein the first and second solvents each containing no ionic substance are each a deionizied water or an aqueous ethanol solution.

7. The producing process according to any one of ¢laims 1 to 6, wherein the second sclivent containing no ionic substance is the deionizied water having a temperature of 35°C or lower, or an aqueous ethanol solution having an ethanol concentration of 30% or less by weight.

8. A polyphenol-containing composition yielded by the preducing process according te any one of claims 1 to 7.

9. A polyphenol-containing composition that a polyphenol content in the solid content is 33% or more by weight, and a theobromine content therein is 1% or less by weight.

1G. An apparatus for producing a polyphenol-containing composition from a polvphenol-containiog plant or a processed product thereof, comprising: an extraction section having a means for obtaining a crude polyphencl extract from the polyphenol-containing plant or the processed product thereol: a purification section providing a first purification and a second purification, wherein the first purification has a means for obtaining a first polyphenol eluate by applying the crude polyphencl extract transferred through a means for transferring from the extraction section to a cation exchange resin subjected beforehand to hydrogen ion substitution, and subsequently passing a first solvent containing no ionic substance through the cation exchange resin, and the second purification has a means for cbtaining a second polyphenol eluate by passing the first polyphenol eluate transferred through the means for transferring from the First purification section again through a cation exchange resin subjected beforehand to hydrogen ion substitution, and subsequently passing a second solvent containing no ionic substance through the cation exchange resin; and a recovery secllon having a means for concentrating or drying the second polyphenol eluate transferred through the means for transferring from the purification section.