WO2022210735A1 - Method for producing polyphenol-containing composition - Google Patents

Method for producing polyphenol-containing composition Download PDF

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Publication number
WO2022210735A1
WO2022210735A1 PCT/JP2022/015549 JP2022015549W WO2022210735A1 WO 2022210735 A1 WO2022210735 A1 WO 2022210735A1 JP 2022015549 W JP2022015549 W JP 2022015549W WO 2022210735 A1 WO2022210735 A1 WO 2022210735A1
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Prior art keywords
polyphenol
containing composition
enzyme
producing
solution
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PCT/JP2022/015549
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French (fr)
Japanese (ja)
Inventor
裕佳 旭
奈津子 村上
知春 八田
基美 造田
寿典 中島
淳 南野
傑 伊藤
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三井製糖株式会社
セルローシック、バイオマス、テクノロジー、カンパニー、リミテッド
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Priority to JP2023511392A priority Critical patent/JPWO2022210735A1/ja
Publication of WO2022210735A1 publication Critical patent/WO2022210735A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07G—COMPOUNDS OF UNKNOWN CONSTITUTION
    • C07G1/00—Lignin; Lignin derivatives
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/22—Preparation of oxygen-containing organic compounds containing a hydroxy group aromatic

Definitions

  • the present invention relates to a method for producing a polyphenol-containing composition from herbaceous biomass.
  • biomass has attracted attention from the viewpoint of problems such as global warming and depletion of petroleum resources, and from the viewpoint of carbon neutrality.
  • One of them is a method of obtaining a polyphenol-containing composition from cellulose-containing biomass that does not compete with food.
  • Cellulose-containing biomass is mainly composed of cellulose and hemicellulose, which are polysaccharides, and lignin, which is an aromatic polymer. A decomposed liquid can be obtained.
  • Patent Document 1 describes a method for efficiently obtaining hydroxycinnamic acid by passing an alkaline aqueous medium through cellulose-containing biomass.
  • Patent Document 2 an extract obtained by treating bagasse, which is sugar cane lees, with an alkaline solution is adjusted to acidity and filtered, and the filtrate is adsorbed with an aromatic synthetic adsorbent to efficiently remove polyphenols.
  • a method of making the containing composition is described.
  • the decomposition solution containing polyphenols obtained by the above-described conventional technology can be used as a deodorant (Patent Document 3), a food discoloration inhibitor (Patent Document 4), an aquatic organism growth promoter (Patent Document 5), and the like.
  • a deodorant Patent Document 3
  • a food discoloration inhibitor Patent Document 4
  • an aquatic organism growth promoter Patent Document 5
  • the pretreatment liquid obtained by treating herbaceous biomass with an alkaline aqueous solution is adjusted to acidity and filtered, and the filtrate is used for aromatic synthesis.
  • There is a method for efficiently producing a polyphenol-containing composition by adsorbing with an adsorbent but poor filterability and micellization-like phenomena occur, and the yield with aromatic synthetic adsorbents decreases. It has become clear from the study of the present inventors that there is a case.
  • one object of the present invention is to provide a new technical means for efficiently producing a polyphenol-containing composition from herbaceous biomass.
  • the extract obtained by treating the herbaceous biomass with an alkali at a temperature of 60°C or higher is cooled to 60°C or lower and then adjusted to pH 3.2 or higher and pH 4.5 or lower to obtain cellobiohydrolase activity. and an enzyme containing xylanase activity, followed by filtration and adsorption on an aromatic synthetic adsorbent, preventing micellization-like phenomena, improving filterability, and increasing the yield of the polyphenol-containing composition. I found out.
  • the present invention comprises the following [1] to [6].
  • [1] A method for producing a polyphenol-containing composition derived from herbaceous biomass, (1) A step of contacting the herbaceous biomass with an alkaline aqueous solution at a temperature of 60° C.
  • [3] The method for producing a polyphenol-containing composition according to [1] or [2], wherein the herbaceous biomass is bagasse.
  • [4] The method for producing a polyphenol-containing composition according to any one of [1] to [3], wherein the alkaline aqueous solution is an aqueous sodium hydroxide solution.
  • [5] The method for producing a polyphenol-containing composition according to [4], wherein the concentration of sodium hydroxide is 0.1 to 10% by mass.
  • [6] The method for producing a polyphenol-containing composition according to any one of [1] to [5], wherein the aromatic resin is a styrene-divinylbenzene resin.
  • a polyphenol-containing composition can be efficiently produced from herbaceous biomass.
  • the present invention can be advantageously used in the production of polyphenol-containing compositions from herbaceous biomass, in suppressing micellization-like phenomena and improving filterability.
  • the present invention can be advantageously used to improve the yield of the polyphenol-containing composition.
  • a method for producing a herbaceous biomass-derived polyphenol-containing composition comprises: (1) A step of contacting the herbaceous biomass with an alkaline aqueous solution at a temperature of 60° C. or higher to obtain an extract; (2) a step of cooling the liquid extract obtained in step (1) to 60°C or less to obtain a cooled liquid extract; (3) a step of adjusting the cooled extract obtained in step (2) to pH 3.2 or higher and 4.5 or lower, and reacting it with an enzyme containing cellobiohydrolase activity and xylanase activity to obtain an enzyme reaction liquid; (4) a step of obtaining a clarified solution by coarsely filtering the enzyme reaction solution obtained in step (3); (5) The clarified liquid obtained in step (4) is passed through a column filled with a synthetic adsorbent made of an aromatic resin that has undergone a special treatment to increase the specific surface area, and the aromatic synthetic adsorbent A step of eluting the components adsorbed to with a mixed solvent of
  • step (1) herbaceous biomass is brought into contact with an alkaline aqueous solution to obtain an extract.
  • examples of herbaceous biomass include sugarcane pomace bagasse, switchgrass, napiergrass, erianthus, corn stover, corn husks, wheat husks, soybean husks, rice straw, wheat straw. , oil palm empty fruit bunches, etc., but not limited to these.
  • herbaceous biomass with a lignin content of 5% or more.
  • bagasse, napier grass, erianthus, corn stover, and rice straw are preferable, and bagasse is more preferable.
  • the lignin content can be determined by measuring Clason lignin, which is obtained by subtracting ash from the acid hydrolysis residue.
  • the shape of the herbaceous biomass is not particularly limited, but it is preferably pulverized.
  • the pulverization means is not particularly limited, and machines commonly used for coarse pulverization of various materials such as ball mills, vibration mills, cutter mills, hammer mills, Willey mills and jet mills can be used.
  • This mechanical pulverization may be either dry or wet, preferably dry pulverization.
  • the water content of the herbaceous biomass is not particularly limited, but a preferable range is, for example, about 3% or more, about 3% or more and about 75% or less, about 5% or more, and about 5%.
  • a preferable range is, for example, about 3% or more, about 3% or more and about 75% or less, about 5% or more, and about 5%.
  • polyphenols may include one or more of hydroxycinnamic acids such as coumaric acid and ferulic acid, and lignin degradation products, and can be measured, for example, by the Folin-Ciocalteu method.
  • the Folin-Ciocalteu method was originally developed for the purpose of analyzing aromatic amino acids such as tyrosine and tryptophan, and proteins containing them.
  • the phenolic hydroxyl group is alkaline and the blue color produced by reduction of phosphotungstic acid and molybdic acid is colorimetrically determined at 700 to 770 nm.
  • a similar operation is performed using a specific reference substance such as gallic acid or catechin, and a quantitative value can be shown in terms of the compound, and the value in terms of catechin is used in the present invention.
  • the alkaline aqueous solution is ammonia, aqueous ammonia, alkali metal hydroxides, alkali metal oxides, alkaline earth metal oxides, alkali metal carbonates, alkaline earth metal carbonates, water
  • An alkaline aqueous solution containing at least one selected from quaternary ammonium oxide and the like can be mentioned, but an aqueous medium containing at least one hydroxide selected from sodium hydroxide and potassium hydroxide is preferred. From the viewpoint of being inexpensive and being used in the food manufacturing process, an aqueous sodium hydroxide solution is more preferable.
  • the alkali concentration of the alkaline aqueous solution is not particularly limited, but from the viewpoint of shortening the treatment time of the pretreatment, it is preferably 0.1% by weight or more, more preferably 0.2% by weight or more, and still more preferably 0% by weight. .3% by weight or more. From the viewpoint of improving extraction efficiency, the concentration of the alkaline solution is preferably 10% by weight or less, more preferably 5% by weight or less, and even more preferably 1.0% by weight or less. From the same viewpoint, the concentration of the alkaline solution is 0.1 to 10% by weight, 0.1 to 5% by weight, 0.1 to 1.0% by weight, 0.2 to 10% by weight, 0.2 to 5% by weight. % by weight, 0.2-1.0% by weight, 0.3-10% by weight, 0.3-5% by weight, or 0.3-1.0% by weight.
  • the lower limit of the pH of the alkaline aqueous solution is not particularly limited as long as it is alkaline, but is pH 7 or higher, preferably pH 8 or higher, more preferably pH 9 or higher, and still more preferably pH 10 or higher.
  • the upper limit of the pH is not particularly limited as long as it is less than pH 14, but from the viewpoint of reducing the amount of alkali used, it can be set at pH 12 or less.
  • a preferable pH range is, for example, 7 or more and 13.5 or less, 8 or more and 13.5 or less, a more preferable pH range is 9 or more and 13.5 or less, and a further preferable pH range is 10 or more and 12 or less. be.
  • the temperature at which the alkaline aqueous solution and herbaceous biomass are brought into contact is 60°C or higher, preferably higher than 60°C.
  • a pressure exceeding normal pressure is more than 100°C or less, more preferably 80°C or more and 100°C or less.
  • the weight ratio of the alkaline aqueous solution and the herbaceous biomass is not particularly limited, but the preferred range is, for example, 100:1 to 2:1, 90:1 to 3:1, 50:1-5:1, 30:1-5:1, 25:1-7:1, 25:1-7:1, 25:1-5:1, 20:1-5: 1.
  • the method of contacting the herbaceous biomass with the alkaline aqueous solution is not particularly limited, but examples thereof include a method of contacting the herbaceous biomass with the alkaline aqueous solution by spraying, immersing, or passing a liquid through the herbaceous biomass. It may be stirred or the container may be rotated so that the herbaceous biomass and the herbaceous biomass are in sufficient contact with each other.
  • the contact time between the alkaline aqueous solution and the herbaceous biomass is not particularly limited, but is preferably about 20 minutes or more and about 72 hours or less, about 20 minutes or more and about 48 hours or less, about 20 minutes or more and about 24 hours or less, or about 30 minutes or more. About 48 hours or less, about 30 minutes or more and about 24 hours or less, about 30 minutes or more and about 12 hours or less, about 30 minutes or more and about 6 hours or less, or about 30 minutes or more and about 3 hours or less.
  • An extract can be obtained by solid-liquid separation of the herbaceous biomass and the alkaline aqueous solution.
  • solid-liquid separators include screw presses and centrifuges. A strainer or the like may be used to remove fine particles.
  • the liquid after passing may be used as it is as an extract liquid, but the reactant of the herbaceous biomass is separated by a solid-liquid separation device. is preferable from the viewpoint of recovery of the extract.
  • the extract obtained by contacting the herbaceous biomass and the alkaline aqueous solution is cooled to 60°C or less.
  • the cooling method is not limited to the time, and the herbaceous biomass and the alkaline aqueous solution may be cooled in a state of being in contact with each other, or may be cooled after the solid-liquid separation described above, but cooling after the solid-liquid separation is more efficient. It is preferable because it can be cooled to If the pH of the extract is adjusted by adding an acidic substance while the extract is at a high temperature, a micelle-like phenomenon is likely to occur. In some cases, the increase in viscosity reduces filterability. Therefore, the temperature is preferably 20° C. to 60° C., preferably 20° C. to 55° C., more preferably 25° C. to 50° C., and more preferably 30° C. to 50° C.
  • step (2) the liquid extract obtained in step (1) is cooled to 60° C. or lower to obtain a cooled liquid extract.
  • step (3) the cooled extract obtained in step (2) is adjusted to pH 3.2 or more and 4.5 or less, and cellobiohydrolase activity and xylanase activity to obtain an enzyme reaction solution.
  • an acidic substance is added to the extract obtained in the step of obtaining the extract to adjust the pH to the acidic range as described above.
  • Acidic substances include, but are not limited to, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, lactic acid, acetic acid, formic acid, citric acid, etc., but are preferably hydrochloric acid, sulfuric acid, nitric acid, and more preferably hydrochloric acid.
  • the method of adjusting the pH using an acidic substance is not particularly limited, but an example is a method of adding and mixing an appropriate concentration of an acidic substance while checking the pH.
  • a continuous system in which the alkaline extract is continuously added during pH adjustment and the liquid after pH adjustment is continuously withdrawn, or a batch system may be used.
  • the temperature during pH adjustment is not particularly limited, it is preferably 20 to 100°C, more preferably 20 to 60°C, and still more preferably 30 to 60°C.
  • the pH adjustment range is usually 3.2 or more and 4.5 or less, preferably pH 3.3 or more and 4.0 or less, more preferably pH 3.5 or more and 4.0 or less.
  • An enzyme with cellobiohydrolase activity is an exo-enzyme that produces cellobiose by degrading the cellulose chain with ⁇ -1,4-linked glucose from the end.
  • the cellobiohydrolase activity can be measured as an enzymatic activity that decomposes 4-nitrophenyl- ⁇ -D-lactopyranoside.
  • the amount of enzyme that produces 1 ⁇ mol of 4-nitrophenol per minute is defined as 1 U.
  • Enzyme activity is measured by a method according to the procedure described in Reference Example 2 below.
  • an enzyme having a cellobiohydrolase activity of 5 U/g or more is defined as an enzyme having a cellobiohydrolase activity. 1000 U/g, more preferably 5 to 500 U/g, still more preferably 10 to 500 U/g, particularly preferably 20 to 300 U/g.
  • Enzymes with xylanase activity are endo-type enzymes that randomly degrade xylans with ⁇ -1,4-linked xylose.
  • Xylanase activity can be determined by measuring the amount of reducing sugar contained in the reaction solution after the reaction using a commercially available xylan reagent (e.g., Birchwood xylan) as a substrate. Kit (XylX6 method)” is preferably used.
  • the XylX6 reagent is decomposed by a combination of the xylanase in the measurement target and the auxiliary reagent ⁇ -xylosidase to generate 4-nitrophenol, thereby measuring xylanase activity. be able to.
  • the amount of enzyme that produces 1 ⁇ mol of 4-nitrophenol per minute is defined as 1 U.
  • the enzymatic activity is measured according to the procedure described in Reference Example 3 below.
  • an enzyme having a xylanase activity of 400 U/g or more is defined as an enzyme having xylanase activity. More preferably 500 to 50,000 U/g, still more preferably 1,000 to 50,000 U/g, particularly preferably 3,000 to 45,000 U/g.
  • microorganisms are produced by microorganisms, and may be produced by, for example, a single microorganism or multiple microorganisms.
  • Microorganisms that produce cellobiohydrolase and xylanase include Trichoderma, Aspergillus, Cellulomonas, Clostridium, Streptomyces, and Humicola. , Acremonium, Irpex, Mucor, Talaromyces, and the like, preferably Trichoderma.
  • Trichoderma microorganism is not particularly limited, Trichoderma reesei is preferred, and specifically Trichoderma reesei QM9414, Trichoderma reesei QM9123, Trichoderma reesei RutC-30 Trichoderma reesei Rut C-30), Trichoderma reesei PC3-7, Trichoderma reesei CL-847, Trichoderma reesei MCG77 (Trichoderma reesei MCG80/G77) Trichoderma reesei MCG80) and Trichoderma viride QM9123 can be exemplified.
  • the microorganisms derived from the genus Trichoderma described above may be mutant strains that have been subjected to mutation treatment with a mutating agent or ultraviolet irradiation to improve cellulase productivity.
  • the purified enzyme may be added, the culture solution may be added as a crude enzyme, or a commercially available cellulase agent or xylanase agent may be used. Enzymes other than hydrase and xylanase may be included. For example, ⁇ -glucosidase, ⁇ -xylosidase, endoglucanase, mannanase and the like may be included.
  • cellulase agents and xylanase agents include, for example, Novozyme's "Selic-Seetech” (registered trademark) and “Seric-Seetech 2" (registered trademark), and Danisco Japan's “Accellace” (registered trademark) 1000. , “Accel Race” (registered trademark) 1500, “Accel Race” (registered trademark) Duet, and Sigma-Aldrich's "Cellulase from Trichoderma reesei ATCC 26921", “Cellulase from Trichoderma viride”, and “Cellulase from Trichoderma longibrachiatum”.
  • the amount to be added may be changed as appropriate depending on the enzyme to be added, and is not particularly limited. Preferably 0.005 to 20 parts by weight, more preferably 0.005 to 5 parts by weight.
  • Adjusting the extract to pH 3.2 or more and 4.5 or less and reacting it with the enzyme to obtain an enzyme reaction solution means that the enzyme is in a state where the extract is adjusted to pH 3.2 or more and 4.5 or less. It is to be an enzyme reaction solution that exists in the liquid, and the enzyme may be added during pH adjustment, but it is preferable to add it after adjusting the pH to 3.2 or more and 4.5 or less. Addition of the above enzyme may be carried out in a continuous manner or in a batch manner.
  • the time for reacting the enzyme means that cellobiohydrolase and xylanase are present at a pH of 3.2 or more and 4.5 or less, and solid-liquid separation is performed to obtain a clarified liquid. It is the time until it is obtained, and in the case of a continuous method, the residence time until the above-mentioned enzyme exists in a state of pH 3.2 or more and 4.5 or less and solid-liquid separation is performed to obtain a clarified liquid. is.
  • the reaction time of the enzyme at pH 3.2 or more and 4.5 or less is not particularly limited, but is preferably 5 minutes or more and 8 hours or less, more preferably 5 minutes or more and 6 hours or less, still more preferably 5 minutes or more and 4 hours or less. More preferably 10 minutes or more and 4 hours or less, particularly preferably 10 minutes or more and 2 hours or less.
  • the temperature at which the enzyme is reacted may be appropriately changed according to the enzyme used, and is not particularly limited, but is preferably 15 to 100°C, more preferably 30 to 60°C, and even more preferably 35 to 55°C.
  • step (4) the enzymatic reaction solution obtained in step (3) is coarsely filtered to obtain a clarified liquid.
  • Crude filtration includes filtration with woven fabric and nonwoven fabric, but is not limited to this, but filtration with woven fabric is preferred.
  • Apparatuses for filtration treatment by coarse filtration include belt presses, belt filters, and filter presses, but are not limited to these, but filter presses are preferred.
  • a filter aid may be used when filtering by coarse filtration.
  • Filter aids include diatomaceous earth, perlite, cellulose, activated carbon and the like, but are not limited to these, but diatomaceous earth is preferred.
  • the filter aid may be added from the step of obtaining the extract to the time of filtering the enzymatic reaction solution to obtain the clarified solution, and the timing of addition is not particularly limited.
  • the amount of the filter aid is not particularly limited, but is 0.05 to 10 parts by weight, preferably 0.1 to 5 parts by weight, per 100 parts by weight of the enzyme reaction solution.
  • the clarified liquid obtained in the step (4) is filled with a synthetic adsorbent made of an aromatic resin that is specially treated to increase the specific surface area.
  • the liquid is passed through the column, and the component adsorbed on the aromatic synthetic adsorbent is eluted with a mixed solvent of ethanol and water to obtain an eluted fraction as a polyphenol-containing composition.
  • the aromatic resin constituting the aromatic synthetic adsorbent is not particularly limited as long as it can adsorb polyphenol components, but from the viewpoint of efficiently adsorbing polyphenol-containing compositions, styrene-divinylbenzene is preferably used.
  • Styrene-divinylbenzene aromatic resins include, for example, aromatic resins having hydrophobic substituents, unsubstituted aromatic resins, and unsubstituted aromatic resins that have undergone special treatment. and porous resins.
  • the specific surface area of the synthetic aromatic adsorbent is preferably 500 m 2 /g or more, more preferably 700 m 2 /g or more, as a dry mass, from the viewpoint of improving the adsorption rate.
  • the specific surface area of the synthetic aromatic adsorbent can be calculated by applying the measured value of the gas adsorption method to the BET formula.
  • the mode pore diameter (mode pore diameter) of the aromatic synthetic adsorbent is preferably 600 angstroms or less, more preferably 300 angstroms or less, and still more preferably, from the viewpoint of high separation and high adsorption properties. is less than 200 Angstroms. The most frequent pore diameter can be measured by a gas adsorption method.
  • Such synthetic adsorbents are commercially available. family resins, all trade names, manufactured by Mitsubishi Chemical Corporation); SP-825, SP-800, SP-850, SP-875, SP-70, SP-700 Aromatic resin applied, both trade names, manufactured by Mitsubishi Chemical Corporation); SP-900 (aromatic resin, trade name, manufactured by Mitsubishi Chemical Corporation); Amberlite (trademark) XAD-2, XAD-4 , XAD-16, XAD-18, XAD-2000 (above, aromatic resins, all trade names, manufactured by Organo Co., Ltd.); Diaion (trademark) SP-205, SP-206, SP-207 (above, Aromatic resins having hydrophobic substituents, both trade names, manufactured by Mitsubishi Chemical Corporation); HP-2MG, EX-0021 (above, aromatic resins having hydrophobic substituents, both trade names, Mitsubishi Chemical Co., Ltd.) and the like. Among them, Diaion (trademark) SP-850 is preferable. These synthetic adsorbents may be used singly or in
  • the amount of aromatic synthetic adsorbent to be packed in the column can be appropriately determined depending on the size of the column, the type of synthetic adsorbent, and the like.
  • the temperature of the filtrate may be 25 to 45°C.
  • the amount and rate of passage of the filtrate through the column can be appropriately determined according to the type of synthetic aromatic adsorbent.
  • the mixed volume ratio (ethanol/water) of the mixed solvent of ethanol and water may be 50/50 to 99/1, and from the viewpoint of improving the elution efficiency, it is preferably 50/50 to 70/30. Within range.
  • the elution rate can be appropriately determined depending on the size of the column, the type of synthetic aromatic adsorbent, and the like. In order to efficiently elute the components adsorbed on the column, it is preferable to wash the inside of the column with water before passing the filtrate through the column.
  • the polyphenol-containing composition obtained as an eluted fraction may be concentrated as necessary. Concentration may be carried out, for example, by using a centrifugal thin film vacuum evaporator to concentrate 5 to 20 times. Thereby, a concentrate containing a polyphenol-containing composition can be obtained.
  • the polyphenol-containing composition obtained by the above method is particularly suitable as a food material because it is obtained by using an aromatic synthetic adsorbent in the elution step and eluting using a mixed solvent of ethanol and water. can be used.
  • JIS Japanese Industrial Standards
  • micellization-like phenomenon ++: micelle formation (state in which liquid becomes cloudy and precipitation cannot occur), +: partial micelle formation (state in which turbidity does not precipitate), -: no micelle formation (turbidity A state in which the quality quickly settles) was evaluated as an index.
  • the extract after pH adjustment was subjected to solid-liquid separation by a filter press.
  • a small filter device MO-4 manufactured by Yabuta Sangyo Co., Ltd. was used as a filter press. Filtrate volume was measured after 5 minutes.
  • SV aromatic synthetic adsorbent
  • Examples 1 and 2 Effect of enzyme addition
  • the temperature of the bagasse alkaline extract prepared according to Reference Example 5 was cooled to 45 ° C. (Example 1) and 60 ° C. (Example 2), and diatomaceous earth was added to 100 1 part by weight was added, and the pH was adjusted to 3.5 using 35% (w/w) hydrochloric acid.
  • Enzyme 1 having cellobiohydrolase activity and xylanase activity: "CelBx" (manufactured by Siam Victory Chemicals Limited) was added.
  • the amount added was 0.1 parts by weight (Example 1) and 0.5 parts by weight (Example 2) with respect to 100 parts by weight of the extract after pH adjustment.
  • micellization-like phenomenon ++: micelle formation (state in which liquid becomes cloudy and precipitation cannot occur), +: partial micelle formation (state in which turbidity does not precipitate), -: no micelle formation (turbidity A state in which the quality quickly settles) was evaluated as an index.
  • cellobiohydrolase activity and xylanase activity of the enzyme were measured according to Reference Examples 2 and 3.
  • the value measured according to Reference Example 1 was used as the protein concentration.
  • the extract after the enzymatic reaction was solid-liquid separated by a filter press.
  • a small filter device MO-4 manufactured by Yabuta Sangyo Co., Ltd. was used as a filter press. Filtrate volume was measured after 5 minutes.
  • an aromatic synthetic adsorbent Diaion SP-850, manufactured by Mitsubishi Chemical Corporation
  • Enzyme 2 (cellobiohydrolase activity, no xylanase activity): “Pectinase from Aspergillus aculeatus” (liquid, manufactured by Sigma-Aldrich), Enzyme 3 (cellobiohydrolase activity, no xylanase activity): “Cellulosine GM5′′ (powdered, manufactured by HI Co., Ltd.) was added to each. The amount added was 0.1 part by weight per 100 parts by weight of the extract after pH adjustment. After the addition, the mixture was reacted at 50° C. for 1 hour while stirring.
  • micellization-like phenomenon ++: micelle formation (state in which liquid becomes cloudy and precipitation cannot occur), +: partial micelle formation (state in which turbidity does not precipitate), -: no micelle formation (turbidity A state in which the quality quickly settles) was evaluated as an index.
  • cellobiohydrolase activity and xylanase activity of the enzyme were measured according to Reference Examples 2 and 3. The value measured according to Reference Example 1 was used as the protein concentration.
  • the extract after the enzymatic reaction was solid-liquid separated by a filter press in the same manner as in Comparative Examples 1 and 2.

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Abstract

The present invention provides a technical means for efficiently producing a polyphenol-containing composition from grass-based biomass. More specifically, the present invention provides a method for producing a polyphenol-containing composition derived from grass-based biomass, the method including: (1) a step in which an extraction liquid is obtained by bringing grass-based biomass into contact with an alkaline aqueous solution of 60°C or higher; (2) a step in which the extraction liquid obtained in step (1) is cooled to 60°C or lower to obtain a cooled extract solution; (3) a step in which the pH of the cooled extraction liquid obtained in step (2) is adjusted to a pH of 3.2-4.5, and the cooled extraction liquid is reacted with an enzyme having cellobiohydrolase activity and xylanase activity to obtain an enzyme-reacted solution; (4) a step in which a clarified liquid is obtained by rough filtration of the enzyme-reacted solution obtained in step (3); (5) a step in which the clarified liquid obtained in step (4) is passed through a column filled with an aromatic synthetic adsorbent composed of an aromatic resin subjected to a special treatment for increasing the specific surface area thereof, and the component adsorbed to the aromatic synthetic adsorbent is eluted using a mixed solvent of ethanol and water so as to obtain an eluted fraction as a polyphenol-containing composition.

Description

ポリフェノール含有組成物の製造方法Method for producing polyphenol-containing composition 関連出願の参照Reference to Related Applications
　本特許出願は、２０２１年３月３０日に出願された日本国特許出願２０２１－５８７３６号に基づく優先権の主張を伴うものであり、かかる先の特許出願における全開示内容は、引用することにより本明細書の一部とされる。 This patent application claims priority based on Japanese Patent Application No. 2021-58736 filed on March 30, 2021, and the entire disclosure content in such earlier patent application is incorporated by reference. incorporated herein.
　本発明は、草本系バイオマスからのポリフェノール含有組成物の製造方法に関する。 The present invention relates to a method for producing a polyphenol-containing composition from herbaceous biomass.
　近年、地球温暖化や石油資源の枯渇といった問題やカーボンニュートラルの観点から、バイオマスの活用が注目されている。その中の一つとして、食糧と競合しないセルロース含有バイオマスからポリフェノール含有組成物を得る方法がある。
　セルロース含有バイオマスは、主に多糖であるセルロース、ヘミセルロース、芳香族ポリマーであるリグニンで構成されており、セルロース含有バイオマス中のリグニンやリグニンと多糖との接続部が分解されることによってポリフェノールを含有する分解液を得ることができる。 In recent years, the use of biomass has attracted attention from the viewpoint of problems such as global warming and depletion of petroleum resources, and from the viewpoint of carbon neutrality. One of them is a method of obtaining a polyphenol-containing composition from cellulose-containing biomass that does not compete with food.
Cellulose-containing biomass is mainly composed of cellulose and hemicellulose, which are polysaccharides, and lignin, which is an aromatic polymer. A decomposed liquid can be obtained.
　例えば、特許文献１には、セルロース含有バイオマスにアルカリ性水性媒体を通液させて効率的にヒドロキシ桂皮酸を得る方法が記載されている。また、特許文献２には、さとうきびの搾り粕であるバガスをアルカリ溶液で処理した抽出液を酸性に調整して濾過し、その濾液を芳香族系合成吸着剤で吸着させることで効率的にポリフェノール含有組成物を製造する方法が記載されている。 For example, Patent Document 1 describes a method for efficiently obtaining hydroxycinnamic acid by passing an alkaline aqueous medium through cellulose-containing biomass. In addition, in Patent Document 2, an extract obtained by treating bagasse, which is sugar cane lees, with an alkaline solution is adjusted to acidity and filtered, and the filtrate is adsorbed with an aromatic synthetic adsorbent to efficiently remove polyphenols. A method of making the containing composition is described.
　従来、こうしたセルロース含有バイオマスのアルカリ処理は、セルロース含有バイオマスから糖液を得やすくするために、前処理として行われ、アルカリ処理後の液分は廃棄されてきたが、上述のようにポリフェノールが含有するため、有効利用が望まれている。
　上述のような従来技術によって得られるポリフェノールを含む分解液は、消臭剤（特許文献３）、食品用変色防止剤（特許文献４）、水産生物成長促進剤（特許文献５）などとして利用が知られている。また、代表的なポリフェノールとしては、クマル酸、フェルラ酸を含有していることが知られている。 Conventionally, such alkali treatment of cellulose-containing biomass has been performed as a pretreatment in order to make it easier to obtain a sugar solution from cellulose-containing biomass, and the liquid after alkali treatment has been discarded. Therefore, effective utilization is desired.
The decomposition solution containing polyphenols obtained by the above-described conventional technology can be used as a deodorant (Patent Document 3), a food discoloration inhibitor (Patent Document 4), an aquatic organism growth promoter (Patent Document 5), and the like. Are known. Moreover, it is known to contain coumaric acid and ferulic acid as representative polyphenols.
ＷＯ２０１７／１７０５４９号WO2017/170549 ＷＯ２０１９／２３０８０３号WO2019/230803 特開２０２０－９３０８０号公報Japanese Unexamined Patent Application Publication No. 2020-93080 ＷＯ２０１８／０７９６４０号WO2018/079640 ＷＯ２０１８／０７９６４１号WO2018/079641
　上述のように、草本系バイオマスからポリフェノール含有組成物を効率的に製造する方法として草本系バイオマスをアルカリ性水溶液で処理した前処理液を酸性に調整して濾過し、その濾過液を芳香族系合成吸着剤で吸着させることで効率的にポリフェノール含有組成物を製造する方法があるが、濾過性が悪いことや、ミセル化様の現象が起き、芳香族系合成吸着剤での収率が低下する場合があることが本発明者らの検討から明らかとなった。 As described above, as a method for efficiently producing a polyphenol-containing composition from herbaceous biomass, the pretreatment liquid obtained by treating herbaceous biomass with an alkaline aqueous solution is adjusted to acidity and filtered, and the filtrate is used for aromatic synthesis. There is a method for efficiently producing a polyphenol-containing composition by adsorbing with an adsorbent, but poor filterability and micellization-like phenomena occur, and the yield with aromatic synthetic adsorbents decreases. It has become clear from the study of the present inventors that there is a case.
　したがって、本発明は、草本系バイオマスからポリフェノール含有組成物を効率的に製造する新たな技術的手段を提供することを一つの目的としている。 Therefore, one object of the present invention is to provide a new technical means for efficiently producing a polyphenol-containing composition from herbaceous biomass.
　本発明者は鋭意検討した結果、草本系バイオマスを６０℃以上の温度でアルカリ処理した抽出液を、６０℃以下に冷却後、ｐＨ３．２以上ｐＨ４．５以下に調整してセロビオハイドラーゼ活性およびキシラナーゼ活性を含む酵素と反応させた後に濾過し、芳香族系合成吸着剤に吸着させることで、ミセル化様の現象を防ぎ、濾過性が改善され、ポリフェノール含有組成物の収率が向上することを見出した。 As a result of intensive studies by the present inventors, the extract obtained by treating the herbaceous biomass with an alkali at a temperature of 60°C or higher is cooled to 60°C or lower and then adjusted to pH 3.2 or higher and pH 4.5 or lower to obtain cellobiohydrolase activity. and an enzyme containing xylanase activity, followed by filtration and adsorption on an aromatic synthetic adsorbent, preventing micellization-like phenomena, improving filterability, and increasing the yield of the polyphenol-containing composition. I found out.
　すなわち、本発明は、以下の［１］から［６］で構成される。
［１］草本系バイオマス由来のポリフェノール含有組成物の製造方法であって、
（１）草本系バイオマスを６０℃以上の温度のアルカリ性水溶液に接触させて抽出液を得る工程、
（２）工程（１）で得られた抽出液を６０℃以下に冷却して冷却抽出液を得る工程、
（３）工程（２）で得られた冷却抽出液をｐＨ３．２以上４．５以下に調整し、セロビオハイドラーゼ活性およびキシラナーゼ活性を含む酵素と反応させ酵素反応液を得る工程、
（４）工程（３）で得られた酵素反応液を粗ろ過により清澄液を得る工程、
（５）工程（４）で得られた清澄液を高比表面積化の特殊処理を施した芳香族系樹脂からなる合成吸着剤が充填されたカラムに通液し、上記芳香族系合成吸着剤に吸着した成分をエタノールおよび水の混合溶媒で溶出して溶出画分をポリフェノール含有組成物として得る工程、
を含むポリフェノール含有組成物の製造方法。
［２］上記セロビオハイドラーゼ活性およびキシラナーゼ活性を含む酵素がトリコデルマ属微生物由来である、［１］に記載のポリフェノール含有組成物の製造方法。
［３］上記草本系バイオマスがバガスである、［１］または［２］に記載のポリフェノール含有組成物の製造方法。
［４］上記アルカリ性水溶液が水酸化ナトリウム水溶液である、［１］から［３］のいずれかに記載のポリフェノール含有組成物の製造方法。
［５］上記水酸化ナトリウムの濃度が０．１から１０質量％である、［４］に記載のポリフェノール含有組成物の製造方法。
［６］上記芳香族系樹脂がスチレン－ジビニルベンゼン系樹脂である、［１］から［５］のいずれかに記載のポリフェノール含有組成物の製造方法。 That is, the present invention comprises the following [1] to [6].
[1] A method for producing a polyphenol-containing composition derived from herbaceous biomass,
(1) A step of contacting the herbaceous biomass with an alkaline aqueous solution at a temperature of 60° C. or higher to obtain an extract;
(2) a step of cooling the liquid extract obtained in step (1) to 60°C or less to obtain a cooled liquid extract;
(3) a step of adjusting the cooled extract obtained in step (2) to pH 3.2 or higher and 4.5 or lower, and reacting it with an enzyme containing cellobiohydrolase activity and xylanase activity to obtain an enzyme reaction liquid;
(4) a step of obtaining a clarified solution by coarsely filtering the enzyme reaction solution obtained in step (3);
(5) The clarified liquid obtained in step (4) is passed through a column filled with a synthetic adsorbent made of an aromatic resin that has undergone a special treatment to increase the specific surface area, and the aromatic synthetic adsorbent A step of eluting the components adsorbed to with a mixed solvent of ethanol and water to obtain an eluted fraction as a polyphenol-containing composition;
A method for producing a polyphenol-containing composition comprising:
[2] The method for producing a polyphenol-containing composition according to [1], wherein the enzyme containing cellobiohydrolase activity and xylanase activity is derived from a Trichoderma microorganism.
[3] The method for producing a polyphenol-containing composition according to [1] or [2], wherein the herbaceous biomass is bagasse.
[4] The method for producing a polyphenol-containing composition according to any one of [1] to [3], wherein the alkaline aqueous solution is an aqueous sodium hydroxide solution.
[5] The method for producing a polyphenol-containing composition according to [4], wherein the concentration of sodium hydroxide is 0.1 to 10% by mass.
[6] The method for producing a polyphenol-containing composition according to any one of [1] to [5], wherein the aromatic resin is a styrene-divinylbenzene resin.
　本発明によれば、草本系バイオマスからポリフェノール含有組成物を効率的に製造することができる。また、本発明は、草本系バイオマスからポリフェノール含有組成物の製造において、ミセル化様の現象の抑制や濾過性の改善において有利に利用することができる。かかる本発明によれば、ポリフェノール含有組成物の収率を向上する上で有利に利用することができる。 According to the present invention, a polyphenol-containing composition can be efficiently produced from herbaceous biomass. In addition, the present invention can be advantageously used in the production of polyphenol-containing compositions from herbaceous biomass, in suppressing micellization-like phenomena and improving filterability. The present invention can be advantageously used to improve the yield of the polyphenol-containing composition.
　本発明の一実施形態によれば、草本系バイオマス由来のポリフェノール含有組成物の製造方法は、
（１）草本系バイオマスを６０℃以上の温度のアルカリ性水溶液に接触させて抽出液を得る工程、
（２）工程（１）で得られた抽出液を６０℃以下に冷却して冷却抽出液を得る工程、
（３）工程（２）で得られた冷却抽出液をｐＨ３．２以上４．５以下に調整し、セロビオハイドラーゼ活性およびキシラナーゼ活性を含む酵素と反応させ酵素反応液を得る工程、
（４）工程（３）で得られた酵素反応液を粗ろ過により清澄液を得る工程、
（５）工程（４）で得られた清澄液を高比表面積化の特殊処理を施した芳香族系樹脂からなる合成吸着剤が充填されたカラムに通液し、上記芳香族系合成吸着剤に吸着した成分をエタノールおよび水の混合溶媒で溶出して溶出画分をポリフェノール含有組成物として得る工程、
を含むことを特徴としている。 According to one embodiment of the present invention, a method for producing a herbaceous biomass-derived polyphenol-containing composition comprises:
(1) A step of contacting the herbaceous biomass with an alkaline aqueous solution at a temperature of 60° C. or higher to obtain an extract;
(2) a step of cooling the liquid extract obtained in step (1) to 60°C or less to obtain a cooled liquid extract;
(3) a step of adjusting the cooled extract obtained in step (2) to pH 3.2 or higher and 4.5 or lower, and reacting it with an enzyme containing cellobiohydrolase activity and xylanase activity to obtain an enzyme reaction liquid;
(4) a step of obtaining a clarified solution by coarsely filtering the enzyme reaction solution obtained in step (3);
(5) The clarified liquid obtained in step (4) is passed through a column filled with a synthetic adsorbent made of an aromatic resin that has undergone a special treatment to increase the specific surface area, and the aromatic synthetic adsorbent A step of eluting the components adsorbed to with a mixed solvent of ethanol and water to obtain an eluted fraction as a polyphenol-containing composition;
is characterized by including
　以下、本発明を実施するための形態について説明する。
＜工程（１）＞
　本発明の一実施形態によれば、上述の通り、工程（１）において、草本系バイオマスをアルカリ性水溶液に接触させて抽出液を得る。 EMBODIMENT OF THE INVENTION Hereinafter, the form for implementing this invention is demonstrated.
<Step (1)>
According to one embodiment of the present invention, as described above, in step (1), herbaceous biomass is brought into contact with an alkaline aqueous solution to obtain an extract.
　本発明の一実施形態によれば、草本バイオマスの例としては、サトウキビの搾り粕であるバガス、スイッチグラス、ネピアグラス、エリアンサス、コーンストーバー、コーン外皮、小麦外皮、大豆外皮、稲わら、麦わら、油椰子空果房などが挙げられるがこれに限定されない。ポリフェノール製造の観点から、リグニン含有率が５％以上の草本系バイオマスを使用することが好ましく、具体的には、バガス、ネピアグラス、エリアンサス、コーンストーバー、稲わらが好ましく、バガスがより好ましい。なお、リグニン含有率は、酸加水分解の残渣から灰分を引いたものであるクラーソンリグニンを測定することで求めることができる。
　本発明の一実施形態によれば、草本系バイオマスの形状は特に限定されないが、粉砕処理されていることが好ましい。粉砕手段は特に限定されず、ボールミル、振動ミル、カッターミル、ハンマーミル、ウィレーミル、ジェットミルなど各種材料の粗粉砕に慣用されている機械を用いて行うことができる。この機械的な粉砕は乾式および湿式のいずれでもよいが、好ましくは乾式粉砕である。 According to one embodiment of the present invention, examples of herbaceous biomass include sugarcane pomace bagasse, switchgrass, napiergrass, erianthus, corn stover, corn husks, wheat husks, soybean husks, rice straw, wheat straw. , oil palm empty fruit bunches, etc., but not limited to these. From the viewpoint of polyphenol production, it is preferable to use herbaceous biomass with a lignin content of 5% or more. Specifically, bagasse, napier grass, erianthus, corn stover, and rice straw are preferable, and bagasse is more preferable. The lignin content can be determined by measuring Clason lignin, which is obtained by subtracting ash from the acid hydrolysis residue.
According to one embodiment of the present invention, the shape of the herbaceous biomass is not particularly limited, but it is preferably pulverized. The pulverization means is not particularly limited, and machines commonly used for coarse pulverization of various materials such as ball mills, vibration mills, cutter mills, hammer mills, Willey mills and jet mills can be used. This mechanical pulverization may be either dry or wet, preferably dry pulverization.
　本発明の一実施形態によれば、草本系バイオマスの含水率は特に限定されないが、好ましい範囲は、例えば、３％程度以上、３％程度以上７５％程度以下、５％程度以上、５％程度以上７０％程度以下、５％程度以上６５％程度以下、５％程度以上５５％程度以下である。 According to one embodiment of the present invention, the water content of the herbaceous biomass is not particularly limited, but a preferable range is, for example, about 3% or more, about 3% or more and about 75% or less, about 5% or more, and about 5%. About 70% or more, about 5% or more and about 65% or less, about 5% or more and about 55% or less.
　本発明の一実施形態によれば、ポリフェノールとは、クマル酸やフェルラ酸といったヒドロキシ桂皮酸、リグニン分解物の1種以上を含んでもよく、例えば、フォーリンチオカルト法によって測定することができる。フォーリンチオカルト法は、元来、チロシン、トリプトファン等の芳香族アミノ酸やこれらを有するたんぱく質の分析を目的に開発された方法である。フェノール性水酸基がアルカリ性でリンタングステン酸、モリブデン酸を還元して生ずる青色を７００～７７０ｎｍで比色定量する方法である。没食子酸、カテキン等特定の基準物質で同様の操作を行い、その化合物換算で定量値を示すことができ、本発明ではカテキン換算での値を用いる。 According to one embodiment of the present invention, polyphenols may include one or more of hydroxycinnamic acids such as coumaric acid and ferulic acid, and lignin degradation products, and can be measured, for example, by the Folin-Ciocalteu method. The Folin-Ciocalteu method was originally developed for the purpose of analyzing aromatic amino acids such as tyrosine and tryptophan, and proteins containing them. In this method, the phenolic hydroxyl group is alkaline and the blue color produced by reduction of phosphotungstic acid and molybdic acid is colorimetrically determined at 700 to 770 nm. A similar operation is performed using a specific reference substance such as gallic acid or catechin, and a quantitative value can be shown in terms of the compound, and the value in terms of catechin is used in the present invention.
　本発明の一実施形態によれば、アルカリ性水溶液は、アンモニア、アンモニア水、アルカリ金属水酸化物、アルカリ金属酸化物、アルカリ土類金属酸化物、アルカリ金属炭酸塩、アルカリ土類金属炭酸塩、水酸化四級アンモニウム等から選択される少なくとも１つを含むアルカリ性水溶液が挙げられるが、好ましくは水酸化ナトリウムおよび水酸化カリウムから選択される少なくとも一つの水酸化物を含む水性媒体である。安価であり、食品製造工程で用いられる観点から、さらに好ましくは水酸化ナトリウム水溶液である。 According to one embodiment of the invention, the alkaline aqueous solution is ammonia, aqueous ammonia, alkali metal hydroxides, alkali metal oxides, alkaline earth metal oxides, alkali metal carbonates, alkaline earth metal carbonates, water An alkaline aqueous solution containing at least one selected from quaternary ammonium oxide and the like can be mentioned, but an aqueous medium containing at least one hydroxide selected from sodium hydroxide and potassium hydroxide is preferred. From the viewpoint of being inexpensive and being used in the food manufacturing process, an aqueous sodium hydroxide solution is more preferable.
　アルカリ性水溶液のアルカリ濃度は、特に限定されないが、前処理の処理時間を短縮する観点から、好ましくは０．１重量％以上であり、より好ましくは０．２重量％以上であり、さらに好ましくは０．３重量％以上である。アルカリ溶液の濃度は、抽出効率を向上させる観点から、好ましくは１０重量％以下であり、より好ましくは５重量％以下であり、さらに好ましくは１．０重量％以下である。アルカリ溶液の濃度は、同様の観点から、０．１～１０重量％、０．１～５重量％、０．１～１．０重量％、０．２～１０重量％、０．２～５重量％、０．２～１．０重量％、０．３～１０重量％、０．３～５重量％、または０．３～１．０重量％であってもよい。 The alkali concentration of the alkaline aqueous solution is not particularly limited, but from the viewpoint of shortening the treatment time of the pretreatment, it is preferably 0.1% by weight or more, more preferably 0.2% by weight or more, and still more preferably 0% by weight. .3% by weight or more. From the viewpoint of improving extraction efficiency, the concentration of the alkaline solution is preferably 10% by weight or less, more preferably 5% by weight or less, and even more preferably 1.0% by weight or less. From the same viewpoint, the concentration of the alkaline solution is 0.1 to 10% by weight, 0.1 to 5% by weight, 0.1 to 1.0% by weight, 0.2 to 10% by weight, 0.2 to 5% by weight. % by weight, 0.2-1.0% by weight, 0.3-10% by weight, 0.3-5% by weight, or 0.3-1.0% by weight.
　また、アルカリ性水溶液のｐＨの下限値は、アルカリ性である限り特に限定されないが、ｐＨ７以上、好ましくはｐＨ８以上、より好ましくはｐＨ９以上、さらに好ましくはｐＨ１０以上である。ｐＨの上限値は、ｐＨ１４未満であれば、特に限定はされないが、アルカリの使用量を少なくする観点で、ｐＨ１２以下で設定することができる。また、好ましいｐＨの範囲は、例えば、７以上１３．５以下、８以上１３．５以下、より好ましいｐＨの範囲は９以上１３．５以下、さらに好ましいｐＨの範囲は１０以上１２以下の範囲である。
　アルカリ性水溶液と草本系バイオマスを接触させる際の温度は６０℃以上、好ましくは６０℃超の温度である。また、アルカリ処理物を１００℃より高く保持するためには、アルカリ処理物に常圧を超える圧力を加える必要があり、高圧設備が必要となるため、生産コストの面から、より好ましくは６０℃超１００℃以下、さらに好ましくは８０℃以上１００℃以下である。 The lower limit of the pH of the alkaline aqueous solution is not particularly limited as long as it is alkaline, but is pH 7 or higher, preferably pH 8 or higher, more preferably pH 9 or higher, and still more preferably pH 10 or higher. The upper limit of the pH is not particularly limited as long as it is less than pH 14, but from the viewpoint of reducing the amount of alkali used, it can be set at pH 12 or less. In addition, a preferable pH range is, for example, 7 or more and 13.5 or less, 8 or more and 13.5 or less, a more preferable pH range is 9 or more and 13.5 or less, and a further preferable pH range is 10 or more and 12 or less. be.
The temperature at which the alkaline aqueous solution and herbaceous biomass are brought into contact is 60°C or higher, preferably higher than 60°C. In addition, in order to keep the alkali-treated product at a temperature higher than 100°C, it is necessary to apply a pressure exceeding normal pressure to the alkali-treated product, which requires high-pressure equipment. It is more than 100°C or less, more preferably 80°C or more and 100°C or less.
　本発明の一実施形態によれば、アルカリ性水溶液と、草本系バイオマス（乾燥重量）との重量割合は、特に限定されないが、好ましい範囲は、例えば、１００：１～２：１、９０：１～３：１、５０：１～５：１、３０：１～５：１、２５：１～７：１、２５：１～７：１、２５：１～５：１、２０：１～５：１である。 According to one embodiment of the present invention, the weight ratio of the alkaline aqueous solution and the herbaceous biomass (dry weight) is not particularly limited, but the preferred range is, for example, 100:1 to 2:1, 90:1 to 3:1, 50:1-5:1, 30:1-5:1, 25:1-7:1, 25:1-7:1, 25:1-5:1, 20:1-5: 1.
　草本系バイオマスとアルカリ性水溶液との接触方法は特に限定されないが、例えばアルカリ性水溶液を草本系バイオマスに噴霧したり、浸漬させたり、通液させたりして接触させる方法が挙げられ、その際にアルカリ性水溶液と草本系バイオマスが十分接触するように、撹拌したり、容器を回転させたりしてもよい。 The method of contacting the herbaceous biomass with the alkaline aqueous solution is not particularly limited, but examples thereof include a method of contacting the herbaceous biomass with the alkaline aqueous solution by spraying, immersing, or passing a liquid through the herbaceous biomass. It may be stirred or the container may be rotated so that the herbaceous biomass and the herbaceous biomass are in sufficient contact with each other.
　アルカリ性水溶液と草本系バイオマスの接触時間は、特に限定されないが、好ましくは、２０分程度以上７２時間程度以下、２０分程度以上４８時間程度以下、２０分程度以上２４時間程度以下、３０分程度以上４８時間程度以下、３０分程度以上２４時間程度以下、３０分程度以上１２時間程度以下、３０分程度以上６時間程度以下または３０分程度以上３時間程度以下ある。 The contact time between the alkaline aqueous solution and the herbaceous biomass is not particularly limited, but is preferably about 20 minutes or more and about 72 hours or less, about 20 minutes or more and about 48 hours or less, about 20 minutes or more and about 24 hours or less, or about 30 minutes or more. About 48 hours or less, about 30 minutes or more and about 24 hours or less, about 30 minutes or more and about 12 hours or less, about 30 minutes or more and about 6 hours or less, or about 30 minutes or more and about 3 hours or less.
　草本系バイオマスとアルカリ性水溶液を固液分離することによって抽出液を得ることができる。固液分離装置としては、スクリュープレス、遠心分離機などが挙げられる。微粒子除去のためにストレイナーなどを使用しても良い。また、アルカリ性水溶液との接触時に草本系バイオマスにアルカリ性水溶液の通液させた場合は、通液後の液を抽出液としてそのまま使用しても良いが、固液分離装置によって草本系バイオマスの反応物を搾ることが抽出液の回収の観点から好ましい。 An extract can be obtained by solid-liquid separation of the herbaceous biomass and the alkaline aqueous solution. Examples of solid-liquid separators include screw presses and centrifuges. A strainer or the like may be used to remove fine particles. In addition, when the herbaceous biomass is passed through the alkaline aqueous solution at the time of contact with the alkaline aqueous solution, the liquid after passing may be used as it is as an extract liquid, but the reactant of the herbaceous biomass is separated by a solid-liquid separation device. is preferable from the viewpoint of recovery of the extract.
　草本系バイオマスとアルカリ性水溶液を接触させた抽出液は６０℃以下になるよう冷却する。冷却方法は時に限定されず、草本系バイオマスとアルカリ性水溶液を接触させた状態で冷却しても良いし、上述の固液分離後に冷却しても良いが、固液分離後に冷却することが効率的に冷却できることから好ましい。抽出液が高温の状態で酸性物質を加えてｐＨ調整を行うと、ミセル化様の現象が起こりやすく、得られた抽出液によって冷却抽出液の温度は適宜変更しても良いが、温度が低い場合は、粘度増加によって濾過性が低下する。そのため、２０℃以上６０℃以下、２０℃以上５５℃以下が好ましく、２５℃以上５０℃以下、３０℃以上５０℃以下がより好ましい。 The extract obtained by contacting the herbaceous biomass and the alkaline aqueous solution is cooled to 60°C or less. The cooling method is not limited to the time, and the herbaceous biomass and the alkaline aqueous solution may be cooled in a state of being in contact with each other, or may be cooled after the solid-liquid separation described above, but cooling after the solid-liquid separation is more efficient. It is preferable because it can be cooled to If the pH of the extract is adjusted by adding an acidic substance while the extract is at a high temperature, a micelle-like phenomenon is likely to occur. In some cases, the increase in viscosity reduces filterability. Therefore, the temperature is preferably 20° C. to 60° C., preferably 20° C. to 55° C., more preferably 25° C. to 50° C., and more preferably 30° C. to 50° C.
＜工程（２）＞
　本発明の一実施形態によれば、工程（２）において、工程（１）で得られた抽出液を６０℃以下に冷却して冷却抽出液を得る。 <Step (2)>
According to one embodiment of the present invention, in step (2), the liquid extract obtained in step (1) is cooled to 60° C. or lower to obtain a cooled liquid extract.
＜工程（３）＞
　さらに、本発明の一実施形態によれば、工程（３）において、工程（２）で得られた冷却抽出液をｐＨ３．２以上４．５以下に調整し、セロビオハイドラーゼ活性およびキシラナーゼ活性を含む酵素と反応させ酵素反応液を得る。 <Step (3)>
Furthermore, according to one embodiment of the present invention, in step (3), the cooled extract obtained in step (2) is adjusted to pH 3.2 or more and 4.5 or less, and cellobiohydrolase activity and xylanase activity to obtain an enzyme reaction solution.
　本発明の一実施形態によれば、上記抽出液を得る工程で得られた抽出液に酸性物質を加えてｐＨを上述のような酸性の範囲に調整する。 According to one embodiment of the present invention, an acidic substance is added to the extract obtained in the step of obtaining the extract to adjust the pH to the acidic range as described above.
　酸性物質としては、塩酸、硫酸、硝酸、リン酸、乳酸、酢酸、ギ酸、クエン酸などが挙げられるが、これに限定されないが、好ましくは、塩酸、硫酸、硝酸、より好ましくは塩酸である。 Acidic substances include, but are not limited to, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, lactic acid, acetic acid, formic acid, citric acid, etc., but are preferably hydrochloric acid, sulfuric acid, nitric acid, and more preferably hydrochloric acid.
　酸性物質によるｐＨ調整方法は特に限定されないが、ｐＨを確認しながら適当な濃度の酸性物質を適宜添加し、混合する方法が挙げられる。ｐＨ調整中にアルカリ抽出液を連続的に投入してｐＨ調整後の液を連続的に抜き出す連続式でも良いし、バッチ式でも良い。 The method of adjusting the pH using an acidic substance is not particularly limited, but an example is a method of adding and mixing an appropriate concentration of an acidic substance while checking the pH. A continuous system in which the alkaline extract is continuously added during pH adjustment and the liquid after pH adjustment is continuously withdrawn, or a batch system may be used.
　ｐＨ調整時の温度は特に限定されないが、好ましくは２０～１００℃、より好ましくは２０～６０℃、さらに好ましくは３０～６０℃である。 Although the temperature during pH adjustment is not particularly limited, it is preferably 20 to 100°C, more preferably 20 to 60°C, and still more preferably 30 to 60°C.
　ｐＨの調整範囲としては、通常、３．２以上４．５以下であるが、好ましくはｐＨ３．３以上４．０以下、さらに好ましくはｐＨ３．５以上４．０以下である。 The pH adjustment range is usually 3.2 or more and 4.5 or less, preferably pH 3.3 or more and 4.0 or less, more preferably pH 3.5 or more and 4.0 or less.
　セロビオハイドラーゼ活性を含む酵素とは、グルコースがβ－１，４結合したセルロース鎖を末端から分解してセロビオースを産生するエキソ型酵素である。セロビオハイドラーゼの活性は、４－ニトロフェニル－β－Ｄ－ラクトピラノシドを分解する酵素活性として測定することができる。１分間に１μｍｏlの４－ニトロフェノールを生成する酵素量を１Ｕと定義する。酵素活性は、後述の参考例２に記載の手順に準じた方法で測定する。本発明では、セロビオハイドラーゼ活性が５Ｕ／ｇ以上である酵素を、セロビオハイドラーゼ活性を有する酵素として定義し、本発明で使用する酵素のセロビオハイドラーゼ活性値は、好ましくは５～１０００Ｕ／ｇ、より好ましくは５～５００Ｕ／ｇ、さらに好ましくは１０～５００Ｕ／ｇ、特に好ましくは２０～３００Ｕ／ｇである。 An enzyme with cellobiohydrolase activity is an exo-enzyme that produces cellobiose by degrading the cellulose chain with β-1,4-linked glucose from the end. The cellobiohydrolase activity can be measured as an enzymatic activity that decomposes 4-nitrophenyl-β-D-lactopyranoside. The amount of enzyme that produces 1 μmol of 4-nitrophenol per minute is defined as 1 U. Enzyme activity is measured by a method according to the procedure described in Reference Example 2 below. In the present invention, an enzyme having a cellobiohydrolase activity of 5 U/g or more is defined as an enzyme having a cellobiohydrolase activity. 1000 U/g, more preferably 5 to 500 U/g, still more preferably 10 to 500 U/g, particularly preferably 20 to 300 U/g.
　キシラナーゼ活性を含む酵素とは、キシロースがβ－１，４結合したキシランをランダムに分解するエンド型酵素である。キシラナーゼの活性は、試薬として市販されているキシラン（例えば、Ｂｉｒｃｈｗｏｏｄ　ｘｙｌａｎ）を基質として、反応後の反応液に含まれる還元糖量を測定することで求めてもよいが、Ｍｅｇａｚｙｍｅ社の“キシラナーゼ分析キット（ＸｙｌＸ６法）”を用いることが好ましい。“キシラナーゼ分析キット（ＸｙｌＸ６法）”では、ＸｙｌＸ６試薬が測定対象中のキシラナーゼと補助試薬であるβ－キシロシダーゼとの組み合わせにより分解されて、４－ニトロフェノールを生成することでキシラナーゼの活性を測定することができる。１分間に１μｍｏlの４－ニトロフェノールを生成する酵素量を１Ｕと定義する。酵素活性は、後述の参考例３に記載の手順に準じた方法で測定する。本発明では、キシラナーゼ活性が４００Ｕ／ｇ以上である酵素を、キシラナーゼ活性を有する酵素として定義し、本発明の一実施形態によれば、酵素のキシラナーゼ活性値は、好ましくは４００～５００００Ｕ／ｇ、より好ましくは５００～５００００Ｕ／ｇ、さらに好ましくは１０００～５００００Ｕ／ｇ、特に好ましくは３０００～４５０００Ｕ／ｇである。 Enzymes with xylanase activity are endo-type enzymes that randomly degrade xylans with β-1,4-linked xylose. Xylanase activity can be determined by measuring the amount of reducing sugar contained in the reaction solution after the reaction using a commercially available xylan reagent (e.g., Birchwood xylan) as a substrate. Kit (XylX6 method)” is preferably used. In the "xylanase assay kit (XylX6 method)", the XylX6 reagent is decomposed by a combination of the xylanase in the measurement target and the auxiliary reagent β-xylosidase to generate 4-nitrophenol, thereby measuring xylanase activity. be able to. The amount of enzyme that produces 1 μmol of 4-nitrophenol per minute is defined as 1 U. The enzymatic activity is measured according to the procedure described in Reference Example 3 below. In the present invention, an enzyme having a xylanase activity of 400 U/g or more is defined as an enzyme having xylanase activity. More preferably 500 to 50,000 U/g, still more preferably 1,000 to 50,000 U/g, particularly preferably 3,000 to 45,000 U/g.
　上記酵素は、微生物により産生され、例えば単一の微生物が産生するものであっても、複数の微生物が産生するものであっても良い。セロビオハイドラーゼおよびキシラナーゼを産生する微生物としては、トリコデルマ属（Ｔｒｉｃｈｏｄｅｒｍａ）、アスペルギルス属（Ａｓｐｅｒｇｉｌｌｕｓ）、セルロモナス属（Ｃｅｌｌｕｌｏｍｏｎａｓ）、クロストリジウム属（Ｃｌｏｓｔｒｉｄｉｕｍ）、ストレプトマイセス属（Ｓｔｒｅｐｔｏｍｙｃｅｓ）、フミコラ属（Ｈｕｍｉｃｏｌａ）、アクレモニウム属（Ａｃｒｅｍｏｎｉｕｍ）、イルペックス属（Ｉｒｐｅｘ）、ムコール属（Ｍｕｃｏｒ）、タラロマイセス属（Ｔａｌａｒｏｍｙｃｅｓ）、などの微生物を挙げることができるが、トリコデルマ属であることが好ましい。 The above enzymes are produced by microorganisms, and may be produced by, for example, a single microorganism or multiple microorganisms. Microorganisms that produce cellobiohydrolase and xylanase include Trichoderma, Aspergillus, Cellulomonas, Clostridium, Streptomyces, and Humicola. , Acremonium, Irpex, Mucor, Talaromyces, and the like, preferably Trichoderma.
　トリコデルマ属微生物は特に限定されないが、トリコデルマ・リーセイ（Ｔｒｉｃｈｏｄｅｒｍａ　ｒｅｅｓｅｉ）が好ましく、具体的にはトリコデルマ・リーセイＱＭ９４１４（Ｔｒｉｃｈｏｄｅｒｍａ　ｒｅｅｓｅｉ　ＱＭ９４１４）、トリコデルマ・リーセイＱＭ９１２３（Ｔｒｉｃｈｏｄｅｒｍａ　ｒｅｅｓｅｉ　ＱＭ９１２３）、トリコデルマ・リーセイＲｕｔＣ－３０（Ｔｒｉｃｈｏｄｅｒｍａ　ｒｅｅｓｅｉＲｕｔ　Ｃ－３０）、トリコデルマ・リーセイＰＣ３－７（Ｔｒｉｃｈｏｄｅｒｍａ　ｒｅｅｓｅｉ　ＰＣ３－７）、トリコデルマ・リーセイＣＬ－８４７（Ｔｒｉｃｈｏｄｅｒｍａ　ｒｅｅｓｅｉ　ＣＬ－８４７）、トリコデルマ・リーセイＭＣＧ７７（Ｔｒｉｃｈｏｄｅｒｍａ　ｒｅｅｓｅｉ　ＭＣＧ７７）、トリコデルマ・リーセイＭＣＧ８０（Ｔｒｉｃｈｏｄｅｒｍａ　ｒｅｅｓｅｉ　ＭＣＧ８０）、トリコデルマ・ビリデＱＭ９１２３（Ｔｒｉｃｈｏｄｅｒｍａ　ｖｉｒｉｄｅ　ＱＭ９１２３）を例示することができる。また、上述のトリコデルマ属に由来する微生物であって、これらを変異剤あるいは紫外線照射などで変異処理を施し、セルラーゼ生産性が向上した変異株であってもよい。 Although the Trichoderma microorganism is not particularly limited, Trichoderma reesei is preferred, and specifically Trichoderma reesei QM9414, Trichoderma reesei QM9123, Trichoderma reesei RutC-30 Trichoderma reesei Rut C-30), Trichoderma reesei PC3-7, Trichoderma reesei CL-847, Trichoderma reesei MCG77 (Trichoderma reesei MCG80/G77) Trichoderma reesei MCG80) and Trichoderma viride QM9123 can be exemplified. Also, the microorganisms derived from the genus Trichoderma described above may be mutant strains that have been subjected to mutation treatment with a mutating agent or ultraviolet irradiation to improve cellulase productivity.
　上記酵素は上記の活性を満たしていれば、精製されたものを添加しても良く、培養液を粗酵素として加えても良く、市販のセルラーゼ剤やキシラナーゼ剤を使用しても良く、セロビオハイドラーゼおよびキシラナーゼ以外の酵素を含んでも良い。例えば、β－グルコシダーゼ、β－キシロシダーゼ、エンドグルカナーゼ、マンナナーゼなどを含んでも良い。 As long as the enzyme satisfies the above activity, the purified enzyme may be added, the culture solution may be added as a crude enzyme, or a commercially available cellulase agent or xylanase agent may be used. Enzymes other than hydrase and xylanase may be included. For example, β-glucosidase, β-xylosidase, endoglucanase, mannanase and the like may be included.
　市販のセルラーゼ剤、キシラナーゼ剤としては、例えばノボザイム社の“セリック・シーテック”（登録商標）、“セリック・シーテック２”（登録商標）や、ダニスコ・ジャパン社の“アクセルレース”（登録商標）１０００、“アクセルレース”（登録商標）１５００、“アクセルレース”（登録商標）デュエットや、シグマ・アルドリッチ社の“セルラーゼ　ｆｒｏｍ　Ｔｒｉｃｈｏｄｅｒｍａ　ｒｅｅｓｅｉ　ＡＴＣＣ　２６９２１”、“セルラーゼ　ｆｒｏｍ　Ｔｒｉｃｈｏｄｅｒｍａ　ｖｉｒｉｄｅ”、および“Ｃｅｌｌｕｌａｓｅ　ｆｒｏｍ　Ｔｒｉｃｈｏｄｅｒｍａ　ｌｏｎｇｉｂｒａｃｈｉａｔｕｍ”、エイチビィアイ社の“セルロシンＴＰ２５”、“セルロシンＨＣ１００”、Ｓｉａｍ　Ｖｉｃｔｏｒｙ　Ｃｈｅｍｉｃａｌｓ　Ｌｉｍｉｔｅｄ社の“ＣｅｌＢｘ”が挙げられるが、これに限定されない。
　添加量については、添加する酵素によって適宜変更しても良く、特に限定されないが、粗酵素や酵素剤として、ｐＨを調整した抽出液１００重量部に対して０．００１重量部から５０重量部、好ましくは、０．００５重量部から２０重量部、さらに好ましくは０．００５重量部から５重量部である。 Commercially available cellulase agents and xylanase agents include, for example, Novozyme's "Selic-Seetech" (registered trademark) and "Seric-Seetech 2" (registered trademark), and Danisco Japan's "Accellace" (registered trademark) 1000. , "Accel Race" (registered trademark) 1500, "Accel Race" (registered trademark) Duet, and Sigma-Aldrich's "Cellulase from Trichoderma reesei ATCC 26921", "Cellulase from Trichoderma viride", and "Cellulase from Trichoderma longibrachiatum". , HBI "Cellulosine TP25", "Cellulosine HC100", Siam Victory Chemicals Limited "CelBx".
The amount to be added may be changed as appropriate depending on the enzyme to be added, and is not particularly limited. Preferably 0.005 to 20 parts by weight, more preferably 0.005 to 5 parts by weight.
　上記抽出液をｐＨ３．２以上４．５以下に調整し、上記酵素と反応させ酵素反応液を得るとは、上記抽出液がｐＨ３．２以上４．５以下に調整された状態で上記酵素が液中に存在する酵素反応液とすることであり、ｐＨ調整中に上記酵素を添加しても良いが、ｐＨ３．２以上４．５以下に調整後に添加することが好ましい。上記酵素の添加は連続式でも良いし、バッチ式でも良い。 Adjusting the extract to pH 3.2 or more and 4.5 or less and reacting it with the enzyme to obtain an enzyme reaction solution means that the enzyme is in a state where the extract is adjusted to pH 3.2 or more and 4.5 or less. It is to be an enzyme reaction solution that exists in the liquid, and the enzyme may be added during pH adjustment, but it is preferable to add it after adjusting the pH to 3.2 or more and 4.5 or less. Addition of the above enzyme may be carried out in a continuous manner or in a batch manner.
　本発明の一実施形態によれば、上記酵素を反応させる時間とは、ｐＨ３．２以上４．５以下の状態でセロビオハイドラーゼおよびキシラナーゼが存在し、固液分離処理を行って清澄液を得るまでの時間のことであり、連続式で行う場合は、ｐＨ３．２以上４．５以下の状態で上記酵素が存在し、固液分離処理を行って清澄液を得るまでの滞留時間のことである。ｐＨ３．２以上４．５以下で上記酵素を反応させる時間は特に限定されないが、好ましくは５分以上８時間以下、より好ましくは５分以上６時間以下、さらに好ましくは５分以上４時間以下、よりさらに好ましくは１０分以上４時間以下、特に好ましくは１０分以上２時間以下である。 According to one embodiment of the present invention, the time for reacting the enzyme means that cellobiohydrolase and xylanase are present at a pH of 3.2 or more and 4.5 or less, and solid-liquid separation is performed to obtain a clarified liquid. It is the time until it is obtained, and in the case of a continuous method, the residence time until the above-mentioned enzyme exists in a state of pH 3.2 or more and 4.5 or less and solid-liquid separation is performed to obtain a clarified liquid. is. The reaction time of the enzyme at pH 3.2 or more and 4.5 or less is not particularly limited, but is preferably 5 minutes or more and 8 hours or less, more preferably 5 minutes or more and 6 hours or less, still more preferably 5 minutes or more and 4 hours or less. More preferably 10 minutes or more and 4 hours or less, particularly preferably 10 minutes or more and 2 hours or less.
　上記酵素を反応させる温度は、使用する酵素に応じて適宜変更して良く、特に限定されないが、好ましくは１５～１００℃、より好ましくは３０～６０℃、さらに好ましくは３５～５５℃である。 The temperature at which the enzyme is reacted may be appropriately changed according to the enzyme used, and is not particularly limited, but is preferably 15 to 100°C, more preferably 30 to 60°C, and even more preferably 35 to 55°C.
＜工程（４）＞
　本発明の一実施形態によれば、工程（４）において、工程（３）で得られた酵素反応液を粗ろ過により清澄液を得る。 <Step (4)>
According to one embodiment of the present invention, in step (4), the enzymatic reaction solution obtained in step (3) is coarsely filtered to obtain a clarified liquid.
　粗ろ過には織布及び不織布によるろ過が挙げられ、これに限定されないが、織布によるろ過が好ましい。粗ろ過により濾過処理する装置としては、ベルトプレス、ベルトフィルター、フィルタープレスが挙げられ、これに限定されないが、フィルタープレスが好ましい。 Crude filtration includes filtration with woven fabric and nonwoven fabric, but is not limited to this, but filtration with woven fabric is preferred. Apparatuses for filtration treatment by coarse filtration include belt presses, belt filters, and filter presses, but are not limited to these, but filter presses are preferred.
　粗ろ過により濾過処理する際は、濾過助剤を使用しても良い。濾過助剤としては、珪藻土、パーライト、セルロース、活性炭などが挙げられ、これに限定されないが、珪藻土であることが好ましい。濾過助剤は、抽出液を得る工程から酵素反応液を濾過処理して清澄液を得るまでに添加されれば良く、添加のタイミングは特に限定されない。濾過助剤の量は、特に限定されないが、酵素反応液１００重量部に対して０．０５重量部から１０重量部、好ましくは０．１重量部から５重量部である。 A filter aid may be used when filtering by coarse filtration. Filter aids include diatomaceous earth, perlite, cellulose, activated carbon and the like, but are not limited to these, but diatomaceous earth is preferred. The filter aid may be added from the step of obtaining the extract to the time of filtering the enzymatic reaction solution to obtain the clarified solution, and the timing of addition is not particularly limited. The amount of the filter aid is not particularly limited, but is 0.05 to 10 parts by weight, preferably 0.1 to 5 parts by weight, per 100 parts by weight of the enzyme reaction solution.
＜工程（５）＞
　本発明の一実施形態によれば、工程（５）において、工程（４）で得られた清澄液を高比表面積化の特殊処理を施した芳香族系樹脂からなる合成吸着剤が充填されたカラムに通液し、上記芳香族系合成吸着剤に吸着した成分をエタノールおよび水の混合溶媒で溶出して溶出画分をポリフェノール含有組成物として得る。 <Step (5)>
According to one embodiment of the present invention, in the step (5), the clarified liquid obtained in the step (4) is filled with a synthetic adsorbent made of an aromatic resin that is specially treated to increase the specific surface area. The liquid is passed through the column, and the component adsorbed on the aromatic synthetic adsorbent is eluted with a mixed solvent of ethanol and water to obtain an eluted fraction as a polyphenol-containing composition.
　上記の芳香族系合成吸着剤を構成する芳香族系樹脂は、ポリフェノール成分を吸着しうるものであれば特に制限はないが、ポリフェノール含有組成物を効率よく吸着させる観点から、スチレン－ジビニルベンゼン系の芳香族樹脂が好ましく用いられる。スチレン－ジビニルベンゼン系の芳香族樹脂としては、例えば、疎水性置換基を有する芳香族系樹脂、無置換基型の芳香族系樹脂、無置換基型に特殊処理を施した芳香族系樹脂等の多孔性樹脂等が挙げられる。 The aromatic resin constituting the aromatic synthetic adsorbent is not particularly limited as long as it can adsorb polyphenol components, but from the viewpoint of efficiently adsorbing polyphenol-containing compositions, styrene-divinylbenzene is preferably used. Styrene-divinylbenzene aromatic resins include, for example, aromatic resins having hydrophobic substituents, unsubstituted aromatic resins, and unsubstituted aromatic resins that have undergone special treatment. and porous resins.
　上記の芳香族系合成吸着剤の比表面積は、吸着率を向上させる観点から、乾燥質量として、好ましくは５００ｍ２/ｇ以上であり、より好ましくは７００ｍ２/ｇ以上である。芳香族系合成吸着剤の比表面積は、ガス吸着法の測定値をＢＥＴの式に当てはめることより算出することができる。芳香族系合成吸着剤の最頻度細孔直径（最頻細孔径）は、高分離性および高吸着性の観点から、好ましくは６００オングストローム以下であり、より好ましくは３００オングストローム以下であり、更に好ましくは２００オングストローム以下である。最頻度細孔直径は、ガス吸着法により測定することができる。 The specific surface area of the synthetic aromatic adsorbent is preferably 500 m 2 /g or more, more preferably 700 m 2 /g or more, as a dry mass, from the viewpoint of improving the adsorption rate. The specific surface area of the synthetic aromatic adsorbent can be calculated by applying the measured value of the gas adsorption method to the BET formula. The mode pore diameter (mode pore diameter) of the aromatic synthetic adsorbent is preferably 600 angstroms or less, more preferably 300 angstroms or less, and still more preferably, from the viewpoint of high separation and high adsorption properties. is less than 200 Angstroms. The most frequent pore diameter can be measured by a gas adsorption method.
　このような合成吸着剤は市販されており、例えばダイヤイオン（商標）ＨＰ－１０、ＨＰ－２０、ＨＰ－２１、ＨＰ－３０、ＨＰ－４０、ＨＰ－５０（以上、無置換基型の芳香族系樹脂、いずれも商品名、三菱ケミカル株式会社製）；ＳＰ－８２５、ＳＰ－８００、ＳＰ－８５０、ＳＰ－８７５、ＳＰ－７０、ＳＰ－７００（以上、無置換基型に特殊処理を施した芳香族系樹脂、いずれも商品名、三菱ケミカル株式会社製）；ＳＰ－９００（芳香族系樹脂、商品名、三菱ケミカル株式会社製）；アンバーライト（商標）ＸＡＤ－２、ＸＡＤ－４、ＸＡＤ－１６、ＸＡＤ－１８、ＸＡＤ－２０００（以上、芳香族系樹脂、いずれも商品名、株式会社オルガノ製）；ダイヤイオン（商標）ＳＰ－２０５、ＳＰ－２０６、ＳＰ－２０７（以上、疎水性置換基を有する芳香族系樹脂、いずれも商品名、三菱ケミカル株式会社製）；ＨＰ－２ＭＧ、ＥＸ－００２１（以上、疎水性置換基を有する芳香族系樹脂、いずれも商品名、三菱ケミカル株式会社製）などが挙げられる。その中でも、ダイヤイオン（商標）ＳＰ－８５０が好ましい。これらの合成吸着剤は、１種単独でも、２種以上を組み合わせて用いてもよい。 Such synthetic adsorbents are commercially available. family resins, all trade names, manufactured by Mitsubishi Chemical Corporation); SP-825, SP-800, SP-850, SP-875, SP-70, SP-700 Aromatic resin applied, both trade names, manufactured by Mitsubishi Chemical Corporation); SP-900 (aromatic resin, trade name, manufactured by Mitsubishi Chemical Corporation); Amberlite (trademark) XAD-2, XAD-4 , XAD-16, XAD-18, XAD-2000 (above, aromatic resins, all trade names, manufactured by Organo Co., Ltd.); Diaion (trademark) SP-205, SP-206, SP-207 (above, Aromatic resins having hydrophobic substituents, both trade names, manufactured by Mitsubishi Chemical Corporation); HP-2MG, EX-0021 (above, aromatic resins having hydrophobic substituents, both trade names, Mitsubishi Chemical Co., Ltd.) and the like. Among them, Diaion (trademark) SP-850 is preferable. These synthetic adsorbents may be used singly or in combination of two or more.
　上記カラムに充填する芳香族系合成吸着剤の量は、カラムの大きさ、合成吸着剤の種類等によって適宜決定することができる。 The amount of aromatic synthetic adsorbent to be packed in the column can be appropriately determined depending on the size of the column, the type of synthetic adsorbent, and the like.
　上記清澄液を上記カラムに通液する際、濾液の温度は、２５～４５℃であってよい。濾液をカラムに通液する時の通液量および通液速度は、芳香族系合成吸着剤の種類等によって適宜決定することができる。 When the clarified liquid is passed through the column, the temperature of the filtrate may be 25 to 45°C. The amount and rate of passage of the filtrate through the column can be appropriately determined according to the type of synthetic aromatic adsorbent.
　上記エタノールおよび水の混合溶媒の混合体積比（エタノール／水）は、５０／５０～９９／１であってよく、溶出効率を向上させる観点からは、好ましくは、５０／５０～７０／３０の範囲内である。溶出速度は、カラムの大きさ、芳香族系合成吸着剤の種類等によって適宜決定することができる。なお、カラムに吸着された成分を効率的に溶出させるために、濾液をカラムに通液する前に、カラム内を水洗することが好ましい。 The mixed volume ratio (ethanol/water) of the mixed solvent of ethanol and water may be 50/50 to 99/1, and from the viewpoint of improving the elution efficiency, it is preferably 50/50 to 70/30. Within range. The elution rate can be appropriately determined depending on the size of the column, the type of synthetic aromatic adsorbent, and the like. In order to efficiently elute the components adsorbed on the column, it is preferable to wash the inside of the column with water before passing the filtrate through the column.
　溶出画分として得られたポリフェノール含有組成物は、必要に応じて濃縮しても良い。濃縮は、例えば、遠心式薄膜真空蒸発装置を用いて、５～２０倍に濃縮すればよい。これにより、ポリフェノール含有組成物を含む濃縮液を得ることができる。 The polyphenol-containing composition obtained as an eluted fraction may be concentrated as necessary. Concentration may be carried out, for example, by using a centrifugal thin film vacuum evaporator to concentrate 5 to 20 times. Thereby, a concentrate containing a polyphenol-containing composition can be obtained.
　上述の方法によって得られたポリフェノール含有組成物は、特に、溶出工程において芳香族系合成吸着剤を用い、エタノールおよび水の混合溶媒を使用して溶出することによって得られるため、食品素材として好適に利用することができる。 The polyphenol-containing composition obtained by the above method is particularly suitable as a food material because it is obtained by using an aromatic synthetic adsorbent in the elution step and eluting using a mixed solvent of ethanol and water. can be used.
　以下、本発明を実施例により具体的に説明するが、本発明はこれらの実施例に限定されない。なお、特段の記載のない限り、本願明細書に記載の測定方法および単位は日本工業規格（ＪＩＳ）の規定に従う。 The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples. Unless otherwise specified, the measurement methods and units described in this specification conform to the Japanese Industrial Standards (JIS).
　（参考例１）タンパク質濃度の測定
　水溶液中のタンパク質の濃度は、ブラッドフォード法による測定キット（Ｑｕｉｃｋ　Ｓｔａｒｔ　Ｂｒａｄｆｏｒｄ　Ｐｒｏｔｅｉｎ　Ａｓｓａｙ、Ｂｉｏ－Ｒａｄ社製）を使用して測定した。 (Reference Example 1) Measurement of Protein Concentration The protein concentration in the aqueous solution was measured using a measurement kit according to the Bradford method (Quick Start Bradford Protein Assay, Bio-Rad).
　（参考例２）セロビオハイドラーゼ活性の測定
　５０ｍＭ酢酸ナトリウム緩衝液（ｐＨ５．０）に、１ｍＭとなるように４－ニトロフェニル－β－Ｄ－ラクトピラノシド（Ｓｉｇｍａ－Ａｌｄｒｉｃｈ社製）を溶解したものを基質溶液とした。９０μＬの基質溶液に適宜希釈した酵素液１０μＬを添加して、３０℃で静置反応させた。６０分後に炭酸ナトリウム溶液１０μＬを添加し、反応停止および遊離した４－ニトロフェノールを発色させ、４０５ｎｍで吸光度を測定した。ブランクは、炭酸ナトリウム溶液添加後に酵素液を添加する以外は同様の方法で反応させたものを用いた。酵素１単位（１Ｕ）は、上記反応条件下において１分間に１μｍｏｌの４－ニトロフェノールを生成する量と定義し、タンパク質量１ｇあたりの活性は以下の式で算出する。 (Reference Example 2) Measurement of cellobiohydrolase activity 4-Nitrophenyl-β-D-lactopyranoside (manufactured by Sigma-Aldrich) dissolved in 50 mM sodium acetate buffer (pH 5.0) to 1 mM was used as the substrate solution. 10 μL of appropriately diluted enzyme solution was added to 90 μL of the substrate solution, and the reaction was allowed to stand at 30°C. After 60 minutes, 10 μL of sodium carbonate solution was added to stop the reaction, develop the released 4-nitrophenol, and measure the absorbance at 405 nm. A blank was prepared by reacting in the same manner except that the enzyme solution was added after adding the sodium carbonate solution. One enzyme unit (1 U) is defined as the amount that produces 1 μmol of 4-nitrophenol per minute under the above reaction conditions, and the activity per 1 g of protein is calculated by the following formula.
　酵素液あたりのセロビオハイドラーゼの活性〔Ｕ／ｍＬ〕＝（４－ニトロフェノール〔μｍｏｌ／ｍＬ〕×反応液量〔μＬ〕）／（反応時間〔ｍｉｎ〕×酵素液量〔μＬ〕）×希釈倍率（倍） Activity of cellobiohydrolase per enzyme solution [U/mL]=(4-nitrophenol [μmol/mL]×reaction volume [μL])/(reaction time [min]×enzyme volume [μL])× Dilution ratio (times)
　酵素のタンパク質量１ｇあたりのセロビオハイドラーゼ活性〔Ｕ／ｇ〕＝（酵素液あたりの活性〔Ｕ／ｍＬ〕／酵素液のタンパク濃度〔ｇ／Ｌ〕）×１０００ 　Cellobiohydrolase activity [U/g] per gram of enzyme protein = (activity per enzyme solution [U/mL]/protein concentration of enzyme solution [g/L]) x 1000
　（参考例３）キシラナーゼ活性の測定
　Ｍｅｇａｚｙｍｅ社“キシラナーゼ分析キット（ＸｙｌＸ６法）”を使用して測定した。１Ｍ酢酸ナトリウム緩衝液（ｐＨ５．０）２．５μＬ、キットに従って調製したＸｙｌＸ６反応溶液２５μＬ、ミリＱ水１２．５μＬを混合し、適宜希釈した酵素液１０μＬを添加して、３０℃で静置反応させた。１０分後に炭酸ナトリウム溶液１００μＬを添加し、反応停止および遊離した４－ニトロフェノールを発色させ、４０５ｎｍで吸光度を測定した。ブランクは、炭酸ナトリウム溶液添加後に酵素液を添加する以外は同様の方法で反応させたものを用いた。酵素１単位（１Ｕ）は、上記反応条件下において１分間に１μｍｏｌの４－ニトロフェノールを生成する量と定義し、タンパク質量１ｇあたりの活性は以下の式で算出する。 (Reference Example 3) Measurement of xylanase activity Measured using Megazyme's "Xylanase Analysis Kit (XylX6 method)". 2.5 μL of 1M sodium acetate buffer (pH 5.0), 25 μL of XylX6 reaction solution prepared according to the kit, and 12.5 μL of milli-Q water are mixed, 10 μL of appropriately diluted enzyme solution is added, and the reaction is allowed to stand at 30°C. let me After 10 minutes, 100 μL of sodium carbonate solution was added to stop the reaction, develop the released 4-nitrophenol, and measure the absorbance at 405 nm. A blank was prepared by reacting in the same manner except that the enzyme solution was added after adding the sodium carbonate solution. One enzyme unit (1 U) is defined as the amount that produces 1 μmol of 4-nitrophenol per minute under the above reaction conditions, and the activity per 1 g of protein is calculated by the following formula.
　酵素液あたりのキシラナーゼの活性〔Ｕ／ｍＬ〕＝（４－ニトロフェノール〔μｍｏｌ／ｍＬ〕×反応液量〔μＬ〕）／（反応時間〔ｍｉｎ〕×酵素液量〔μＬ〕）×希釈倍率（倍） Xylanase activity per enzyme solution [U/mL] = (4-nitrophenol [μmol/mL] x reaction volume [μL]) / (reaction time [min] x enzyme volume [μL]) x dilution ratio ( times)
　酵素のタンパク質量１ｇあたりのキシラナーゼ活性〔Ｕ／ｇ〕＝（酵素液あたりの活性〔Ｕ／ｍＬ〕／酵素液のタンパク濃度〔ｇ／Ｌ〕）×１０００ 　Xylanase activity [U/g] per gram of enzyme protein = (activity per enzyme solution [U/mL]/protein concentration of enzyme solution [g/L]) x 1000
　（参考例４）ポリフェノール量の測定
　ポリフェノール量の測定はフォーリンチオカルト法により以下の条件で実施した。適宜希釈した測定試料１．０ｍＬ、フェノール試液（ナカライテスク社）１．０ｍＬ、水５ｍＬを２５ｍＬのメスフラスコに入れて５分間室温で放置し、これに７％炭酸ナトリウム水溶液１０ｍＬを加える。更に水を加えて２５ｍＬとして混合し、２時間室温で放置する。反応液の一部を取り、φ０．４５μｍのＰＴＦＥフィルターで濾過し、７５０ｎｍで吸光度を測定する（吸光度は０．６ＡＢＳ以下となるようにサンプルを適宜希釈）。標準物質としてカテキン試薬（シグマ社、純度９８％以上）を用い、カテキン換算値として算出した。 (Reference Example 4) Measurement of amount of polyphenol The amount of polyphenol was measured by the Folin-Ciocalteu method under the following conditions. 1.0 mL of an appropriately diluted measurement sample, 1.0 mL of phenol test solution (Nacalai Tesque) and 5 mL of water are placed in a 25 mL volumetric flask and allowed to stand at room temperature for 5 minutes. Add more water to make 25 mL and mix and let stand at room temperature for 2 hours. A part of the reaction solution is taken, filtered through a φ0.45 μm PTFE filter, and the absorbance is measured at 750 nm (the sample is appropriately diluted so that the absorbance is 0.6 ABS or less). A catechin reagent (Sigma, purity 98% or higher) was used as a standard substance and calculated as a catechin conversion value.
　（参考例５）バガスのアルカリ抽出液の調製
　サトウキビの搾り粕であるバガスを０．４５（ｗｔ／ｗｔ）％水酸化ナトリウム水溶液に乾燥重量で５ｗｔ％添加・混合し、９０℃、３時間反応させ、固形分と液分を分離して、液分として、アルカリ抽出液を得た。 (Reference Example 5) Preparation of alkaline extract of bagasse Bagasse, which is sugar cane lees, was added to and mixed with 0.45 (wt/wt)% sodium hydroxide aqueous solution at 5 wt% in terms of dry weight, and reacted at 90°C for 3 hours. Then, the solid content and the liquid content were separated to obtain an alkaline extract as the liquid content.
　（比較例１～３）中和時の温度と液性状
　参考例５に従って調製したバガスのアルカリ抽出液の温度を３８℃（比較例１）、４５℃（比較例２）、６０℃（比較例３）まで冷却し、珪藻土をアルカリ抽出液１００重量部に対して１重量部加え、３５％（ｗ／ｗ）塩酸を用いて、ｐＨ３．５に調整した。このとき、ミセル化様の現象の評価として、＋＋：ミセル化（液が白濁し、沈殿ができない状態）、＋：一部ミセル化（濁質が沈殿しない状態）、－：ミセル化しない（濁質がすぐに沈殿する状態）を指標として評価した。 (Comparative Examples 1 to 3) Temperature and liquid properties during neutralization 3), 1 part by weight of diatomaceous earth was added to 100 parts by weight of the alkaline extract, and the pH was adjusted to 3.5 using 35% (w/w) hydrochloric acid. At this time, as an evaluation of micellization-like phenomenon, ++: micelle formation (state in which liquid becomes cloudy and precipitation cannot occur), +: partial micelle formation (state in which turbidity does not precipitate), -: no micelle formation (turbidity A state in which the quality quickly settles) was evaluated as an index.
　ｐＨ調整後の抽出液を、フィルタープレスにより固液分離した。フィルタープレスは薮田産業株式会社製の小型濾過装置ＭＯ－４を使用した。５分後の濾液量を測定した。
　フィルタープレスの濾液は、芳香族系合成吸着剤（ダイヤイオンＳＰ－８５０、三菱ケミカル株式会社製）を充填したカラムに、流速７．６Ｌ／ｈ（ＳＶ＝２０）の条件で通液した。このとき、樹脂のカラムの詰まりについて、＋＋：詰まりが発生し、通液不能となる、＋：流速が低下するが全量通液できた、－：詰まりが発生せず、流速７．６Ｌ／ｈで全量通液できたとして評価した。 The extract after pH adjustment was subjected to solid-liquid separation by a filter press. A small filter device MO-4 manufactured by Yabuta Sangyo Co., Ltd. was used as a filter press. Filtrate volume was measured after 5 minutes.
The filtrate from the filter press was passed through a column filled with an aromatic synthetic adsorbent (Diaion SP-850, manufactured by Mitsubishi Chemical Corporation) at a flow rate of 7.6 L/h (SV=20). At this time, regarding the clogging of the resin column, ++: clogging occurred and the liquid could not be passed, +: the flow rate decreased but the entire amount of liquid could be passed, -: no clogging occurred and the flow rate was 7.6 L/h. It was evaluated that the entire amount of liquid could be passed through.
　その後、合成吸着剤の体積の１０倍量の水で洗浄し、６０％（ｖ／ｖ）エタノール水溶液をＳＶ＝２で通液して溶出し、溶出画分を得た。溶出画分中のポリフェノール量を参考例４に従って測定し、比較例１でのポリフェノール量を１００％として、ポリフェノール量（％）を求めた。 After that, it was washed with water 10 times the volume of the synthetic adsorbent, and eluted by passing 60% (v/v) ethanol aqueous solution at SV = 2 to obtain an eluted fraction. The amount of polyphenol in the eluted fraction was measured according to Reference Example 4, and the amount of polyphenol (%) was calculated with the amount of polyphenol in Comparative Example 1 as 100%.
　結果を表１に示す。中和時の温度が高いとミセル化様の現象が起き、フィルタープレスの５分後の濾液の量も少なかった。ミセル化が起きた場合では樹脂カラムの詰まりも発生した。また、溶出画分中のポリフェノール量もミセル化が起きたものでは低かった。 The results are shown in Table 1. When the temperature during neutralization was high, a micelle-like phenomenon occurred, and the amount of filtrate after 5 minutes of filter pressing was also small. When micellization occurred, clogging of the resin column also occurred. In addition, the amount of polyphenols in the eluted fraction was low in those with micellization.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　（実施例１および２）酵素添加による効果
　参考例５に従って調製したバガスのアルカリ抽出液の温度を４５℃（実施例１）、６０℃（実施例２）まで冷却し、珪藻土をアルカリ抽出液１００重量部に対して１重量部加え、３５％（ｗ／ｗ）塩酸を用いて、ｐＨ３．５に調整した。その後、酵素１（セロビオハイドラーゼ活性あり、キシラナーゼ活性あり）：“ＣｅｌＢｘ”（Ｓｉａｍ　Ｖｉｃｔｏｒｙ　Ｃｈｅｍｉｃａｌｓ　Ｌｉｍｉｔｅｄ社製）を添加した。添加量はｐＨ調整後の抽出液１００重量部に対して０．１重量部（実施例１）、０．５重量部（実施例２）とした。添加後は、攪拌しながら５０℃で１時間反応させた。このとき、ミセル化様の現象の評価として、＋＋：ミセル化（液が白濁し、沈殿ができない状態）、＋：一部ミセル化（濁質が沈殿しない状態）、－：ミセル化しない（濁質がすぐに沈殿する状態）を指標として評価した。 (Examples 1 and 2) Effect of enzyme addition The temperature of the bagasse alkaline extract prepared according to Reference Example 5 was cooled to 45 ° C. (Example 1) and 60 ° C. (Example 2), and diatomaceous earth was added to 100 1 part by weight was added, and the pH was adjusted to 3.5 using 35% (w/w) hydrochloric acid. After that, Enzyme 1 (having cellobiohydrolase activity and xylanase activity): "CelBx" (manufactured by Siam Victory Chemicals Limited) was added. The amount added was 0.1 parts by weight (Example 1) and 0.5 parts by weight (Example 2) with respect to 100 parts by weight of the extract after pH adjustment. After the addition, the mixture was reacted at 50° C. for 1 hour while stirring. At this time, as an evaluation of micellization-like phenomenon, ++: micelle formation (state in which liquid becomes cloudy and precipitation cannot occur), +: partial micelle formation (state in which turbidity does not precipitate), -: no micelle formation (turbidity A state in which the quality quickly settles) was evaluated as an index.
　また、酵素のセロビオハイドラーゼ活性とキシラナーゼ活性は参考例２、３にしたがって測定した。タンパク濃度は参考例１にしたがって測定した値を使用した。 In addition, the cellobiohydrolase activity and xylanase activity of the enzyme were measured according to Reference Examples 2 and 3. The value measured according to Reference Example 1 was used as the protein concentration.
　酵素反応後の抽出液は、フィルタープレスによる固液分離を行った。フィルタープレスは薮田産業株式会社製の小型濾過装置ＭＯ－４を使用した。５分後の濾液量を測定した。 The extract after the enzymatic reaction was solid-liquid separated by a filter press. A small filter device MO-4 manufactured by Yabuta Sangyo Co., Ltd. was used as a filter press. Filtrate volume was measured after 5 minutes.
　フィルタープレスの濾液は、芳香族系合成吸着剤（ダイヤイオンＳＰ－８５０、三菱ケミカル株式会社製）を充填したカラムに、流速７．６Ｌ／ｈ（ＳＶ＝２０）の条件で通液した。このとき、樹脂のカラムの詰まりについて、＋＋：詰まりが発生し、通液不能となる、＋：流速が低下するが全量通液できた、－：詰まりが発生せず、流速７．６Ｌ／ｈで全量通液できたとして評価した。 The filtrate from the filter press was passed through a column packed with an aromatic synthetic adsorbent (Diaion SP-850, manufactured by Mitsubishi Chemical Corporation) at a flow rate of 7.6 L/h (SV=20). At this time, regarding the clogging of the resin column, ++: clogging occurred and the liquid could not be passed, +: the flow rate decreased but the entire amount of liquid could be passed, -: no clogging occurred and the flow rate was 7.6 L/h. It was evaluated that the entire amount of liquid could be passed through.
　その後、合成吸着剤の体積の１０倍量の水で洗浄し、６０％（ｖ／ｖ）エタノール水溶液をＳＶ＝２で通液して溶出し、溶出画分を得た。溶出画分中のポリフェノール量を参考例４に従って測定し、比較例１でのポリフェノール量を１００％として、ポリフェノール量（％）を求めた。 After that, it was washed with water 10 times the volume of the synthetic adsorbent, and eluted by passing 60% (v/v) ethanol aqueous solution at SV = 2 to obtain an eluted fraction. The amount of polyphenol in the eluted fraction was measured according to Reference Example 4, and the amount of polyphenol (%) was calculated with the amount of polyphenol in Comparative Example 1 as 100%.
　結果を表２に示す。酵素を反応させることにより、ミセル化様の現象が起こらず、フィルタープレスの５分後の濾液の量も多く、濾過性が改善された。また、樹脂カラムの詰まりも発生せず、ポリフェノール量も改善した。 The results are shown in Table 2. By reacting the enzyme, a micelle-like phenomenon did not occur, and the amount of the filtrate after 5 minutes of filter pressing was large, and the filterability was improved. In addition, clogging of the resin column did not occur, and the amount of polyphenol was also improved.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　（比較例４および５）酵素による違い
　参考例５に従って調製したバガスのアルカリ抽出液の温度を４５℃（比較例４）、６０℃（比較例５）まで冷却し、珪藻土をアルカリ抽出液１００重量部に対して１重量部加え、３５％（ｗ／ｗ）塩酸を用いて、ｐＨ３．５に調製した。その後、酵素２（セロビオハイドラーゼ活性あり、キシラナーゼ活性なし）：”Ｐｅｃｔｉｎａｓｅ　ｆｒｏｍ　Ａｓｐｅｒｇｉｌｌｕｓ　ａｃｕｌｅａｔｕｓ”（液状、Ｓｉｇｍａ－Ａｌｄｒｉｃｈ社製）、酵素３（セロビオハイドラーゼ活性あり、キシラナーゼ活性なし）：“セルロシンＧＭ５”（粉末状、エイチビィアイ社製）をそれぞれ添加した。添加量はｐＨ調整後の抽出液１００重量部に対して０．１重量部とした。添加後は、攪拌しながら５０℃で１時間反応させた。このとき、ミセル化様の現象の評価として、＋＋：ミセル化（液が白濁し、沈殿ができない状態）、＋：一部ミセル化（濁質が沈殿しない状態）、－：ミセル化しない（濁質がすぐに沈殿する状態）を指標として評価した。また、酵素のセロビオハイドラーゼ活性とキシラナーゼ活性は参考例２および３にしたがって測定した。タンパク濃度は参考例１にしたがって測定した値を使用した。
　酵素反応後の抽出液は、比較例１および２と同様にフィルタープレスにより固液分離した。 (Comparative Examples 4 and 5) Differences due to enzymes The temperature of the bagasse alkaline extract prepared according to Reference Example 5 was cooled to 45 ° C. (Comparative Example 4) and 60 ° C. (Comparative Example 5), and diatomaceous earth was added to 100 weight of the alkaline extract. 1 part by weight was added to each part, and the pH was adjusted to 3.5 using 35% (w/w) hydrochloric acid. Then, Enzyme 2 (cellobiohydrolase activity, no xylanase activity): "Pectinase from Aspergillus aculeatus" (liquid, manufactured by Sigma-Aldrich), Enzyme 3 (cellobiohydrolase activity, no xylanase activity): "Cellulosine GM5″ (powdered, manufactured by HI Co., Ltd.) was added to each. The amount added was 0.1 part by weight per 100 parts by weight of the extract after pH adjustment. After the addition, the mixture was reacted at 50° C. for 1 hour while stirring. At this time, as an evaluation of micellization-like phenomenon, ++: micelle formation (state in which liquid becomes cloudy and precipitation cannot occur), +: partial micelle formation (state in which turbidity does not precipitate), -: no micelle formation (turbidity A state in which the quality quickly settles) was evaluated as an index. In addition, cellobiohydrolase activity and xylanase activity of the enzyme were measured according to Reference Examples 2 and 3. The value measured according to Reference Example 1 was used as the protein concentration.
The extract after the enzymatic reaction was solid-liquid separated by a filter press in the same manner as in Comparative Examples 1 and 2.
　結果を表３に示す。酵素２および３ではミセル化が起こり、フィルタープレスでの５分後の濾液量も少なかった。 The results are shown in Table 3. Enzymes 2 and 3 resulted in micellization and low filtrate volumes after 5 minutes on the filter press.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
　（比較例６、および実施例３～５）ｐＨによる違い
　ｐＨを３．０（比較例６）、３．３（実施例３）、４．０（実施例４）、４．５（実施例５）としたこと以外は実施例１と同様にしてミセル化の評価を行った。結果を表４に示すとおりであり、アルカリ抽出液のｐＨ調整後のｐＨが本発明の範囲外である場合は酵素反応後にミセル化した。 (Comparative Example 6 and Examples 3 to 5) Differences due to pH Evaluation of micelle formation was performed in the same manner as in Example 1 except that 5) was performed. The results are shown in Table 4. When the pH of the alkaline extract after pH adjustment was outside the range of the present invention, micellization was performed after the enzymatic reaction.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004

Claims (6)

  1. 　草本系バイオマス由来のポリフェノール含有組成物の製造方法であって、
    （１）草本系バイオマスを６０℃以上のアルカリ性水溶液に接触させて抽出液を得る工程、
    （２）工程（１）で得られた抽出液を６０℃以下に冷却して冷却抽出液を得る工程、
    （３）工程（２）で得られた冷却抽出液をｐＨ３．２以上４．５以下に調整し、セロビオハイドラーゼ活性およびキシラナーゼ活性を含む酵素と反応させ酵素反応液を得る工程、
    （４）工程（３）で得られた酵素反応液を粗ろ過により清澄液を得る工程、
    （５）工程（４）で得られた清澄液を高比表面積化の特殊処理を施した芳香族系樹脂からなる合成吸着剤が充填されたカラムに通液し、前記芳香族系樹脂からなる合成吸着剤に吸着した成分をエタノールおよび水の混合溶媒で溶出して溶出画分をポリフェノール含有組成物として得る工程、
    を含むポリフェノール含有組成物の製造方法。     A method for producing a polyphenol-containing composition derived from herbaceous biomass,
    (1) A step of contacting the herbaceous biomass with an alkaline aqueous solution at 60°C or higher to obtain an extract;
    (2) a step of cooling the liquid extract obtained in step (1) to 60°C or less to obtain a cooled liquid extract;
    (3) a step of adjusting the cooled extract obtained in step (2) to pH 3.2 or higher and 4.5 or lower, and reacting it with an enzyme containing cellobiohydrolase activity and xylanase activity to obtain an enzyme reaction liquid;
    (4) a step of obtaining a clarified solution by coarsely filtering the enzyme reaction solution obtained in step (3);
    (5) The clarified liquid obtained in step (4) is passed through a column filled with a synthetic adsorbent made of an aromatic resin that has undergone a special treatment to increase the specific surface area, and the aromatic resin made of the A step of eluting the component adsorbed on the synthetic adsorbent with a mixed solvent of ethanol and water to obtain an eluted fraction as a polyphenol-containing composition;
    A method for producing a polyphenol-containing composition comprising:
  2. 　前記セロビオハイドラーゼ活性およびキシラナーゼ活性を含む酵素がトリコデルマ属微生物由来である、請求項１に記載のポリフェノール含有組成物の製造方法。 The method for producing a polyphenol-containing composition according to claim 1, wherein the enzyme containing cellobiohydrolase activity and xylanase activity is derived from a Trichoderma microorganism.
  3. 　前記草本系バイオマスがバガスである、請求項１または２に記載のポリフェノール含有組成物の製造方法。 The method for producing a polyphenol-containing composition according to claim 1 or 2, wherein the herbaceous biomass is bagasse.
  4. 　前記アルカリ性水溶液が水酸化ナトリウム水溶液である、請求項１から３のいずれかに記載のポリフェノール含有組成物の製造方法。 The method for producing a polyphenol-containing composition according to any one of claims 1 to 3, wherein the alkaline aqueous solution is an aqueous sodium hydroxide solution.
  5. 　前記水酸化ナトリウムの濃度が０．１から１０重量％である、請求項４に記載のポリフェノール含有組成物の製造方法。 The method for producing a polyphenol-containing composition according to claim 4, wherein the concentration of sodium hydroxide is 0.1 to 10% by weight.
  6. 　前記芳香族系樹脂がスチレン－ジビニルベンゼン系樹脂である、請求項１から５のいずれかに記載のポリフェノールの製造方法。 The method for producing polyphenol according to any one of claims 1 to 5, wherein the aromatic resin is a styrene-divinylbenzene resin.
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WO2013018678A1 (en) * 2011-07-29 2013-02-07 東レ株式会社 Method of manufacturing filter aid
WO2019230803A1 (en) * 2018-05-29 2019-12-05 三井製糖株式会社 Method for producing polyphenol composition from bagasse

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013018678A1 (en) * 2011-07-29 2013-02-07 東レ株式会社 Method of manufacturing filter aid
WO2019230803A1 (en) * 2018-05-29 2019-12-05 三井製糖株式会社 Method for producing polyphenol composition from bagasse

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