Method for producing a polyphenol-containing composition

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

Citation of Related Applications

[0001] This patent application claims the right of priority based on Japanese Patent Application No. 2021-58736 filed on March 30, 2021, and all the disclosure contents in such prior patent application are incorporated herein by reference and made a part of this specification.

Technical Field

[0002] The present invention relates to a method for producing a polyphenol-containing composition from herbaceous biomass.

Background Art

[0003] In recent years, from the perspectives of problems such as global warming and depletion of oil resources, and carbon neutrality, the utilization of biomass has attracted attention. Among them, there is a method for obtaining a polyphenol-containing composition from cellulose-containing biomass that does not compete with food. Cellulose-containing biomass is mainly composed of cellulose, a polysaccharide, hemicellulose, and lignin, an aromatic polymer. By decomposing lignin in the cellulose-containing biomass and the connection part between lignin and polysaccharide, a decomposition liquid containing polyphenols can be obtained.

[0004] For example, Patent Document 1 describes a method for efficiently obtaining hydroxycinnamic acid by passing an alkaline aqueous medium through cellulose-containing biomass. Further, Patent Document 2 describes a method for efficiently producing a polyphenol-containing composition by adjusting the pH of an extract obtained by treating bagasse, which is sugarcane press cake, with an alkaline solution to be acidic, filtering it, and adsorbing the filtrate with an aromatic synthetic adsorbent.

[0005] Conventionally, such an alkaline treatment of cellulose-containing biomass has been performed as a pretreatment to facilitate obtaining a sugar solution from cellulose-containing biomass, and the liquid component after the alkaline treatment has been discarded. However, since it contains polyphenols as described above, effective utilization is desired. The decomposition liquids containing polyphenols obtained by the conventional technologies described above are known to be used as deodorants (Patent Document 3), food discoloration inhibitors (Patent Document 4), and aquatic organism growth promoters (Patent Document 5). Furthermore, coumaric acid and ferulic acid are known to be representative polyphenols contained within these liquids. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] WO2017 / 170549 [Patent Document 2] WO2019 / 230803 issue [Patent Document 3] Japanese Patent Publication No. 2020-93080 [Patent Document 4] WO2018 / 079640 issue [Patent Document 5] WO2018 / 079641 [Overview of the project] [Problems that the invention aims to solve]

[0007] As described above, one method for efficiently producing polyphenol-containing compositions from herbaceous biomass involves treating herbaceous biomass with an alkaline aqueous solution, adjusting the pretreatment solution to an acidic state, filtering it, and adsorbing the resulting filtrate with an aromatic synthetic adsorbent. However, our investigations have revealed that this method can have poor filterability, and micellar-like phenomena can occur, sometimes leading to a decrease in the yield of the aromatic synthetic adsorbent.

[0008] Therefore, one objective of the present invention is to provide a new technical means for efficiently producing polyphenol-containing compositions from herbaceous biomass. [Means for solving the problem]

[0009] As a result of diligent research, the inventors have found that by treating herbaceous biomass with alkali at a temperature of 60°C or higher, cooling the extract to below 60°C, adjusting the pH to between 3.2 and 4.5, reacting it with enzymes containing cellobiohydrase activity and xylanase activity, filtering it, and then adsorbing it onto an aromatic synthetic adsorbent, micellization-like phenomena are prevented, filterability is improved, and the yield of the polyphenol-containing composition is increased.

[0010] In other words, the present invention consists of the following [1] to [6]. [1] A method for producing a polyphenol-containing composition derived from herbaceous biomass, (1) A step of obtaining an extract by contacting herbaceous biomass with an alkaline aqueous solution at a temperature of 60°C or higher. (2) A step of cooling the extract obtained in step (1) to 60°C or below to obtain a cooled extract. (3) Adjust the cooled extract obtained in step (2) to a pH of 3.2 or higher and 4.5 or lower, and react it with an enzyme containing cellobiohydrolase activity and xylanase activity to obtain an enzyme reaction solution. (4) A step to obtain a clarified solution by coarse filtration of the enzyme reaction solution obtained in step (3), (5) The clarified liquid obtained in step (4) is passed through a column packed with a synthetic adsorbent made of an aromatic resin that has been specially treated to increase its specific surface area, and the components adsorbed on the aromatic synthetic adsorbent are eluted with a mixed solvent of ethanol and water to obtain the eluted fraction as a polyphenol-containing composition. A method for producing a polyphenol-containing composition. [2] A method for producing the polyphenol-containing composition according to [1], wherein the enzyme containing the cellobiohydrolase activity and xylanase activity is derived from a microorganism of the genus Trichoderma. [3] A method for producing the polyphenol-containing composition according to [1] or [2], wherein the herbaceous biomass is bagasse. [4] A 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. The method for producing a polyphenol-containing composition according to [4], wherein the concentration of the sodium hydroxide is 0.1 to 10% by mass. 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. [Advantages of the Invention]

[0011] According to the present invention, a polyphenol-containing composition can be efficiently produced from herbaceous biomass. Further, the present invention can be advantageously used for suppressing a micellization-like phenomenon and improving the filterability in the production of a polyphenol-containing composition from herbaceous biomass. According to such the present invention, it can be advantageously used for improving the yield of the polyphenol-containing composition. [Embodiments for Carrying Out the Invention]

[0012] According to one embodiment of the present invention, a method for producing a polyphenol-containing composition derived from herbaceous biomass comprises: (1) a step of bringing herbaceous biomass into contact with an alkaline aqueous solution at a temperature of 60° C. or higher to obtain an extract; (2) a step of cooling the extract obtained in step (1) to 60° C. or lower to obtain a cooled extract; (3) a step of adjusting the cooled extract obtained in step (2) to a pH of 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 solution; (4) a step of obtaining a clarified solution by rough filtration of the enzyme reaction solution obtained in step (3); (5) a step of passing the clarified solution obtained in step (4) through a column filled with a synthetic adsorbent composed of an aromatic resin subjected to a special treatment for increasing the specific surface area, and eluting the components adsorbed on the aromatic synthetic adsorbent with a mixed solvent of ethanol and water to obtain an elution fraction as a polyphenol-containing composition. It is characterized by including the above steps.

[0013] Hereinafter, embodiments for carrying out the present invention will be described. [Step (1)] According to one embodiment of the present invention, as described above, in step (1), the herbaceous biomass is brought into contact with an alkaline aqueous solution to obtain an extract.

[0014] According to one embodiment of the present invention, examples of herbaceous biomass include, but are not limited to, bagasse which is the squeezed residue of sugarcane, switchgrass, napier grass, miscanthus, corn stover, corn husk, wheat bran, soybean hull, rice straw, wheat straw, empty fruit bunches of oil palm, etc. From the perspective of polyphenol production, it is preferable to use herbaceous biomass having a lignin content of 5% or more. Specifically, bagasse, napier grass, miscanthus, corn stover, and rice straw are preferable, and bagasse is more preferable. The lignin content can be determined by measuring Klason lignin which is the residue obtained by acid hydrolysis minus the ash content. 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 it can be carried out using machines commonly used for coarse pulverization of various materials such as ball mills, vibration mills, cutter mills, hammer mills, Wiley mills, jet mills, etc. This mechanical pulverization may be either dry or wet, but dry pulverization is preferable.

[0015] According to one embodiment of the present invention, the moisture content of the herbaceous biomass is not particularly limited, but the preferable range is, for example, about 3% or more, about 3% or more and about 75% or less, about 5% or more, about 5% or more and about 70% or less, about 5% or more and about 65% or less, about 5% or more and about 55% or less.

[0016] According to one embodiment of the present invention, polyphenols may include one or more hydroxycinnamic acids and lignin degradation products such as coumaric acid and ferulic acid, and can be measured, for example, by the Forlinthiocalt method. The Forlinthiocalt method was originally developed for the purpose of analyzing aromatic amino acids such as tyrosine and tryptophan, and proteins containing them. It is a method of colorimetric quantitative determination at 700-770 nm by reducing phosphotungstic acid and molybdic acid with phenolic hydroxyl groups in an alkaline environment to produce a blue color. The same operation can be performed with specific reference substances such as gallic acid and catechin, and quantitative values ​​can be shown in terms of the compound, and in this invention, the value in terms of catechin is used.

[0017] According to one embodiment of the present invention, an alkaline aqueous solution may include an alkaline aqueous solution containing at least one selected from ammonia, aqueous ammonia, alkali metal hydroxide, alkali metal oxide, alkaline earth metal oxide, alkali metal carbonate, alkaline earth metal carbonate, quaternary ammonium hydroxide, etc., but preferably an aqueous medium containing at least one hydroxide selected from sodium hydroxide and potassium hydroxide. From the viewpoint of being inexpensive and used in food manufacturing processes, an aqueous sodium hydroxide solution is even more preferable.

[0018] The alkalinity of the alkaline aqueous solution is not particularly limited, but from the viewpoint of shortening the pretreatment time, it is preferably 0.1% by weight or more, more preferably 0.2% by weight or more, and even more preferably 0.3% 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 a similar viewpoint, the concentration of the alkaline solution may be 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, 0.2 to 1.0% by weight, 0.3 to 10% by weight, 0.3 to 5% by weight, or 0.3 to 1.0% by weight.

[0019] Furthermore, the lower limit of the pH of the alkaline aqueous solution is not particularly limited as long as it is alkaline, but it is preferably pH 7 or higher, preferably pH 8 or higher, more preferably pH 9 or higher, and even 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 to pH 12 or lower. Furthermore, preferred pH ranges are, for example, 7 to 13.5, 8 to 13.5, a more preferred pH range is 9 to 13.5, and an even more preferred pH range is 10 to 12. The temperature at which the alkaline aqueous solution is brought into contact with herbaceous biomass is 60°C or higher, preferably above 60°C. Furthermore, in order to maintain the alkaline-treated material at a temperature higher than 100°C, it is necessary to apply pressure exceeding atmospheric pressure to the alkaline-treated material, requiring high-pressure equipment. Therefore, from the standpoint of production costs, the temperature is more preferably between 60°C and 100°C, and even more preferably between 80°C and 100°C.

[0020] According to one embodiment of the present invention, the weight ratio of the alkaline aqueous solution to the herbaceous biomass (dry weight) is not particularly limited, but preferred ranges are, for example, 100:1 to 2:1, 90:1 to 3:1, 50:1 to 5:1, 30:1 to 5:1, 25:1 to 7:1, 25:1 to 7:1, 25:1 to 5:1, and 20:1 to 5:1.

[0021] The method of contact between herbaceous biomass and alkaline aqueous solution is not particularly limited, but examples include spraying, immersing, or passing the alkaline aqueous solution through the herbaceous biomass to bring them into contact. In such cases, stirring or rotating the container may be done to ensure sufficient contact between the alkaline aqueous solution and the herbaceous biomass.

[0022] The contact time between the alkaline aqueous solution and the herbaceous biomass is not particularly limited, but is preferably between 20 minutes and 72 hours, 20 minutes and 48 hours, 20 minutes and 24 hours, 30 minutes and 48 hours, 30 minutes and 24 hours, 30 minutes and 12 hours, 30 minutes and 6 hours, or 30 minutes and 3 hours.

[0023] An extract can be obtained by solid-liquid separation of herbaceous biomass and an alkaline aqueous solution. Examples of solid-liquid separation devices include screw presses and centrifuges. A strainer may also be used to remove fine particles. Furthermore, if the alkaline aqueous solution is passed through the herbaceous biomass during contact with the alkaline aqueous solution, the resulting liquid may be used directly as the extract. However, from the viewpoint of extract recovery, it is preferable to extract the reaction products from the herbaceous biomass using a solid-liquid separation device.

[0024] The extract obtained by contacting herbaceous biomass with an alkaline aqueous solution is cooled to a temperature of 60°C or lower. The cooling method is not limited to this; it may be cooled while the herbaceous biomass and alkaline aqueous solution are in contact, or it may be cooled after the solid-liquid separation described above. However, cooling after solid-liquid separation is preferable because it allows for more efficient cooling. If an acidic substance is added to adjust the pH of the extract at a high temperature, a micelle-like phenomenon is likely to occur. The temperature of the cooled extract may be changed as appropriate depending on the obtained extract, but if the temperature is too low, the filterability will decrease due to increased viscosity. Therefore, a temperature of 20°C to 60°C, 20°C to 55°C is preferable, and 25°C to 50°C, and 30°C to 50°C are more preferable.

[0025] <Process (2)> According to one embodiment of the present invention, in step (2), the extract obtained in step (1) is cooled to 60°C or below to obtain a cooled extract.

[0026] <Process (3)> Furthermore, according to one embodiment of the present invention, in step (3), the cooled extract obtained in step (2) is adjusted to a pH of 3.2 or higher and 4.5 or lower, and reacted with an enzyme containing cellobiohydrolase activity and xylanase activity to obtain an enzyme reaction solution.

[0027] According to one embodiment of the present invention, an acidic substance is added to the extract obtained in the step of obtaining the above extract to adjust the pH to the acidic range described above.

[0028] Examples of acidic substances include, but are not limited to, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, lactic acid, acetic acid, formic acid, and citric acid. Preferably, hydrochloric acid, sulfuric acid, or nitric acid is used, and more preferably, hydrochloric acid.

[0029] The method of pH adjustment using acidic substances is not particularly limited, but one method involves adding an appropriate concentration of acidic substance while monitoring the pH and then mixing it. A continuous method, in which alkaline extract is continuously added during pH adjustment and the pH-adjusted solution is continuously withdrawn, or a batch method may be used.

[0030] The temperature during pH adjustment is not particularly limited, but is preferably 20 to 100°C, more preferably 20 to 60°C, and even more preferably 30 to 60°C.

[0031] The pH adjustment range is usually 3.2 to 4.5, but preferably 3.3 to 4.0, and more preferably 3.5 to 4.0.

[0032] An enzyme containing cellobiohydrase activity is an exo-type enzyme that breaks down cellulose chains, in which glucose is linked by β-1,4 bonds, from the terminals to produce cellobiose. Cellobiohydrase activity can be measured as the enzyme activity that breaks down 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 the method described in Reference Example 2 below. In this invention, an enzyme with cellobiohydrase activity of 5 U / g or more is defined as an enzyme possessing cellobiohydrase activity, and the cellobiohydrase activity value of the enzyme used in this invention is preferably 5 to 1000 U / g, more preferably 5 to 500 U / g, even more preferably 10 to 500 U / g, and particularly preferably 20 to 300 U / g.

[0033] Enzymes containing xylanase activity are endo-type enzymes that randomly degrade xylan, which is a β-1,4 linked xylose molecule. Xylanase activity can be determined by measuring the amount of reducing sugar in the reaction solution after using commercially available xylan (e.g., Birchwood xylan) as a substrate, but it is preferable to use Megazyme's "Xylanase Analysis Kit (XylX6 Method)". In the "Xylanase Analysis Kit (XylX6 Method)", the XylX6 reagent is degraded by the combination of xylanase in the sample and the auxiliary reagent β-xylosidase to produce 4-nitrophenol, thereby measuring xylanase activity. The amount of enzyme that produces 1 μmol of 4-nitrophenol per minute is defined as 1 U. Enzyme activity is measured by the method described in Reference Example 3 below. In this invention, an enzyme having xylanase activity of 400 U / g or more is defined as an enzyme having xylanase activity. According to one embodiment of the present invention, the xylanase activity value of the enzyme is preferably 400 to 50,000 U / g, more preferably 500 to 50,000 U / g, even more preferably 1,000 to 50,000 U / g, and particularly preferably 3,000 to 45,000 U / g.

[0034] The above enzymes are produced by microorganisms, and may be produced by a single microorganism or by multiple microorganisms. Examples of microorganisms that produce cellobiohydrolase and xylanase include those of the genera Trichoderma, Aspergillus, Cellulomonas, Clostridium, Streptomyces, Humicola, Acremonium, Irpex, Mucor, and Talaromyces, but Trichoderma is preferred.

[0035] While the genus Trichoderma is not particularly limited, Trichoderma reesei is preferred, specifically Trichoderma reesei QM9414, Trichoderma reesei QM9123, Trichoderma reesei Rut C-30, Trichoderma reesei PC3-7, Trichoderma reesei CL-847, Trichoderma reesei MCG77, Trichoderma reesei MCG80, and Trichoderma viride QM9123. QM9123) can be used as an example. Alternatively, the above-mentioned microorganisms derived from the genus Trichoderma may be mutant strains in which cellulase productivity has been improved by mutagenesis treatment with mutagens or ultraviolet irradiation.

[0036] The above enzymes may be added in a purified form, the culture medium may be added as crude enzymes, commercially available cellulase or xylanase agents may be used, and enzymes other than cellobiohydrolase and xylanase may also be included. For example, β-glucosidase, β-xylosidase, endoglucanase, mannanase, etc. may be included.

[0037] Examples of commercially available cellulase and xylanase agents include, but are not limited to, Novozyme's "Ceric C-Tech" (registered trademark) and "Ceric C-Tech 2" (registered trademark), Danisco Japan's "AccelLase" (registered trademark) 1000, "AccelLase" (registered trademark) 1500, and "AccelLase" (registered trademark) Duet, Sigma-Aldrich's "Cellulase from Trichoderma reesei ATCC 26921," "Cellulase from Trichoderma viride," and "Cellulase from Trichoderma longibrachiatum," HBI's "Cellulosin TP25" and "Cellulosin HC100," and Siam Victory Chemicals Limited's "CelBx." The amount to be added is not particularly limited and may be changed as appropriate depending on the enzyme being added, but as crude enzyme or enzyme preparation, it is 0.001 to 50 parts by weight, preferably 0.005 to 20 parts by weight, and more preferably 0.005 to 5 parts by weight, per 100 parts by weight of pH-adjusted extract.

[0038] Adjusting the above extract to a pH of 3.2 to 4.5 and reacting it with the above enzyme to obtain an enzyme reaction solution means that the above extract is adjusted to a pH of 3.2 to 4.5 and the above enzyme is present in the solution. The above enzyme may be added during pH adjustment, but it is preferable to add it after adjusting the pH to 3.2 to 4.5. The above enzyme may be added in a continuous or batch manner.

[0039] According to one embodiment of the present invention, the reaction time of the enzyme is the time from when cellobiohydrolase and xylanase are present at a pH of 3.2 to 4.5 until a clarified liquid is obtained by solid-liquid separation treatment. In the case of a continuous process, it is the residence time from when the enzyme is present at a pH of 3.2 to 4.5 until a clarified liquid is obtained by solid-liquid separation treatment. The reaction time of the enzyme at a pH of 3.2 to 4.5 is not particularly limited, but is preferably 5 minutes to 8 hours, more preferably 5 minutes to 6 hours, even more preferably 5 minutes to 4 hours, even more preferably 10 minutes to 4 hours, and especially preferably 10 minutes to 2 hours.

[0040] The reaction temperature of the above enzymes can be appropriately changed depending on 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.

[0041] <Process (4)> According to one embodiment of the present invention, in step (4), the enzyme reaction solution obtained in step (3) is subjected to coarse filtration to obtain a clarified solution.

[0042] Coarse filtration includes, but is not limited to, filtration with woven fabric and non-woven fabric, but filtration with woven fabric is preferred. Apparatus for filtration by coarse filtration includes, but is not limited to, belt presses, belt filters, and filter presses, but a filter press is preferred.

[0043] When filtration is performed by coarse filtration, a filter aid may be used. Examples of filter aids include, but are not limited to, diatomaceous earth, perlite, cellulose, and activated carbon, although diatomaceous earth is preferred. The filter aid can be added from the step of obtaining the extract to the step of filtering the enzyme reaction solution to obtain the clarified solution, and the timing of addition is not particularly limited. The amount of 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.

[0044] <Process (5)> According to one embodiment of the present invention, in step (5), the clarified liquid obtained in step (4) is passed through a column packed with a synthetic adsorbent made of an aromatic resin that has been specially treated to increase its specific surface area, and the components adsorbed on the aromatic synthetic adsorbent are eluted with a mixed solvent of ethanol and water to obtain a polyphenol-containing composition from the eluted fraction.

[0045] The aromatic resin constituting the above-mentioned 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 aromatic resins are preferably used. Examples of styrene-divinylbenzene aromatic resins include porous resins such as aromatic resins having hydrophobic substituents, unsubstituted aromatic resins, and unsubstituted aromatic resins that have undergone special treatment.

[0046] From the viewpoint of improving the adsorption rate, the specific surface area of ​​the above aromatic synthetic adsorbent is preferably 500 m² as a dry mass. 2 It is 700m or more / g, and more preferably 700m 2 The value is 1 / g or more. The specific surface area of ​​the aromatic synthetic adsorbent can be calculated by applying the measured value by the gas adsorption method to the BET formula. The most frequent pore diameter (mode pore size) of the aromatic synthetic adsorbent is preferably 600 angstroms or less, more preferably 300 angstroms or less, and even more preferably 200 angstroms or less, from the viewpoint of high separation and high adsorption. The most frequent pore diameter can be measured by the gas adsorption method.

[0047] Such synthetic adsorbents are commercially available, for example, Diaion (trademark) HP-10, HP-20, HP-21, HP-30, HP-40, HP-50 (all are substituent-free aromatic resins, all are trade names, manufactured by Mitsubishi Chemical Corporation); SP-825, SP-800, SP-850, SP-875, SP-70, SP-700 (all are substituent-free aromatic resins that have undergone special treatment, all are trade names, manufactured by Mitsubishi Chemical Corporation); SP-900 (aromatic resin, trade name, manufactured by Mitsubishi Chemical Corporation). Examples include: Amberlite (trademark) XAD-2, XAD-4, XAD-16, XAD-18, XAD-2000 (all aromatic resins, all trade names, manufactured by Organo Corporation); Diaion (trademark) SP-205, SP-206, SP-207 (all aromatic resins having hydrophobic substituents, all trade names, manufactured by Mitsubishi Chemical Corporation); HP-2MG, EX-0021 (all aromatic resins having hydrophobic substituents, all trade names, manufactured by Mitsubishi Chemical Corporation). Among these, Diaion (trademark) SP-850 is preferred. These synthetic adsorbents may be used individually or in combination of two or more.

[0048] The amount of aromatic synthetic adsorbent packed into the above column can be appropriately determined depending on the size of the column, the type of synthetic adsorbent, etc.

[0049] When the clarified liquid is passed through the column, the temperature of the filtrate may be between 25 and 45°C. The flow rate and flow rate of the filtrate through the column can be appropriately determined depending on the type of aromatic synthetic adsorbent, etc.

[0050] The mixed volume ratio (ethanol / water) of the above ethanol and water mixed solvent may be 50 / 50 to 99 / 1, and from the viewpoint of improving elution efficiency, it is preferably in the range of 50 / 50 to 70 / 30. The elution rate can be appropriately determined depending on the size of the column, the type of aromatic synthetic adsorbent, etc. 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.

[0051] The polyphenol-containing composition obtained as an eluted fraction may be concentrated as needed. Concentration can be performed, for example, by using a centrifugal thin-film vacuum evaporator to a concentration of 5 to 20 times. This will yield a concentrated solution containing the polyphenol-containing composition.

[0052] The polyphenol-containing composition obtained by the above method can be suitably used as a food ingredient, especially since it is obtained by using an aromatic synthetic adsorbent in the elution step and eluting it using a mixed solvent of ethanol and water. [Examples]

[0053] The present invention will be described in detail 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 shall conform to the provisions of the Japanese Industrial Standards (JIS).

[0054] (Reference Example 1) Measurement of Protein Concentration The protein concentration in the aqueous solution was measured using a Bradford assay kit (Quick Start Bradford Protein Assay, Bio-Rad).

[0055] (Reference Example 2) Measurement of cellobiohydrolase activity A substrate solution was prepared by dissolving 4-nitrophenyl-β-D-lactopyranoside (Sigma-Aldrich) in 50 mM sodium acetate buffer (pH 5.0) to a concentration of 1 mM. 10 μL of enzyme solution, appropriately diluted, was added to 90 μL of 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 and allow the liberated 4-nitrophenol to develop color. The absorbance was measured at 405 nm. A blank sample was prepared using the same method except that the enzyme solution was added after the sodium carbonate solution. One unit of enzyme (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 gram of protein is calculated using the following formula.

[0056] Cellobiohydrolase activity per enzyme solution [U / mL] = (4-nitrophenol [μmol / mL] × reaction solution volume [μL]) / (reaction time [min] × enzyme solution volume [μL]) × dilution factor (times)

[0057] Cellobiohydrolase activity per gram of enzyme protein [U / g] = (Activity per enzyme solution [U / mL] / Protein concentration of enzyme solution [g / L]) × 1000

[0058] (Reference Example 3) Measurement of xylanase activity The analysis was performed using the Megazyme Xylanase Analysis Kit (XylX6 method). 2.5 μL of 1 M 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 were mixed. 10 μL of enzyme solution, diluted as appropriate, was added, and the mixture was allowed to stand at 30°C. After 10 minutes, 100 μL of sodium carbonate solution was added to stop the reaction and allow the liberated 4-nitrophenol to develop color. The absorbance was measured at 405 nm. A blank sample was prepared using the same method except that the enzyme solution was added after the sodium carbonate solution. One unit of enzyme (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 gram of protein is calculated using the following formula.

[0059] Xylanase activity per enzyme solution [U / mL] = (4-nitrophenol [μmol / mL] × reaction solution volume [μL]) / (reaction time [min] × enzyme solution volume [μL]) × dilution factor (times)

[0060] Xylanase activity per gram of enzyme protein [U / g] = (Activity per enzyme solution [U / mL] / Protein concentration of enzyme solution [g / L]) × 1000

[0061] (Reference Example 4) Measurement of Polyphenol Content The amount of polyphenols was measured using the Forin-Ciocalt method under the following conditions: 1.0 mL of the sample, diluted appropriately, 1.0 mL of phenol reagent (Nacalai Tesque), and 5 mL of water were placed in a 25 mL volumetric flask and left at room temperature for 5 minutes. Then, 10 mL of 7% sodium carbonate aqueous solution was added. Further water was added to make a total volume of 25 mL, and the mixture was mixed and left at room temperature for 2 hours. A portion of the reaction solution was taken and filtered through a φ0.45 μm PTFE filter, and the absorbance was measured at 750 nm (the sample was appropriately diluted so that the absorbance was 0.6 ABS or less). Catechin reagent (Sigma-Ace, purity 98% or higher) was used as a standard substance, and the value was calculated as a catechin equivalent.

[0062] (Reference Example 5) Preparation of alkaline extract from bagasse Bagasse, the residue from sugarcane extraction, was added and mixed at a dry weight of 5 wt% to a 0.45 (wt / wt)% sodium hydroxide aqueous solution. The mixture was reacted at 90°C for 3 hours, and the solid and liquid components were separated to obtain an alkaline extract as the liquid component.

[0063] (Comparative Examples 1-3) Temperature and Liquid Properties during Neutralization The alkaline extract of bagasse prepared according to Reference Example 5 was cooled to 38°C (Comparative Example 1), 45°C (Comparative Example 2), and 60°C (Comparative Example 3). Diatomaceous earth was added at a rate of 1 part by weight per 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, the micellar-like phenomenon was evaluated using the following indicators: ++: Micellarization (the liquid becomes cloudy and no precipitate forms), +: Partial micellization (the turbidity does not precipitate), -: No micellization (the turbidity precipitates immediately).

[0064] The pH-adjusted extract was subjected to solid-liquid separation using a filter press. A small filtration device, model MO-4, manufactured by Yabuta Sangyo Co., Ltd., was used as the filter press. The volume of filtrate was measured after 5 minutes. 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, the clogging of the resin column was evaluated as follows: ++: clogging occurred and liquid flow was impossible, +: flow rate decreased but the entire volume could be passed, -: no clogging occurred and the entire volume could be passed at a flow rate of 7.6 L / h.

[0065] Subsequently, the synthetic adsorbent was washed with 10 times its volume of water, and eluted by passing a 60% (v / v) ethanol aqueous solution at SV=2 to obtain the eluted fraction. The amount of polyphenols in the eluted fraction was measured according to Reference Example 4, and the amount of polyphenols (%) was determined by setting the amount of polyphenols in Comparative Example 1 as 100%.

[0066] The results are shown in Table 1. When the neutralization temperature was high, a micellar-like phenomenon occurred, and the amount of filtrate after 5 minutes of filter pressing was also low. When micellar occurred, clogging of the resin column also occurred. In addition, the amount of polyphenols in the eluted fraction was lower in those where micellar occurred.

[0067] [Table 1]

[0068] (Examples 1 and 2) Effects of enzyme addition The alkaline extract of bagasse prepared according to Reference Example 5 was cooled to 45°C (Example 1) and 60°C (Example 2). Diatomaceous earth was added to 1 part by weight per 100 parts by weight of the alkaline extract, and the pH was adjusted to 3.5 using 35% (w / w) hydrochloric acid. Then, enzyme 1 (with 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) per 100 parts by weight of the pH-adjusted extract. After addition, the mixture was reacted at 50°C for 1 hour with stirring. At this time, the micellar-like phenomenon was evaluated using the following indicators: ++: micellar (liquid becomes cloudy, and no precipitate forms), +: partial micellar (turbidity does not precipitate), -: no micellar (turbidity precipitates immediately).

[0069] Furthermore, the cellobiohydrolase and xylanase activities of the enzymes were measured according to Reference Examples 2 and 3. The protein concentration used was the value measured according to Reference Example 1.

[0070] The extract after the enzymatic reaction was subjected to solid-liquid separation using a filter press. A small filtration device, model MO-4, manufactured by Yabuta Sangyo Co., Ltd., was used as the filter press. The volume of filtrate was measured after 5 minutes.

[0071] 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, the clogging of the resin column was evaluated as follows: ++: clogging occurred and liquid flow was impossible, +: flow rate decreased but the entire volume could be passed, -: no clogging occurred and the entire volume could be passed at a flow rate of 7.6 L / h.

[0072] Subsequently, the synthetic adsorbent was washed with 10 times its volume of water, and eluted by passing a 60% (v / v) ethanol aqueous solution at SV=2 to obtain the eluted fraction. The amount of polyphenols in the eluted fraction was measured according to Reference Example 4, and the amount of polyphenols (%) was determined by setting the amount of polyphenols in Comparative Example 1 as 100%.

[0073] The results are shown in Table 2. By reacting with the enzyme, micellization-like phenomena did not occur, the amount of filtrate after 5 minutes of filter pressing was increased, and the filtration efficiency was improved. Furthermore, clogging of the resin column did not occur, and the polyphenol content also improved.

[0074] [Table 2]

[0075] (Comparative Examples 4 and 5) Differences due to enzymes The alkaline extract of bagasse prepared according to Reference Example 5 was cooled to 45°C (Comparative Example 4) and 60°C (Comparative Example 5). Diatomaceous earth was added at a rate of 1 part by weight per 100 parts by weight of the alkaline extract, and the pH was adjusted to 3.5 using 35% (w / w) hydrochloric acid. Then, enzyme 2 (with cellobiohydrase activity, without xylanase activity): "Pectinase from Aspergillus aculeatus" (liquid, manufactured by Sigma-Aldrich) and enzyme 3 (with cellobiohydrase activity, without xylanase activity): "Cellulosin GM5" (powder, manufactured by HBI) were added. The amount added was 0.1 parts by weight per 100 parts by weight of the pH-adjusted extract. After addition, the mixture was reacted at 50°C for 1 hour while stirring. In this study, the micellization-like phenomenon was evaluated using the following indicators: ++: Micellization (the liquid becomes cloudy and no precipitate forms), +: Partial micellization (the turbidity does not precipitate), and -: No micellization (the turbidity precipitates immediately). The cellobiohydrolase activity and xylanase activity of the enzymes were measured according to Reference Examples 2 and 3. The protein concentration used was the value measured according to Reference Example 1. The extract after the enzymatic reaction was subjected to solid-liquid separation by filter press, as in Comparative Examples 1 and 2.

[0076] The results are shown in Table 3. Enzymes 2 and 3 resulted in micelle formation, and the filtrate volume after 5 minutes in the filter press was also low.

[0077] [Table 3]

[0078] (Comparative Example 6, and Examples 3-5) Differences due to pH The micellation was evaluated in the same manner as in Example 1, except that the pH was set to 3.0 (Comparative Example 6), 3.3 (Example 3), 4.0 (Example 4), and 4.5 (Example 5). 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, micellation occurred after the enzymatic reaction.

[0079] [Table 4]

Claims

1. A method for producing a polyphenol-containing composition derived from herbaceous biomass, (1) A step of obtaining an extract by contacting herbaceous biomass with an alkaline aqueous solution at 60°C or higher and separating the herbaceous biomass and the alkaline aqueous solution using solid-liquid separation. (2) A step of cooling the extract obtained in step (1) to 60°C or below to obtain a cooled extract, (3) Adjust the pH of the cooled extract obtained in step (2) to 3.2 or higher and 4.5 or lower, add an enzyme solution containing an enzyme having cellobiohydrolase activity and xylanase activity, and react with the enzyme to obtain an enzyme reaction solution. (4) A step to obtain a clarified solution by coarse filtration of the enzyme reaction solution obtained in step (3), A method for producing a polyphenol-containing composition, comprising the steps of: (5) passing the clarified liquid obtained in step (4) through a column packed with a synthetic adsorbent made of an aromatic resin that has been specially treated to increase its specific surface area, and eluting the components adsorbed on the synthetic adsorbent made of the aromatic resin with a mixed solvent of ethanol and water to obtain the eluted fraction as a polyphenol-containing composition.

2. A method for producing a polyphenol-containing composition according to claim 1, wherein the enzyme comprising the cellobiohydrolase activity and xylanase activity is derived from a microorganism of the genus Trichoderma.

3. A method for producing a polyphenol-containing composition according to claim 1 or 2, wherein the herbaceous biomass is bagasse.

4. A method for producing a polyphenol-containing composition according to any one of claims 1 to 3, wherein the alkaline aqueous solution is an aqueous solution of sodium hydroxide.

5. A method for producing the polyphenol-containing composition according to claim 4, wherein the concentration of the sodium hydroxide is 0.1 to 10% by weight.

6. The method for producing polyphenols according to any one of claims 1 to 5, wherein the aromatic resin is a styrene-divinylbenzene resin.

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