Method for producing 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 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; (2) a step in which the pH of the extraction liquid obtained in step (1) is adjusted to a pH of 3.2-4.5, and the extraction liquid is reacted with an enzyme having cellobiohydrolase activity and xylanase activity to obtain an enzyme-reacted solution; and (3) a step in which a clarified liquid containing a polyphenol-containing composition is obtained by performing solid-liquid separation of the enzyme-reacted solution obtained in step (2).

Description

Method for producing polyphenol-containing composition REFERENCE TO RELATED APPLICATIONS

[0001] This patent application claims priority to Japanese Patent Application No. 2021-58651, filed on March 30, 2021, the entire disclosure of which is incorporated herein by reference.

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

[0003] In recent years, the use of biomass has been attracting attention due to issues such as global warming, the depletion of petroleum resources, and from the perspective of carbon neutrality. One of the methods for obtaining polyphenol compositions from cellulose-containing biomass is one that does not compete with food.

[0004] Cellulose-containing biomass is mainly composed of the polysaccharides cellulose and hemicellulose, and the aromatic polymer lignin. By decomposing the lignin in the cellulose-containing biomass and the connections between the lignin and the polysaccharides, a decomposition liquid containing polyphenols can be obtained.

[0005] For example, Patent Document 1 describes a method for efficiently obtaining hydroxycinnamic acids by passing an alkaline aqueous medium through cellulose-containing biomass, and Patent Document 2 describes a method for recovering acid-insoluble lignin from an alkaline extract of a plant. Furthermore, Patent Document 3 describes a method for efficiently producing a polyphenol composition by treating bagasse, the residue of sugarcane, with an alkaline solution to obtain an extract, adjusting the extract to an acidic state, filtering the extract, and adsorbing the filtrate with an aromatic synthetic adsorbent.

[0006] Conventionally, such alkali treatment of cellulose-containing biomass has been carried out as a pretreatment to facilitate the production of sugar solution from cellulose-containing biomass, and the liquid fraction after alkali treatment has been discarded. However, as described above, because it contains polyphenols, there is a need for effective utilization of the liquid fraction.

[0007] The polyphenol-containing hydrolyzed liquid obtained by the above-mentioned conventional techniques is known to be used as a deodorizer (Patent Document 4), a food discoloration inhibitor (Patent Document 5), a growth promoter for aquatic organisms (Patent Document 6), etc. Furthermore, it is known that representative polyphenols contained therein include coumaric acid and ferulic acid.

[0008] WO2017 / 170549, WO2012 / 120184, WO2019 / 230803, JP2020-93080A, WO2018 / 079640, WO2018 / 079641

[0009] As described above, a conventional method for efficiently producing a polyphenol-containing composition from herbaceous biomass involves treating herbaceous biomass with an alkaline solution, adjusting the pretreatment liquid to an acidic state, and then subjecting the liquid to solid-liquid separation. However, the inventors' investigations have revealed that this method can result in poor solid-liquid separability or the occurrence of a micellization-like phenomenon, making solid-liquid separation impossible. Furthermore, the inventors' investigations have revealed that membrane separation treatments, which are used for purifying or concentrating the liquid after solid-liquid separation, can also result in poor filterability.

[0010] Therefore, one object of the present invention is to provide a new technical means for efficiently producing a polyphenol-containing composition from herbaceous biomass.

[0011] As a result of extensive research, the present inventors have discovered that a polyphenol-containing composition can be efficiently produced from herbaceous biomass by treating herbaceous biomass with alkali, adjusting the pH of the extract, and reacting it with a specific enzyme, thereby completing the present invention.

[0012] That is, according to one embodiment of the present invention, the following [1] to

[13] are provided: [1] A method for producing a polyphenol-containing composition derived from herbaceous biomass, comprising: (1) contacting herbaceous biomass with an alkaline aqueous solution to obtain an extract; (2) adjusting the pH of the extract obtained in step (1) to between 3.2 and 4.5, and reacting the extract with an enzyme having cellobiohydrolase activity and xylanase activity to obtain an enzymatic reaction solution; and (3) performing solid-liquid separation on the enzymatic reaction solution obtained in step (2) to obtain a clarified liquid containing the polyphenol-containing composition. [2] A method for producing a polyphenol-containing composition according to [1], wherein the extract obtained in step (1) is adjusted to a pH of between 3.5 and 4.0. [3] A method for producing a polyphenol-containing composition according to [1] or [2], wherein the alkaline aqueous solution used in step (1) is at least 60°C. [4] The method for producing a polyphenol-containing composition according to any one of [1] to [3], further comprising step (4) of filtering the clarified solution obtained in step (3) through one or more separation membranes selected from the group consisting of microfiltration membranes, ultrafiltration membranes, nanofiltration membranes, and reverse osmosis membranes to obtain a permeate and / or retentate containing the polyphenol-containing composition. [5] The method for producing a polyphenol-containing composition according to [4], wherein the separation membrane is a microfiltration membrane and a permeate containing the polyphenol-containing composition is obtained. [6] The method for producing a polyphenol-containing composition according to [4] or [5], wherein the permeate obtained by filtering the clarified solution through a microfiltration membrane in step (4) is filtered through one or more separation membranes selected from the group consisting of ultrafiltration membranes, nanofiltration membranes, and reverse osmosis membranes to recover a retentate containing the polyphenol-containing composition. [7] The method for producing a polyphenol-containing composition according to any one of [4] to [6], wherein the clarified solution or the permeate obtained by filtering through a microfiltration membrane in step (4) is filtered through a nanofiltration membrane to obtain a permeate containing the polyphenol-containing composition. [8] The method for producing a polyphenol-containing composition according to [7], wherein the molecular weight cutoff of the nanofiltration membrane is 300 to 1000. [9] The method for producing a polyphenol-containing composition according to any one of [1] to [8], wherein the reaction time of the enzyme in step (2) is 10 minutes to 2 hours.

[10] A method for producing a polyphenol-containing composition according to any one of [1] to [9], wherein the herbaceous biomass is bagasse.

[11] A method for producing a polyphenol-containing composition according to any one of [1] to

[10] , wherein the enzyme is derived from a microorganism of the genus Trichoderma.

[12] A polyphenol-containing composition comprising coumaric acid and xylose, wherein the xylose content relative to the coumaric acid is 1 to 200% (w / w).

[13] The polyphenol-containing composition according to

[12] , wherein the xylose content relative to the coumaric acid is 2 to 50% (w / w).

[0013] According to the present invention, a polyphenol-containing composition can be efficiently produced from herbaceous biomass. Furthermore, according to the present invention, when producing a polyphenol-containing composition from herbaceous biomass, a micellization-like phenomenon can be prevented and the efficiency of solid-liquid separation can be improved. Furthermore, according to the present invention, the filterability of the product can be improved in the separation membrane treatment after the solid-liquid separation.

[0014] According to one embodiment of the present invention, a method for producing a polyphenol-containing composition derived from herbaceous biomass includes the steps of: (1) contacting herbaceous biomass with an alkaline aqueous solution to obtain an extract; (2) adjusting the pH of the extract obtained in step (1) to between 3.2 and 4.5, and reacting the extract with an enzyme having cellobiohydrolase activity and xylanase activity to obtain an enzyme reaction solution; and (3) performing solid-liquid separation of the enzyme reaction solution obtained in step (2) to obtain a clarified liquid containing the polyphenol-containing composition.

[0015] DETAILED DESCRIPTION OF THE INVENTION The following describes an embodiment of the present invention. <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.

[0016] According to one embodiment of the present invention, examples of herbaceous biomass include, but are not limited to, bagasse (sugarcane pomace), switchgrass, napier grass, erianthus, corn stover, corn husks, wheat husks, soybean husks, rice straw, wheat straw, and oil palm empty fruit bunches. From the perspective 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 preferred, with bagasse being more preferred. The lignin content can be determined by measuring Klason lignin, which is the residue obtained by subtracting ash from the acid hydrolysis residue.

[0017] 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 method is not particularly limited, and it can be carried out using machines commonly used for coarse pulverization of various materials, such as a ball mill, vibration mill, cutter mill, hammer mill, Willey mill, or jet mill. This mechanical pulverization may be either dry or wet, but dry pulverization is preferred.

[0018] According to one embodiment of the present invention, the moisture content of the herbaceous biomass is not particularly limited, but preferred ranges are, for example, about 3% or more, about 3% to about 75%, about 5% or more, about 5% to about 70%, about 5% to about 65%, and about 5% to about 55%.

[0019] 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. They can be measured, for example, by the Folin-Ciocalteu method. The Folin-Ciocalteu method was originally developed for the analysis of aromatic amino acids such as tyrosine and tryptophan, as well as proteins containing these. The method involves colorimetric quantification of the blue color produced by the reduction of phosphotungstic acid and molybdic acid in alkaline conditions using phenolic hydroxyl groups at 700-770 nm. Similar procedures can be performed using specific reference substances such as gallic acid and catechin, and quantitative values ​​can be expressed in terms of the corresponding compounds. In the present invention, values ​​in terms of catechin are used.

[0020] According to one embodiment of the present invention, the alkaline aqueous solution may be an alkaline aqueous solution containing at least one selected from ammonia, ammonia water, alkali metal hydroxides, alkali metal oxides, alkaline earth metal oxides, alkali metal carbonates, alkaline earth metal carbonates, quaternary ammonium hydroxides, etc., but is preferably an aqueous medium containing at least one hydroxide selected from sodium hydroxide and potassium hydroxide, and more preferably an aqueous sodium hydroxide solution or an aqueous potassium hydroxide solution.

[0021] The alkali concentration of the alkaline aqueous solution is not particularly limited, but is preferably 0.05% by weight to 10% by weight, more preferably 0.05% by weight to 5% by weight, even more preferably 0.1% by weight to 5% by weight, even more preferably 0.1% by weight to 3% by weight, even more preferably 0.1% by weight to 2% by weight.

[0022] The lower limit of the pH of the alkaline aqueous solution is not particularly limited as long as it is alkaline, but 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. The preferred pH range is, for example, from 7 to 13.5, or from 8 to 13.5, more preferably from 9 to 13.5, and even more preferably from 10 to 12.

[0023] According to one embodiment of the present invention, the temperature when the alkaline aqueous solution is brought into contact with the herbaceous biomass is 60° C. or higher. Furthermore, in order to maintain the alkali-treated product at a temperature higher than 100° C., it is necessary to apply a pressure exceeding atmospheric pressure to the alkali-treated product, which requires high-pressure equipment. Therefore, from the standpoint of production costs, the temperature is preferably 60 to 100° C., more preferably 80 to 100° C., and even more preferably 60° C. or higher but lower than 100° C., or 80° C. or higher but lower than 100° C.

[0024] Furthermore, 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.

[0025] The method for contacting herbaceous biomass with the alkaline aqueous solution is not particularly limited, but examples include methods of contacting the herbaceous biomass by spraying, immersing, or passing the alkaline aqueous solution over the herbaceous biomass, and during this process, the alkaline aqueous solution may be stirred or the container may be rotated to ensure sufficient contact between the herbaceous biomass and the alkaline aqueous solution.

[0026] The contact time between the alkaline aqueous solution and the herbaceous biomass is not particularly limited, but is preferably from about 20 minutes to about 72 hours, from about 20 minutes to about 48 hours, from about 20 minutes to about 24 hours, from about 30 minutes to about 48 hours, from about 30 minutes to about 24 hours, from about 30 minutes to about 12 hours, from about 30 minutes to about 6 hours, or from about 30 minutes to about 3 hours.

[0027] An extract can be obtained by subjecting herbaceous biomass and an alkaline aqueous solution to solid-liquid separation. Examples of solid-liquid separators include a screw press and a centrifuge. A strainer or the like may be used to remove fine particles. Furthermore, when the alkaline aqueous solution is passed through the herbaceous biomass during contact with the alkaline aqueous solution, the liquid after passing through may be used as the extract as is, but from the viewpoint of recovering the extract, it is preferable to squeeze the reaction product of the herbaceous biomass using a solid-liquid separator.

[0028] <Step (2)> According to one embodiment of the present invention, in step (2), the extract obtained in step (1) is adjusted to a pH of 3.2 to 4.5, and reacted with an enzyme having cellobiohydrolase activity and xylanase activity to obtain an enzyme reaction solution.

[0029] 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.

[0030] 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. Preferred are hydrochloric acid, sulfuric acid, and nitric acid, and more preferred are hydrochloric acid.

[0031] The method for adjusting the pH using an acidic substance is not particularly limited, but examples include a method in which an acidic substance of an appropriate concentration is appropriately added and mixed while checking the pH. A continuous method in which an alkaline extract is continuously added during the pH adjustment and the liquid after the pH adjustment is continuously withdrawn may be used, or a batch method may be used.

[0032] The temperature during the pH adjustment is not particularly limited, but is preferably 20°C or higher and 100°C or lower, more preferably 20°C or higher and 60°C or lower, and more preferably 30°C or higher and 60°C or lower.

[0033] The pH is usually adjusted to a range of 3.2 to 4.5, preferably 3.3 to 4.5, more preferably 3.3 to less than 4.0, more preferably 3.5 to 4.0, and even more preferably 3.5 to less than 4.0.

[0034] An enzyme having cellobiohydrolase activity is an exoenzyme that degrades cellulose chains with β-1,4-linked glucose from the terminal to produce cellobiose. Cellobiohydrolase activity can be measured as the enzyme activity for degrading 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 according to the procedure described in Reference Example 2 below. In the present invention, an enzyme having cellobiohydrolase activity of 5 U / g or more is defined as an enzyme having cellobiohydrolase activity, and the cellobiohydrolase activity value of the enzyme used in the present invention is preferably 5 to 1,000 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.

[0035] An enzyme with xylanase activity is an endo-enzyme that randomly degrades xylans in which xylose is β-1,4-linked. Xylanase activity can be determined by measuring the amount of reducing sugars contained in the reaction solution after the reaction using commercially available xylan (e.g., Birchwood xylan) as a substrate. However, it is preferable to use Megazyme's "Xylanase Analysis Kit (XylX6 Method)." With the "Xylanase Analysis Kit (XylX6 Method)," the XylX6 reagent is decomposed by a combination of the xylanase being measured 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 using a method similar to that described in Reference Example 3 below. In the present 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 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.

[0036] According to one embodiment of the present invention, the enzymes are produced by microorganisms, and may be produced by, for example, a single microorganism or multiple microorganisms. Examples of microorganisms that produce cellobiohydrolase and xylanase include microorganisms of the genus Trichoderma, Aspergillus, Cellulomonas, Clostridium, Streptomyces, Humicola, Acremonium, Irpex, Mucor, and Talaromyces, with the genus Trichoderma being preferred.

[0037] The Trichoderma microorganism is not particularly limited, but Trichoderma reesei is preferred, and specifically, Trichoderma reesei QM9414, Trichoderma reesei QM9123, Trichoderma reesei Rut C-30, Trichoderma reesei PC3-7, Trichoderma reesei CL-847, Trichoderma reesei MCG7 ... Examples of the microorganisms include Trichoderma reesei MCG77, Trichoderma reesei MCG80, and Trichoderma viride QM9123. The microorganisms may also be mutant strains of the above-mentioned microorganisms derived from the genus Trichoderma that have been subjected to a mutation treatment using a mutagen or ultraviolet irradiation, etc., to improve cellulase productivity.

[0038] According to one embodiment of the present invention, as long as the enzyme satisfies the above activity, it may be added in the form of a purified enzyme, or the culture solution may be added as a crude enzyme, or a commercially available cellulase or xylanase preparation may be used, or an enzyme other than cellobiohydrolase or xylanase, such as β-glucosidase, β-xylosidase, endoglucanase, or mannanase, may be included.

[0039] Examples of commercially available cellulase and xylanase agents include Novozymes' "Cellic C-Tech" (registered trademark) and "Cellic C-Tech 2" (registered trademark), Danisco Japan's "Accelrace" (registered trademark) 1000, "Accelrace" (registered trademark) 1500, and "Accelrace" (registered trademark) Duet, Sigma-Aldrich's "Cellulase from Trichoderma reesei ATCC 26921," "Cellulase from Trichoderma viride," and "Cellulase from Trichoderma longibrachiatum," and HIBI's "Cellulosin TP25" and "Cellulosin HC100." However, these are not limited thereto.

[0040] The amount of the enzyme to be added may be changed appropriately depending on the enzyme to be added and is not particularly limited, but as a 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 the pH-adjusted extract.

[0041] "Adjusting the extract to a pH within the predetermined range and reacting with the enzyme to obtain an enzyme reaction solution" means preparing an enzyme reaction solution in which the extract is adjusted to a pH within the predetermined range and the enzyme is present in the solution, and although the enzyme may be added during the pH adjustment, it is preferable to add it after adjusting the pH to the predetermined range. The enzyme may be added continuously or batchwise.

[0042] According to one embodiment of the present invention, the enzyme reaction time refers to the time during which the enzyme is present at a pH within the predetermined range and a clear liquid is obtained by solid-liquid separation treatment, and in the case of a continuous system, it refers to the residence time during which the enzyme is present at a pH within the predetermined range and a clear liquid is obtained by solid-liquid separation treatment. The enzyme reaction time at a pH within the predetermined range 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, still more preferably 10 minutes to 4 hours, and particularly preferably 10 minutes to 2 hours.

[0043] The temperature at which the enzyme is reacted may 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.

[0044] <Step (3)> According to one embodiment of the present invention, in step (3), the enzyme reaction solution obtained in step (2) is subjected to solid-liquid separation to obtain a clear liquid containing a polyphenol-containing composition.

[0045] In the solid-liquid separation treatment of the above-mentioned enzyme reaction solution, the method of solid-liquid separation treatment is not particularly limited, and methods such as filtration, centrifugation, and sedimentation can be used, or a combination thereof can be used. As the solid-liquid separation device, a sedimentation device, a decanter-type centrifugal settler, a disc-type centrifugal settler, a screw press, a filter press, a belt press, a belt screen, etc. can be used, or a combination thereof can be used. Preferred are a filter press, a decanter-type centrifugal settler, and a disc-type centrifugal settler.

[0046] Depending on the solid-liquid separation method, a filter aid may be used. Examples of filter aids include, but are not limited to, diatomaceous earth, perlite, cellulose, activated carbon, etc., and diatomaceous earth is preferred. The filter aid may be added from the step of obtaining an extract to the step of performing solid-liquid separation of the enzyme reaction solution to obtain a 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.

[0047] <Step (4)> According to one embodiment of the present invention, the clarified liquid obtained in step (3) is filtered through one or more separation membranes selected from the group consisting of microfiltration membranes, ultrafiltration membranes, nanofiltration membranes, and reverse osmosis membranes to obtain a permeate and / or a retentate containing a polyphenol-containing composition.

[0048] According to a preferred embodiment of the present invention, in step (4), the permeate obtained by filtering the clarified liquid through a microfiltration membrane is filtered through one or more separation membranes selected from the group consisting of ultrafiltration membranes, nanofiltration membranes, and reverse osmosis membranes, and the retentate containing the polyphenol-containing composition is recovered.

[0049] In addition, according to a more preferred embodiment of the present invention, in step (4), the clarified liquid or the permeate obtained by filtering through a microfiltration membrane is filtered through a nanofiltration membrane to obtain a permeate containing a polyphenol-containing composition.

[0050] Hereinafter, the step of filtering the clarified liquid through one or more separation membranes selected from the group consisting of microfiltration membranes, ultrafiltration membranes, and nanofiltration membranes to recover a polyphenol-containing composition will be described.

[0051] A microfiltration membrane is a membrane with an average pore size of 0.01 μm to 5 mm, and is also abbreviated as a microfiltration membrane or MF membrane. In order to concentrate solids on the membrane surface and prevent clogging inside the membrane, the average pore size is preferably 0.45 μm or less, and more preferably 0.22 μm or less.

[0052] The material of the microfiltration membrane is not particularly limited, but examples thereof include celluloses, aromatic polyamides, polyvinyl alcohol, polysulfone, polyvinylidene fluoride, polyethylene, polyacrylonitrile, polypropylene, polycarbonate, polytetrafluoroethylene, ceramics, and metals.

[0053] Examples of the form of the microfiltration membrane include, but are not limited to, hollow fiber, tubular, flat membrane, and spiral types. The filtration method may be dead-end filtration, cross-flow filtration, constant pressure filtration, or constant flow rate filtration. When clogging of the microfiltration membrane occurs, backwashing may be performed by passing a cleaning liquid from the permeate side to the non-permeate side of the membrane, or air washing may be performed by supplying a gas to the non-permeate side of the membrane to peel off the cake formed on the membrane surface. Examples of cleaning liquids for backwashing include the filtrate of the microfiltration membrane, water, and chemical solutions.

[0054] An ultrafiltration membrane is a membrane with a molecular weight cutoff greater than 1,000 and less than 200,000, and is abbreviated as ultrafiltration or UF membrane. Here, the pore size of an ultrafiltration membrane is so small that it is difficult to measure the pore size on the membrane surface using an electron microscope or the like. Therefore, instead of the average pore size, the molecular weight cutoff is used as an indicator of the pore size. The molecular weight cutoff is described on page 92 of Membrane Science Experiment Series, Vol. III, Artificial Membrane, edited by the Membrane Society of Japan, edited by Kimura Takashi, Nakao Shinichi, Oya Haruhiko, and Nakagawa Tsutomu (1993, Kyoritsu Shuppan), as follows: "A plot of data with the molecular weight of the solute on the horizontal axis and the rejection rate on the vertical axis is called a molecular weight cutoff curve. The molecular weight at which the rejection rate is 90% is called the molecular weight cutoff of the membrane." This is an indicator of the membrane performance of an ultrafiltration membrane, and is well known to those skilled in the art.

[0055] The material of the ultrafiltration membrane is not particularly limited, but examples thereof include celluloses, aromatic polyamides, polyvinyl alcohol, polysulfone, polyvinylidene fluoride, polyethylene, polyacrylonitrile, polypropylene, polycarbonate, polytetrafluoroethylene, ceramics, and metals.

[0056] The material of the ultrafiltration membrane is not particularly limited, but examples thereof include celluloses, aromatic polyamides, polyvinyl alcohol, polysulfone, polyvinylidene fluoride, polyethylene, polyacrylonitrile, polypropylene, polycarbonate, polytetrafluoroethylene, ceramics, and metals.

[0057] The ultrafiltration membrane may be of a hollow fiber type, a tubular type, a flat membrane type, a spiral type, etc., but is not limited to these. The filtration method may be dead-end filtration, cross-flow filtration, constant pressure filtration, or constant flow rate filtration.

[0058] Nanofiltration membranes, also known as nanofilters (nanofiltration membranes, NF membranes), are generally defined as membranes that allow monovalent ions to pass through and block divalent ions. These membranes are thought to have tiny pores on the order of a few nanometers, and are primarily used to block microparticles, molecules, ions, salts, etc. in water.

[0059] The nanofiltration membrane may be made of a polymeric material such as cellulose acetate polymer, polyamide, polyester, polyimide, or vinyl polymer, or may be made of a plurality of membrane materials.

[0060] The nanofiltration membrane may have any of a hollow fiber, tubular, flat membrane, spiral wound, etc. The filtration method may be dead-end filtration, cross-flow filtration, constant pressure filtration, or constant flow rate filtration.

[0061] The molecular weight cutoff of the nanofiltration membrane is generally 100 to 1000, preferably 150 to 1000, and more preferably 300 to 1000. By filtering through a nanofiltration membrane with a molecular weight cutoff of 300 or more and 1000 or less, colored substances in the polyphenol components can be concentrated on the non-permeated side, and polyphenol components with reduced coloration can be obtained on the permeated side.

[0062] When the clarified liquid is filtered through one or more separation membranes selected from the group consisting of microfiltration membranes, ultrafiltration membranes, and nanofiltration membranes, the polyphenol-containing composition can be recovered as the permeate or non-permeate of the separation membrane. It is preferable to recover the polyphenol-containing composition as the permeate of the separation membrane. Furthermore, filtering the clarified liquid through a microfiltration membrane before filtering it through an ultrafiltration or nanofiltration membrane can prevent clogging of the ultrafiltration or nanofiltration membrane. Furthermore, components contained in the clarified liquid can be fractionated and / or purified by combining ultrafiltration and / or nanofiltration membranes with different pore sizes.

[0063] The process of filtering the clarified liquid through a reverse osmosis membrane to recover the polyphenol-containing composition will be described below.

[0064] Reverse osmosis membranes, also known as RO membranes, are generally defined as membranes with desalination capabilities, including for monovalent ions. These membranes are believed to have ultra-fine pores ranging from a few angstroms to a few nanometers in size and are primarily used to remove ionic components, such as in seawater desalination and ultrapure water production. Furthermore, they have a molecular weight cutoff of less than 100.

[0065] According to one embodiment of the present invention, the reverse osmosis membrane may be made of a polymeric material such as cellulose acetate polymer, polyamide, polyester, polyimide, or vinyl polymer, or may be made of a plurality of membrane materials.

[0066] The reverse osmosis membrane may be of a hollow fiber type, a tubular type, a flat membrane type, a spiral type, etc., but is not limited to these. The filtration method may be dead-end filtration, cross-flow filtration, constant pressure filtration, or constant flow rate filtration.

[0067] The clarified liquid can be filtered through a reverse osmosis membrane to concentrate the polyphenol components in the non-permeated side. Furthermore, depending on the type and operating conditions of the reverse osmosis membrane, impurities other than the polyphenol components can be removed in the permeated side, allowing the polyphenol components to be concentrated and purified. That is, when the clarified liquid is filtered through a reverse osmosis membrane, the polyphenol-containing composition is recovered as the non-permeated liquid of the separation membrane.

[0068] The clarified liquid may be filtered through one or more separation membranes selected from the group consisting of microfiltration membranes, ultrafiltration membranes, and nanofiltration membranes, and the resulting permeate may be filtered through a reverse osmosis membrane to recover the polyphenol-containing composition as a non-permeate. By filtering the permeate obtained by filtering through a microfiltration membrane and / or an ultrafiltration membrane through a reverse osmosis membrane, polyphenol components can be concentrated on the non-permeate side of the reverse osmosis membrane while preventing clogging of the reverse osmosis membrane. However, it is preferable to filter at least the permeate filtered through the microfiltration membrane through a reverse osmosis membrane to concentrate the polyphenol components on the non-permeate side of the reverse osmosis membrane. Alternatively, the polyphenol components may be fractionated or purified by filtering the permeate through a nanofiltration membrane through a reverse osmosis membrane.

[0069] <Polyphenol-Containing Composition> According to one embodiment of the present invention, a polyphenol-containing composition derived from herbaceous biomass can be obtained by the above-described production method. According to one embodiment of the present invention, the polyphenol-containing composition derived from herbaceous biomass is characterized by containing at least coumaric acid as a polyphenol component and further containing xylose as a component other than polyphenols. The composition ratio of these components is not particularly limited, but a xylose content (% (w / w)) relative to coumaric acid of preferably 1 to 200% (w / w), more preferably 2 to 100% (w / w), and even more preferably 2 to 50% (w / w) is effective in preventing discoloration over time. The degree of discoloration can be evaluated by absorbance at 550 nm; the lower the absorbance, the lower the degree of discoloration.

[0070] The polyphenol-containing composition may be in the form of a liquid or a powder, but a polyphenol-containing composition having a xylose content of 2 to 50% (w / w) relative to coumaric acid is preferably in the form of a powder, since it does not feel sticky when touched with the hand. The method for producing the powder is not particularly limited, but spray drying is preferred.

[0071] The present invention will be described in more detail below with reference to examples, but is not limited to these examples. Unless otherwise specified, the measurement methods and units described in this specification are in accordance with the provisions of the Japanese Industrial Standards (JIS).

[0072] 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, manufactured by Bio-Rad).

[0073] Reference Example 2: Measurement of cellobiohydrolase activity. 4-Nitrophenyl-β-D-lactopyranoside (Sigma-Aldrich) was dissolved in 50 mM sodium acetate buffer (pH 5.0) to a concentration of 1 mM to prepare a substrate solution. 10 μL of appropriately diluted enzyme solution was added to 90 μL of the substrate solution, and the mixture was allowed to react 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, and the absorbance was measured at 405 nm. A blank was prepared by the same reaction method except that the enzyme solution was added after the addition of the sodium carbonate solution. One unit (1 U) of enzyme is defined as the amount of enzyme required to produce 1 μmol of 4-nitrophenol per minute under the above reaction conditions, and the activity per 1 g of protein was calculated using the following formula:

[0074] Cellobiohydrolase activity per enzyme solution [U / mL] = (4-nitrophenol [µmol / mL] × reaction solution volume [µL]) / (reaction time [min] × enzyme solution volume [µL]) × dilution ratio (fold)

[0075] Cellobiohydrolase activity per 1 g of enzyme protein [U / g] = (activity per enzyme solution [U / mL] / protein concentration of enzyme solution [g / L]) × 1000

[0076] Reference Example 3: Measurement of Xylanase Activity Measurement 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, and 10 μL of appropriately diluted enzyme solution was added. 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, and the absorbance was measured at 405 nm. A blank was prepared by the same reaction method except that the enzyme solution was added after the addition of the sodium carbonate solution. One unit (1 U) of enzyme is defined as the amount of enzyme required to produce 1 μmol of 4-nitrophenol per minute under the above reaction conditions, and the activity per gram of protein was calculated using the following formula:

[0077] Xylanase activity per enzyme solution [U / mL] = (4-nitrophenol [μmol / mL] × reaction solution volume [μL]) / (reaction time [min] × enzyme solution volume [μL]) × dilution ratio (fold) Xylanase activity per 1 g of enzyme protein [U / g] = (activity per enzyme solution [U / mL] / protein concentration of enzyme solution [g / L]) × 1000

[0078] (Reference Example 4) Measurement of aromatic compounds The concentrations of aromatic compounds such as coumaric acid and ferulic acid were measured under the following conditions: Instrument: Hitachi high-performance liquid chromatogram LaChrom Eite Column: Synergi 2.5μ Hydro-RP100A 100×3.00 mm (Phenomenex) Mobile phase: 0.1% phosphoric acid:acetonitrile = gradient from 93:7 to 5:95 Detector: Diode Array Flow rate: 0.6 mL / min Temperature: 40°C

[0079] (Reference Example 5) Measurement of sugar concentration The xylose concentration was quantified by comparison with a standard sample under the following HPLC conditions: Column: AQUITY UPLC BEH Amide (Waters) Mobile phase A: 80% acetonitrile + 0.1% TFA Mobile phase B: 30% acetonitrile + 0.1% TFA Flow rate: 0.12 mL / min

[0080] The proportion of mobile phase B was gradually increased from 0 to 40% over 10 minutes, and at 10.01 minutes, only mobile phase A was used again, and analysis was continued until 20 minutes. Detection method: ELSD (Evaporative Light Scattering Detector) Temperature: 55°C

[0081] (Reference Example 6) Preparation of alkaline extract of bagasse Bagasse, which is sugarcane pomace, was added to and mixed with a 0.45 (wt / wt) % aqueous sodium hydroxide solution in an amount of 5 wt % on a dry basis, and the mixture was reacted at 90°C for 3 hours. The solid and liquid fractions were separated, and an alkaline extract was obtained as the liquid fraction.

[0082] (Reference Example 7) Measurement of turbidity The turbidity of the supernatant obtained by centrifuging the saccharified solution was measured using an indoor high-speed turbidity meter (2100N) manufactured by HACH.

[0083] (Comparative Examples 1 to 4, Examples 1 and 2) Solid-Liquid Separation by Sedimentation The alkaline extract of bagasse prepared in accordance with Reference Example 6 was adjusted to pH 3.5 with 35% (w / w) hydrochloric acid. Enzyme 1 (has cellobiohydrolase activity, has no xylanase activity): "Pectinase from Aspergillus aculeatus" (liquid, manufactured by Sigma-Aldrich), Enzyme 2 (has no cellobiohydrolase activity, has no xylanase activity): "Amyloglucosidase from Aspergillus aculeatus" (liquid, manufactured by Sigma-Aldrich), and Enzyme 3 (has no cellobiohydrolase activity, has no xylanase activity) were added. niger (liquid, manufactured by Sigma-Aldrich), enzyme 3 (has cellobiohydrolase activity, no xylanase activity): "Cellulosin GM5" (powder, manufactured by HI), enzyme 4 (has cellobiohydrolase activity and xylanase activity): "Cellulosin TP25" (powder, manufactured by HI), and enzyme 5 (has cellobiohydrolase activity and xylanase activity): "Accelrace Duet" (liquid, manufactured by Danisco Japan) were added. The amounts of enzymes added were 0.03 g per 30 g of extract adjusted to pH 3.5.

[0084] After addition, the mixture was reacted at 50°C for 1 hour with stirring. The mixture was then allowed to stand for 15 minutes, and the supernatant after precipitation was collected and its turbidity was measured according to Reference Example 7. The same procedure was also performed without adding the enzyme. If no turbid substance precipitated, 15 mL of the mixture was collected and its turbidity was measured. The cellobiohydrolase activity and xylanase activity of each enzyme were measured according to Reference Examples 2 and 3.

[0085] The results are shown in Table 1. In Comparative Examples 1 to 4, the turbid matter generated by pH adjustment did not settle, but instead became cloudy and micellized, resulting in a turbidity of 1000 or more, making measurement impossible. On the other hand, in Examples 1 and 2, the turbidity of the supernatant was significantly lower than in Comparative Examples 1 to 4, and solid-liquid separation by settling upon standing was improved.

[0086]

[0087] (Comparative Examples 5 and 6, Examples 3 to 6) Solid-liquid separation properties due to differences in pH The enzyme was added to the alkaline extract of bagasse in the same manner as in Example 1, except that the pH was adjusted to 3.0, 3.3, 3.8, 4.0, 4.5, or 5.0. The extract was allowed to stand for 15 minutes, and the supernatant after sedimentation was collected and measured for turbidity in the same manner as in Reference Example 7.

[0088] The results are shown in Table 2. As shown in Table 2, the turbidity of the supernatant decreased at pH 3.3 to 4.5, and solid-liquid separation by sedimentation upon standing improved.

[0089]

[0090] (Comparative Example 7, Examples 7 and 8) Solid-Liquid Separation Using a Filter Press Diatomaceous earth was added to the alkaline extract of bagasse prepared according to Reference Example 6 in an amount of 1 part by weight per 100 parts by weight of the alkaline extract, and the pH was adjusted to 3.5 and 4.5 using 35% (w / w) hydrochloric acid. To 3 L of the pH-adjusted extract, 3 mL of Enzyme 5 (having cellobiohydrolase activity and xylanase activity): "Accelrace Duet" (liquid, manufactured by Danisco Japan) was added, and the mixture was reacted at 50°C for 30 minutes with stirring. After the reaction, solid-liquid separation was performed using a filter press. A compact filtration device, MO-4, manufactured by Yabuta Sangyo Co., Ltd. was used as the filter press. Solid-liquid separation using a filter press was also performed using the same method, except that no enzyme was added to the alkaline treatment solution adjusted to pH 3.5. The results of a 5-minute treatment are shown in Table 3.

[0091] As shown in Table 3, in Examples 7 and 8, the amount of filtrate after 5 minutes was larger than in Comparative Example 7, and solid-liquid separation by filter press was improved. In addition, the amount of filtrate was larger in Example 7 than in Example 8, and solid-liquid separation by filter press was improved.

[0092]

[0093] (Comparative Examples 8 to 10, Example 9) Filtration Treatment Using a Microfiltration Membrane The alkaline extract of bagasse prepared in accordance with Reference Example 6 was adjusted to pH 3.5 and pH 5.0 using 35% (w / w) hydrochloric acid. To 30 mL of the pH-adjusted extract, 30 μL of Enzyme 5 (having cellobiohydrolase activity and xylanase activity): "Accelrace Duet" (liquid, manufactured by Danisco Japan) was added, and the mixture was allowed to react at 50° C. for 30 minutes with stirring.

[0094] After the reaction, the mixture was centrifuged at 1500 G for 10 minutes, and 2 mL of the supernatant was microfiltrated by centrifuging at 500 G for 1 minute using an Ultrafree-CL (MILLPORE) filter, and the amount of permeate was measured. Microfiltration was also performed under conditions without the addition of enzyme using the same method, except that no enzyme was added. The results are shown in Table 4.

[0095] As shown in Table 4, in Example 9, in which the enzyme was added to the extract adjusted to pH 3.5, the filterability through the microfiltration membrane was significantly improved compared to when the enzyme was not added. On the other hand, when the enzyme was added at pH 5.0, there was almost no change in the filterability through the microfiltration membrane.

[0096]

[0097] (Comparative Examples 11-15, Examples 10-14) Total circulation filtration treatment using an ultrafiltration membrane, a nanofiltration membrane, and a reverse osmosis membrane The alkaline extract of bagasse prepared according to Reference Example 6 was adjusted to pH 3.5 using 35% (w / w) hydrochloric acid. To 10 L of the pH-adjusted extract, 10 mL of enzyme 5 (having cellobiohydrolase activity and xylanase activity): "Accelrace Duet" (liquid, manufactured by Danisco Japan) was added, and the mixture was reacted at 50°C for 30 minutes with stirring. After the reaction, the mixture was allowed to stand, and the supernatant was obtained. The supernatant was filtered using a microfiltration membrane "SPV0.2" (manufactured by Synder, pore size 0.2 μm) at a membrane surface linear velocity of 20 cm / sec.

[0098] The permeate of the microfiltration membrane was filtered using, respectively, ultrafiltration membrane: "SPE50" (manufactured by Synder, molecular weight cutoff 50,000 Da), nanofiltration membrane 1: "SPE1" (manufactured by Synder, molecular weight cutoff 1,000 Da), nanofiltration membrane 2: "NFG" (manufactured by Synder, molecular weight cutoff 600-800 Da), nanofiltration membrane 3: "NFW" (manufactured by Synder, molecular weight cutoff 300-500 Da), reverse osmosis membrane: "BW60-1812-75" (obtained from a Filmtec module, salt rejection rate 99%). With all types of separation membranes, total circulation filtration was performed in which the filtrate was returned to the supply tank at a membrane surface linear velocity of 20 cm / sec and a filtration flux of 0.5 m / D, and the operating pressure at the start of filtration and the operating pressure 30 minutes after the start of filtration were measured.

[0099] In addition, filtration was also carried out using a microfiltration membrane under conditions in which no enzyme was added, using the same method, and the permeate of the microfiltration membrane was filtered using the same type of membrane.

[0100] The results are shown in Table 5. As shown in Table 5, the operating pressure at the start of filtration and after 30 minutes were lower in the samples with enzyme addition than in the samples without enzyme addition, and the addition of enzyme improved filterability in filtration using ultrafiltration membranes, nanofiltration membranes, and reverse osmosis membranes. Furthermore, although not shown in the table, the filtration rate in the microfiltration membrane samples with enzyme addition was about 10 times that of the samples without enzyme addition.

[0101]

[0102] (Comparative Examples 16-19, Examples 15-18) Filtration Treatment Using Microfiltration Membrane, Nanofiltration Membrane, and Reverse Osmosis Membrane The alkaline extract of bagasse prepared according to Reference Example 6 was adjusted to pH 3.5 and pH 5.0 using 35% (w / w) hydrochloric acid. To 10 L of the pH-adjusted extract, 10 mL of Enzyme 5 (having cellobiohydrolase activity and xylanase activity): "Accelrace Duet" (liquid, manufactured by Danisco Japan) was added, and the mixture was reacted at 50°C for 30 minutes with stirring. After the reaction, the mixture was allowed to stand, and the supernatant was obtained. The supernatant was filtered using a microfiltration membrane "SPV0.2" (manufactured by Synder, pore size 0.2 μm) at a membrane surface linear velocity of 20 cm / sec.

[0103] The permeates of the microfiltration membrane were filtered using nanofiltration membrane 2: "NFG" (manufactured by Synder, molecular weight cutoff 600-800 Da), nanofiltration membrane 3: "NFW" (manufactured by Synder, molecular weight cutoff 300-500 Da), and reverse osmosis membrane: "BW60-1812-75" (obtained from a module manufactured by Filmtec, salt rejection rate 99%). With all types of separation membranes, constant flow rate filtration was performed at a membrane surface linear velocity of 20 cm / sec and a filtration flux of 0.2 m / D, with the operating pressure set at an upper limit of 6 MPa. When the pressure reached 6 MPa, the system was switched to constant pressure filtration at 6 MPa and concentrated. In addition, the permeate of the microfiltration membrane was filtered using the same method except that no enzyme was added, and the permeate of the microfiltration membrane was filtered using the same type of membrane.

[0104] The permeate of the microfiltration membrane, the permeate of the nanofiltration membranes 2 and 3, and the non-permeate of the reverse osmosis membrane were visually evaluated for coloration and measured using a spectrophotometer to quantify the coloration. Measurements were performed immediately after preparation and 7 days after refrigerated storage. The absorbance of the liquid was measured using a spectrophotometer UV-1280 (manufactured by Shimadzu Corporation), using the wavelength of 550 nm, which was a characteristic peak for this color measurement difference, as an index.

[0105] The permeate from the microfiltration membrane, the permeate from nanofiltration membranes 2 and 3, and the retentate from the reverse osmosis membrane were powdered by spray drying. To evaluate the stickiness of the resulting powders, the powders were left on a laboratory desk for one hour, and then the stickiness of the powders was evaluated by pressing the powder with a finger and determining whether it adhered to the hand.

[0106] Furthermore, the concentrations of coumaric acid and xylose were measured for the permeate of the microfiltration membrane, the permeate of the nanofiltration membranes 2 and 3, and the retentate of the reverse osmosis membrane using the methods of Reference Examples 4 and 5, and the xylose content relative to coumaric acid (% (w / w)) was calculated according to the following formula.

[0107] Xylose content (%) relative to coumaric acid = xylose concentration (w / v) / coumaric acid concentration (w / v) × 100

[0108] The results are shown in Table 6. As shown in Table 6, by filtering through a nanofiltration membrane with a molecular weight cutoff of less than 1000, the permeate through the nanofiltration membrane was not visually colored, and the absorption amount measured by a spectrophotometer increased, indicating decolorization. Furthermore, the sample containing the enzyme showed a smaller change in absorption amount measured by a spectrophotometer after preparation than the sample containing no enzyme. Furthermore, the dried powder obtained with the enzyme added was not sticky when touched.

Claims

1. A method for producing a polyphenol-containing composition derived from herbaceous biomass, comprising: (1) contacting herbaceous biomass with an alkaline aqueous solution to obtain an extract; (2) adjusting the pH of the extract obtained in step (1) to 3.2 to 4.5 and reacting it with an enzyme having cellobiohydrolase activity and xylanase activity to obtain an enzyme reaction solution; (3) A step of subjecting the enzyme reaction solution obtained in step (2) to solid-liquid separation to obtain a clear liquid containing a polyphenol-containing composition; A method for producing a polyphenol-containing composition comprising the steps of:

2. The method for producing a polyphenol-containing composition according to claim 1, wherein the extract obtained in the step (1) is adjusted to a pH of 3.5 or more and less than 4.

0.

3. The method for producing a polyphenol-containing composition according to claim 1 or 2, wherein the alkaline aqueous solution used in step (1) has a temperature of 60° C. or higher.

4. The method for producing a polyphenol-containing composition according to any one of claims 1 to 3 further comprises a step (4) of filtering the clarified liquid obtained in the step (3) through one or more separation membranes selected from the group consisting of a microfiltration membrane, an ultrafiltration membrane, a nanofiltration membrane and a reverse osmosis membrane to obtain a permeate and / or a non-permeate containing the polyphenol-containing composition.

5. The method for producing a polyphenol-containing composition according to claim 4, wherein the separation membrane is a microfiltration membrane and a permeate containing the polyphenol-containing composition is obtained.

6. 6. The method for producing a polyphenol-containing composition according to claim 4 or 5, wherein the permeate obtained by filtering the clarified liquid through a microfiltration membrane in step (4) is filtered through one or more separation membranes selected from the group consisting of ultrafiltration membranes, nanofiltration membranes and reverse osmosis membranes, and a non-permeate containing the polyphenol-containing composition is recovered.

7. The method for producing a polyphenol-containing composition according to any one of claims 4 to 6, wherein the clarified liquid or the permeate obtained by filtering through a microfiltration membrane in the step (4) is filtered through a nanofiltration membrane to obtain a permeate containing a polyphenol-containing composition.

8. The method for producing a polyphenol-containing composition according to claim 7, wherein the molecular weight cut-off of the nanofiltration membrane is 300 to 1,000.

9. The method for producing a polyphenol-containing composition according to any one of claims 1 to 8, wherein the reaction time of the enzyme in the step (2) is from 10 minutes to 2 hours.

10. The method for producing a polyphenol-containing composition according to any one of claims 1 to 9, wherein the herbaceous biomass is bagasse.

11. The method for producing a polyphenol-containing composition according to any one of claims 1 to 10, wherein the enzyme is derived from a microorganism of the genus Trichoderma.

12. A polyphenol-containing composition which is an alkaline extract of herbaceous biomass, contains coumaric acid and xylose, and the xylose content relative to the coumaric acid is 1 to 200% (w / w).

13. The polyphenol-containing composition according to claim 12, wherein the xylose content relative to the coumaric acid is 2 to 50% (w / w).

14. The polyphenol-containing composition described in claim 12 or 13, wherein the alkaline extract of the herbaceous biomass is an enzyme-treated product of the alkaline extract, and the enzyme is an enzyme having cellobiohydrolase and xylanase activity.