Production method of sugar solution

Abstract

A production method of a sugar solution that includes the following steps (1) to (3) whereby a sugar solution containing concentrated sugar derived from a cassava pulp can be efficiently produced. Step (1): a step for enzymatically hydrolyzing a cassava pulp to obtain a saccharified liquid; Step (2): a step for filtering the saccharified liquid obtained in Step (1) through a separation membrane having a molecular weight cut off of more than 100,000 Da and not more than 300,000 Da to thereby block polymer components derived from the cassava pulp and having a weight-average molecular weight of 10,000-20,000 Da on the non-permeate side; and Step (3) for filtering the filtrate obtained in Step (2) through a nanofiltration membrane and / or a reverse osmosis membrane to recover the sugar solution from the non-permeate side.

Description

Sugar solution manufacturing method

[0001] The present invention relates to a method for producing a sugar solution by hydrolyzing cassava meal.

[0002] As an alternative to fossil fuels from an environmental perspective, the production of chemicals by fermentation using sugars produced from plant biomass is being considered, and the production of fuel ethanol using sugars obtained from plant biomass is being promoted in the United States, Brazil, Thailand, etc. However, because starch and sucrose are also food sources, from an ethical perspective, there is a demand to convert plant biomass to inedible parts.

[0003] Cassava meal is attracting attention as a raw material that is easier to handle than the inedible parts of plant biomass and does not compete with food. Cassava rhizomes contain a lot of starch and have long been used as a raw material for starch production, but cassava meal is the residue left after the starch is extracted from the cassava rhizomes. Cassava meal contains a lot of starch in addition to cellulose fiber, and although it can be used as animal feed, its usefulness is low and most of it is currently discarded.

[0004] As a method for producing sugars using inedible parts of plant biomass as a raw material, Patent Document 1 discloses a method in which a saccharified solution obtained by hydrolyzing cellulose-containing biomass is passed through a microfiltration membrane having an average pore size of 0.01 μm to 5 mm and / or an ultrafiltration membrane having a molecular weight cutoff of 1,000 to 200,000 to remove fine particles and polymeric components, and then filtered through a nanofiltration membrane and / or a reverse osmosis membrane, whereby a purified and concentrated sugar solution is recovered from the retentate side and fermentation inhibitors are removed from the permeate side.

[0005] Among methods for producing sugars using inedible parts of plant biomass as a raw material, Patent Document 2 describes a conventional technique using cassava meal as a raw material, which involves hydrolyzing a hot water treatment product obtained by hot water treatment of cassava meal so that the solid content concentration of the cassava meal is 10% by weight or less, and then separating the hydrolyzate into solid and liquid. The resulting liquid fraction is filtered through an ultrafiltration membrane with a molecular weight cutoff of 300 to 200,000 Da, and a saccharification enzyme is recovered from the retentate side and a saccharified solution is recovered from the permeate side, thereby producing a glucose sugar solution. Patent Document 3 discloses a method for producing a sugar solution, in which cassava meal is hydrolyzed with a saccharifying enzyme having polygalacturonic acid decomposition activity, the resulting saccharified solution is filtered through a separation membrane with a molecular cutoff of 2,000 to 100,000 Da, the saccharifying enzyme is recovered from the retentate side, the saccharified solution is recovered from the permeate side, and the recovered saccharified solution is further filtered through a separation membrane with a molecular cutoff of 150 to 1,000 Da, thereby separating and removing galacturonic acid, a fermentation inhibitor, from the retentate side, and a purified sugar solution is recovered from the permeate side.

[0006] WO2010 / 067785 WO2019 / 189650 WO2019 / 189651

[0007] The present inventors have newly discovered a problem in that when attempting to concentrate sugars derived from cassava meal by filtering the saccharified solution obtained by enzymatic hydrolysis of cassava meal through a nanofiltration membrane and / or a reverse osmosis membrane, clogging of the membrane occurs.

[0008] As a result of intensive research to solve the above-mentioned problems, the inventors discovered that the saccharified solution obtained by enzymatic hydrolysis of cassava meal contains high molecular weight components (weight average molecular weight 10,000 to 20,000 Da) derived from cassava meal, which become membrane-clogging components in the filtration process using nanofiltration membranes and / or reverse osmosis membranes to concentrate sugar, and further discovered that by filtering the saccharified solution through a separation membrane with a molecular weight cutoff of more than 100,000 Da and not more than 300,000 Da, the high molecular weight components derived from cassava meal can be prevented from reaching the non-permeated liquid side, thereby completing the present invention.

[0009] That is, the present invention comprises the following [1] to

[10] . [1] A method for producing a sugar solution, comprising the following steps (1) to (3): step (1): enzymatically hydrolyzing cassava meal to obtain a sugar solution, step (2): filtering the sugar solution obtained in step (1) through a separation membrane having a molecular weight cutoff of more than 100,000 Da and not more than 300,000 Da to block high molecular weight components derived from the cassava meal having a weight average molecular weight of 10,000 to 20,000 Da in the retentate side, and step (3): filtering the filtrate obtained in step (2) through a nanofiltration membrane and / or a reverse osmosis membrane to recover the sugar solution from the retentate side. [2] The method for producing a sugar solution according to [1], wherein the separation membrane used in step (2) has a molecular weight cutoff of more than 100,000 Da and not more than 200,000 Da. [3] The method for producing a sugar solution according to [1] or [2], wherein the separation membrane in step (2) is a hollow fiber membrane. [4] The method for producing a sugar solution according to any of [1] to [3], wherein the enzyme in step (1) has at least cellulase activity and / or amylase activity. [5] The method for producing a sugar solution according to any of [1] to [4], wherein step (1) is a step of solid-liquid separation of a saccharified solution obtained by enzymatic hydrolysis of cassava meal. [6] The method for producing a sugar solution according to [5], wherein the solid-liquid separation is press filtration. [7] The method for producing a sugar solution according to [5] or [6], wherein the turbidity of the saccharified solution obtained by solid-liquid separation in step (1) is 300 NTU or less. [8] A sugar solution containing glucose derived from cassava meal and a high molecular weight component derived from cassava meal having a weight-average molecular weight of 10,000 to 20,000 Da, wherein the peak area ratio of the high molecular weight component derived from cassava meal having a weight-average molecular weight of 10,000 to 20,000 Da is 0.1 to 2% of the total peak area in gel filtration chromatography analysis using a differential refractometer and pullulan as the standard substance. [9] The sugar solution according to [8], further containing a component derived from cassava meal of less than 10,000, wherein the peak area ratio of the component derived from cassava meal of less than 10,000 Da is 98 to 99.9% of the total peak area in gel filtration chromatography analysis using a differential refractometer and pullulan as the standard substance.

[10] The sugar solution according to [8] or [9], wherein the glucose concentration is 80 g / L or more.

[0010] According to the present invention, by filtering a saccharified liquid obtained by enzymatic hydrolysis of cassava meal through a separation membrane having a molecular weight cutoff of more than 100,000 Da and not more than 300,000 Da, it is possible to remove high molecular weight components (weight average molecular weight of 10,000 to 20,000 Da) derived from the cassava meal. This prevents clogging of the membrane in the subsequent step of filtering through a nanofiltration membrane and / or reverse osmosis membrane, making it possible to efficiently produce a concentrated sugar liquid.

[0011] Furthermore, the sugar solution obtained by the present invention can not only be used as a fermentation raw material for producing chemical products, but also can increase the productivity of chemical products.

[0012] Cassava meal is a by-product of starch production from cassava roots (cassava tubers), and is the residue discarded after starch extraction. More specifically, when producing starch from cassava tubers, the cassava tubers are washed, peeled, and coarsely crushed, then ground and dispersed, and sieved into starch and fiber. Cassava meal is the fiber sieved out. Cassava meal can be produced by purchasing cassava tubers using the method described above, or it can be purchased from cassava meal producers or commercially available for use as feed.

[0013] Cassava meal can be either wet, where moisture remains, or dried, where the wet product has been dried. Either can be used in the present invention. Alternatively, either one can be used alone, or they can be mixed. When used in the present invention, the particle size can be adjusted by pulverization or the like.

[0014] Hereinafter, the method for producing a sugar solution of the present invention using cassava meal as a raw material will be described step by step.

[0015] [Step (1)] Step (1) is a step of enzymatically hydrolyzing cassava meal to obtain a saccharified solution containing a hydrolysate containing sugar. As described above, cassava meal is a fibrous residue discharged after starch extraction, and therefore contains cellulose as a fibrous component, and also contains starch. Therefore, an enzyme capable of hydrolyzing cellulose and / or starch is used as the enzyme in this step.

[0016] An example of an enzyme capable of hydrolyzing the cellulose contained in cassava meal is cellulase, which is an enzyme composition that contains enzyme components such as cellobiohydrolase, endoglucanase, exoglucanase, β-glucosidase, endoxylanase, and xylosidase and has the activity of hydrolyzing and saccharifying cellulose.

[0017] The cellulase may be a commercially available cellulase preparation, a culture solution of a cellulase-producing microorganism may be used directly, or cellulase may be purified from the culture solution and used. Also, these may be used in combination.

[0018] Examples of commercially available cellulase preparations include Cellulase enzyme blend (manufactured by Sigma-Aldrich Japan LLC), Cellulosin TP25 (manufactured by HIBI Corporation), Acremonium Cellulase (manufactured by Meiji Seika Pharma Co., Ltd.), and Meicelase (manufactured by Meiji Seika Pharma Co., Ltd.).

[0019] Examples of cellulase-producing microorganisms include filamentous fungal microorganisms such as those of the genera Trichoderma, Acremonium, Talaromyces, Aspergillus, Cellulomonas, Clostridium, Streptomyces, Humicola, Irpex, Mucor, Phanerochaete, white-rot fungi, and brown-rot fungi. Furthermore, the cellulase may be derived from a mutant strain of these microorganisms that has been subjected to a mutation treatment using a mutagen or ultraviolet irradiation to improve cellulase productivity. Among these cellulases derived from filamentous fungi, it is preferable to use cellulases derived from the genus Trichoderma and / or cellulases derived from the genus Acremonium, which produce large amounts of enzyme components with high specific activity in cellulose hydrolysis in the culture medium.

[0020] The amount of cellulase added to hydrolyze the cellulose contained in cassava meal can be appropriately set within a reasonable range that allows cellulose hydrolysis to be carried out effectively and economically. For example, 0.001 U to 10 U per 1 g of dry weight of cassava meal is preferred, and 0.01 to 5 U is more preferred. The dry weight of cassava meal is the weight of the cassava meal (Wa (g)) minus the weight of the water contained in the cassava meal. Specifically, the moisture content Rw (wt%) is measured using an infrared moisture meter, and the value calculated using the following formula (1) is used. The infrared moisture meter used is an FD-720 (manufactured by Kett Electric Laboratory Co., Ltd.), and the value measured in the automatic stop mode, which stops measurement when the moisture change over 30 seconds at a drying temperature of 105 ° C becomes 0.05% or less, is used.

[0021] Dry weight (g)=Wa−Wa×Rw (Formula 1).

[0022] The enzymatic activity of cellulase was measured in filter paper units (FPU) according to the method described below, which is based on the Measurement of Cellulase Activities, Laboratory Analytical Procedure (LAP) by the National Renewable Energy Laboratory (NREL). Filter paper cut to 1 cm length and 6 cm width and 1.0 ml of 50 mM citrate buffer (pH 4.8) were placed in a test tube and heated at 50 ° C for 5 minutes. 0.5 ml of cellulase diluted to produce 2 mg of glucose per 0.5 ml of enzyme solution was added to the test tube and heated at 50 ° C for 60 minutes. The reaction was stopped by adding 3 ml of dinitrosalicylic acid reagent and boiling for 5 minutes. 0.2 ml of the colored reaction solution is mixed with 2.5 ml of water, and the absorbance at 540 nm is measured. For the calibration curve, a similar procedure is performed using 2-6.7 g / L glucose standard solutions instead of filter paper and enzyme solution, resulting in color development. A reagent blank is prepared by performing the same procedure using only the citrate buffer solution. An enzyme blank is prepared by performing the same procedure using the citrate buffer solution and the diluted cellulase. The activity per enzyme solution is calculated using the following formula (2). The activity per mg of protein is calculated using the following formulas (2) and (3).

[0023] Activity per enzyme solution (U / ml) = 0.37 / cellulase concentration producing 2 mg of glucose... (Equation 2).

[0024] Activity per mg of protein (U / mg) = Activity per enzyme solution (U / ml) / Protein concentration of enzyme solution (mg / ml) (Equation 3).

[0025] Protein concentration can be measured by existing methods such as the Bradford method.

[0026] Examples of enzymes that can hydrolyze the starch contained in cassava meal include amylase. Amylase is an enzyme composition that contains enzyme components such as α-amylase, β-amylase, glucoamylase, and α-glucosidase and has the activity of hydrolyzing starch (amylose) to saccharify it. Among these enzyme activities, it is preferable to treat this process with an amylase that has at least α-amylase and / or glucoamylase activity. In this case, it is also preferable to promote the hydration reaction of the starch by, for example, heating the cassava meal to 60°C or higher in advance.

[0027] The amount of amylase added to hydrolyze the starch contained in cassava meal may be set within a reasonable range that allows effective and economical starch hydrolysis, but the enzyme activity per 1 g of dry weight of cassava meal is preferably 0.01 U or more, more preferably 0.1 U or more, and even more preferably 0.2 U or more. There is no upper limit, but from an economical standpoint, it can be set to, for example, 100 U or less.

[0028] The enzyme activity of α-amylase was measured as the decomposition activity of 2-chloro-4-nitrophenyl 65-azido-65-deoxy-β-maltopentaoside (N3-G5-β-CNP). The "65" in "2-chloro-4-nitrophenyl 65-azido-65-deoxy-β-maltopentaoside" indicates that the hydroxyl group at the 6th position of the fifth glucose residue from the reducing end of the glucose constituting the maltooligosaccharide has been substituted. Specifically, an appropriately diluted enzyme solution was added to a mixture of N3-G5-β-CNP solution, glucoamylase, and β-glucosidase solutions, and the mixture was incubated at 37°C for 10 minutes. Sodium carbonate solution was added to terminate the reaction and color the liberated 2-chloro-4-nitrophenol (CNP), and the absorbance was measured at 400 nm. A blank was prepared by the same method except that the enzyme solution was added after the addition of the sodium carbonate solution. One unit (1 U) of enzyme is the amount required to produce 1 μmol of CNP per minute under the above reaction conditions, and is calculated using the following formula 4. The activity per mg of protein is calculated using the following formulas 4 and 5.

[0029] Activity per enzyme solution (U / ml) = (Es - Eb) x 0.179 x Df (Equation 4).

[0030] Activity per mg of protein (U / mg) = Activity per enzyme solution (U / ml) / Protein concentration of enzyme solution (mg / ml) (Equation 5).

[0031] In formula (4), Es represents the absorbance of the measurement sample, Eb represents the absorbance of the blank, and Df represents the dilution factor of the measurement sample.

[0032] The enzymatic activity of glucoamylase is measured as the decomposition activity of 4-nitrophenyl-β-maltoside (G2-β-PNP). Specifically, an appropriately diluted enzyme solution is added to a mixture of G2-β-PNP solution and β-glucosidase solution, and the mixture is allowed to react at 37°C for 10 minutes. Sodium carbonate solution is added to stop the reaction and to color the liberated 4-nitrophenol (PNP), and the absorbance is measured at 400 nm. A blank is prepared by the same reaction method except that the enzyme solution is added after the addition of the sodium carbonate solution. One unit of enzyme (1 U) is the amount required to produce 1 μmol of PNP per minute under the above reaction conditions, and is calculated using the following formula (6). The activity per mg of protein is calculated using the following formulas (6) and (7).

[0033] Activity per enzyme solution (U / ml) = (Es - Eb) x 0.171 x Df (Equation 6).

[0034] Activity per mg of protein (U / mg) = activity per enzyme solution (U / ml) / protein concentration of enzyme solution (mg / ml) (Equation 7).

[0035] In the formula (6), Es represents the absorbance of the measurement sample, Eb represents the absorbance of the blank, and Df represents the dilution factor of the measurement sample.

[0036] The amylase may be a commercially available amylase preparation, a culture medium of an amylase-producing microorganism may be used directly, or amylase may be purified from the culture medium. Alternatively, a mixture of these enzymes may be used.

[0037] Examples of commercially available amylase preparations include glucoamylase preparations such as Amyloglucosidase from Aspergillus niger (manufactured by Sigma-Aldrich Japan LLC), Gluczyme AF6 (manufactured by Amano Enzyme Inc.), Gluczyme NL4.2 ​​(manufactured by Amano Enzyme Inc.), and AMYLOGLUCOSIDASE (manufactured by MEGAZYME).

[0038] In addition to cellulase and amylase, other enzymes such as pectinase may be added. Pectinase is a general term for enzymes that hydrolyze pectin, and refers to an enzyme composition that has the activity of hydrolyzing pectin and contains enzyme components such as pectinesterase, pectin lyase, polygalacturonase, and pectin methylesterase. These enzyme treatments may be carried out separately or simultaneously if the enzyme treatment conditions are suitable.

[0039] The temperature conditions for the enzymatic hydrolysis reaction are not particularly limited as long as they are within the optimum temperature range for the enzyme used, but a temperature of 30 to 60°C is preferred, for example.

[0040] The pH during the enzymatic hydrolysis reaction is not particularly limited as long as it is within the optimum pH range for the enzyme used, and is preferably, for example, pH 4 to 7. As the pH adjuster, a conventional acid or alkali can be used, and examples of acids that can be used include hydrochloric acid and sulfuric acid, and examples of alkalis that can be used include sodium hydroxide and ammonia.

[0041] The treatment time for the enzymatic hydrolysis reaction may be set within a range in which the enzymatic decomposition reaction is sufficiently carried out, and is preferably 1 to 24 hours, more preferably 2 to 22 hours, and even more preferably 4 to 20 hours. If the treatment time is less than 1 hour, the reaction will not proceed sufficiently, and if it is longer than 24 hours, contamination will occur.

[0042] Prior to the enzymatic hydrolysis of cassava meal, pretreatment may be performed to increase the reactivity of the enzyme to the cassava meal. The pretreatment method may be a known pretreatment method, such as acid treatment with sulfuric acid, acetic acid, etc., alkali treatment with caustic soda, ammonia, etc., hydrothermal treatment, subcritical water treatment, steaming treatment, etc. These treatment methods may be performed alone or in combination.

[0043] The saccharified solution obtained in step (1) contains sugars and insoluble solids produced by hydrolysis of cellulose and / or starch contained in the cassava meal. The saccharified solution containing sugars and insoluble solids may be directly subjected to the subsequent step (2), but it is preferable to subject the saccharified solution to solid-liquid separation to reduce the insoluble solids, and then to the subsequent step (2).

[0044] The solid-liquid separation method is not particularly limited, and examples include centrifugation using a screw decanter, a plate-type centrifuge, a cyclone, etc., sedimentation, compression separation using a screw press, etc., and filtration separation using a filter press, a belt press, a belt filter, a precoat filter, etc., or a combination thereof. Solid-liquid separation methods include continuous and batch methods, and either method is acceptable. Cassava meal saccharification liquid contains fibers floating in water and fibers sinking in water. Press filtration using a filter press or a belt press, which can remove both fibers in one step, is preferred, with a filter press being more preferred. The filter press may be vertical or horizontal. When using a filter press, the liquid may be pumped or pumped using compressed gas. The compression pressure is not particularly limited as long as the filtrate can be recovered, but is preferably 0.01 to 3 MPa, and more preferably 0.05 to 1 MPa.

[0045] The solid-liquid separation temperature is not particularly limited as long as it is within the range of temperatures applicable to the apparatus used, but a temperature of 20 to 50°C is preferred, for example.

[0046] The recovery rate of the saccharified solution by solid-liquid separation measured by the following (Equation 8) is preferably 50% or more.

[0047] Recovery rate %=(weight of saccharified solution recovered after solid-liquid separation) / (weight of saccharified solution before solid-liquid separation)×100 (Equation 8).

[0048] By subjecting the saccharified solution obtained in step (1) to solid-liquid separation, the amount of insoluble solids in the saccharified solution after solid-liquid separation is reduced. Turbidity can be used as a method for evaluating the amount of insoluble solids. Turbidity is defined in JIS K0101, "Testing Methods for Industrial Water," as follows: "Turbidity represents the degree of cloudiness of water, and is classified and displayed as visual turbidity, transmitted light turbidity, scattered light turbidity, and integrating sphere turbidity. When measured in comparison with a kaolin standard solution, the unit is "degree (kaolin)," and when measured in comparison with a formazin standard solution, the unit is "degree (formazin)."" In the present invention, a formazin standard solution, which has superior reproducibility and stability compared to a kaolin standard solution, is used, and the turbidity "NTU" measured by the scattered light measurement method is used. NTU stands for "Nephelometric Turbidity Unit." The turbidity of the saccharified solution obtained by solid-liquid separation in step (1) is preferably as low as possible in consideration of subsequent steps, specifically, preferably 1,000 NTU or less, more preferably 500 NTU or less, even more preferably 300 NTU or less, and particularly preferably 150 NTU or less. If the turbidity of the saccharified solution exceeds 1,000 NTU, there is a risk of reduced filterability during membrane separation in the subsequent step (2).

[0049] As described above, the insoluble solids content in the saccharified solution can be reduced by performing solid-liquid separation of the saccharified solution containing insoluble solids obtained in step (1), but by adding an acidic substance to the saccharified solution before solid-liquid separation, the insoluble solids content can be reduced without using a solid-liquid separation device well known to those skilled in the art, thereby reducing the burden of solid-liquid separation. Furthermore, by performing solid-liquid separation after adding an acidic substance, the insoluble solids content in the saccharified solution after solid-liquid separation can be further reduced.

[0050] The insoluble solids contained in the saccharified solution obtained in step (1) are insoluble solids that have not been subjected to the hydrolysis reaction by the enzyme in step (1), and are thought to include components such as polysaccharides, pectin, and lignin, but are not limited to these.

[0051] Whether the insoluble solids content has been reduced can be evaluated by comparing the amount of insoluble solids before and after the addition of the acidic substance. The insoluble solids content can be quantified according to the method specified in JIS K0102 14.1 Minimum Suspended Solids (MLSS) (2019).

[0052] The acidic substance is not particularly limited as long as it can reduce the insoluble solid content in the saccharified solution, but a monovalent acidic substance is preferred, and hydrochloric acid, nitric acid, acetic acid, lactic acid, or formic acid is more preferred, with hydrochloric acid being even more preferred.

[0053] The pH of the saccharified solution after the addition of the acidic substance is not particularly limited, but is preferably from 3.0 to less than 4.5, and more preferably from 3.0 to 4.0. A pH of less than 3.0 may adversely affect the growth of microorganisms when the produced sugar solution is used for fermentation, while a pH of less than 4.5 can reduce the insoluble solid content, which is a technical feature of the present invention.

[0054] [Step (2)] In the present invention, a separation membrane with a molecular weight cutoff of more than 100,000 Da but not more than 300,000 Da is used in step (2). The molecular weight cutoff in the present invention refers to the molecular weight at which the solute rejection rate is 90% in a molecular weight cutoff curve plotted with the molecular weight of the solute on the horizontal axis and the rejection rate on the vertical axis. The molecular weight cutoff can be determined by filtering various dextran standard samples or protein standard samples with known molecular weights through a separation membrane, evaluating their rejection rates, and plotting the resulting separation curve. Specifically, a separation membrane with a molecular weight cutoff of 100,000 Da refers to a separation membrane that rejects 90% of molecules with a molecular weight of 100,000 Da.

[0055] The present inventors have discovered that the saccharified solution obtained in step (1) of the present invention contains high molecular weight components (hereinafter simply referred to as "high molecular weight components") derived from cassava meal and having a weight-average molecular weight of 10,000 to 20,000 Da, and that the high molecular weight components cause clogging of the nanofiltration membrane and / or reverse osmosis membrane in the subsequent filtration process. The high molecular weight components are presumed to be by-products of enzymatic hydrolysis of cassava meal, and are primarily composed of polysaccharides. When a saccharified solution containing high-molecular-weight components is filtered through a separation membrane having a molecular weight cutoff of more than 100,000 Da but not more than 300,000 Da, preferably more than 100,000 Da but not more than 200,000 Da, or 120,000 Da or more but not more than 300,000 Da, and more preferably 120,000 Da or more but not more than 200,000 Da, it is expected that the high-molecular-weight components will also permeate the separation membrane. However, in step (2) of the present invention, contrary to expectations, a separation membrane having a molecular weight cutoff of more than 100,000 Da but not more than 300,000 Da can block the high-molecular-weight components in the retentate side. Note that a molecular weight cutoff of a separation membrane exceeding 300,000 Da reduces the amount of high-molecular-weight components that can be blocked in the retentate side and increases the amount of high-molecular-weight components in the permeate side, which is undesirable because it clogs the nanofiltration membrane and / or reverse osmosis membrane in step (3). Furthermore, if a separation membrane with a molecular weight cutoff of 100,000 Da or less is used, the separation membrane will immediately become clogged in step (2), which is not preferable.

[0056] The separation characteristics of the separation membrane in step (2) are determined by a dense layer called the functional layer. Materials that can be used for the functional layer of the separation membrane used in step (2) include polyethersulfone (PES), polysulfone (PS), polyacrylonitrile (PAN), polyvinylidene fluoride (PVDF), regenerated cellulose, cellulose, cellulose ester, sulfonated polysulfone, sulfonated polyethersulfone, polyolefin, polyvinyl alcohol, polymethyl methacrylate, and polyethylene tetrafluoride. However, because regenerated cellulose, cellulose, and cellulose ester are susceptible to degradation by cellulase, it is preferable to use a separation membrane whose functional layer is made of a synthetic polymer compound such as PES or PVDF.

[0057] The flux in the filtration through the separation membrane in step (2) may be determined taking membrane fouling into consideration, and is preferably, for example, 0.01 to 2.0 m / day, more preferably 0.05 to 2.0 m / day, and even more preferably 0.1 to 2.0 m / day. If the flux exceeds 2.0 m / day, a significant increase in the transmembrane pressure difference of the separation membrane and rapid membrane fouling may occur. If the flux is less than 0.01 m / day, the number of membranes required for membrane separation increases, resulting in higher equipment costs.

[0058] Flux (m / day) refers to the amount of water filtered through the membrane per unit membrane area and unit time, and can be calculated using the following formula (Formula 9):

[0059] Flux (m / day) = Amount of water filtered through the membrane per day (m ３ / day) / membrane area (m ２ )...(Formula 9).

[0060] The separation membrane used in step (2) can be of any suitable type, such as a hollow fiber, tubular, flat membrane, or spiral membrane. However, because the saccharified solution contains insoluble solids, a separation membrane that is resistant to membrane fouling is preferred. Specifically, a hollow fiber or tubular membrane is preferred, with a hollow fiber membrane (i.e., a hollow fiber membrane) being more preferred. A hollow fiber membrane refers to a separation membrane that is hollow and has a functional layer on the inside or outside. Hollow fiber membranes can be broadly classified into internal pressure hollow fiber membranes, which allow raw water to pass through the inside of the hollow fiber membrane and obtain filtrate on the outside of the hollow fiber membrane, and external pressure hollow fiber membranes, which allow raw water to pass through the outside of the hollow fiber membrane and obtain filtrate on the inside of the hollow fiber membrane. External pressure hollow fiber membranes are more resistant to membrane fouling by solids and easier to clean than internal pressure hollow fiber membranes, and are therefore preferably used in step (2).

[0061] Specific examples of the separation membrane in step (2) include SPE200, SPE300, LV, BX, and V5 manufactured by Synder, "Microza" (registered trademark) UF series manufactured by Asahi Kasei Corporation, UV200 from NADIR, and "Treyfil" (registered trademark) HFU series, HSU series, "Toray UF" HFU series, and HSU series manufactured by Toray Industries, Inc.

[0062] The membrane separation method in step (2) may be dead-end filtration or cross-flow filtration, but cross-flow filtration is preferred. Furthermore, the membrane separation method in step (2) may be constant-pressure filtration or constant-flow filtration. When clogging of the separation 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 remove the cake formed on the membrane surface. Examples of backwashing cleaning liquids include the filtrate from the separation membrane, water, and chemical solutions.

[0063] [Step (3)] The filtrate obtained in step (2) has a reduced content of polymeric components that clog nanofiltration membranes and reverse osmosis membranes. Therefore, by further filtering the filtrate through a nanofiltration membrane and / or a reverse osmosis membrane, it is possible to recover a sugar solution containing concentrated sugars derived from the cassava meal from the non-permeated side of each separation membrane.

[0064] 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.

[0065] As the material for the nanofiltration membrane, polymeric materials such as cellulose acetate polymers, polyamides, polyesters, polyimides, and vinyl polymers can be used, but if a cellulose acetate membrane is used for a long period of time, the enzymes used in step (1), particularly a portion of the cellulase component, may permeate in step (2) and decompose the cellulose, which is the membrane material, so a nanofiltration membrane made of a polyamide material is preferred. The material for the nanofiltration membrane is not limited to membranes made of the above-mentioned single material, but may also be a membrane containing multiple membrane materials.

[0066] Examples of the nanofiltration membrane type include, but are not limited to, hollow fiber, tubular, flat membrane, and spiral membrane types. The filtration method may be dead-end filtration, cross-flow filtration, constant pressure filtration, or constant flow rate filtration.

[0067] Reverse osmosis membranes, also known as RO membranes, are generally defined as membranes capable of desalination, including of monovalent ions. These membranes are believed to have ultra-micropores ranging from a few angstroms to a few nanometers in size, and are primarily used to remove ionic components in processes such as seawater desalination and ultrapure water production.

[0068] As the material for the reverse osmosis membrane, polymeric materials such as cellulose acetate polymers, polyamides, polyesters, polyimides, and vinyl polymers can be used, but if a cellulose acetate membrane is used for a long period of time, the enzymes used in step (1), particularly a portion of the cellulase component, may permeate in step (2) and decompose the cellulose, which is the membrane material, so a nanofiltration membrane made of a polyamide material is preferred. The material for the reverse osmosis membrane is not limited to membranes made of the above-mentioned single material, but may also be a membrane containing multiple membrane materials.

[0069] Examples of the reverse osmosis membrane include, but are not limited to, hollow fiber, tubular, flat membrane, and spiral membrane types. The filtration method may be dead-end filtration, cross-flow filtration, constant pressure filtration, or constant flow rate filtration.

[0070] The filtration treatment using a nanofiltration membrane and / or a reverse osmosis membrane can be carried out according to the method described in WO2010 / 067785.

[0071] The sugar solution may be passed through a nanofiltration membrane and / or a reverse osmosis membrane and then recovered from the retentate side, and then further concentrated to further increase the sugar concentration. The concentration method is not particularly limited, and may be membrane concentration, evaporation concentration, or a combination of these.

[0072] [Concentrated sugar solution] The sugar solution obtained in step (3) (hereinafter, the sugar solution recovered from the retentate side of the nanofiltration membrane and / or reverse osmosis membrane, and the sugar solution obtained by further concentrating this sugar solution, are collectively referred to as "concentrated sugar solution") has a high concentration of monosaccharides derived from cassava meal, particularly glucose, and can be preferably used as a raw material for microbial fermentation. The glucose concentration of the concentrated sugar solution is preferably 80 g / L or more, more preferably 100 g / L or more, and even more preferably 120 g / L or more. There is no particular upper limit for the glucose concentration of the concentrated sugar solution, but because glucose is more likely to precipitate at high concentrations, it is preferably 700 g / L or less, and more preferably 650 g / L or less.

[0073] The concentrated sugar solution can also be characterized by its molecular weight distribution, which is measured by gel filtration chromatography using a differential refractometer and pullulan as a standard.

[0074] The concentrated sugar solution is characterized in that, as a result of analysis of molecular weight distribution by gel filtration chromatography using the above-mentioned differential refractometer and pullulan as a standard substance, it contains a cassava meal-derived polymer component having a weight-average molecular weight of 10,000 to 20,000 Da, and the peak area ratio of the cassava meal-derived polymer component having a weight-average molecular weight of 10,000 to 20,000 Da to the total peak area is 0.1 to 2%, preferably 0.2 to 2%, more preferably 0.2 to 1.5%, even more preferably 0.2 to 1%, even more preferably 0.2 to 0.6%, and particularly preferably 0.3 to 0.6%. This technical feature not only contributes to reducing membrane clogging during the filtration treatment of the nanofiltration membrane and / or reverse osmosis membrane in step (3), but also contributes to improving the productivity of chemical products when the concentrated sugar solution is used as a fermentation raw material for producing chemical products.

[0075] Further, the concentrated sugar liquid contains a cassava meal-derived component having a weight average molecular weight of less than 10,000 as determined by the analysis of molecular weight distribution by gel filtration chromatography using the above-mentioned differential refractometer and pullulan as a standard substance, and the area ratio of the peak of the cassava meal-derived component having a weight average molecular weight of less than 10,000 in the total peak area is preferably 98 to 99.9%, more preferably 98.5 to 99.8%, even more preferably 99 to 99.8%, particularly preferably 99.4 to 99.8%, and most preferably 99.4 to 99.7%.

[0076] The concentrated sugar solution can be used as a fermentation feedstock to produce chemical products. Microorganisms that can be used to produce chemical products using the concentrated sugar solution as a fermentation feedstock include yeasts such as baker's yeast, bacteria such as Escherichia coli and coryneform bacteria, filamentous fungi, and actinomycetes. The microorganisms may be isolated from the natural environment, or may have some properties modified by mutation or genetic recombination.

[0077] Chemical products obtainable by fermentation using concentrated sugar solution as a fermentation feedstock are not particularly limited as long as they are substances produced by microorganisms or cultured cells. Specific examples include substances mass-produced in the fermentation industry, such as alcohols, organic acids, amino acids, and nucleic acids. Examples of alcohols include ethanol, butanol, 2,3-butanediol, 1,4-butanediol, and glycerol; examples of organic acids include acetic acid, lactic acid, succinic acid, and malic acid; examples of amino acids include lysine and glutamic acid; and examples of nucleic acids include inosinic acid, guanylic acid, inosine, and guanosine. The present invention can also be applied to the production of substances such as proteins, enzymes, and antibiotics. Therefore, a variety of chemical products can be produced using the present invention.

[0078] (Reference Example 1) Method for analyzing glucose concentration The concentration of glucose contained in the sugar solution was quantified by comparison with a standard sample under the following high performance liquid chromatography (HPLC) conditions: (HPLC conditions) Column: Shodex Sugar Series SH1011 (manufactured by Resonac Co., Ltd.) Mobile phase: 5 mM sulfuric acid (flow rate: 0.6 mL / min) Reaction solution: None Detection method: RI (differential refractive index) Temperature: 65°C.

[0079] (Reference Example 2) Preparation of cassava cake saccharification liquid 8.5 kg of cassava cake (water content 83%) was mixed with 3.5 L of RO water and 0.02 U of thermostable amylase (Sigma-Aldrich Japan LLC) as α-amylase, and 4 N sodium hydroxide (Nacalai Tesque Co., Ltd.) was added to adjust the pH to 5.0, and then autoclaved at 90 ° C. for 2 hours. 0.2 U of amyloglucosidase from Aspergillus niger (Sigma-Aldrich Japan LLC) was added as glucoamylase, and 0.2 U of Acremonium cellulase (Meiji Seika Pharma Co., Ltd.) was added as cellulase per 1 g of dry weight of cassava cake. The enzymatic reaction was carried out at 50 ° C. for 8 hours with stirring to obtain a saccharification liquid.

[0080] The obtained saccharified solution was subjected to solid-liquid separation using a filter press to remove residue, thereby obtaining a filter press filtrate, which was a saccharified solution with a reduced insoluble solid content.

[0081] The turbidity of the filter press filtrate was analyzed using a portable turbidity meter 2100P (manufactured by HACH) and found to be 150 NTU. The glucose concentration of the filter press filtrate was measured by the method of Reference Example 1 and found to be 60 g / L.

[0082] (Reference Example 3) Reduction of insoluble solids by adding acidic substance to cassava cake saccharification liquid 8.5 kg of cassava cake (moisture content 83%) was mixed with 3.5 L of RO water and 0.02 U of thermostable amylase (manufactured by Sigma-Aldrich Japan LLC) as α-amylase, and 4 N sodium hydroxide (manufactured by Nacalai Tesque Inc.) was added to adjust the pH to 5.0, and then autoclaved at 90 ° C. for 2 hours. Cassava meal was charged so that the dry weight was 12% by weight, and 0.2 U of amyloglucosidase from Aspergillus niger (Sigma-Aldrich Japan LLC) was used as a glucoamylase per 1 g of dry weight of cassava meal. 1 U of Acremonium cellulase (manufactured by Meiji Seika Pharma Co., Ltd.) was added as a cellulase per 1 g of dry weight of cassava meal. The enzyme reaction was carried out at 50 ° C. for 24 hours while mixing and stirring to obtain a cassava meal saccharified liquid.

[0083] The obtained cassava saccharified liquid was centrifuged (1,500 x G, 3 minutes) or filtered under the conditions of without and with the addition of an acidic substance, and the amount of insoluble solids in the cassava saccharified liquid, centrifuged liquid fraction, and filter press filtrate was compared. With the addition of an acidic substance, 10 N hydrochloric acid was added to the cassava saccharified liquid to adjust the pH to 3.5, and the pH of the saccharified liquid without the addition of an acidic substance was 4.5 (the pH of the cassava saccharified liquid decreased from the start of hydrolysis due to the production of galacturonic acid during hydrolysis). The insoluble solids were measured according to JIS K0102 14.1 Suspended Solids (MLSS) (2019) and quantitatively evaluated by converting the value to ppm. The results are shown in Table 1. It was confirmed that the addition of acidic substances reduced the insoluble solids in the cassava cake saccharification liquid, centrifuged liquid fraction, and filter press filtrate.

[0084] Reference Example 4: Effect of Addition of Acidic Substance to Bagasse Saccharified Solution on Insoluble Solids Content To 1.82 kg of bagasse (water content 45%), 6.5 L of RO water and 562 mL of 4 N sodium hydroxide (manufactured by Nacalai Tesque, Inc.) were added, and the mixture was reacted at 90° C. for 2 hours. 10 N hydrochloric acid was then added to adjust the pH at the start of saccharification to 5.0, and the mixture was charged to a dry weight of 12% bagasse. 0.2 U of amyloglucosidase from Aspergillus niger (Sigma-Aldrich Japan, LLC) per 1 g of dry weight of bagasse was added as a glucoamylase, and 1 U of Acremonium cellulase (manufactured by Meiji Seika Pharma Co., Ltd.) per 1 g of dry weight of bagasse was added as a cellulase. The mixture was then subjected to an enzymatic reaction at 50° C. for 24 hours with mixing and stirring to obtain a bagasse saccharified solution.

[0085] The obtained bagasse saccharified solution was centrifuged or filtered through a filter press with or without the addition of an acidic substance, as in Reference Example 3, and the amounts of insoluble solids in the bagasse saccharified solution, the centrifuged liquid fraction, and the filter press filtrate were compared. Note that, in the case of the addition of an acidic substance, the bagasse saccharified solution was adjusted to pH 3.5 by adding 10 N hydrochloric acid. The pH of the saccharified solution without the addition of an acidic substance was 4.5 (pH 4.5 after hydrolysis. During saccharification, some sugars were converted to lactic acid by microorganisms, causing the pH to drop from the start of hydrolysis. However, the drop in pH suppressed microbial growth, and the pH stabilized at 4.5). The insoluble solids were also measured as in Reference Example 3. The results are shown in Table 1. The addition of acidic substances reduced the insoluble solids content in the cassava saccharification liquid, the centrifuged liquid fraction of the cassava saccharification liquid, and the filter press filtrate of the cassava saccharification liquid, but increased the insoluble solids content in the bagasse saccharification liquid, the centrifuged liquid fraction of the bagasse saccharification liquid, and the filter press filtrate of the bagasse saccharification liquid.

[0086]

[0087] (Example 1) Production of concentrated sugar solution from cassava pulp saccharification liquid using a hollow fiber membrane and a reverse osmosis membrane with a molecular weight cutoff of 150,000 Da Ultrafiltration membrane module "Toray UF" HFU (manufactured by Toray Industries, Inc.) A polyvinylidene fluoride hollow fiber membrane with a nominal pore diameter of 0.01 μm and a nominal molecular weight cutoff of 150,000 Da was cut out, and a miniature module (hereinafter referred to as membrane A) with an inner diameter of 10 mm and a length of 320 mm consisting of 22 hollow fiber membranes was prepared. 1.5 L of the filter press filtrate obtained in Reference Example 2 was supplied to membrane A using a tube pump at a temperature of 35 ° C. and a membrane surface linear velocity of 20 cm / sec. As a result of performing the first membrane separation by cross-flow filtration, 1.4 L of filtrate was obtained without clogging the membrane.

[0088] A second membrane separation was carried out on 1 L of the filtrate obtained by membrane separation using Membrane A, using a UTC-70 (molecular weight cutoff: 65, Toray Industries, Inc.) (hereinafter referred to as Membrane B) as a reverse osmosis membrane to concentrate the filtrate by two times. The membrane separation device was a "SEPA" (registered trademark) CF-II (effective membrane area: 140 cm). ２Filtration was carried out using a filtration column (GE W&PT) at an operating temperature of 35°C and a membrane surface linear velocity of 20 cm / sec until the filtration pressure reached 4 MPa. As a result, 0.5 L of concentrated sugar solution, which was concentrated twice as much as the unpermeated solution, was obtained without clogging the membrane. The glucose concentration of the concentrated sugar solution was measured by the method of Reference Example 1 and was found to be 120 g / L.

[0089] Example 2: Production of concentrated sugar solution from cassava cake saccharification liquid using a hollow fiber membrane with a molecular weight cutoff of 150,000 Da and a nanofiltration membrane. In order to double the concentration of 1 L of filtrate obtained using Membrane A obtained by the same method as in the first membrane separation in Example 1, a second membrane separation was carried out using an NFS (molecular weight cutoff 100-250 Da, Synder) (hereinafter referred to as Membrane C) nanofiltration membrane under the same conditions as in Example 1. As a result, 0.5 L of concentrated sugar solution was obtained, which was double the concentration in the retentate side, without clogging the membrane. The glucose concentration of the concentrated sugar solution was measured by the method in Reference Example 1 and was found to be 120 g / L.

[0090] (Comparative Example 1) Production of concentrated sugar solution from cassava pulp saccharification liquid using a hollow fiber membrane with a pore size of 0.05 μm and a reverse osmosis membrane A polyvinylidene fluoride hollow fiber membrane with a nominal pore size of 0.05 μm used in the microfiltration membrane module "Trefil" (registered trademark) HFS manufactured by Toray Industries, Inc. was cut out, and a miniature module (hereinafter referred to as membrane D) with an inner diameter of 10 mm and a length of 320 mm consisting of 22 hollow fiber membranes was prepared. 1.5 L of the filter press filtrate obtained in Reference Example 2 was supplied to membrane D using a tube pump at a temperature of 35 ° C. and a membrane surface linear velocity of 20 cm / sec. As a result of performing the first membrane separation by cross-flow filtration, 1.4 L of filtrate was obtained without clogging the membrane.

[0091] In order to concentrate 1 L of the filtrate obtained by membrane separation using membrane D by a second membrane separation using membrane B in the same manner as in Example 1, 0.7 L of concentrated sugar solution was obtained on the retentate side, but due to clogging of the membrane, the concentration was only 1.5 times. The glucose concentration of the concentrated sugar solution was measured by the method of Reference Example 1 and was found to be 90 g / L.

[0092] (Comparative Example 2) Membrane separation of cassava pulp saccharified liquid using a flat membrane with a molecular weight cutoff of 50,000 Da. 1.5 L of filter press filtrate prepared by the method described in Reference Example 2 was filtered using a flat membrane SPE50 (manufactured by Synder, molecular weight cutoff 50,000 Da) (hereinafter referred to as Membrane E). The membrane separation device was a "SEPA" (registered trademark) CF-II (effective membrane area 140 cm). ２ Filtration was carried out using a filtration filter (GE W&PT) at an operating temperature of 35°C and a membrane surface linear velocity of 20 cm / sec until the filtration pressure reached 0.8 MPa, the upper limit of the operating pressure listed in the catalog, and no more filtrate was obtained. As a result, only 0.5 L of filtrate was obtained due to clogging of the membrane.

[0093]

[0094] (Example 3) Evaporation and concentration of concentrated sugar solution The concentrated sugar solution obtained in Example 1 was concentrated with an evaporator to prepare an evaporated and concentrated sugar solution with a glucose concentration of 500 g / L.

[0095] (Reference Example 5) Analysis of Molecular Weight Distribution by Gel Filtration Chromatography (GFC) The saccharified solution, and the retentate and permeate from membrane separation were diluted 10-fold with 0.2 M aqueous sodium nitrate and filtered through a disk filter with a pore size of 0.45 μm to prepare a sample solution for measurement. The molecular weight in the sample solution was measured by the following method, and the average molecular weight was calculated as the average molecular weight converted to a standard (pullulan). When the value was outside the measurement range for the standard, the average molecular weight was calculated by extrapolation of the calibration curve. When the separation of each peak was insufficient, vertical division was performed at the peak valley. Apparatus: "Prominence" (Shimadzu Corporation) Column: OHpakSB-G + SB-805HQ + SB-804HQ (Resonac Corporation) Column temperature: 40°C Mobile phase: 0.2 M aqueous sodium nitrate solution Flow rate: 0.5 mL / min Detector: differential refractometer (RI) Injection volume: 100 μL Standard: pullulan.

[0096] (Reference Example 6) Analysis of molecular weight distribution of cassava cake saccharification liquid The cassava cake saccharification liquid (filter press filtrate) obtained in Reference Example 2 was analyzed for molecular weight distribution by the method described in Reference Example 5. The results are shown in Table 3. The cassava cake saccharification liquid contained a monosaccharide component with a weight-average molecular weight of 250 Da and a polymer component whose main component was thought to be a polysaccharide with a weight-average molecular weight of 14,000 Da.

[0097]

[0098] (Reference Example 7) Analysis of turbidity and molecular weight distribution of retentate and permeate obtained by membrane separation of cassava pulp saccharification liquid. The retentate and permeate obtained by membrane separation using Membrane A in Example 1, and the retentate and permeate obtained by membrane separation using Membrane D in Comparative Example 1 were analyzed for turbidity and molecular weight distribution. Turbidity was measured using a portable turbidimeter 2100P (manufactured by HACH), and molecular weight distribution was analyzed using the method of Reference Example 5. The ratio (%) of the peak area of ​​the polymer component with a weight average molecular weight of 10,000 to 20,000 Da to the total peak area was calculated. The results are shown in Table 4. In membrane separation using Membrane A, turbidity and polymer components with a weight average molecular weight of 10,000 to 20,000 Da were blocked on the retentate side. On the other hand, in membrane separation using Membrane D, turbidity was blocked on the retentate side, but polymer components with a weight average molecular weight of 10,000 to 20,000 Da were not blocked and permeated.

[0099]

[0100] Example 4: Molecular weight analysis of the cassava pulp saccharification liquid, the retentate, permeate, and concentrated sugar solution obtained by membrane separation of the cassava pulp saccharification liquid, and the evaporated and concentrated sugar solution. The molecular weight distribution of the cassava pulp saccharification liquid (filter press filtrate) obtained in Reference Example 2, the retentate and permeate obtained by membrane separation using Membrane A in Example 1, the concentrated sugar solution (the retentate) obtained by membrane separation using Membrane B in Example 1, and the evaporated and concentrated sugar solution obtained in Example 3 were analyzed by the method described in Reference Example 5. The evaporated and concentrated sugar solution was diluted 5-fold with water and then with a 0.2 M aqueous sodium nitrate solution as described in Reference Example 5, and then analyzed for molecular weight distribution. For comparison, the molecular weight distribution of the retentate and permeate obtained by membrane separation using Membrane D in Comparative Example 1 was also analyzed. Table 5 shows the calculated ratios (%) of the peak area of ​​high molecular weight components with a weight-average molecular weight of 10,000 to 20,000 Da to the total peak area and the ratio (%) of the peak area of ​​high molecular weight components with a weight-average molecular weight of less than 10,000 to the total peak area.

[0101] In the retentate and permeate obtained by membrane separation using Membrane A in Example 1, high molecular weight components having a weight-average molecular weight of 10,000 to 20,000 Da were blocked by Membrane A, and therefore the high molecular weight components were concentrated in the retentate and significantly reduced in the permeate. Furthermore, the results of molecular weight analysis of the concentrated sugar solution obtained by membrane separation using Membrane A and the evaporated concentrate of the concentrated sugar solution were the same as those of the permeate obtained by membrane separation using Membrane A.

[0102] On the other hand, the molecular weight analysis results of the retentate and permeate obtained by membrane separation using Membrane D in Comparative Example 1 were almost the same as the analysis results of the saccharified solution in Reference Example 2, and it was confirmed that high molecular weight components with a weight-average molecular weight of 10,000 to 20,000 Da were not blocked by Membrane D.

[0103]

[0104] (Example 5) Membrane separation of cassava pulp saccharification liquid using a flat membrane with a molecular weight cutoff of 200,000 Da. 1.5 L of the filter press filtrate obtained in Reference Example 2 was subjected to total circulation operation in which the permeate was returned to the supply tank using a flat membrane LV (manufactured by Synder, molecular weight cutoff 200,000 Da) (hereinafter referred to as Membrane F) as the separation membrane. The membrane separation device was a "SEPA" (registered trademark) CF-II (effective membrane area 140 cm). ２ The operating temperature was 35°C and the membrane surface linear velocity was 20 cm / sec. The retentate and permeate were sampled and analyzed for molecular weight distribution by the method of Reference Example 5, and the ratio (%) of the peak area of ​​polymer components with a weight-average molecular weight of 10,000 to 20,000 Da to the total peak area was determined. The turbidity of the permeate was also analyzed using a portable turbidimeter 2100P (manufactured by HACH).

[0105] The amount of the polymer component having a weight-average molecular weight of 10,000 to 20,000 Da that permeated the separation membrane was calculated by dividing the ratio (%) of the peak area of ​​the polymer component having a weight-average molecular weight of 10,000 to 20,000 Da in the permeated liquid to the total peak area by the ratio (%) of the peak area of ​​the polymer component having a weight-average molecular weight of 10,000 to 20,000 Da in the non-permeated liquid to the total peak area, resulting in a value of 0.08, confirming that the polymer component having a weight-average molecular weight of 10,000 to 20,000 Da was blocked on the non-permeated liquid side of Membrane F. In addition, the turbidity of the permeated liquid was 50 NTU or less, confirming that turbid matter was also blocked on the non-permeated liquid side.

[0106] (Reference Example 8) Method for Analyzing Ethanol Ethanol in the culture medium was quantified by comparison with a standard under the following HPLC conditions: Column: Shodex Sugar Series SH1011 (manufactured by Resonac Co., Ltd.) Column temperature: 65°C Detection method: differential refractive index detector Detector temperature: 40°C Mobile phase: 0.005 MH ２ SO ４ Flow rate: 0.6 mL / min.

[0107] (Example 6) Ethanol fermentation using concentrated sugar solution Ethanol was produced by microbial fermentation using the concentrated sugar solution obtained in Example 1 as a fermentation raw material, and the ethanol fermentation rate was evaluated.

[0108] An ethanol-fermenting microorganism (Saccharomyces cerevisiae (OC-2 strain)) was inoculated into 5 mL of YPD medium and cultured at 30°C for 16 hours (preculture). The concentrated sugar solution was filtered through a filter (Stericup 0.22 μm, Merck Millipore), and 9 mL of the filtered solution was weighed out and placed in a test tube. 1 mL of corn steep liquor (Oji Cornstarch Co., Ltd.) adjusted to 20% by weight was added and mixed. The preculture solution was added to each test tube so that the turbidity reached 0.5. Culture was carried out at 30°C, and the ethanol concentration after 24 hours was measured by the method described in Reference Example 8. The results of the ethanol fermentation rate are shown in Table 6.

[0109] (Comparative Example 3) Ethanol fermentation using a reagent glucose solution Ethanol fermentation using a reagent glucose solution was carried out in the same manner as in Example 6, except that a reagent glucose solution prepared by dissolving glucose (D(+) glucose, manufactured by Wako Pure Chemical Industries, Ltd.) in pure water to a concentration of 120 g / L (the same glucose concentration as in the concentrated sugar solution) was used instead of the concentrated sugar solution, and the ethanol concentration after 24 hours was measured by the method of Reference Example 8. The results of the ethanol fermentation rate are shown in Table 6, and the ethanol fermentation rate was improved in the ethanol fermentation using the concentrated sugar solution compared to when the reagent glucose solution was used.

[0110]

Claims

1. A method for producing a sugar solution, comprising the following steps (1) to (3): Step (1): Enzymatically hydrolyzing cassava meal to obtain a saccharified solution; Step (2): Filtering the saccharified solution obtained in Step (1) through a separation membrane with a molecular weight cutoff of more than 100,000 Da and not more than 300,000 Da to block cassava meal-derived high molecular weight components with a weight average molecular weight of 10,000 to 20,000 Da in the retentate side; and Step (3): Filtering the filtrate obtained in Step (2) through a nanofiltration membrane and / or a reverse osmosis membrane to recover a sugar solution from the retentate side.

2. The method for producing a sugar solution according to claim 1, wherein the separation membrane used in step (2) has a molecular weight cutoff of more than 100,000 Da and not more than 200,000 Da.

3. The method for producing a sugar solution according to claim 1 or 2, wherein the separation membrane in step (2) is a hollow fiber membrane.

4. The method for producing a sugar solution according to claim 1 or 2, wherein the enzyme in step (1) has at least cellulase activity and / or amylase activity.

5. The method for producing a sugar solution according to claim 1 or 2, wherein the step (1) is a step of separating a sugar solution obtained by enzymatic hydrolysis of cassava meal into solid and liquid.

6. The method for producing a sugar solution according to claim 5, wherein the solid-liquid separation is press filtration.

7. The method for producing a sugar solution according to claim 5, wherein the turbidity of the sugar solution obtained by the solid-liquid separation in step (1) is 300 NTU or less.

8. A sugar solution containing cassava meal-derived glucose and a cassava meal-derived polymer component having a weight-average molecular weight of 10,000 to 20,000 Da, wherein the area ratio of the peak of the cassava meal-derived polymer component having a weight-average molecular weight of 10,000 to 20,000 Da to the total peak area in gel filtration chromatographic analysis using a differential refractometer and pullulan as a standard substance is 0.1 to 2%.

9. The sugar solution according to claim 8, further comprising a cassava meal-derived component having a weight-average molecular weight of less than 10,000, wherein the peak area ratio of the cassava meal-derived component having a weight-average molecular weight of less than 10,000 to the total peak area in gel filtration chromatographic analysis using a differential refractometer and pullulan as the standard substance is 98 to 99.9%.

10. The sugar solution according to claim 8 or 9, having a glucose concentration of 80 g / L or more.