Method for producing sugar solution

Abstract

A sugar solution in which sugar derived from cassava meal is concentrated can be efficiently produced by a method for producing a sugar solution comprising the following steps (1)-(3). Step (1): a step for obtaining a saccharified liquid by enzymatic hydrolysis of cassava meal; step (2): a step in which the saccharified liquid obtained in step (1) is filtered through a separation membrane having a molecular weight cut-off of more than 100,000 Da but 300,000 Da or less, and a cassava meal-derived polymer component having a weight-average molecular weight of 10,000-20,000 Da is blocked on the non-permeate side; and step (3): a step for filtering the filtrate obtained in step (2) through a nanofiltration membrane and / or a reverse osmosis membrane, and collecting the sugar solution from the non-permeate side.

Description

Technical Field

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

[0002] As a substitute for fossil fuels from the perspective of environmental protection, the production of chemicals by fermentation using sugars produced from plant biomass as raw materials has been studied, and the production of fuel ethanol using sugars obtained from plant biomass as raw materials has been promoted in the United States, Brazil, Thailand, etc. On the other hand, since starch and sucrose are also sources of food, from an ethical point of view, conversion to inedible parts of plant biomass is required.

[0003] Therefore, cassava meal has attracted attention as a raw material that is easier to handle than the inedible parts of plant biomass and does not compete with food. The rhizome of cassava has been used as a raw material for making starch since ancient times because it contains a large amount of starch. Cassava meal is the residue after the starch is taken out from the rhizome of cassava. In addition to the cellulose-based fiber components, a large amount of starch remains in cassava meal, which is sometimes used as feed, but the current situation is that the utilization value is low and most of it is discarded.

[0004] As a method for producing sugars using the inedible parts of plant biomass as raw materials, Patent Document 1 discloses a method in which a saccharified liquid obtained by hydrolyzing a biomass containing cellulose is passed through a microfiltration membrane having an average pore diameter 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 particulates and polymer components, the liquid is filtered through a nanofiltration membrane and / or a reverse osmosis membrane, a purified / concentrated sugar liquid is recovered from the non-permeate side, and fermentation inhibitors are removed from the permeate side.

[0005] In a method for producing sugars using inedible parts of plant biomass as raw materials, as a prior art using cassava meal as a raw material, Patent Document 2 discloses a method for producing a glucose solution by hydrolyzing a hot water treated product obtained by subjecting cassava meal to a hot water treatment so that the solid content concentration of the cassava meal becomes 10% by weight or less, filtering a liquid fraction obtained by further subjecting the hydrolyzate to solid-liquid separation through an ultrafiltration membrane having a molecular weight cutoff of 300 to 200,000 Da, recovering a saccharifying enzyme from the non-permeated liquid side, and recovering a saccharified liquid from the permeated liquid side. Patent Document 3 discloses a method for producing a sugar liquid, wherein a saccharified liquid obtained by hydrolyzing cassava meal with a saccharifying enzyme having polygalacturonic acid-degrading activity is filtered through a separation membrane having a molecular weight cutoff in the range of 2,000 to 100,000 Da, the saccharifying enzyme is recovered from the non-permeate side, the saccharified liquid is recovered from the permeate side, the recovered saccharified liquid is further filtered through a separation membrane having a molecular weight cutoff of 150 to 1,000 Da, galacturonic acid, which is a fermentation inhibitor, is separated and removed on the non-permeate side, and a refined sugar liquid is recovered from the permeate side.

[0006] Prior art literature

[0007] Patent Literature

[0008] Patent document 1: WO2010 / 067785

[0009] Patent Document 2: WO2019 / 189650

[0010] Patent document 3: WO2019 / 189651 Summary of the invention

[0011] Problems to be solved by the invention

[0012] The present inventors have newly discovered that when an attempt is made to concentrate sugars derived from cassava meal by filtering a saccharified liquid obtained by enzymatic hydrolysis of cassava meal through a nanofiltration membrane and / or a reverse osmosis membrane, clogging of the membrane occurs.

[0013] Means for solving problems

[0014] In order to solve the above-mentioned problems, intensive studies were conducted, and as a result, it was found that the saccharified liquid obtained by enzymatic hydrolysis of cassava meal contains high-molecular components (weight-average molecular weight 10,000 to 20,000 Da) derived from cassava meal, which become clogging components of the membrane in the filtration treatment step using a nanofiltration membrane and / or a reverse osmosis membrane for concentrating sugars. Furthermore, by filtering the saccharified liquid through a separation membrane with a molecular weight cutoff of more than 100,000 Da and less than 300,000 Da, the high-molecular components derived from cassava meal can be blocked on the non-permeate side, thereby completing the present invention.

[0015] That is, the present invention is constituted by the following [1] to

[10] .

[0016] [1] A method for producing a sugar solution, comprising the following steps (1) to (3).

[0017] Step (1): a step of enzymatically hydrolyzing cassava meal to obtain a saccharified liquid;

[0018] Step (2): filtering the saccharified liquid obtained in step (1) through a separation membrane having a molecular weight cutoff of more than 100,000 Da and less than 300,000 Da, thereby blocking high molecular weight components derived from cassava meal having a weight average molecular weight of 10,000 to 20,000 Da on the non-permeate side; and

[0019] Step (3): A step of filtering the filtrate obtained in step (2) through a nanofiltration membrane and / or a reverse osmosis membrane to recover a sugar solution from the non-permeated liquid side.

[0020] [2] The method for producing a sugar solution according to [1], wherein the molecular weight cutoff of the separation membrane in the step (2) is greater than 100,000 Da and less than 200,000 Da.

[0021] [3] The method for producing a sugar solution according to [1] or [2], wherein the separation membrane in the step (2) is a hollow fiber membrane.

[0022] [4] The method for producing a sugar solution according to any one of [1] to [3], wherein the enzyme in the step (1) has at least cellulase activity and / or amylase activity.

[0023] [5] The method for producing a sugar solution according to any one of [1] to [4], wherein the step (1) is a step of enzymatically hydrolyzing cassava meal and subjecting the obtained saccharified solution to solid-liquid separation.

[0024] [6] The method for producing a sugar solution according to [5], wherein the solid-liquid separation is filter press.

[0025] [7] The method for producing a sugar solution according to [5] or [6], wherein the turbidity of the saccharified liquid obtained by the solid-liquid separation in the step (1) is 300 NTU or less.

[0026] [8] A sugar solution comprising 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 area ratio of the peak of the high molecular weight component derived from cassava meal having a weight average molecular weight of 10,000 to 20,000 Da in the total peak area in gel filtration chromatography analysis using pullulan as a standard substance using a differential refractometer is 0.1 to 2%.

[0027] [9] The sugar solution according to [8], further comprising a component derived from cassava meal having a weight average molecular weight of less than 10,000, wherein the area ratio of the peak of the component derived from cassava meal having a weight average molecular weight of less than 10,000 in the area of ​​all peaks in gel filtration chromatography analysis using pullulan as a standard substance using a differential refractometer is 98 to 99.9%.

[0028]

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

[0029] Effects of the Invention

[0030] According to the present invention, since the saccharified liquid obtained by enzymatic hydrolysis of cassava meal is filtered through a separation membrane with a molecular weight cutoff of more than 100,000 Da and less than 300,000 Da, high molecular components (weight average molecular weight 10,000 to 20,000 Da) derived from cassava meal can be removed, clogging of the membrane is not caused in the subsequent filtration step through the nanofiltration membrane and / or reverse osmosis membrane, and a concentrated sugar liquid can be produced efficiently.

[0031] 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 improve the productivity of chemical products. DETAILED DESCRIPTION

[0032] Cassava meal is a byproduct of starch production from cassava rhizomes (cassava tubers), and is the residue discharged after the starch is extracted. More specifically, when starch is produced from cassava tubers, the cassava tubers are washed / peeled and then roughly crushed, then ground and dispersed, and sieved to separate into starch and fiber components, and the fiber component that is sieved and separated is cassava meal. Cassava meal can be produced by purchasing cassava tubers and using the above method, or it can be purchased from a cassava meal discharger, or purchased as a commercially available material for feed.

[0033] There are wet products in which water remains in the cassava dregs, and dry products obtained by drying the wet products, but both can be used in the present invention. In addition, only one of them can be used, or a mixture can be used. In addition, when used in the present invention, the particle size can be adjusted by pulverizing treatment, etc.

[0034] Hereinafter, each step of the method for producing the sugar solution of the present invention using cassava meal as a raw material will be described.

[0035] [Process (1)]

[0036] Step (1) is a step of hydrolyzing cassava meal with an enzyme to obtain a saccharified liquid containing a hydrolyzate containing sugar. As described above, cassava meal is a residue containing fiber components that is discharged after starch is removed, so it contains cellulose as a fiber component, and further, starch also remains. Therefore, as the enzyme in this step, an enzyme that can hydrolyze cellulose and / or starch is used.

[0037] As an enzyme that can hydrolyze cellulose contained in cassava meal, cellulase can be mentioned. Cellulase is an enzyme composition containing enzyme components such as cellobiohydrolase, endoglucanase, exoglucanase, β-glucosidase, endoxylanase, and xylosidase, and having the activity of hydrolyzing cellulose to saccharify it.

[0038] As the cellulase, a commercially available cellulase preparation may be used, or a culture solution of a cellulase-producing microorganism may be used directly, or cellulase purified from the culture solution may be used. Furthermore, these may be used as a mixture.

[0039] Examples of commercially available cellulase preparations include cellulase enzyme blend (manufactured by Sigmar Drizzle Japan Co., Ltd.), Celosin TP25 (manufactured by Echibiyi Co., Ltd.), Acremonium cellulase (manufactured by Meiji Seika Phalma Co., Ltd.), and Meicelase (manufactured by Meiji Seika Phalma Co., Ltd.).

[0040] As cellulase producing microorganisms, filamentous fungi microorganisms such as Trichoderma, Acremonium, Talaromyces, Aspergillus, Cellulomonas, Clostridium, Streptomyces, Humicola, Irpex, Mucor, Phanerochaete, white rot fungi, and brown rot fungi can be exemplified. In addition, it can be cellulase derived from mutant strains in which cellulase productivity is improved by mutagenization treatment of these microorganisms with mutagens or ultraviolet irradiation. Among such cellulases derived from filamentous fungi, it is preferred to use cellulases derived from Trichoderma and / or cellulases derived from Acremonium that produce enzyme components with high specific activity in the hydrolysis of cellulose in large quantities in the culture solution.

[0041] As for the amount of cellulase added for hydrolyzing the cellulose contained in the cassava meal, it is sufficient to appropriately set an appropriate range that can effectively and economically implement the hydrolysis of cellulose. For example, 0.001U to 10U per 1g of dry weight of cassava meal is preferred, and 0.01 to 5U is more preferred. It should be noted that the dry weight of cassava meal is the weight obtained by subtracting the weight of the water contained in the cassava meal from the weight of the cassava meal (Wa (g)). Specifically, use: use an infrared moisture meter to measure the moisture ratio Rw (weight %), and the value calculated by the following (Formula 1). Use: Use an infrared moisture meter FD-720 (manufactured by Kit Scientific Laboratory Co., Ltd.) with a drying temperature of 105°C and an automatic stop mode in which the measurement is stopped as soon as the moisture change in 30 seconds becomes less than 0.05%.

[0042] Dry weight (g) = Wa - Wa × Rw···(Formula 1).

[0043] The enzymatic activity of cellulase is measured using the filter paper units (FPU) according to the method of measuring cellulase activity of the National Renewable Energy Laboratory (NREL) (NREL), the Laboratory Analytical Procedure (LAP), and the method described below. Filter paper cut into 1 cm in length and 6 cm in width and 1.0 ml of 50 mM citric acid buffer (pH 4.8) are added to a test tube and heated at 50 ° C for 5 minutes. 0.5 ml of cellulase diluted in a manner that the amount of glucose generated per 0.5 ml of enzyme solution becomes 2 mg is added to the test tube and heated at 50 ° C for 60 minutes. 3 ml of dinitrosalicylic acid reagent is added to stop the reaction and boil for 5 minutes. 0.2 ml of the reaction solution that has developed color is mixed with 2.5 ml of water and the absorbance at 540 nm is measured. The standard curve was developed by performing the same operation using a 2-6.7 g / L glucose standard solution instead of filter paper and enzyme solution. The reagent blank was developed by performing the same operation using only the above-mentioned citric acid buffer solution. The enzyme blank was developed by performing the same operation using the above-mentioned citric acid buffer solution and the above-mentioned diluted cellulase. The activity per unit of enzyme solution was calculated by the following (Formula 2). The activity per 1 mg of protein was calculated by the following (Formula 2) and (Formula 3).

[0044] Activity per unit of enzyme solution (U / ml) = 0.37 / cellulase concentration that produces 2 mg of glucose... (Formula 2).

[0045] Activity per 1 mg of protein (U / mg) = activity per unit of enzyme solution (U / ml) / protein concentration of enzyme solution (mg / ml) (Formula 3).

[0046] The protein concentration can be measured by existing methods such as the Bradford method.

[0047] As an enzyme that can hydrolyze the starch contained in the cassava meal, amylase can be mentioned. The so-called amylase is an enzyme composition containing enzyme components such as α-amylase, β-amylase, glucoamylase, α-glucosidase, and having the activity of hydrolyzing starch (amylose) and saccharifying. Among these enzyme activities, it is preferred to treat with an amylase containing at least α-amylase and / or glucoamylase activity in this step. In addition, in this case, it is preferred to promote the hydration reaction of starch by heating the cassava meal to 60°C or above in advance.

[0048] The amount of amylase added for hydrolyzing starch contained in cassava meal may be set within a suitable range that allows for effective and economical hydrolysis of starch, and 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 further preferably 0.2 U or more. There is no upper limit, but from the viewpoint of economic efficiency, for example, it may be 100 U or less.

[0049] The enzyme activity of α-amylase uses the value measured as the decomposition activity of 2-chloro-4-nitrophenyl 65-azido-65-deoxy-β-maltopentanoside (N3-G5-β-CNP). The "65" of "2-chloro-4-nitrophenyl 65-azido-65-deoxy-β-maltopentanoside" means that the 6-hydroxyl group of the 5th glucose residue from the reducing end side of the glucose constituting the oligomaltose is substituted. Specifically, an appropriately diluted enzyme solution is added to a mixed solution of N3-G5-β-CNP solution and a glucoamylase and a β-glucosidase solution, and the mixture is reacted at 37°C for 10 minutes. In order to stop the reaction and to color the free 2-chloro-4-nitrophenol (CNP), a sodium carbonate solution is added, and the absorbance is measured at 400nm. The blank uses a substance that has been reacted by the same method except that the enzyme solution is added after the sodium carbonate solution is added. One unit (1U) of enzyme was defined as the amount of CNP produced in 1 μmol per minute under the above reaction conditions, and was calculated by the following (Formula 4). The activity per 1 mg of protein was calculated by the following (Formula 4) and (Formula 5).

[0050] The activity of each unit of enzyme solution (U / ml) = (Es-Eb) x 0.179 x Df (Formula 4).

[0051] Activity per 1 mg of protein (U / mg) = activity per unit of enzyme solution (U / ml) / protein concentration of enzyme solution (mg / ml) (Formula 5).

[0052] In (Formula 4), Es represents the absorbance of the measurement sample, Eb represents the absorbance of the blank, and Df represents the dilution ratio of the measurement sample.

[0053] 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 a G2-β-PNP solution and a β-glucosidase solution, and the mixture is reacted at 37°C for 10 minutes. In order to stop the reaction and to color the free 4-nitrophenol (PNP), a sodium carbonate solution is added, and the absorbance is measured at 400nm. The blank uses a substance that has been reacted in the same manner except that the enzyme solution is added after the sodium carbonate solution is added. One unit (1U) of the enzyme is set to the amount of 1 μmol of PNP generated in 1 minute under the above reaction conditions, and is calculated by the following (Formula 6). The activity per 1 mg of protein is calculated by the following (Formula 6) and (Formula 7).

[0054] Activity per unit of enzyme solution (U / ml) = (Es-Eb)×0.171×Df···(Formula 6).

[0055] Activity per 1 mg of protein (U / mg) = activity per unit of enzyme solution (U / ml) / protein concentration of enzyme solution (mg / ml) (Formula 7).

[0056] In addition, in (Formula 6), Es represents the absorbance of the measurement sample, Eb represents the absorbance of the blank, and Df represents the dilution ratio of the measurement sample.

[0057] As amylase, a commercially available amylase preparation may be used, or a culture solution of an amylase-producing microorganism may be used directly, or amylase purified from the culture solution may be used. In addition, these plural enzymes may be used in a mixture.

[0058] Examples of commercially available amylase preparations include glucoamylase preparations such as amyloglucosidase from Aspergillus niger (manufactured by Sigamodrich Japan Co., Ltd.), Glucose Plan AF6 (manufactured by Amano Enzyme Co., Ltd.), Glucose Plan NL4.2 ​​(manufactured by Amano Enzyme Co., Ltd.), and amyloglucosidase (manufactured by Megazyme).

[0059] In addition, in addition to cellulase and amylase, other enzymes such as pectinase can be further added. The so-called pectinase is a general term for enzymes that hydrolyze pectin, and is an enzyme composition that contains enzyme components such as pectinesterase, pectin lyase, polygalacturonase, pectin methylesterase, and has the activity of hydrolyzing pectin. These enzyme treatments can be implemented separately, or they can be implemented simultaneously if the enzyme treatment conditions meet the requirements.

[0060] The temperature conditions for the hydrolysis reaction using an enzyme are not particularly limited as long as they are within the range of the most suitable temperature for the enzyme used, and are preferably 30 to 60°C, for example.

[0061] The pH of the enzymatic hydrolysis reaction is not particularly limited as long as it is within the optimal pH range for the enzyme used, and is preferably pH 4 to 7. The pH adjuster may be an existing acid or base, and as an acid, for example, hydrochloric acid, sulfuric acid, etc. may be used, and as an base, for example, sodium hydroxide, ammonia, etc. may be used.

[0062] The treatment time of the hydrolysis reaction using the enzyme can be set within the range of fully carrying out the decomposition reaction using the enzyme, for example, preferably 1 to 24 hours, more preferably 2 to 22 hours, and further preferably 4 to 20 hours. If the treatment time is less than 1 hour, the reaction is not fully carried out, and if it is longer than 24 hours, it becomes the cause of pollution.

[0063] Before the enzymatic hydrolysis reaction of the cassava meal, a pretreatment for improving the reactivity of the enzyme to the cassava meal can be performed. The pretreatment method can be a known pretreatment method, and examples include acid treatment using sulfuric acid, acetic acid, etc., alkali treatment using caustic soda, ammonia, etc., hydrothermal treatment, subcritical water treatment, cooking treatment, etc. These treatment methods can be implemented alone, or a plurality of treatment methods can be combined.

[0064] The saccharified liquid obtained in step (1) contains sugars and insoluble solids generated by hydrolyzing cellulose and / or starch contained in cassava pulp. The saccharified liquid containing sugars and insoluble solids may be directly supplied to the subsequent step (2), but it is preferred that the saccharified liquid be subjected to solid-liquid separation to reduce the insoluble solids before being supplied to the subsequent step (2).

[0065] The method of solid-liquid separation is not particularly limited, and examples thereof include centrifugal separation using a spiral decanter, a separation plate type centrifuge, a cyclone separator, etc., sedimentation separation, pressing separation using a screw press, etc., filtration separation using a filter press, a belt press, a belt filter, a pre-coat filter, etc., and these may be combined. As methods for solid-liquid separation, there are continuous and intermittent methods, and either method may be used. The saccharified liquid of cassava meal contains fibers suspended in water and fibers settled in water, and preferably a filter press, a belt press, etc., which can be removed in one step, is used, and a filter press is more preferred. The type of filter press may be vertical or horizontal. The method of delivering liquid in the case of filter pressing may be performed using a pump, or may be delivered by pressure using compressed gas. The pressing pressure is not particularly limited as long as the filtrate can be recovered, and for example, is preferably 0.01 to 3 MPa, and more preferably 0.05 to 1 MPa.

[0066] The solid-liquid separation temperature is not particularly limited as long as it is within the applicable temperature range of the device used, and is preferably 20 to 50°C, for example.

[0067] The recovery rate of the saccharified liquid by solid-liquid separation measured by the following (Formula 8) is preferably 50% or more.

[0068] Recovery rate %=(weight of saccharified liquid recovered after solid-liquid separation) / (weight of saccharified liquid before solid-liquid separation)×100···(Formula 8).

[0069] The amount of insoluble solids in the saccharified liquid obtained in step (1) is reduced by subjecting the saccharified liquid to solid-liquid separation. As a method for evaluating the amount of insoluble solids, turbidity can be used. The so-called turbidity (Tubidity) is defined in "The so-called turbidity indicates the degree of turbidity of water, and is expressed by visual turbidity, transmitted light turbidity, scattered light turbidity and integrating sphere turbidity. When measured in comparison with a kaolin standard solution, "degree (kaolin)" is used as the unit, and when measured in comparison with a formazin standard solution, "degree (formazin)" is used as the unit." and JISK0101 "Industrial Water Testing Method (Industrial Water Testing Method)". In the present invention, the turbidity "NTU" measured by the scattered light measurement method using a formazin standard solution that has excellent reproducibility and stability compared to a kaolin standard solution is used. The so-called NTU refers to the "Nephelometric Tubidity Unit". In consideration of the subsequent steps, the turbidity of the saccharified liquid obtained by the solid-liquid separation in the present step (1) is preferably as low as possible, and specifically, it is preferably 1,000 NTU or less, more preferably 500 NTU or less, further preferably 300 NTU or less, and particularly preferably 150 NTU or less. If the turbidity of the saccharified liquid exceeds 1,000 NTU, the filterability by membrane separation in the subsequent step (2) may be reduced.

[0070] Although the insoluble solid content in the saccharified liquid can be reduced by subjecting the saccharified liquid containing the insoluble solid content obtained in step (1) to solid-liquid separation as described above, by adding an acidic substance to the saccharified liquid before solid-liquid separation, the insoluble solid content can be reduced without using a solid-liquid separation device well known to those skilled in the art, and the burden of solid-liquid separation can be reduced. Furthermore, by subjecting the solid-liquid separation to the addition of the acidic substance, the insoluble solid content in the saccharified liquid after the solid-liquid separation can be further reduced.

[0071] The insoluble solid content contained in the saccharified liquid obtained in step (1) is an insoluble solid content that has not been subjected to the hydrolysis reaction using the enzyme in step (1), and is observed to contain, for example, polysaccharides, pectin, lignin, etc., but is not limited thereto.

[0072] Whether the insoluble solid content is reduced can be evaluated by comparing the insoluble solid content before and after the addition of the acidic substance. As a quantitative method for the insoluble solid content, it can be quantified according to the method specified in the standard of JIS K0102 14.1 Suspended solids (MLSS) (2019).

[0073] The acidic substance is not particularly limited as long as it can reduce the insoluble solid content contained in the saccharified liquid, but is preferably a monobasic acidic substance, more preferably hydrochloric acid, nitric acid, acetic acid, lactic acid or formic acid, and still more preferably hydrochloric acid.

[0074] The pH of the saccharified liquid after the addition of the acidic substance is not particularly limited, but is preferably pH 3.0 or higher and less than 4.5, and more preferably pH 3.0 or higher and less than 4.0. If the pH is less than 3.0, the growth of microorganisms may be adversely affected when the produced sugar liquid is fermented and utilized, while the insoluble solid content, which is the technical feature of the present invention, can be reduced by setting the pH to less than 4.5.

[0075] [Step (2)]

[0076] In the present invention, in step (2), a separation membrane with a molecular weight cutoff of more than 100,000Da and less than 300,000Da is used. The so-called molecular weight cutoff in the present invention is the molecular weight at which the rejection rate of the solute becomes 90% in a molecular weight cutoff curve in which data is plotted with the molecular weight of the solute as the horizontal axis and the rejection rate as the vertical axis. As a method for determining the molecular weight cutoff, various dextran standard samples and protein standard samples with clear molecular weights are filtered through a separation membrane, and their rejection rates are evaluated. The separation curve is plotted to determine the molecular weight cutoff. Specifically, a separation membrane with a molecular weight cutoff of 100,000Da refers to a separation membrane that blocks 90% of molecules with a molecular weight of 100,000Da.

[0077] The present inventors have found that the saccharified liquid obtained in step (1) of the present invention contains a high molecular weight component (hereinafter referred to as "high molecular weight component") having a weight average molecular weight of 10,000 to 20,000 Da derived from cassava meal, and that the high molecular weight component causes membrane clogging in subsequent filtration treatment of nanofiltration membranes and / or reverse osmosis membranes. The high molecular weight component is presumed to be a byproduct of enzymatic hydrolysis of cassava meal, and contains polysaccharides as a main component. If the saccharified liquid containing high molecular weight components is filtered through a separation membrane having a molecular weight cutoff of more than 100,000 Da and less than 300,000 Da, preferably more than 100,000 Da and less than 200,000 Da or more than 120,000 Da and less than 300,000 Da, and more preferably more than 120,000 Da and less than 200,000 Da, it is expected that the high molecular weight components will also pass through the separation membrane. However, in step (2) of the present invention, contrary to expectation, the high molecular weight components can be blocked on the non-permeate side by the separation membrane having a molecular weight cutoff of more than 100,000 Da and less than 300,000 Da. It should be noted that if the molecular weight cutoff of the separation membrane exceeds 300,000 Da, the amount of high molecular weight components that can be blocked on the non-permeate side decreases, and the amount of high molecular weight components on the permeate side increases, so that the nanofiltration membrane and / or the reverse osmosis membrane is blocked in step (3), which is not preferred. Furthermore, when a separation membrane having a molecular weight cutoff of 100,000 Da or less is used, this is not preferred because the separation membrane immediately becomes blocked in step (2).

[0078] The separation characteristics of the separation membrane in step (2) are determined by a dense layer called a functional layer. As the raw material of the functional layer of the separation membrane used in step (2), raw materials such as polyethersulfone (PES), polysulfone (PS), polyacrylonitrile (PAN), polyvinylidene fluoride (PVDF), regenerated cellulose, cellulose, cellulose ester, sulfonated polysulfone, sulfonated polyethersulfone, polyolefin, polyvinyl alcohol, polymethyl methacrylate, and polytetrafluoroethylene can be used. However, since regenerated cellulose, cellulose, and cellulose ester are decomposed by cellulase, it is preferable to use a separation membrane using a synthetic polymer compound such as PES and PVDF as the raw material of the functional layer.

[0079] The flux in the filtration using the separation membrane in step (2) can be determined by taking into account the fouling of the membrane, and is, for example, preferably 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 pressure difference between the membranes of the separation membrane and rapid fouling of the membrane may sometimes be caused. If the flux is less than 0.01 m / day, the number of membrane strips required for membrane separation increases, and the equipment cost rises.

[0080] The flux (m / day) refers to the amount of water filtered through the membrane per unit membrane area per unit time, and can be calculated using the following formula (Formula 9).

[0081] Flux (m / day) = the amount of water filtered through the membrane per day (m 3 / day) / membrane area (m 2 )···(Formula 9).

[0082] The separation membrane of step (2) can use a material of appropriate form such as a hollow fiber type, a tube type, a flat membrane type, a spiral type, etc., but since the saccharified liquid contains insoluble solid components, it is preferably a separation membrane resistant to membrane fouling, specifically, preferably a hollow fiber type or a tube type, and more preferably a hollow fiber type (i.e., a hollow fiber membrane). The so-called hollow fiber membrane refers to a separation membrane with a functional layer on its inner or outer side, which is hollow inside. The hollow fiber membrane can be roughly divided into an internal pressure type hollow fiber membrane that allows filtered raw water to pass through the inner side of the hollow fiber membrane and obtains a filtrate on the outer side of the hollow fiber membrane, and an external pressure type hollow fiber membrane that allows filtered raw water to pass through the outer side of the hollow fiber membrane and obtains a filtrate on the inner side of the hollow fiber membrane, but the external pressure type hollow fiber membrane has the characteristics of being resistant to membrane fouling caused by solid substances compared with the internal pressure type, and is also easy to wash, so it is preferably used in this step (2).

[0083] Specific examples of the separation membrane for step (2) include SPE200, SPE300, LV, BX and V5 manufactured by Synder, "Microzer" (registered trademark) UF series manufactured by Asahi Kasei Corporation, UV200 manufactured by NADIR, "Toray Fil" (registered trademark) HFU series and HSU series manufactured by Toray Corporation, "Toray UF" HFU series and HSU series manufactured by Toray Corporation, etc.

[0084] The membrane separation method of step (2) may be full filtration or cross flow filtration, but cross flow filtration is preferred. In addition, the membrane separation method of step (2) may be constant pressure filtration or constant flow filtration. In the case where the separation membrane is clogged, backwashing may be performed by passing a washing liquid from the permeable side of the membrane to the non-permeable side, or by supplying gas to the non-permeable side of the membrane to remove the filter cake formed on the membrane surface. The washing liquid for backwashing may include a filtrate of the separation membrane, water, or a liquid medicine.

[0085] [Step (3)]

[0086] Since the content of high molecular weight components that become membrane clogging components of the nanofiltration membrane and the reverse osmosis membrane in the filtrate obtained in step (2) is reduced, by further filtering through the nanofiltration membrane and / or the reverse osmosis membrane, a sugar solution in which the sugar derived from the cassava meal is concentrated can be recovered from the non-permeate liquid side of each separation membrane.

[0087] Nanofiltration membrane is also called nanofilter (nanofiltration membrane, NF membrane), which is generally defined as a membrane that allows monovalent ions to pass through and blocks divalent ions. It is a membrane that is believed to have tiny gaps of several nanometers and is mainly used to block tiny particles, molecules, ions, salts, etc. in water.

[0088] As raw materials for the nanofiltration membrane, polymer raw materials such as cellulose acetate polymers, polyamides, polyesters, polyimides, and vinyl polymers can be used. However, if the cellulose acetate membrane is used for a long time, a part of the enzyme used in step (1), especially the cellulase component, may permeate in step (2) to decompose the cellulose as the membrane raw material. Therefore, a nanofiltration membrane having a polyamide material is preferred. The raw material of the nanofiltration membrane is not limited to a membrane composed of one of the above raw materials, and may be a membrane containing a plurality of membrane raw materials.

[0089] As the form of nanofiltration membrane, hollow fiber type, tubular type, flat membrane type, spiral type etc. can be enumerated, but are not limited to these. In addition, as filtering mode, it can be full amount filtration, it can also be cross flow filtration, it can also be constant pressure filtration and constant flow filtration.

[0090] Reverse osmosis membranes, also known as RO membranes, are generally defined as "membranes with a desalination function that includes monovalent ions." They are membranes that are believed to have ultra-fine pores ranging from several angstroms to several nanometers, and are mainly used to remove plasma components in seawater desalination and ultrapure water production.

[0091] As the raw material of the reverse osmosis membrane, polymer raw materials such as cellulose acetate polymers, polyamides, polyesters, polyimides, and vinyl polymers can be used. However, if the cellulose acetate membrane is used for a long time, a part of the enzyme used in step (1), especially the cellulase component, may permeate in step (2) to decompose the cellulose as the membrane raw material. Therefore, a nanofiltration membrane having a polyamide material is preferred. The raw material of the reverse osmosis membrane is not limited to a membrane composed of one of the above raw materials, and may be a membrane composed of a plurality of membrane raw materials.

[0092] As the form of reverse osmosis membrane, hollow fiber type, tubular type, flat membrane type, spiral type etc. can be enumerated, but are not limited to these. In addition, as filtering mode, it can be full amount filtration, it can also be cross flow filtration, it can be constant pressure filtration and it can also be constant flow filtration.

[0093] The filtration treatment using a nanofiltration membrane and / or a reverse osmosis membrane can be performed according to the method described in WO2010 / 067785.

[0094] In order to further increase the sugar concentration of the sugar solution recovered from the non-permeated liquid side after passing through the nanofiltration membrane and / or the reverse osmosis membrane, it may be further concentrated. The concentration method is not particularly limited and may be membrane concentration, evaporation concentration, or a combination of these.

[0095] [Concentrated sugar solution]

[0096] The sugar solution obtained in step (3) (hereinafter, the sugar solution recovered from the non-permeated liquid side of the nanofiltration membrane and / or the reverse osmosis membrane, and the sugar solution obtained by further concentrating the sugar solution are collectively referred to as "concentrated sugar solution") has a high concentration of monosaccharides derived from cassava meal, especially glucose, and can be preferably used as a raw material for fermentation of microorganisms. 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 on the glucose concentration of the concentrated sugar solution. If it is a high concentration, glucose is easy to precipitate, so it is preferably 700 g / L or less, and more preferably 650 g / L or less.

[0097] Furthermore, the concentrated sugar solution can be characterized by a molecular weight distribution determined by gel filtration chromatography using a differential refractometer with pullulan as a standard substance.

[0098] The concentrated sugar solution is characterized by comprising a high molecular weight component derived from cassava meal having a weight average molecular weight of 10,000 to 20,000 Da according to the analysis result of the molecular weight distribution in the gel filtration chromatography using pullulan as a standard substance using a differential refractometer, and 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 in the area of ​​all peaks is 0.1 to 2%, preferably 0.2 to 2%, more preferably 0.2 to 1.5%, further preferably 0.2 to 1%, further preferably 0.2 to 0.6%, and particularly preferably 0.3 to 0.6%. In addition to contributing to reducing membrane clogging in the filtration treatment of the nanofiltration membrane and / or the reverse osmosis membrane in the above step (3), this technical feature also contributes to improving the productivity of the chemical product when the concentrated sugar solution is used as a fermentation raw material for producing the chemical product.

[0099] Furthermore, the analysis result of the molecular weight distribution of the concentrated sugar solution in the gel filtration chromatography using pullulan as a standard substance using a differential refractometer is that the concentrated sugar solution includes components derived from cassava meal having a weight average molecular weight of less than 10,000, and the area ratio of the peaks of components derived from cassava meal having a weight average molecular weight of less than 10,000 in the area of ​​all peaks is preferably 98 to 99.9%, more preferably 98 to 99.8%, further preferably 98.5 to 99.8%, further preferably 99 to 99.8%, particularly preferably 99.4 to 99.8%, and most preferably 99.4 to 99.7%.

[0100] As the purpose of the concentrated sugar solution, the concentrated sugar solution can be used as a fermentation raw material to manufacture chemicals. Microorganisms used when using the concentrated sugar solution as a fermentation raw material to manufacture chemicals include yeasts such as baker's yeast, bacteria such as Escherichia coli and Corynebacterium glutamicum, filamentous fungi, actinomycetes, etc. The microorganism can be a substance separated from the natural environment, and can also be a substance whose properties have been partially changed by mutation or genetic recombination.

[0101] As chemicals obtained by fermentation using concentrated sugar solution as fermentation raw material, there is no particular limitation as long as it is a substance produced by microorganisms and cultured cells. As a specific example, substances produced in large quantities in the fermentation industry such as alcohols, organic acids, amino acids, nucleic acids, etc. can be cited. For example, as alcohols, ethanol, butanol, 2,3-butanediol, 1,4-butanediol, glycerol, etc. can be cited, as organic acids, acetic acid, lactic acid, succinic acid, malic acid, etc. can be cited, as amino acids, lysine, glutamic acid, etc. can be cited, as nucleic acids, inosinic acid, guanylic acid, inosine, guanosine, etc. can be cited. In addition, it can also be applied to the production of substances such as proteins, antibiotics, etc. such as enzymes. Therefore, various chemicals can be manufactured by the present invention.

[0102] Example

[0103] (Reference Example 1) Glucose concentration analysis method

[0104] The concentration of glucose contained in the sugar solution was quantified by comparison with a standard substance under the conditions of high performance liquid chromatography (HPLC) shown below.

[0105] (HPLC conditions)

[0106] Column: Shodex Sugar series SH1011 (manufactured by Rakuten Co., Ltd.)

[0107] Mobile phase: sulfuric acid 5mM (flow rate 0.6mL / min)

[0108] Reaction liquid: None

[0109] Detection method: RI (differential refractive index)

[0110] Temperature: 65℃.

[0111] (Reference Example 2) Preparation of cassava meal saccharification liquid

[0112] 3.5 L of RO water and 0.02 U of a thermostable amylase (manufactured by Shigma Aldrich Japan) as α-amylase were added to 8.5 kg of cassava meal (water content 83%), mixed, 4N sodium hydroxide (manufactured by Nacalai Test 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 derived from Aspergillus niger (manufactured by Shigma Aldrich Japan) as glucoamylase and 0.2 U of Acremonium cellulase (manufactured by Meiji Seika Falma Co., Ltd.) as a cellulase were added per 1 g of dry weight of cassava meal, and an enzyme reaction was performed at 50° C. for 8 hours while stirring to obtain a saccharified liquid.

[0113] The obtained saccharified liquid was subjected to solid-liquid separation by a filter press to remove the residue, thereby obtaining a filter press filtrate which is a saccharified liquid with reduced insoluble solid content.

[0114] The turbidity of the filter press filtrate was analyzed by a portable turbidity meter 2100P (manufactured by HACH) and the result was 150 NTU. In addition, the glucose concentration of the filter press filtrate was measured by the method of Reference Example 1 and the result was 60 g / L.

[0115] (Reference Example 3) Reduction of insoluble solid content by adding an acidic substance to cassava meal saccharification liquid

[0116] 8.5 kg of cassava meal (water content: 83%) was mixed with 3.5 L of RO water and 0.02 U of a thermostable α-amylase (manufactured by Sigma-Aldrich Jampan Co., Ltd.), and the mixture was adjusted to pH 5.0 by adding 4N sodium hydroxide (manufactured by Nacalai Test Co., Ltd.), followed by autoclaving at 90°C for 2 hours. Cassava meal was added in such a manner that the dry weight of the meal became 12% by weight, and 0.2 U of amyloglucosidase derived from Aspergillus niger (manufactured by Shigma Aldrich Japan) as a glucoamylase and 1 U of Acremonium cellulase (manufactured by Meiji Seika Fluma) as a cellulase were added per 1 g of the dry weight of the cassava meal, and an enzyme reaction was carried out at 50°C for 24 hours while mixing / stirring to obtain a cassava meal saccharified liquid.

[0117] The obtained cassava meal saccharification liquid was centrifuged (1,500×G, 3 minutes) or filtered by a filter press under various conditions with and without the addition of acidic substances, and the amount of insoluble solid content in the cassava meal saccharification liquid, centrifugal separation liquid fraction, and filter press filtrate was compared. It should be noted that for the addition of acidic substances, 10N hydrochloric acid was added to the cassava meal saccharification liquid to adjust the pH to 3.5, and the pH of the saccharification liquid without the addition of acidic substances was 4.5 (the pH of cassava meal was lower than that at the beginning of hydrolysis because galacturonic acid was generated during hydrolysis). The determination of the insoluble solid content was carried out by calculating MLSS (mg / L) in accordance with JISK0102 14.1 Suspended solids (MLSS) (2019), and converting the value into ppm for quantitative evaluation. The results are shown in Table 1, confirming that the insoluble solid content in the cassava meal saccharification liquid, centrifugal separation liquid fraction, and filter press filtrate was reduced by adding acidic substances.

[0118] (Reference Example 4) Effect of adding an acidic substance to bagasse saccharification liquid on insoluble solid content

[0119] RO water 6.5 L and 4N sodium hydroxide (manufactured by Nakalai Tesque Co., Ltd.) 562 mL were added to 1.82 kg of bagasse (water content 45%), and the mixture was reacted at 90°C for 2 hours. Then, 10N hydrochloric acid was added to adjust the pH at the start of saccharification to 5.0, and the mixture was added so as to be 12% by weight as a dry weight of bagasse, and 0.2 U of amyloglucosidase derived from Aspergillus niger (Sigma Aldrich Japan) as a glucoamylase per 1 g of dry weight of bagasse and 1 U of Acremonium cellulase (manufactured by Meiji Seika Fluma Co., Ltd.) as a cellulase per 1 g of dry weight of bagasse were added, and the mixture was subjected to enzyme reaction at 50°C for 24 hours while mixing and stirring to obtain a bagasse saccharified liquid.

[0120] The obtained bagasse saccharification liquid was centrifuged or filtered by a filter press in the same manner as in Reference Example 3 under various conditions with and without the addition of an acidic substance, and the amounts of insoluble solid components in the bagasse saccharification liquid, the centrifuged liquid fraction, and the filter press filtrate were compared. It should be noted that for the condition where an acidic substance was added, 10N hydrochloric acid was added to the bagasse saccharification liquid to adjust the pH to 3.5. The pH of the saccharification liquid without the addition of an acidic substance was 4.5 (pH 4.5 after hydrolysis. During saccharification, a portion of the sugars were converted to lactic acid by microorganisms, and the pH was lower than at the beginning of hydrolysis. However, the proliferation of microorganisms was inhibited by the reduction in pH, and the pH was stabilized at 4.5.). In addition, the insoluble solid content was measured in the same manner as in Reference Example 3. The results are shown in Table 1. Although the insoluble solid content of cassava meal saccharified liquid, the centrifugal separation liquid fraction of cassava meal saccharified liquid and the filter press filtrate of cassava meal saccharified liquid was reduced by adding acidic substances, the insoluble solid content of bagasse saccharified liquid, the centrifugal separation liquid fraction of bagasse saccharified liquid and the filter press filtrate of bagasse saccharified liquid was increased by adding acidic substances.

[0121] [Table 1]

[0122] Table 1

[0123]

[0124] (Example 1) Production of concentrated sugar solution of cassava meal saccharified liquid using a hollow fiber membrane with a molecular weight cutoff of 150,000 Da and a reverse osmosis membrane

[0125] A polyvinylidene fluoride hollow fiber membrane with a nominal pore size of 0.01 μm and a nominal molecular weight cutoff of 150,000 Da used in the ultrafiltration membrane module "Toray UF" HFU (manufactured by Toray Co., Ltd.) was cut out to produce a micromodule (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. 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, and the first membrane separation was performed by cross-flow filtration. As a result, the membrane was not clogged and 1.4 L of filtrate was obtained.

[0126] The filtrate 1L obtained by membrane separation using membrane A was subjected to second membrane separation using UTC-70 (molecular weight cutoff 65, Toray Co., Ltd.) (hereinafter referred to as membrane B) as a reverse osmosis membrane in order to perform a 2-fold concentration. The membrane separation apparatus used was "SEPA" (registered trademark) CF-II (effective membrane area 140 cm 2, GE W&PT), the operating temperature was set to 35°C, the membrane surface linear velocity was set to 20 cm / sec, and the filtration process was performed until the filtration pressure reached 4 MPa. As a result, the membrane was not clogged and 0.5 L of concentrated sugar solution was obtained on the non-permeate side, which was concentrated by 2 times. The glucose concentration of the concentrated sugar solution was measured by the method of Reference Example 1 and the result was 120 g / L.

[0127] (Example 2) Production of concentrated sugar solution of cassava meal saccharified liquid using hollow fiber membrane with molecular weight cutoff of 150,000 Da and nanofiltration membrane

[0128] In order to double-concentrate 1 L of the filtrate obtained by the same method as the first membrane separation in Example 1 using membrane A, a second membrane separation was performed using NFS (molecular weight cutoff 100-250 Da, Synder) (hereinafter referred to as membrane C) as a nanofiltration membrane under the same conditions as in Example 1. As a result, the membrane was not clogged and 0.5 L of a concentrated sugar solution that was double-concentrated was obtained on the non-permeate side. The glucose concentration of the concentrated sugar solution was measured by the method of Reference Example 1 and the result was 120 g / L.

[0129] (Comparative Example 1) Production of Concentrated Sugar Solution of Cassava Slag Saccharified Liquid Using Hollow Fiber Membrane with a Pore Diameter of 0.05 μm and Reverse Osmosis Membrane

[0130] A polyvinylidene fluoride hollow fiber membrane with a nominal pore size of 0.05 μm used in the microfiltration membrane module "トレフィル" (registered trademark) HFS manufactured by Toray Co., Ltd. was cut out to produce a micromodule (hereinafter referred to as membrane D) having an inner diameter of 10 mm and a length of 320 mm consisting of 22 hollow fiber membranes. 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, and the first membrane separation was performed by cross-flow filtration. As a result, the membrane was not clogged and 1.4 L of filtrate was obtained.

[0131] The filtrate 1L obtained by membrane separation using membrane D was subjected to second membrane separation using membrane B in the same manner as in Example 1 in order to perform a 2-fold concentration. As a result, 0.7L of concentrated sugar solution was obtained on the non-permeate side, but the concentration was limited to 1.5 times due to clogging of the membrane. 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.

[0132] (Comparative Example 2) Membrane separation of cassava saccharified liquid using a flat membrane with a molecular weight cutoff of 50,000 Da

[0133] 1.5 L of the 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 used was "SEPA" (registered trademark) CF-II (effective membrane area 140 cm 2 , GE W&PT), the operating temperature was set to 35°C, the membrane surface linear velocity was set to 20 cm / sec, the filtration pressure was set to 0.8 MPa, which is the upper limit of the operating pressure in the catalog, and the filtration treatment was carried out until the filtrate stopped flowing out. As a result, the filtrate was limited to 0.5 L due to membrane clogging.

[0134] [Table 2]

[0135]

[0136] (Example 3) Evaporation concentration of concentrated sugar solution

[0137] The concentrated sugar solution obtained in Example 1 was concentrated using an evaporator to prepare an evaporation-concentrated sugar solution having a glucose concentration of 500 g / L.

[0138] (Reference Example 5) Analysis of Molecular Weight Distribution by Gel Filtration Chromatography (GFC)

[0139] The saccharified solution, and the non-permeated solution and permeated solution obtained by membrane separation were diluted 10 times with a 0.2M sodium nitrate aqueous solution, filtered with a disc filter with a pore size of 0.45μm, and a sample solution for measurement was prepared. The molecular weight in the sample solution was measured by the following method, and the molecular weight was calculated as the average molecular weight converted by the standard (pululan). In the case of exceeding the measurement range of the standard, the average molecular weight was calculated by extrapolation of the calibration curve. In the case of insufficient separation of each peak, vertical segmentation was performed in the peak valley part.

[0140] Device: "Prominence" (manufactured by Shimadzu Corporation)

[0141] Column: OHpakSB-G+SB-805HQ+SB-804HQ (Raykin Co., Ltd.)

[0142] Column temperature: 40°C

[0143] Mobile phase: 0.2M sodium nitrate aqueous solution

[0144] Flow rate: 0.5mL / min

[0145] Detector: Differential Refractometer (RI)

[0146] Injection volume: 100 μL

[0147] Standard: Pullulan.

[0148] (Reference Example 6) Analysis of Molecular Weight Distribution of Cassava Dregs Saccharified Liquid

[0149] The cassava meal saccharification liquid (filter press filtrate) obtained in Reference Example 2 was analyzed for molecular weight distribution by the method described in Reference Example 5, and the obtained results are shown in Table 3. The cassava meal saccharification liquid contained a monosaccharide component with a weight average molecular weight of 250 Da and a polysaccharide with a weight average molecular weight of 14,000 Da as a main component of a high molecular weight component.

[0150] [Table 3]

[0151] Table 3

[0152] Weight average molecular weight (D a) Peak 1 (monosaccharide component) 250 Peak 2 (polymer component) 14，0 00

[0153] (Reference Example 7) Analysis of turbidity and molecular weight distribution of non-permeate and permeate obtained by membrane separation of cassava meal saccharification liquid

[0154] 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 Co.), and molecular weight distribution was analyzed by the method of Reference Example 5 to determine the ratio (%) of the peak area of ​​the high molecular weight component with a weight average molecular weight of 10,000 to 20,000 Da to the total peak area. The results are shown in Table 4, and it was confirmed that in the membrane separation using membrane A, turbidity and high molecular weight components with a weight average molecular weight of 10,000 to 20,000 Da were blocked on the retentate side, while in the membrane separation using membrane D, turbidity was blocked on the retentate side, but high molecular weight components with a weight average molecular weight of 10,000 to 20,000 Da were not blocked and permeated.

[0155] [Table 4]

[0156] Table 4

[0157]

[0158] (Example 4) Molecular Weight Analysis of Cassava Meal Saccharification Liquid, Non-permeate Liquid, Permeate Liquid and Concentrated Sugar Liquid Obtained by Membrane Separation of Cassava Meal Saccharification Liquid, and Evaporation Concentrated Sugar Liquid

[0159] The molecular weight distribution of the cassava pulp saccharification liquid (filter press filtrate) obtained in Reference Example 2, the non-permeate and permeate obtained by membrane separation using membrane A in Example 1, the concentrated sugar liquid as the non-permeate obtained by membrane separation using membrane B in Example 1, and the evaporation concentrated sugar liquid obtained in Example 3 were analyzed by the method described in Reference Example 5. The evaporation concentrated sugar liquid was diluted 5 times with water and then diluted with the 0.2M sodium nitrate aqueous solution described in Reference Example 5, and the molecular weight distribution was analyzed. In addition, as a comparative object, the molecular weight distribution of the non-permeate and permeate obtained by membrane separation using membrane D in Comparative Example 1 was also analyzed. The ratio (%) of the peak area of ​​the high molecular weight component 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 with a weight average molecular weight of less than 10,000 to the total peak area were calculated, and the obtained results are shown in Table 5.

[0160] In the non-permeated liquid and the permeated liquid obtained by the membrane separation using the membrane A in Example 1, the high molecular weight components with a weight average molecular weight of 10,000 to 20,000 Da were blocked by the membrane A, so that the high molecular weight components were concentrated in the non-permeated liquid and significantly reduced in the permeated liquid. In addition, the molecular weight analysis results of the concentrated sugar liquid obtained by membrane separation using the membrane A and the evaporation concentrate of the concentrated sugar liquid were the same as those of the permeated liquid obtained by membrane separation using the membrane A.

[0161] On the other hand, it was confirmed that the molecular weight analysis results of the non-permeate and permeate obtained by membrane separation using membrane D in Comparative Example 1 were almost the same as the analysis results of the saccharified liquid in Reference Example 2, and the high molecular weight components with a weight average molecular weight of 10,000 to 20,000 Da were not blocked by membrane D.

[0162] [Table 5]

[0163]

[0164] (Example 5) Membrane separation of cassava saccharified liquid using a flat membrane with a molecular weight cutoff of 200,000 Da

[0165] 1.5 L of the filter press filtrate obtained in Reference Example 2 was subjected to full 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 a separation membrane. The membrane separation device used was "SEPA" (registered trademark) CF-II (effective membrane area 140 cm 2, GE W&PT), the operating temperature was set to 35°C, and the membrane surface linear velocity was set to 20 cm / sec. The non-permeated liquid and the permeated liquid were sampled, and the molecular weight distribution was analyzed by the method of Reference Example 5 to determine the ratio (%) of the peak area of ​​the high molecular weight component with a weight average molecular weight of 10,000 to 20,000 Da to the total peak area. In addition, the turbidity of the permeated liquid was analyzed using a portable turbidity meter 2100P (manufactured by HACH).

[0166] The amount of the high molecular weight component of 10,000 to 20,000 Da that permeated the separation membrane was calculated by dividing the ratio (%) of the peak area of ​​the high molecular weight component of 10,000 to 20,000 Da to the total peak area of ​​the permeate by the ratio (%) of the peak area of ​​the high molecular weight component of 10,000 to 20,000 Da to the total peak area of ​​the non-permeate, and the result was 0.08, confirming that the high molecular weight component of 10,000 to 20,000 Da was blocked on the non-permeate side of the membrane F. In addition, the turbidity of the permeate was 50 NTU or less, confirming that the turbidity was also blocked on the non-permeate side.

[0167] (Reference Example 8) Analysis method of ethanol

[0168] The ethanol in the culture solution was quantified by comparison with a standard substance under the HPLC conditions shown below.

[0169] Column: Shodex Sugar series SH1011 (manufactured by Rakuten Co., Ltd.)

[0170] Column temperature: 65°C

[0171] Detection method: Differential refractometer

[0172] Detector temperature: 40°C

[0173] Mobile phase: 0.005M H2SO4

[0174] Flow rate: 0.6 mL / min.

[0175] (Example 6) Ethanol fermentation using concentrated sugar solution

[0176] Using the concentrated sugar solution obtained in Example 1 as a fermentation raw material, ethanol was produced by fermentation with microorganisms, and the fermentation rate of ethanol was evaluated.

[0177] The ethanol fermentation microorganism (Saccharomyces cerevisiae (OC-2 strain)) was inoculated into 5 mL of YPD medium and cultured at 30°C for 16 hours (pre-culture). 9 mL of the concentrated sugar solution was filtered through a filter (Sterrickettsup 0.22 μm, Melkumipore) in a test tube, and 1 mL of corn syrup (Prince Corn Starter Co., Ltd.) adjusted to 20% by weight was added and mixed. The pre-culture solution was added to each test tube in such a way that the turbidity became 0.5. The culture was carried out at 30°C, and the ethanol concentration after 24 hours was measured by the method of Reference Example 8. The results of the fermentation rate of ethanol are shown in Table 6.

[0178] (Comparative Example 3) Ethanol Fermentation Using Glucose Solution as a Reagent

[0179] Ethanol fermentation using the reagent glucose solution was carried out in the same manner as in Example 6 except that a reagent glucose solution in which glucose (D(+) glucose, manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in pure water to give a glucose concentration of 120 g / L (the same glucose concentration as 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 increased in the ethanol fermentation using the concentrated sugar solution compared to the case where the reagent glucose solution was used.

[0180] [Table 6]

[0181] Table 6

[0182] Ethanol fermentation rate (g / L / hr) Example 6 1.6 Comparative Example 3 1.1 .

Claims

1. A method for producing a sugar solution, comprising the following steps 1 to 3: Step 1: A step of enzymatically hydrolyzing cassava meal to obtain saccharified liquid; Step 2: filtering the saccharified liquid obtained in step 1 through a separation membrane having a molecular weight cutoff of more than 100,000 Da and less than 300,000 Da, thereby blocking high molecular weight components derived from cassava meal having a weight average molecular weight of 10,000 to 20,000 Da on the non-permeate side; and Step 3: A step of filtering the filtrate obtained in step 2 through a nanofiltration membrane and / or a reverse osmosis membrane to recover a sugar solution from the non-permeated liquid side. 2 . The method for producing a sugar solution according to claim 1 , wherein the molecular weight cutoff of the separation membrane in step 2 is more than 100,000 Da and not more than 200,000 Da. 3 . The method for producing a sugar solution according to claim 1 , wherein the separation membrane in step 2 is a hollow fiber membrane. 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 , wherein the step 1 is a step of enzymatically hydrolyzing cassava meal and subjecting the obtained saccharified solution to solid-liquid separation. The method for producing a sugar solution according to claim 5, wherein the solid-liquid separation is filter pressing. 7 . The method for producing a sugar solution according to claim 5 , wherein the turbidity of the saccharified liquid obtained by the solid-liquid separation in the step 1 is 300 NTU or less.

8. A sugar solution comprising 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 area ratio of the peak of the high molecular weight component derived from cassava meal having a weight average molecular weight of 10,000 to 20,000 Da in the total peak area in gel filtration chromatography analysis using pullulan as a standard substance using a differential refractometer is 0.1 to 2%.

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

10. The sugar solution according to claim 8 or 9, wherein The glucose concentration is above 80 g / L.

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