Methods and compositions for processing cellulosic biomass, and products produced thereby.

Abstract

To provide methods for treating cellulosic biomass to produce cellulosic sugars, and products thereof.SOLUTION: A two-step method for activating cellulosic feedstock is described. The feedstock is subjected to a first high-temperature activation step at a temperature above 190°C. and a second activation step at a lower temperature under alkaline conditions. Also described are methods and compositions for the enzymatic hydrolysis of activated cellulose using one or more cellulase enzymes, a surfactant and polyaspartic acid. Also described are products of the methods.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross-reference of prior applications This application claims priority under the Paris Convention to U.S. Patent Application No. 62 / 145,785, filed Apr. 10, 2015, and U.S. Patent Application No. 62 / 246,271, filed Oct. 26, 2015, the entire contents of which are incorporated herein by reference.

[0002] Technical field This application relates to methods for processing cellulosic biomass to produce cellulosic saccharides. In one aspect, methods for activating cellulosic feedstocks and / or enzymatic hydrolysis for producing glucose are provided. Products produced from the activated cellulosic feedstocks and the enzymatic hydrolysis of the activated cellulosic feedstocks are also provided.

Background Art

[0003] The production of saccharides such as glucose from cellulosic biomass has been the focus of much research and development. However, the high cost and low conversion rates of many of the processes limit the widespread application of cellulosic sugar technology.

[0004]

[0005] While high-yield glucose (>90%) based on cellulose has been reported, these yields are typically obtained at low glucose concentrations, usually 2–5%. Even with high enzyme input, achieving both high yield and high concentration of glucose is difficult because the presence of glucose usually reduces cellulase enzyme activity. Since cellulase enzyme activity decreases over time, maintaining yield requires the addition of new enzymes to the enzymatic hydrolysis reaction. However, the high cost of cellulase enzymes can be very expensive. Cellulase enzymes can also bind to recalcitrant cellulose and / or lignin, preventing their use in further hydrolysis of cellulose to glucose. This unproductive binding also hinders the reuse of the enzyme, which would otherwise be desirable to reduce enzyme usage and lower costs.

[0006] Various different processes have been developed for the pretreatment of cellulosic raw materials, and various enzymatic hydrolysis processes have been developed to convert the treated cellulosic raw materials into sugars. For example, Parekh (PCT Publication No. WO2014 / 026154) describes a two-step pretreatment process for lignocellulosic biomass, mainly under acidic conditions. Schiffino et al. (US Patent Application Publication No. 2011 / 0250645) describe a method for improving the release of monosaccharides from alkali-treated biomass. Liu et al. (US Patent Application Publication No. 2011 / 0300586) describe a two-step pretreatment process for lignocellulosic biomass, with the aim of reducing the crystallinity of cellulose and dissociating hemicellulose-cellulose complexes. Embodiments include mild steam treatment or autohydrolysis followed by hydrolysis with dilute acid or hot water. [Overview of the project]

[0007] summary This summary is intended to guide the reader to the more detailed explanation that follows and is not intended to limit or define any claimed or unclaimed invention. One or more inventions may exist in any combination or sub-combination of components or process steps disclosed in any part of this document, including its claims and drawings.

[0008] In one broad embodiment, a method is provided for activating cellulosic raw materials to increase the chemical and / or enzymatic reactivity of cellulose in the raw material. The activated cellulose can then be converted to cellulosic sugars, for example, by enzymatic hydrolysis of the activated cellulose.

[0009] In this embodiment, the cellulosic raw material may be subjected to a first high-temperature activation step and a subsequent second activation step at a lower temperature under alkaline conditions. In this embodiment, the method may include subjecting the raw material to a first activation step in which the raw material is treated at a temperature higher than 190°C and a pressure higher than 200 psig to produce a first activated cellulose stream containing cellulose II and insoluble solids. The insoluble solids may include components of the raw material other than cellulose, such as lignin. Subsequently, the first activated cellulose stream may be subjected to a second activation step in which the first activated cellulose stream is treated with alkali at a lower temperature than the first activation step to produce a second activated cellulose stream containing cellulose IV. Preferably, the first activation step is carried out in the presence of water.

[0010] Without being limited by theory, the first activation step is thought to alter the crystalline state of cellulose in the cellulosic raw material, producing a first activated cellulose stream containing a higher proportion of cellulose II compared to the amount of cellulose II in the cellulosic raw material. The second activation step is thought to further alter the crystalline state of cellulose in the first activated cellulose stream, producing a second activated cellulose stream containing a higher proportion of cellulose IV compared to the amount of cellulose IV in the first activated cellulose stream. In one embodiment, the two-step activation method described herein produces a mixture of cellulose II, hydrated cellulose II, and alkaline cellulose IV. Optionally, the cellulosic material may be treated, for example, by washing and / or filtering after one or each activation step, to remove soluble non-cellulosic components.

[0011] The cellulose activation methods described herein have also been determined to result in activated cellulose with an increase in glucan levels and / or a decrease in the levels of non-cellulosic components of cellulosic raw materials such as lignin. For example, in one embodiment, the method described herein produces activated cellulose containing at least 60%, at least 70%, or at least 75% glucan. In one embodiment, the method described herein produces activated cellulose containing less than 25%, less than 20%, or less than 15% lignin.

[0012] In another broader embodiment, methods and compositions are provided for stabilizing an enzyme, maintaining its activity, and / or obtaining an enzyme reuse stream during enzymatic hydrolysis.

[0013] In another broad embodiment, an enzymatic hydrolysis mixture suitable for use in the enzymatic hydrolysis of cellulose is provided. In a preferred embodiment, the enzymatic hydrolysis mixture is brought into contact with activated cellulose produced according to the method described herein.

[0014] These embodiments provide one or more cellulase enzymes combined with surfactants and / or dispersants for the enzymatic hydrolysis of cellulose. Without being limited by theory, it is thought that the cellulase enzymes may form complexes with surfactants and / or dispersants, which may stabilize the enzymes, help maintain enzymatic activity, prevent enzymatic degradation, and / or promote the recovery of the enzymes after enzymatic hydrolysis. The presence of dispersants such as oligopeptides is also thought to help prevent unproductive binding of the cellulase enzymes due to interactions with lignin and / or other noncellulosic components. In preferred embodiments, the surfactant is a nonionic surfactant such as a polysorbate surfactant. In another preferred embodiment, the surfactant is a mixture of surfactants such as Tween®, alkoxylated glycerides, and nonylphenol. In one embodiment, the dispersant is a non-enzymatic oligopeptide, optionally a polyamino acid, optionally a polyamino acid having a molecular weight of 500-10,000, 1,000-5,000, or 3,500-4,500. In preferred embodiments, the polyamino acid is polyaspartic acid.

[0015] The methods and compositions described herein offer numerous advantages with respect to the activation of cellulose and / or the production of cellulosic sugars, which can be obtained from several embodiments. For example, in some embodiments, the use of the methods and compositions described herein can produce a glucose-rich sugar stream containing more than about 12% glucose, more than about 14% glucose, more than about 16% glucose, or more than about 18% glucose. Furthermore, in these or other embodiments, the methods and compositions described herein can produce high-yielding monosaccharides. For example, in some embodiments, the methods and compositions described herein can produce glucose yields of more than about 70%, more than about 80%, more than about 85%, more than about 90%, or more than about 95% of the theoretical glucose yield. The theoretical glucose yield in an enzymatic hydrolysis reaction can be determined based on the amount of glucan in the activated cellulosic material subjected to enzymatic hydrolysis. In some preferred embodiments, the methods and compositions described herein can produce a glucose-rich sugar stream having both high glucose yield and high concentration. For example, in one embodiment, a glucose-rich sugar stream has more than 12% glucose and a yield of more than 70%, or more than 14% glucose and a yield of more than 80%, or more than 16% glucose and a yield of more than 90%.

[0016] In another broad embodiment, a method is provided for the enzymatic hydrolysis of activated cellulose to produce cellulosic sugars such as glucose. In this embodiment, the enzymatic hydrolysis may be carried out as a batch process or a continuous process. The enzymatic hydrolysis may be carried out using an enzymatic hydrolysis mixture and / or activated cellulose as disclosed herein.

[0017] In another embodiment, a method is provided for processing a glucose-rich sugar stream to remove the enzyme used in enzymatic hydrolysis. Removal and / or reuse of the enzyme used in enzymatic hydrolysis can reduce the amount of enzyme required for enzymatic hydrolysis and, consequently, reduce the cost associated with the production of cellulosic sugars. For example, the methods and compositions described herein may be used to recover at least 60%, at least 70%, at least 80%, or at least 85% of the cellulase enzyme activity in the enzyme reuse stream after enzymatic hydrolysis. The enzyme reuse stream may be reused to continue processing activated cellulose and / or used to process new activated cellulose, such as a second activated cellulose stream as described herein. In some embodiments, the glucose-rich sugar stream undergoes multiple enzyme removal treatments, either two or more identical enzyme removal treatments or different enzyme removal treatments.

[0018] In another embodiment, a glucose-rich sugar stream produced by a method as described herein is provided. In one embodiment, the sugar stream contains more than 12%, more than 14%, more than 16%, or more than 18% glucose. In one embodiment, the sugar stream contains polyaspartic acid. In some embodiments, the polyaspartic acid is present at concentrations from 1 ppb to 10,000 ppm.

[0019] In another embodiment, a method is provided for producing a glucose-rich sugar stream, comprising (a) supplying activated cellulose containing a mixture of cellulose II, hydrated cellulose II, and alkali cellulose IV; and subjecting the activated cellulose to enzymatic hydrolysis using one or more cellulase enzymes, surfactants, and dispersants to produce a glucose-rich sugar stream. Optionally, the activated cellulose may be produced using the method described herein.

[0020] In another embodiment, a glucose-rich sugar stream is provided that further contains non-glucose sugars, where the non-glucose sugars are one or more xylose, xylooligosaccharides, and xylan. In one embodiment, the non-glucose sugars account for about 3-8%, about 4-7%, or about 5-6% of the dry matter of the composition. In a particular embodiment, the glucose-rich sugar stream contains about 5% non-glucose sugars.

[0021] In a further aspect of the present invention, a fructose-rich sugar stream is provided, prepared by the conversion of glucose to fructose in the glucose-rich sugar stream of the present invention. The fructose-rich sugar stream further contains non-fructose sugars, where the non-fructose sugars are one or more xylose, xylooligosaccharides, and xylan. In one embodiment, the non-fructose sugars account for about 1-8%, about 2-7%, or about 3-6% of the dry matter of the composition. In a particular embodiment, the fructose-rich sugar stream contains about 5% non-glucose sugars.

[0022] In another aspect of the present invention, a glucose syrup or fructose syrup with a lower glycemic index is provided, wherein the glucose syrup or fructose syrup contains about 1-8%, about 2-7%, or about 3-6% of one or more xylose, xylooligosaccharides, and xylan, and its glycemic index is lower than that of conventional glucose syrup or fructose syrup produced by conventional methods.

[0023] Other features and advantages of the present disclosure will become apparent from the following detailed description. However, various changes and modifications within the spirit and scope of the present disclosure will be apparent to those skilled in the art from this detailed description, and thus it should be understood that the detailed description and specific examples are provided for purposes of illustration only while showing embodiments of the disclosure. In particular, it will be recognized that any method may be used with all aspects disclosed herein, or with any specific combination or sub - combination of aspects.

Brief Description of the Drawings

[0024] The drawings included in this application are for the purpose of illustrating various embodiments of the methods and compositions of the teachings herein and are not intended to limit the scope of what is taught. The present disclosure will now be described in connection with the following drawings. [Figure 1] A flow diagram of a method according to a preferred embodiment, including two - stage activation of a cellulosic feedstock, enzymatic hydrolysis of the activated feedstock, and an enzyme removal treatment to obtain an enzyme recycle stream and a saccharide stream rich in glucose and low in enzyme.

Mode for Carrying Out the Invention

[0025] Hereinafter, various methods and compositions are described, providing examples of embodiments of the invention according to each claim. The embodiments described hereinafter do not limit the invention according to any claim, and the invention according to any claim may include methods and compositions different from those described hereinafter. The invention according to the claim is not limited to a method and composition having all the features of any one method and composition described hereinafter, or features common to a plurality or all of the following methods and compositions. The methods or compositions described hereinafter may not be embodiments of the invention according to any claim. Any invention disclosed in a method or composition described hereinafter that is not claimed herein may be the subject of another protective document, such as a continuing patent application, and the applicant, inventor, or owner does not intend to abandon, disclaim, or generally dedicate any such invention by its disclosure herein.

[0026] Various methods and compositions useful for the treatment of cellulosic biomass to produce cellulosic saccharides are described herein. In one embodiment, a method of activating a cellulosic feedstock to produce activated cellulose is provided. Subjecting a cellulosic feedstock to a first activation step at high temperature and high pressure followed by a second activation step at a lower temperature using an alkali has been determined to yield activated cellulose having chemical and / or physical properties advantageous for the hydrolysis of cellulose to monosaccharides.

[0027] The methods disclosed herein use a cellulosic feedstock 10. The cellulosic feedstock 10 can be any feedstock known in the field of cellulosic carbohydrate technology. For example, the cellulosic feedstock can include one or more types of straw, corn stover, bagasse, hardwood, softwood, energy crops, and the like.

[0028] The raw agricultural products supplied to the plant may be processed by crushing, grinding, milling, or other means in order to remove rocks, soil, and other substances present in the raw agricultural products, and to reduce the size of the raw agricultural products or forest-derived raw materials supplied to the process.

[0029] As illustrated in Figure 1, the cellulosic raw material 10 may be supplied to a reactor 14, where the cellulosic raw material 10 is subjected to a first activation step to produce a first activated cellulose stream 16. In the first activation step, the cellulosic raw material 10 may be treated at high temperature and pressure to produce a first activated cellulose stream 16 containing cellulose II and insoluble solids.

[0030] Reactor 14 may be a batch reactor or a continuous-process reactor. In the case of a batch reactor, the cellulosic raw material 10 is supplied to reactor 14, which may be a stirred-tank reactor, and can be brought up to operating conditions for a desired time. If reactor 14 is a continuous-flow reactor, it may be a steam-exposed reactor as known in the art, and can be maintained under desired operating conditions.

[0031] The first activation step may be carried out under conditions that increase the amount of cellulose II in the first activated cellulose stream compared to the amount of cellulose II in the raw material.

[0032] The temperature may be higher than 190°C, optionally higher than 210°C, preferably higher than 220°C, and less than approximately 250°C. Therefore, the process may be carried out at temperatures in the range of 190°C to 250°C, 210°C to 250°C, 220°C to 240°C, or 222°C to 230°C.

[0033] The pressure can be higher than 200 psig and optionally less than 500 psig. At a minimum, the pressure in the reactor corresponds to the temperature by saturated steam thermodynamics. In one embodiment, the pressure can be increased above this value by adding pressurized gas or superheating.

[0034] The cellulosic raw material 10 may be subjected to the first activation step for less than 30 minutes, less than 20 minutes, less than 10 minutes, or less than 5 minutes. The length of the processing time will vary depending on many factors, including the degree of the activation step, for example, the temperature and pressure of the reactor 14.

[0035] It will be understood that temperature, pressure, and processing time can be combined in any desired combination. For example, the first activation step may include exposing the raw material to a pressure of 200-500 psig and a temperature of 200-250°C for 1-30 minutes, or to a pressure of 200-500 psig and a temperature of 190-215°C for less than 4 minutes.

[0036] Optionally, the first activation step is carried out in the presence of water. Water can be introduced into reactor 14 by one or more of the following: water present in the cellulosic raw material 10, water present in reactor 14 when the cellulosic raw material is introduced into reactor 14, and water introduced by the feed stream 12. The total amount of water introduced into the reactor may be at least 30% and may reach 90%. In certain embodiments, 50% of the water is present in the reactor.

[0037] The water present in reactor 14 may be in the form of water vapor or liquid water, preferably in the form of liquid water. It will be understood that the temperature and pressure of the first activation step may be selected so that liquid water is present in reactor 14.

[0038] The first activated cellulose stream 16 may have a solid content of approximately 30% to 50% by weight. The solid mainly contains cellulose which can then be subjected to the second activation step. The solid may further contain lignin, hemicellulose, and trace components such as ash, protein, or extract.

[0039] Optionally, the cellulosic material may be subjected to one or more washing steps after the first activation step and / or the second activation step, either under the same conditions or different conditions. For this purpose, the first activated cellulose stream 16 may be subjected to one or more washing steps before the second activation step to remove hemicellulose and some soluble non-cellulosic components such as ash, extracts, and lignin. The first washing removes these soluble substances, and since the soluble substances are at an acidic pH, the washing step also reduces the need for alkali in the second alkaline activation step.

[0040] As illustrated in Figure 1, the first activated cellulose stream 16 and the washing water 20 can be introduced into the washing reactor 18 to produce wastewater 22 and the washed first activated cellulose stream 24.

[0041] The washing water 20 may be hot water, for example, water at a temperature of about 40°C to 100°C or about 50°C to 95°C. The wastewater stream 22 may be treated and reused in the process or elsewhere, or it may be discarded.

[0042] The washing reactor 18 may be any design known in the art. Optionally, the washing reactor 18 may operate countercurrently, and it may be a countercurrent belt filter. Other filtration or separation methods, such as a filter press, twin-wire press, twin-roll press, rotary vacuum filter, or centrifuge, may also be used.

[0043] As illustrated in Figure 1, the washed first activated cellulose stream 24 is supplied to the reactor 26, where it is subjected to a second activation step to produce a second activated cellulose stream 30. In another embodiment, part or all of the first activated cellulose stream may be introduced into the reactor 26. The following description is based on Figure 1 illustrating the use of the first washing step. In the second activation step, the washed first activated cellulose stream 24 may be treated with alkali at a lower temperature than in the first activation step to produce a second activated cellulose stream containing cellulose IV.

[0044] Reactor 26 may be a batch reactor or a continuous-process reactor. In the case of a batch reactor, the washed first activated cellulose stream 24 is supplied to reactor 26, which may be a stirred-tank reactor, and can be brought up to operating conditions for a desired time. If reactor 26 is a continuous-flow reactor, it may be a steam-exposed reactor as known in the art, and can be maintained at desired operating conditions.

[0045] The second activation step may be carried out under conditions that increase the amount of cellulose IV in the second activated cellulose stream compared to the amount of cellulose IV in the washed first activated cellulose stream 24.

[0046] The temperature can be optionally higher than 60°C and less than approximately 180°C, less than approximately 160°C, less than approximately 140°C, less than approximately 120°C, less than approximately 100°C, or less than approximately 80°C. Therefore, this process can be carried out at temperatures within the ranges of 60°C to 180°C, 60°C to 160°C, 60°C to 140°C, 60°C to 120°C, 60°C to 100°C, or 60°C to 80°C.

[0047] Optionally, the second activation step is performed at superatmospheric pressure. For example, superatmospheric pressure can range from approximately 0.1 to 400 psig.

[0048] The washed first activated cellulose stream 24 may be subjected to the second activation step for less than 180 minutes, less than 120 minutes, less than 90 minutes, or less than 60 minutes, or optionally longer than 15 minutes, longer than 30 minutes, or longer than 45 minutes. The length of the processing time will vary depending on the degree of the activation step, and many factors including, for example, the temperature and pressure of the reactor 26.

[0049] It will be understood that temperature, pressure, and processing time can be combined in any desired combination. For example, the second activation step may include exposing the first activated cellulose stream to a temperature of 60–240°C at a pressure of 0–500 psig for 15–120 minutes, or to a temperature of 80–150°C at a pressure of 0–300 psig for at least 60 minutes.

[0050] As illustrated in Figure 1, the second activation step preferably includes treating the first activated cellulose stream in the presence of an alkali. The alkali can be introduced into reactor 26 in any way. For example, as illustrated, an alkali stream 28 is introduced into reactor 26 separately. It will be understood that the alkali stream 28 may be introduced into reactor 26 prior to, simultaneously with, or after the introduction of the washed first activated cellulose stream 24 into reactor 26. Alternatively, the alkali stream 28 may be introduced into the washed first activated cellulose stream 24, and then the combined stream may be introduced into reactor 26.

[0051] The alkali may include one or more of sodium hydroxide, potassium hydroxide, magnesium hydroxide, and ammonia. In one embodiment, the alkali is sodium hydroxide. In one embodiment, the alkali is added to the first activated cellulose stream 24 at an amount of about 10% to 1%, about 7% to 2%, or preferably less than 6% of the total insoluble solids. The alkali swells the cellulose and further breaks the intermolecular and intramolecular hydrogen bonds of the cellulose, thereby further altering its crystalline structure. Optionally, the second activation step may be carried out in the presence of an oxidizing agent and / or an enzyme, such as laccase and / or a lignin-modifying enzyme.

[0052] Examples of oxidizing agents suitable for use in the second activation step include, but are not limited to, hydrogen peroxide (H2O2). In one embodiment, the oxidizing agent is added at a concentration of less than about 2% and / or higher than 0.0001% of the total insoluble solids in the first activated cellulose stream 16 / 24. In one embodiment, the oxidizing agent is added at less than about 1%, less than about 0.1%, or less than about 0.001%, optionally between about 1% and 0.0001%, of the total insoluble solids in the first activated cellulose stream 16 / 24. Examples of enzymes suitable for use in the second activation step include, but are not limited to, lignin-modifying enzymes, such as laccase oxidases.

[0053] The second activated cellulose stream 30 may have a solids content of approximately 5% to 50% by weight, preferably approximately 20% to 35%. The solids mainly consist of cellulose, which can be optionally recovered and reused. Other components are hemicellulose and lignin, both present in amounts of less than 20%.

[0054] Optionally, the second activated cellulose stream 30 may be subjected to one or more post-activation washing steps to remove alkali and solubilized lignin.

[0055] The second activated cellulose stream 30 and the wash water 34 may be introduced into the wash reactor 32 to produce wastewater 36 and a washed second activated cellulose stream 38. The wash reactor 32 may be operated in the same manner as the wash reactor 18, or in a different manner.

[0056] The washing water 34 may be hot water, for example, water at a temperature of about 50°C to 95°C or about 60°C to 95°C. The wastewater stream 36 may be treated and reused in the process or elsewhere, or it may be discarded.

[0057] The washing reactor 32 may be any design known in the art. Optionally, the washing reactor 32 may operate countercurrently, and it may be a countercurrent belt filter. Other filtration or separation methods, such as a filter press, twin-wire press, twin-roll press, rotary vacuum filter, or centrifuge, may also be used.

[0058] The advantage of subjecting cellulosic raw materials to a first activation step at high temperature followed by a second activation step at a lower temperature under alkaline conditions has been shown to increase the level of glycans and decrease the level of lignin in the second activated cellulose stream 30 compared to raw materials that have undergone only the first high-temperature activation step. Without being limited by theory, the two-step activation process described herein is considered to alter the crystallinity of cellulose in the raw materials and improve the physical and / or chemical properties of cellulose for enzymatic hydrolysis. In one embodiment, the two-step activation process yields activated cellulose containing cellulose II and alkali cellulose IV.

[0059] Those skilled in the art will understand that cellulose exists in several different crystalline structures, corresponding to the positions of interchain and intrachain hydrogen bonds. For example, naturally occurring cellulose found in cellulosic biomass has structure I α and I βCellulose I is the cellulose that has the properties of cellulose I. Cellulose in regenerated cellulose fibers is usually cellulose II. Regenerated cellulose fibers refer to fibers produced by the viscose process for viscose production of cellophane or rayon. The conversion of cellulose I to cellulose II is irreversible. The structures of cellulose III and cellulose IV can be produced by various chemical treatments. Different crystalline forms of cellulose can be identified by characteristic X-ray diffraction patterns. Different crystalline structures of cellulose and cellulose are further described in Perez and Samain, “Structure and Engineering of Cellulose,” Advances in Carbohydrate Chemistry and Biochemistry, Vol. 64, Elsevier (2010), which is incorporated herein by reference in its entirety.

[0060] The washed secondary activated cellulose stream 38 can be subjected to enzymatic hydrolysis using one or more cellulase enzymes 42 in an enzymatic hydrolysis reactor 40 to produce a glucose-rich sugar stream 44. Part or all of the secondary activated cellulose stream 30 may be subjected to enzymatic hydrolysis, and therefore, it will be understood that only part or none of the secondary activated cellulose stream 30 may be washed. The following description applies to the secondary activated cellulose stream 30 whether or not it has undergone the washing step.

[0061] Surprisingly, activated cellulose containing cellulose II (which may be a combination of cellulose II and hydrated cellulose II) and cellulose IV (which may be alkali cellulose IV), which can be produced by the two-step activation process disclosed herein, was found to be particularly susceptible to enzymatic hydrolysis. In particular, activated cellulose showed a remarkable ability to adsorb cellulase enzymes. Contact of activated cellulose with one or more cellulase enzymes in a glucose-rich sugar stream can first produce enzymes adsorbed on the activated cellulose. The cellulose can then be removed from the glucose-rich sugar stream and optionally introduced into the enzymatic hydrolysis reactor 40.

[0062] Accordingly, activated cellulose produced by any method disclosed herein can be subjected to enzymatic hydrolysis, breaking down the cellulose into cellulosic sugars such as glucose. Alternatively, the enzymatic hydrolysis step disclosed herein can be used in conjunction with any conventional enzymatically hydrolyzable cellulose-based raw material.

[0063] Therefore, activated cellulose, or optionally a secondary activated cellulose stream as described herein, can generate a glucose-rich sugar stream by contact with one or more cellulase enzymes. As illustrated in Figure 1, the washed secondary activated cellulose stream 30 and enzyme stream 42 are introduced into the enzymatic hydrolysis reactor 40 to generate a glucose-rich sugar stream 44. The washed secondary activated cellulose stream 30 may be introduced into the enzymatic hydrolysis reactor 40 simultaneously with, or after, the introduction of the enzyme stream 42 into the reactor 40. Alternatively, or in addition, the enzyme stream 42 may be introduced into the washed secondary activated cellulose stream 30, and the combined stream may be introduced into the enzymatic hydrolysis reactor 40.

[0064] The enzymatic hydrolysis reactor 40 may be any enzymatic hydrolysis reactor known in the art and may operate in batch or continuously. The enzymatic hydrolysis reactor 40 may operate at any conventional temperature and pressure, cellulose input, enzyme input, etc. For example, the enzymatic hydrolysis reactor 40 may operate in a temperature range of 40°C to 55°C.

[0065] Cellulase enzymes may be selected to break down cellulose into monosaccharides. For example, a cellulase enzyme may be selected to hydrolyze 1,4-β-D-glycosidic bonds into monosaccharides. One or more cellulase enzymes may include enzymes having at least one of cellobiohydrolase, endoglucanase, and β-glucosidase activity. Cellulase enzyme preparations may be isolated from numerous sources, such as natural cultures of bacteria, yeast, or fungi, but those skilled in the art will recognize the use of enzymes produced using recombinant techniques. Examples of commercially available enzymes suitable for use in the methods described herein include, but are not limited to, Novozymes' Ctec 2 or 3 and AB Enzymes' Rohament.

[0066] One or more cellulase enzymes may be added in amounts of 0.1–120 mg, 0.2–60 mg, or 1–30 mg of enzyme protein per gram of glucan. In one embodiment, the cellulase enzyme is added in amounts of 0.1–5 mg of enzyme protein per gram of glucan in the activated cellulose. In one embodiment, one or more cellulase enzymes are added to the activated cellulose in amounts of about 2–60 Filter Paper Units (FPU) / g glucan, or optionally about 2–30 or 1–15 FPU / g glucan. One or more cellulase enzymes may be added independently to the activated cellulose directly, or they may be combined with surfactants and / or dispersants first, as described later.

[0067] To convert cellulose into monosaccharides by enzymatic hydrolysis, one or more cellulase enzymes can be brought into contact with activated cellulose for an appropriate duration (e.g., 24–144 hours, 48–144 hours, 48–60 hours, or 24–72 hours).

[0068] In some embodiments, at least about 70%, 75%, 80%, 85%, 90%, or 95% of the theoretical glucose yield based on the glycan content of activated cellulose is converted to glucose during enzymatic hydrolysis, generating a glucose-rich sugar stream. In some embodiments, enzymatic hydrolysis is carried out for a predetermined time or until a predetermined glucose yield is obtained. After a certain period of time, the rate of glucose production from the enzymatic hydrolysis of cellulose may decrease due to depletion of the cellulose substrate or because the presence of glucose inhibits the activity of the cellulase enzyme.

[0069] Optionally, activated cellulose can be contacted with one or more cellulase enzymes in the presence of a surfactant and / or a dispersant. In preferred embodiments, the dispersant is polyaspartic acid.

[0070] Surprisingly, it has been determined that subjecting activated cellulose, particularly the activated cellulose disclosed herein, to enzymatic hydrolysis in the presence of surfactants and / or dispersants such as polyaspartic acid offers numerous advantages in the production of monosaccharides. For example, the presence of surfactants and / or dispersants can improve the stability of the cellulase enzyme, help protect it from degradation, prevent irreversible binding, and / or improve its activity. The presence of surfactants and / or dispersants is also thought to improve the recovery of the cellulase enzyme into the enzymatic reuse stream after enzymatic hydrolysis. For example, in some embodiments, the cellulase enzyme may be used for the enzymatic hydrolysis of activated cellulose, removed from the resulting glucose-rich sugar stream, and reused in the enzymatic hydrolysis reactor 40 or contacted with new activated cellulose for further enzymatic hydrolysis. In some embodiments, the cellulase enzyme may be used and reused for at least 3 to 4 enzymatic hydrolysis cycles, each lasting 48 to 72 hours.

[0071] The surfactant may be a nonionic surfactant, or optionally a polysorbate surfactant such as Tween. The surfactant may also be a mixture of surfactants. In a preferred embodiment, the surfactant is a mixture of Tween 80, an alkoxylated glyceride, and a nonylphenol. In one embodiment, the surfactant is present in an amount less than about 2% and / or greater than about 0.01%. In one embodiment, the surfactant is present in an amount of 1% to 0.01%, 0.5% to 0.05%, or about 0.1% to 0.2% by weight of the cellulose content in the activated cellulose.

[0072] The dispersant may be an oligopeptide, optionally a non-enzymatic polypeptide having a molecular weight of 500–10,000 or 1,000–5,000. The oligopeptide may be polyaspartic acid. Polyaspartic acid may have a molecular weight of 500–10,000, 1,000–5,000, or 3,500–4,500. Polyaspartic acid may be present in an input amount of less than about 2% and / or higher than about 0.001% by weight of the cellulose content in the activated cellulose. In some embodiments, polyaspartic acid may be present in an input amount of 1%–0.001%, 0.25%–0.025%, or about 0.1% by weight of the cellulose content in the activated cellulose.

[0073] Optionally, the ratio of surfactant to dispersant (e.g., polyaspartic acid) in the enzymatic hydrolysis mixture is 0.1:1 to 10:1, and optionally 0.5:1 to 2:1. Optionally, the molar ratio of a dispersant (e.g., polyaspartic acid) to one or more cellulase enzymes is between 0.01 and 10:1.

[0074] Accordingly, an enzymatic hydrolysis mixture containing one or more cellulase enzymes, one or more surfactants, and one or more dispersants may be used in any enzymatic hydrolysis step, or in connection with any activation and enzymatic hydrolysis steps disclosed herein. The enzymatic hydrolysis mixture is particularly suitable for the enzymatic hydrolysis of activated cellulose containing cellulose II and cellulose IV, as described herein.

[0075] One or more cellulase enzymes, surfactants, and dispersants may be introduced into the enzymatic hydrolysis reactor 40 individually, in combination, or in subcombinations. For example, they may be combined individually with activated cellulose before introducing stream 38 into reactor 40 (for example, each may be added to stream 38 sequentially, or stream 38 may be divided into three streams and the cellulase enzyme, surfactant, and dispersant may be added to one of the divided streams). Alternatively, one or more cellulase enzymes, surfactants, and dispersants may be mixed together to form stream 42 before combining the mixture with activated cellulose (for example, introducing stream 42 into reactor 40, or introducing stream 42 into stream 38 before introducing stream 38 into reactor 40). Mixing the enzymes, surfactants, and dispersants together before contact with activated cellulose is thought to help stabilize the enzymes and promote the formation of a three-component complex that prevents enzyme degradation. Therefore, one or more cellulase enzymes may be mixed with surfactants and dispersants before subjecting activated cellulose to enzymatic hydrolysis. For example, one or more cellulase enzymes may be mixed with a surfactant and a dispersant for at least 5 seconds, at least 10 seconds, at least 30 seconds, or at least 1 minute before contacting them with activated cellulose (e.g., Stream 38) or before subjecting the activated cellulose to enzymatic hydrolysis.

[0076] As illustrated in Figure 1, the glucose-rich sugar stream 44 may be subjected to an enzyme removal step to obtain a glucose-rich, enzyme-low sugar stream 48 and an enzyme reuse stream 50. The enzyme removal step may be any enzyme removal step known in the art and may be carried out in any apparatus known in the art. Optionally, the enzyme removal step may include contacting the glucose-rich sugar stream 44 with cellulose for, for example, a limited time, which may be activated cellulose produced by any method disclosed herein.

[0077] For example, the enzyme removal step may include the following steps: (a) A step of contacting a glucose-rich sugar stream containing an enzyme with cellulose to obtain cellulose having the enzyme adsorbed thereon; and, (b) A step of subjecting a glucose-rich sugar stream to a cellulose removal step to obtain a glucose-rich sugar stream with reduced cellulose levels and an enzyme reuse stream.

[0078] Optionally, step (a) includes contacting a glucose-rich sugar stream with activated cellulose, optionally, a second activated cellulose stream produced according to the method described herein.

[0079] Without being limited by theory, it is thought that enzymes in glucose-rich sugar streams adsorb onto cellulose, and that removing cellulose from glucose-rich sugar streams removes enzymes from the streams, resulting in glucose-rich sugar streams 48 and enzyme reuse streams 50 with fewer enzymes. In a particularly preferred embodiment, the cellulase enzyme in the glucose-rich sugar stream is in the presence of a surfactant and a dispersant, and the enzyme in glucose-rich sugar stream 44 is removed by contacting glucose-rich sugar stream 44 with activated cellulose 16, 24, 30, 38 produced using the method described herein.

[0080] Accordingly, the cellulose stream 52 can be introduced into the reactor 46. The reactor 46 may include any reactor that allows the glucose-rich sugar stream 44 and the cellulose to come into contact with each other, thereby recovering the enzyme from the solution and separating the cellulose together with the enzyme adsorbed thereon. Thus, for example, the reactor 46 may include a stirred-tank reactor or a plugged-stream reactor for mixing the glucose-rich sugar stream and the cellulose to produce a mixed stream 54.

[0081] Glucose-rich sugar streams and cellulose can be in contact together for less than approximately 2 hours, less than approximately 90 minutes, or less than approximately 60 minutes, and can be in contact together for approximately 10 to 60 minutes, or approximately 30 to 90 minutes.

[0082] Next, the mixed stream 54 is subjected to a solid-liquid separation step in a separator 56. The separator 56 can be any separator known in the art. The separator 56 may use any separation technique known in the art, such as filtration, decantation, gravity separation, centrifugation, or the use of a press. For example, the separator 56 may include a filter, a press, and optionally a twin-screw press, a twin-wire press, or a twin-roll press.

[0083] The enzyme recycling stream 50 can be a high-solids stream. For example, the enzyme recycling stream may contain more than approximately 30%, more than approximately 40%, or more than approximately 50% cellulosic solids.

[0084] The enzyme reuse stream 50 can be used to perform enzymatic hydrolysis on new activated cellulose. Alternatively, the enzyme reuse stream 50 can be reused in reactor 40. Thus, when reactor 40 is operating in batch mode, the reuse stream 50 can be obtained by recovering and processing the purge stream of the glucose-rich sugar stream. Surprisingly, it has been determined that the enzymes reused in this process usually retain their activity even after being reused once, twice, three or four times. As a result, the glucose-rich sugar stream 48 can contain more than approximately 12% glucose, more than approximately 14% glucose, more than approximately 16% glucose or more than approximately 18% glucose. Furthermore, glucose yields of more than approximately 70%, more than approximately 80%, more than approximately 85%, more than approximately 90%, or more than approximately 95% of the theoretical glucose yield can be obtained.

[0085] Optionally, the glucose-rich sugar stream 48 may contain detectable levels of polyaspartic acid. In one embodiment, the sugar stream contains approximately 1 ppb to 10,000 ppm of polyaspartic acid.

[0086] Surprisingly, the glucose-rich sugar stream resulting from the enzymatic hydrolysis of activated cellulose disclosed herein was found to contain about 5% non-glucose sugars, one or more of xylose, xylose oligomers (xylooligosaccharides), and xylan. As used herein, xylooligosaccharide refers to a polymer of xylose having a degree of polymerization (dp) of about 2 to about 10. As used herein, xylan refers to a polymer of xylose having a degree of polymerization (dp) of >10.

[0087] In certain embodiments, the enzymatic hydrolysis of activated cellulose may be carried out using a hydrolysis mixture as disclosed herein. In further embodiments, a glucose-rich sugar stream is prepared using the cellulose activation method and / or enzymatic hydrolysis method described herein.

[0088] Glucose obtained using standard methods from sources such as maize is also known to contain approximately 5% non-glucose sugars. However, the non-glucose sugars found in maize glucose are those with higher glycemic indexes, such as maltose, maltotriose, and the higher molecular weight dextrose sugars.

[0089] In one embodiment, the non-glucose sugars found in the glucose-rich sugar stream disclosed herein constitute about 1–8%, about 2–7%, or about 3–6% of the dry material of the composition and are one or more xylose, xylooligosaccharides, and xylan. In a particular embodiment, the dry material of the glucose-rich sugar stream was found to contain 95% glucose, 4% xylose, and 1% xylooligosaccharides.

[0090] The glucose obtained by the methods disclosed herein can be converted to fructose using known methods, such as glucose isomerization to fructose, as described, for example, by SZ Dziedzic et al. in “Handbook of starch hydrolysis products and their derivatives,” December 31, 1995, pp. 55-58 (incorporated herein by reference). The fructose obtained by the conversion of glucose obtained by the methods disclosed herein also contains approximately 3-5% non-glucose sugars, which have been found to be xylose and / or xylose oligomers.

[0091] In a further aspect of the present invention, a fructose-rich sugar stream is provided, prepared by the conversion of glucose to fructose in a glucose-rich sugar stream disclosed herein. The fructose-rich sugar stream also contains non-fructose sugars, which are one or more xylose, xylooligosaccharides, and xylan. In one embodiment, the non-fructose sugars constitute about 1-8%, about 2-7%, or about 3-6% of the dry matter of the composition.

[0092] In another aspect of the present invention, a glucose product or a fructose product with a lower glycemic index is provided, wherein the glucose product or fructose product contains about 1-8%, about 2-7%, or about 3-6%, preferably 5%, of non-glucose or non-fructose sugars, wherein the non-glucose or non-fructose sugars are one or more of xylose, xylooligosaccharides, and xylan.

[0093] In a further embodiment, the glycemic index of the glucose or fructose product is lower than that of conventional glucose or fructose syrup produced by conventional methods. The glycemic index (GI) can be measured using methods known in the art, such as those described in “In vitro method for predicting glycemic index of foods using simulated digestion and an artificial neural network” RL Magaletta et al., Cereal Chemistry vol. 87, no. 4, 2010.

[0094] The glucose or fructose products obtained by the methods described herein may be used in the manufacture of various foods and beverages as a substitute for glucose or fructose with a higher glycemic index to provide products with a lower glycemic index. Foods and beverages with a lower glycemic index may offer health benefits in managing blood glucose and insulin levels, and thus reduce the risk of heart disease and / or diabetes. Foods with a lower glycemic index may also be useful in controlling appetite and weight loss.

[0095] It will be understood that one or more embodiments described herein for the activation of cellulosic raw materials may be used in conjunction with one or more embodiments described herein for the enzymatic hydrolysis of cellulose to produce cellulosic sugars from cellulosic raw materials.

[0096] The foregoing is a description of the present invention and is intended to be non-limiting. Those skilled in the art will understand that other changes and modifications may be made without departing from the scope of the invention as defined in the claims appended herein. The claims should not be limited by preferred embodiments and examples, but should be interpreted in the broadest sense consistent with the description as a whole.

[0097] The above disclosures describe the application in general terms, but a more complete understanding can be obtained by referring to the specific examples below. These examples are provided solely for illustrative purposes and are not intended to limit the scope of the disclosure. Modifications of form and substitution of equivalents are attempted where circumstances suggest or are appropriate. Certain terms are used herein, but such terms are intended to be descriptive and not limiting. The non-limiting examples below are examples of the disclosure. [Examples]

[0098] [Example 1] Treatment of sugarcane bagasse to activate cellulose Various methods for activating cellulose were investigated using sugarcane bagasse. Sugarcane bagasse was subjected to either a first steam treatment step of 220°C with a residence time of 5 minutes followed by hot water washing with 80°C water, or a first steam treatment followed by treatment with alkaline hydrogen peroxide. Alternatively, it could be treated with alkaline washing at 90°C for 60 or 120 minutes, and with 1% peroxide added in solid form.

[0099] As shown in Table 1, the use of a steam treatment step followed by a two-step treatment with alkaline hydrogen peroxide significantly increased glucan levels and decreased lignin levels compared to steam treatment and hot water washing.

[0100] [Table 1]

[0101] [Example 2] Enzymatic hydrolysis of activated sugarcane bagasse The water-insoluble cellulose component prepared in Example 1 was subsequently subjected to enzymatic hydrolysis for 72 hours. Comet additive S-001, which contains a 1:1 mixture of the surfactant Tween 80 and the dispersant polyaspartic acid having a molecular weight of 3500-4500, was also added to the enzymatic hydrolysis mixture for alkali-treated sugarcane bagasse.

[0102] As shown in Table 2, enzymatic hydrolysis of sugarcane bagasse treated with alkali in the presence of Comet additive S-001 resulted in a glucose yield of 105.1 grams of glucose per gram of glucan, which was close to the theoretical yield of approximately 110 grams of glucose per gram of glucan.

[0103] [Table 2]

[0104] Furthermore, as shown in Table 3, enzymatic hydrolysis in the presence of Comet additive S-001 did not alter the yield of monomeric xylose compared to alkali-treated sugarcane bagasse that underwent enzymatic hydrolysis without the additive. Therefore, the additive did not adversely affect the yield.

[0105] [Table 3]

[0106] Table 4 shows the analysis of the percentage of total protein content present in the supernatant 72 hours after enzymatic hydrolysis. The use of Comet additive S-001 resulted in a higher percentage of total protein, indicating higher levels of enzymes in the supernatant and improved enzyme stability.

[0107] [Table 4]

[0108] Alkaline-treated sugarcane bagasse was subjected to multiple reuse hydrolysis cycles in the presence of Comet additive S-001. As shown in Table 5, reuse hydrolysis was able to produce high yields and high concentrations of glucose over the repeated enzymatic hydrolysis cycles while minimizing enzyme loss.

[0109] [Table 5]

[0110] [Example 3] Treatment of wheat straw to activate cellulose Methods for activating cellulosic raw materials were investigated using wheat straw. The wheat straw was subjected to a first steam treatment step at 220°C with a residence time of 5 minutes, followed by hot water washing at 80°C, then alkaline washing at 90°C for 60 minutes and with 1% peroxide added in solid form. As shown in Table 6, the use of a two-step treatment with alkaline hydrogen peroxide resulted in water-insoluble components containing high levels of glucan (75.1%).

[0111] [Table 6]

[0112] [Example 4] Enzymatic hydrolysis of activated wheat straw Water-insoluble cellulose components of steam-treated wheat straw, or wheat straw that was steam-treated followed by alkali treatment, were subjected to enzymatic hydrolysis as shown in Tables 7-9. The surfactant Tween 80 was also added as indicated in the table.

[0113] [Table 7]

[0114] [Table 8]

[0115] [Table 9]

[0116] Further studies of the enzymatic hydrolysis of steam-treated and subsequently alkali-peroxide-treated wheat straw, with the addition of a surfactant (Tween 80), were conducted as shown in Tables 10–12.

[0117] [Table 10]

[0118] [Table 11]

[0119] [Table 12]

[0120] [Example 5] Cellulosic glucose products produced by the activation and enzymatic conversion methods described herein were prepared and measured to have the following specifications.

[0121] Chemical data and physical data Total solids 50-70% Moisture content 30-50%

[0122] Composition (based on dry material) Glucose 95% Xylose 4% Xylooligosaccharide 1% Ash content <0.01%

[0123] pH: 3~5 Conductivity: (30% DS)50 μs / cm Specific gravity: 1.2 Appearance: Transparent solution Scent: Sweet

[0124] Mineral ash content (PPM) Chloride 16 Sulfate <1 Calcium 5 Potassium <1 Magnesium <1 Sodium 2 Lin 2

[0125] Glycemic index data Cellulosic glucose products having the above-described composition were found to have a glycemic index (GI) of 72. In comparison, glucose alone is known to have a glycemic index of 100. Dextrose is also known to have a glycemic index of 100, while maltose and maltodextrin are known to have glycemic indices of 105 and 110, respectively.

[0126] This disclosure describes what are currently considered embodiments, and it should be understood that this disclosure is not limited to the embodiments disclosed. Conversely, this disclosure is intended to include various modifications and equivalent arrangements that fall within the spirit and scope of the appended claims.

[0127] All publications, patents, and patent applications are incorporated herein by reference to the same extent that each individual publication, patent, and patent application is specifically and individually indicated to be incorporated by reference as a whole. This application provides the following: 1. A two-step method for activating cellulosic raw materials, (a) A first activation step in which the raw material is subjected to a temperature higher than 190°C and a pressure higher than 200 psig to produce a first activated cellulose stream containing cellulose II and insoluble solids; (b) A second activation step in which the first activated cellulose stream is treated with alkali at a lower temperature than the first activation step to produce a second activated cellulose stream containing cellulose IV. The above method, including. 2. The method according to 1 above, wherein the first activation step is carried out in the presence of water. 3. The method according to 1 or 2 above, wherein the first activation step includes exposing the raw material to a pressure of 200 to 500 psig and a temperature of 200 to 250°C for 1 to 30 minutes. 4. The method according to 1 or 2 above, wherein the first activation step includes exposing the raw material to a pressure of 200-500 psig and a temperature of 190-215°C for less than 4 minutes. 5. The method according to any one of 1 to 4 above, further comprising the step of washing the first activated cellulose stream to remove soluble non-cellulosic components before the second activation step. 6. The method according to 5 above, wherein the step of washing the first activated cellulose stream includes the use of water at approximately 50-95°C and optionally the use of countercurrent washing. 7. The method according to any one of 1 to 6 above, wherein the primary activated cellulose stream has a solid content of approximately 30-50% (wt / wt). 8. The method according to any one of 1 to 7 above, wherein the second activation step includes exposing the first activated cellulose stream to a temperature of 60 to 240°C for 15 to 120 minutes. 9. The method according to 8 above, wherein the second activation step includes exposing the first activated cellulose stream to a temperature of 80-150°C for at least 60 minutes. 10. The method according to any of items 1 to 9 above, wherein the second activation step is performed at superatmospheric pressure, optionally at approximately 0.1 to 400 psig. 11. The method according to any one of 1 to 10 above, wherein the alkali in the second activation step comprises one or more of sodium hydroxide, potassium hydroxide, magnesium hydroxide, and ammonia. 12. The method according to any one of 1 to 11 above, wherein the alkali in the second activation step contains sodium hydroxide, and the sodium hydroxide is added in an amount of about 10% to 1%, or preferably less than about 6%, of the insoluble solids in the first activated cellulose stream. 13. The method according to any one of 1 to 12 above, wherein the second activation step is carried out in the presence of an oxidizing agent and / or enzyme, optionally laccase or lignin-modifying enzyme. 14. The method according to 13 above, wherein the oxidizing agent is hydrogen peroxide, which is optionally added in an amount of less than 1% of the insoluble solids in the first activated cellulose stream. 15. The method according to any one of 1 to 14 above, wherein the second activated cellulose stream contains a mixture of cellulose II, hydrated cellulose II, and alkali cellulose IV. 16. The method according to any one of 1 to 15 above, further comprising the step of washing the second activated cellulose stream to remove soluble non-cellulosic components. 17. The method according to 16 above, wherein the step of washing the second activated cellulose stream includes the use of water at approximately 60-95°C and optionally the use of countercurrent washing. 18. A method for generating a glucose-rich sugar stream, comprising the step of contacting a second activated cellulose stream obtained from any of the methods described in 1 to 17 above with one or more cellulase enzymes to generate a glucose-rich sugar stream. 19. The method according to 18, wherein one or more cellulase enzymes are selected to hydrolyze the 1,4-β-D-glycosidic bond to a monosaccharide. 20. The method according to 18 or 19 above, wherein one or more cellulase enzymes include enzymes having at least one of cellobiohydrolase, endoglucanase, and β-glucosidase activity, optionally including commercially available Novozymes Ctec 2 or 3, or AB Enzymes Rohament. 21. The method according to any one of 18 to 20 above, further comprising contacting a secondary activated cellulose stream with a surfactant and / or a dispersant. 22. The method according to 21 above, wherein the dispersant is an oligopeptide, optionally a non-enzymatic polypeptide having a molecular weight of 500 to 10,000 or 1,000 to 5,000. 23. The method according to 22 above, wherein the polypeptide is polyaspartic acid. 24. The method according to 22 or 23 above, wherein the polypeptide is added to the second activated cellulose stream in an amount of less than 2%, 1% to 0.001%, 0.25% to 0.025%, or about 0.1% of the weight of the cellulose content of the second activated stream. 25. The method according to any one of the above 21 to 24, wherein the surfactant is a nonionic surfactant, and optionally a polysorbate surfactant such as Tween (trademark). 26. The method according to any one of 21 to 25 above, wherein the surfactant is a mixture of surfactants comprising Tween, alkoxylated glycerides, and nonylphenol. 27. The method according to 25 or 26 above, wherein the surfactant is added to the second activated cellulose stream in an amount of less than 2%, 1% to 0.01%, 0.5% to 0.05%, or about 0.1% to 0.2% by weight of the cellulose content of the second activated stream. 28. The method according to any one of 21 to 27 above, wherein one or more cellulase enzymes, surfactants, and dispersants are individually added to the second activated cellulose stream. 29. The method according to any one of 21 to 27 above, wherein one or more cellulase enzymes are optionally mixed with a surfactant and a dispersant for at least 10 seconds before coming into contact with a second activated cellulose stream. 30. The method according to 29 above, wherein one or more cellulase enzymes form a three-component complex with a surfactant and a dispersant. 31. The method according to any one of the above 18-30, wherein one or more cellulase enzymes are added to a second activated cellulose stream at an input amount of 0.1 to 120, optionally 0.1 to 5 mg of enzyme protein per gram of glucan. 32. The method according to 31 above, wherein one or more cellulase enzymes are added to the second activated cellulose stream at an input amount of approximately 2 to 60 FPU / g glucan. 33. The method according to any one of the above 18 to 32, wherein a second activated cellulose stream is contacted with one or more cellulase enzymes for 24 to 144 hours, 48 ​​to 144 hours, 48 ​​to 60 hours, 24 to 96 hours, less than 60 hours, or less than 48 hours to produce a glucose-rich sugar stream. 34. The method according to any one of 18 to 33 above, further comprising the step of subjecting a glucose-rich sugar stream to an enzymatic removal treatment to obtain a glucose-rich, enzyme-low sugar stream and an enzyme reuse stream. 35. Enzyme removal treatment, (a) A step of contacting a glucose-rich sugar stream containing an enzyme with cellulose to obtain cellulose having the enzyme adsorbed thereon; and, (b) A glucose-rich sugar stream is subjected to a cellulose removal step to obtain a glucose-rich sugar stream with reduced cellulose levels and an enzyme reuse stream. The method described in 34 above, including the method described in 34 above. 36. The method according to 35, wherein a glucose-rich sugar stream is brought into contact with activated cellulose, optionally a second activated cellulose stream produced according to any of the methods described in 1 to 17 above, for optionally less than 60 minutes. 37. The method according to 35 or 36 above, wherein the cellulose removal step includes filtration and optionally the enzyme reuse stream contains about 40 wt% more cellulose solids. 38. The method according to any one of 34 to 37 above, further comprising mixing the enzyme reuse stream with fresh activated cellulose. 39. The method according to any one of the above 35 to 38, further comprising repeating the cellulose removal step described in 35 above. 40. The method according to any one of items 18 to 39 above, wherein the glucose-rich sugar stream contains more than approximately 12% glucose, more than approximately 14% glucose, more than approximately 16% glucose, or more than approximately 18% glucose. 41. The method according to any one of the above 18 to 40, wherein the method produces a glucose yield of more than approximately 70%, more than approximately 80%, more than approximately 85%, more than approximately 90%, or more than approximately 95% of the theoretical glucose yield. 42. A method for producing a glucose-rich sugar stream from activated cellulose, comprising the step of subjecting activated cellulose to enzymatic hydrolysis using one or more cellulase enzymes, a surfactant, and polyaspartic acid to produce a glucose-rich sugar stream. 43. The method according to 42, wherein one or more cellulase enzymes are selected to hydrolyze the 1,4-β-D-glycosidic bond to a monosaccharide. 44. The method according to 42 or 43, wherein one or more cellulase enzymes include an enzyme having at least one of cellobiohydrolase, endoglucanase, and β-glucosidase activity. 45. The method according to any one of the above 42 to 44, wherein the polyaspartic acid has a molecular weight of 500 to 10,000. 46. ​​The method according to any one of the above 42 to 44, wherein the polyaspartic acid has a molecular weight of 1000 to 5000. 47. The method according to any one of the above 42 to 44, wherein the polyaspartic acid has a molecular weight of 3500 to 4500. 48. The method according to any one of 42 to 47 above, wherein polyaspartic acid is present in an input amount of less than 2%, 1% to 0.001%, 0.25% to 0.025%, or about 0.1% of the weight of the cellulose content of the second activated stream. 49. The method according to any one of the above 44 to 48, wherein the surfactant is a nonionic surfactant, and optionally a polysorbate surfactant such as Tween (trademark). 50. The method according to any one of 44 to 49 above, wherein the surfactant is a mixture of surfactants comprising Tween, alkoxylated glycerides, and nonylphenol. 51. The method according to 49 or 50 above, wherein the surfactant is present in an amount of less than 2%, 1% to 0.01%, 0.5% to 0.05%, or about 0.1% to 0.2% by weight of the cellulose content of the second activated stream. 52. The method according to any one of 42 to 51 above, wherein one or more cellulase enzymes, surfactants, and polypeptides are individually mixed with activated cellulose. 53. The method according to any one of 42 to 51 above, wherein one or more cellulase enzymes are optionally mixed with a surfactant and a polypeptide for at least 10 seconds before the activated cellulose is subjected to enzymatic hydrolysis. 54. The method according to 53 above, wherein one or more cellulase enzymes form a three-component complex with a surfactant and polyaspartic acid. 55. The method according to any one of the above 42 to 54, wherein one or more cellulase enzymes are present in an amount of enzyme protein input of 0.1 to 5 mg per gram of glucan. 56. The method according to 55 above, wherein one or more cellulase enzymes are present in 2 to 60 FPU / g glucan. 57. The method according to any one of 42 to 56 above, further comprising the step of subjecting activated cellulose to enzymatic hydrolysis for a period of 24 to 144 hours, 48 ​​to 144 hours, 48 ​​to 60 hours, 24 to 96 hours, less than 60 hours, or less than 48 hours. 58. The method according to any one of 41 to 57 above, further comprising the steps of subjecting a glucose-rich sugar stream to an enzymatic removal treatment to obtain a glucose-rich, enzyme-poor sugar stream and an enzyme reuse stream, and reusing the enzyme reuse stream for enzymatic hydrolysis. 59. The method according to 58, wherein, after introducing an enzyme reuse stream for use in enzymatic hydrolysis, the method produces a glucose yield of more than 70%, more than 80%, more than 85%, more than 90%, or more than 95% of the theoretical yield. 60. (a) A step of contacting a glucose-rich sugar stream with cellulose to obtain cellulose having enzymes adsorbed thereon; and, (b) A glucose-rich sugar stream is subjected to a cellulose removal step to obtain a glucose-rich sugar stream with reduced cellulose levels and an enzyme reuse stream. The method described in 58 or 59 above, including the method described in 58 or 59 above. 61. The method according to 60, wherein a glucose-rich sugar stream is brought into contact with activated cellulose, and optionally a second activated cellulose stream produced according to the method of any one of 1 to 17 above. 62. The method according to 60 or 61 above, wherein the cellulose removal step includes filtration. 63. The method according to any one of 58-62 above, further comprising the step of mixing the enzyme reuse stream with fresh activated cellulose. 64. The method according to any one of the above 58 to 63, further comprising repeating the cellulose removal step described in 58 above. 65. The method according to any one of 42 to 64 above, wherein the glucose-rich sugar stream contains more than approximately 12% glucose, more than approximately 14% glucose, more than approximately 16% glucose, or more than approximately 18% glucose. 66. The method according to any one of the above 42 to 65, wherein the glucose yield is approximately 70%, approximately 80%, approximately 85%, approximately 90%, or approximately 95%. 67. A method for generating a glucose-rich sugar stream, (a) A step of supplying activated cellulose containing a mixture of cellulose II, hydrated cellulose II, and alkaline cellulose IV; (b) The activated cellulose is subjected to enzymatic hydrolysis using one or more cellulase enzymes, a surfactant, and a dispersant to produce a glucose-rich sugar stream. The above method, including. 68. The method according to 67, wherein activated cellulose is produced by the method described in any of 1 to 17 above. 69. The method according to 67 or 68, wherein the dispersant is a non-enzymatic polypeptide having a molecular weight of less than 10,000. 70. The method according to any one of the above 67 to 69, wherein the dispersant is present in an amount of less than 2%, 1% to 0.001%, 0.25% to 0.025%, or about 0.1% of the weight of the cellulose content of the activated cellulose. 71. The method according to 70 above, wherein the dispersant is polyaspartic acid. 72. The method according to 71 above, wherein the polyaspartic acid has a molecular weight of 1000 to 5000, and optionally 3500 to 4500. 73. The method according to any one of paragraphs 67 to 72 above, wherein the surfactant is present in an amount of less than 2%, 1% to 0.01%, 0.5% to 0.05%, or about 0.1% to 0.2% of the weight of the cellulose content of the activated cellulose. 74. The method according to 73 above, wherein the surfactant is a nonionic surfactant, and optionally a polysorbate surfactant such as Tween (trademark). 75. The method according to 74, wherein the surfactant is a mixture of surfactants comprising Tween, alkoxylated glycerides, and nonylphenol. 76. The method according to any one of 67 to 75 above, wherein one or more cellulase enzymes, surfactants, and dispersants are individually added to activated cellulose. 77. The method according to any one of the above 67 to 75, wherein one or more cellulase enzymes are optionally mixed with a surfactant and a dispersant for at least 10 seconds before the activated cellulose is subjected to enzymatic hydrolysis. 78. The method according to any one of 67 to 77, further comprising the step of subjecting a glucose-rich sugar stream to an enzymatic removal treatment to obtain a glucose-rich, enzyme-low sugar stream and an enzyme reuse stream. 79. The method according to any one of 67 to 78 above, wherein the glucose-rich sugar stream contains more than approximately 12% glucose, more than approximately 14% glucose, more than approximately 16% glucose, or more than approximately 18% glucose. 80. The method according to any one of the above 67-75, wherein the glucose yield is greater than approximately 70%, greater than approximately 80%, greater than approximately 85%, greater than approximately 90%, or greater than approximately 95%. 81. (a) One or more cellulase enzymes; (b) surfactants or mixtures of surfactants; and (c) Polyaspartic acid having a molecular weight of 500 to 10,000 A mixture of enzymatic hydrolyzed substances containing [the substance]. 82. The enzymatic hydrolysis mixture according to 81 above, wherein one or more cellulase enzymes hydrolyze the 1,4-β-D-glycosidic bonds to monosaccharides. 83. An enzymatic hydrolysis mixture according to 81 or 82, wherein one or more cellulase enzymes include enzymes having one or more cellobiohydrolase, endoglucanase, and β-glucosidase activity. 84. An enzymatic hydrolysis mixture according to any one of the above 81 to 83, wherein the polyaspartic acid has a molecular weight of 1000 to 5000. 85. The method according to any one of the above 81 to 84, wherein the surfactant is a nonionic surfactant, or optionally a polysorbate surfactant such as Tween (trademark). 86. An enzymatic hydrolysis mixture according to any of items 81 to 85 above, wherein the ratio of surfactant to polyaspartic acid is 0.1:1 to 10:1, and optionally 0.5:1 to 2:1. 87. A glucose-rich sugar stream produced by any of the methods described in items 18-80 above. 88. The glucose-rich sugar stream according to 87, wherein the sugar stream contains more than 12% glucose, more than 14% glucose, more than 16% glucose, or more than 18% glucose. 89. The glucose-rich sugar stream according to 87 or 88, wherein the sugar stream contains polyaspartic acid. 90. A glucose-rich sugar stream as described in any of items 87-89 above, in which polyaspartic acid is present at a concentration of 1 ppb to 10,000 ppm. 91. A glucose-rich sugar stream according to any one of the above 87-89, wherein the glucose-rich sugar stream contains non-glucose sugars. 92. The glucose-rich sugar stream described in 91 above, wherein the non-glucose sugar is one or more of xylose, xylooligosaccharide, or xylan. 93. A glucose-rich sugar stream as described in 91 or 92 above, wherein the dry material composition comprises approximately 95% glucose and approximately 5% non-glucose sugars. 94. A glucose syrup containing approximately 95% glucose and approximately 5% non-glucose sugars, wherein the non-glucose sugars are one or more of xylose, xylooligosaccharides, and xylan. 95. A fructose syrup containing approximately 95% fructose and approximately 5% non-fructose sugars, wherein the non-fructose sugars are one or more of xylose, xylooligosaccharides, and xylan. 96. The fructose syrup according to 95, prepared by glucose isomerization of the glucose-rich sugar stream described in 93 above.

Claims

1. A glucose syrup, wherein the dry substance in the glucose syrup is (a) 95% glucose, and (b) Contains 5% non-glucose sugars, The glucose syrup contains polyaspartic acid at a concentration of 1 ppb to 10,000 ppm, and the non-glucose sugars include one or more of xylose, xylooligosaccharides, and xylan.

2. The glucose syrup according to claim 1, wherein the dry substance in the glucose syrup contains less than 0.01% ash.

3. The glucose syrup according to claim 1, having a pH of 3 to 5.

4. The glucose syrup according to claim 1, having an electrical conductivity of 50 μs / cm.

5. The glucose syrup according to claim 1, having a specific gravity of 1.

2.

6. The glucose syrup according to claim 1, wherein the dry substance in the glucose syrup comprises 95% glucose, 4% xylose, and 1% xylooligosaccharide.

7. The glucose syrup according to claim 1, having a glycemic index of less than 100.

8. The glucose syrup according to claim 7, having a glycemic index of 72.

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