WO2018021782A1 - Method for recovering sugar solution prepared by saccharification of biomass - Google Patents

Abstract

The present invention relates to a method for effectively recovering a sugar solution containing glucose and saccharogenic residues after saccharification using an acid or a saccharogenic enzyme of a wood-based or algae biomass, and a device for implementing the method. More specifically, the present invention relates to a method capable of recovering a sugar solution using minimal equipment and water after aggregating fine particles by adding a protein suspension to a suspension of glucose and saccharogenic residues which are produced by saccharifying cellulose by adding an acid or saccharogenic enzyme to the biomass, while capable of minimizing an amount of saccharide lost in the residues, and a device for implementing the method.

Description

Recovery method of sugar solution prepared by saccharification of biomass

The present invention relates to a method for efficiently recovering a sugar solution from a sugar solution containing glucose and a saccharified residue after saccharification using an acid or a saccharifying enzyme of woody or algal biomass, and more particularly, to an apparatus for implementing the method. Biomass glycosylation step of glycosylation by adding an enzyme or acid to the biomass pretreatment; A fine particle aggregation step of preparing a slurry in which fine particles are aggregated by adding and stirring a glycated residue protein additive for biomass saccharification; And a sugar solution recovery step of separating and recovering a sugar solution by centrifugation or filtration of the slurry in which the fine particles are aggregated, and a method for recovering a sugar solution prepared by saccharification of biomass, which can be implemented. For the device.

This application claims priority based on Korean Patent Application No. 10-2016-0095286 filed on July 27, 2016 and Korean Patent Application No. 10-2016-0140275 filed on October 26, 2016, and the application. All content disclosed in the specification and drawings of the present invention is integrated in this application.

As a substitute for fossil fuels such as petroleum and coal, lignocellulosic biomass is a biobased economy through the conversion of bioalcohol, a transportation fuel, and lignocellulosic sugar, an industrial fermentation sugar. It is being evaluated as a major means of transition. Already, commercial production of bioethanol from woody biomass has already begun in several developed countries, including the United States. Recent reports indicate that Renmatix and Sweetwater in the United States have It is said that commercial production of industrial fermented sugar is started from biomass. The wood-based biomass resources used here are wood and corn refinery by-products, and cellulose, which is a structural component of biomass, is a direct source of bioalcohol or industrial fermented sugar.

In addition, algal biomass such as green algae and diatoms, which are attracting attention as the third generation biomass and steadily being developed for practical use, contain carbohydrates such as starch and cellulose, as well as proteins and oils. It is also promising as a biofuel and food raw material such as bioethanol and biodiesel. Green algae and diatoms mainly containing starch or cellulose in algae biomass do not have lignin in the sieve, unlike wood based biomass, and thus do not require high temperature and high pressure pretreatment applied to wood based biomass. Carbohydrates such as and cellulose are easily converted to monosaccharides by acid or starch degrading enzymes and fibrinase.

In order to convert the cellulose contained in such biomass into glucose, an acid or glycosylase is added to the pretreatment of the cellulose main component obtained by the pretreatment of the biomass and saccharified for a predetermined time at a specific temperature. The glycosylated in this way is in a state in which a sugar solution in which glucose or wood sugar such as glucose or wood sugar produced by hydrolysis of cellulose or hemicellulose is dissolved is mixed with glycosylated residues such as lignin, which are not hydrolyzed and remain in a solid state. Since the glycosylated residue of the lignin main component has a hydrophobic surface, enzyme hydrolysis causes a decrease in enzyme activity due to irreversible adsorption of enzymes. As a result, glycation time increases and yields decrease.

As a technique for solving this problem, US Patent No. US 8,728,320B reduces the adsorption inactivation of enzymes by adsorbing lignin on the surface of lignin by adding proteins from the outside into the reaction system and adsorbing and removing water-soluble lignin on proteins. However, this technique does not yet provide a technical solution for effective solid-liquid separation to prepare a high concentration of sugar solution, but also considers a method for reusing a considerable amount of active enzyme remaining after enzymatic saccharification. I can't.

When bioethanol is produced from biomass as a raw material, monosaccharides such as glucose are converted to ethanol by adding nutrients for culturing various microorganisms, including nitrogen sources such as ammonia, and incubating for a predetermined time after inoculating microorganisms. do. Thereafter, the desired ethanol can be selectively recovered by heating all or part of the fermentation medium to evaporate the ethanol and then condensing the ethanol in the gaseous state again.

On the other hand, when manufacturing fermented sugar (bio sugar) for culturing microorganisms using biomass as a raw material, recovering the sugar solution from the sugars, and preparing a high-density sugar solution for purification to remove impurities other than sugars. A further enrichment process is carried out. In order to recover the sugar solution, insoluble solid particles must be removed from the saccharide, so filtration or centrifugation is common. For example, U.S. Patent Application Publication No. US2015 / 0344921A1 includes a technique of centrifuging or filtering a saccharite as it is for recycling of enzymes after saccharification. The technique also demonstrates the recovery and reuse of enzymes from filtrates.

However, when a centrifuge such as a decanter is used to recover a sugar solution from a sugar that contains a large amount of sugar and has a specific gravity greater than that of water and at the same time as a fine particle, it can be operated for a long time even at high speed (for example, 60 at 1,776 × g per minute). Minutes), so the energy cost is not small, so it is not practical. On the other hand, in order to prepare a clear sugar solution, the filtration is not easy due to the rapid rise in pressure due to the fine particles blocking the membrane or the filter cloth pores, and it is inevitable that further processing such as microfiltration is inevitable because it contains fine particles after filtration. Do. In addition, in the case where the slurry containing fine particles is to be separated by filtration, it is known to use a mineral additive called a filtration aid.

For this reason, polymer coagulants are often added to agglomerate the fine particles and then filtered or centrifuged. In this case, the polymer flocculant used is a synthetic chemical substance having an ionic or nonionic molecular weight of several hundred thousand or more, and the amount of particles to be aggregated increases the amount of usage. Therefore, when the sugars prepared from wood-based biomass as raw materials contain high concentrations of sugars and insoluble saccharified residues up to several%, the required amount of polymer flocculant increases, which inevitably increases the manufacturing cost of the resulting sugar solution. . In addition, it is not possible to exclude the possibility of contamination of the sugar solution by the remaining polymer compound and the restriction of its use.

On the other hand, when the biomass is glycosylated by enzymes, the saccharification may be performed by heating the saccharification after enzyme saccharification to denature the enzymes already contained, and then perform solid-liquid separation (US Patent Publication No. US 2015 / 0354017A1). However, glycosylated glycosylated enzymes of woody biomass pre-treated products still contain inexpensive glycosylase (fibrin hydrolase), and these enzymes maintain enzymatic activity even after saccharification. Known (Novozymes' Product Sheet, Special Food / 2001-08524-03.pdf). Therefore, as described in the US Patent Publication No. US2015 / 0344921A1, the technique for separating and recovering enzymes by ultrafiltration and recycling them in the manufacturing process of the wood-based biomass raw material fermented sugars has already been proposed by many people. It is not preferable to heat the glycosylation to thermally denature the glycosylase and to use the cohesive force of the denatured protein for solid-liquid separation, resulting in abandonment of the considerable amount of active enzyme use remaining after enzyme glycosylation.

Therefore, the present inventors easily prepare a high concentration of sugar solution by separating the biomass saccharification without using the cohesion by denaturation enzyme, and recovers the sugar solution containing the enzyme by such an effective solid-liquid separation to remain after the enzyme saccharification After much effort has been made to develop a technology that can reuse a significant amount of active enzymes, the present invention has been completed.

The present inventors have therefore used a small number of processes and minimal energy to separate biomass sacchalytes containing high concentrations of monosaccharides and several percent insoluble particles into clear sugar solutions and insoluble saccharification residues containing little microparticles. We wanted to develop a method that can be used and how to use the device to implement this method.

In addition, by the effective solid-liquid separation to recover the sugar solution containing glycosylase to develop a technique that can reuse a significant amount of the active enzyme remaining after the enzyme saccharification.

In order to solve the above problems, the present invention by adding a water solution or suspension which can be suspended in water or a protein solution for agglutination of the glycosylated residues to the saccharification and inducing mutual coagulation of the microparticles to produce macroparticles The present invention provides a method for centrifugation or filtration and an apparatus for implementing the same.

More specifically, the present invention comprises the steps of inducing agglomeration of microparticles and converting them into macroparticles by adding and stirring an aqueous solution or suspension of a vegetable or animal protein having a hydrophobic surface to a biomass saccharide; A first solid-liquid separation step of recovering the sugar solution by centrifuging or filtering the slurry converted into the macroparticles at a low rotational speed for a short time; Recovering the first solid-liquid residue, ie, the precipitate that has been settled by centrifugation or the remaining solid content in the filter cloth after filtration, and adding water to gently stir to prepare a residue hydrous slurry; The present invention provides a method for efficiently recovering a sugar solution containing an enzyme as it is from a biomass saccharose comprising the step of recovering the remaining sugar solution by high-speed centrifugation or pressure filtration of the residue hydrolyzate.

In addition, the present invention is added to an aqueous solution or suspension having a hydrophobic surface in the sugar solution and stirred and then continuously centrifuged using a decanter, added to the water while transferring the discharged solid content, stirred and increased by a filter Provided is a sugar solution recovery apparatus which is injected into a press and filtered under pressure.

According to the present invention, a sugar solution containing an enzyme is recovered from a suspension containing glucose and saccharified residue produced by saccharification of biomass by solid-liquid separation using minimal equipment and water, thereby maximizing the recovery of sugar and using it. By drastically reducing the number of devices and uptime, equipment and operating costs can be reduced.

In addition, it is possible to reuse a significant amount of the active enzyme remaining after the enzyme saccharification, it is possible to produce a higher concentration of sugar solution at a lower cost.

1 is a conceptual diagram of a flowchart and a process of a method for efficiently recovering a sugar solution from a biomass saccharide according to an embodiment of the present invention.

FIG. 2 is a standard curve diagram of an enzyme activity in a sugar solution of a biomass saccharin according to an embodiment of the present invention instead of the glycosylation rate. FIG.

Hereinafter, the present invention will be described in detail. The terms or words used in this specification and claims are not to be construed as limiting in their usual or dictionary meanings, and the inventors may appropriately define the concept of terms in order to best explain their invention in the best way possible. It should be interpreted as meaning and concept corresponding to the technical idea of the present invention based on the principle that the present invention.

As used throughout this specification, the term "biomass glycosylated means a slurry in which monosaccharides and insoluble solid particles obtained by hydrolyzing wood-based biomass or algal biomass with saccharifying enzyme or acid are dispersed in water. In addition, the "glycosylated residue" is a solid insoluble in water in which the components such as lignin, which cannot be hydrolyzed any more after the biomass is hydrolyzed by enzymes or acids and converted to monosaccharides. Means.

In the present invention, the glycosylated residue aggregation protein additive (hereinafter, weakly referred to as protein additive) added for solid-liquid separation of the glycosylated biomass includes a general protein except the fibrin hydrolase used for enzymatic glycosylation of biomass. Means an additive. The main component is a protein having a hydrophobic surface, and at least some of the polypeptides are not particularly limited. The most effective protein additives for solid-liquid separation of the glycosylated compounds of the present invention are globular proteins as the main constituents and are denatured by applying heat or changing pH (pH) to expose the hydrophobic surface to the surface, It is desirable to have the property of being adsorbed on a hydrophobic surface. Examples include soy protein, egg albumin, ovalbumin, human serum albumin, bovine serum albumin and globulin. Etc. can be mentioned. Another protein additive that can be used for the solid-liquid separation of the present invention is one having fibrous protein or scleroprotein as a main component and having a hydrophobic surface in the protein, thereby having the property of aggregation with each other. Such protein additives include additives containing keratin, collagen, fibroin, elastin, gluten, which is one of the storage proteins, and the like.

In the present invention, the biomass sacchalate preparation step of glycosylation by adding acid or glycosylase to the biomass pretreatment; A fine particle aggregation step of preparing a slurry in which fine particles are aggregated by adding and stirring a glycated residue protein additive for biomass saccharification; And a sugar solution recovery step of separating the sugar solution by centrifugation or filtration of the slurry in which the fine particles are aggregated, thereby providing a recovery method of the sugar solution prepared by saccharification of biomass.

In the method for recovering the sugar solution prepared by saccharification of the biomass according to the present invention, the fine particle aggregation step may add a protein additive for aggregation of glycated residues in an aqueous solution or suspension.

In the method for recovering a sugar solution prepared by saccharification of biomass according to the present invention, the protein additive for aggregating glycated residues has a hydrophobic surface at least in the molecular structure of the protein, so that the acidity (pH) of the aqueous solution is dissolved after dissolving in water. It may be to form a suspension when adjusted or heated.

In the present invention, a method of preparing a protein additive added to a saccharide for solid-liquid separation of biomass saccharide is to first dissolve or suspend the protein in water. And prior to addition to the cargo per heating the protein solution or suspension at 80 to 121 ^o C suspension was made to induce thermal denaturation it is more effective. In this case, a method of controlling the acidity (pH) of a sugar solution containing an aqueous protein solution or suspension or agglutinating protein may be used for effective denaturation of the protein. The protein concentration in the aqueous protein solution or suspension is preferably 100 mg / L to 100 g / L, more preferably 500 mg / L to 50 g / L. The total amount of protein added to the saccharin is preferably increased according to the amount of saccharified residue, which is a water insoluble solid in the saccharified substance, and the amount may be used in an amount of 0.01 g to 100 g per 1 kg of saccharified residue, and 0.1 per kg of saccharified residue. It is preferable to use it in the ratio of g-10g.

In the present invention, the method of agglomerating microparticles in the sugars most effectively while minimizing the loss of saccharase by using an aqueous solution or a suspension, which is a protein additive prepared for solid-liquid separation of biomass glycosylated sugars, has a sugar at a rate such that precipitation does not occur. Slowly adding an aqueous protein solution or suspension while stirring the cargo and maintaining the stirring state for a certain time.

In the method for recovering the sugar solution prepared by saccharification of the biomass according to the present invention, the sugar solution recovery step may use any one of a centrifuge, a filter press or a decanter.

The present invention further provides a method for recovering a sugar solution from the biomass saccharide with a minimum loss of sugar even with a minimum process. To this end, 1) by adding an aqueous solution or suspension of water soluble or suspended in water to the glycosylated residue containing the saccharified residue and agglomerated into fine particles by agglomerating the fine particles in the saccharification, and 2) converted to the macroparticles The sugar solution is transferred to a centrifuge or a filter, and the sugar solution is recovered by centrifugation at a low rotational speed for a short time or filtered without being pressurized at a high pressure. 3) The saccharified residue that has been settled by centrifugation or the remaining saccharification residue on the filter cloth after filtration. And dilute and increase the sugar solution in the saccharified residue by stirring with water, and 4) transfer the increased sugar solution to a centrifuge or filter, and centrifuge or filter to recover the remaining sugar solution. .

In the present invention, the glycosylated to separate the solid and the liquid, respectively, is produced by hydrolyzing fibrin such as cellulose and hemicellulose using fibrin hydrolase, and the sugar solution contains a large amount of saccharified residue, for example, corn. Agricultural byproducts such as stems, sunflower stems, palm fruits and palm trees, energy crops such as silver grass and reeds, enzymatic hydrolysates of woody biomass including woody biomass such as eucalyptus, acacia, willow, poplar hybrids, chlorella, etc. Enzyme hydrolyzate of algae biomass containing diatoms such as green algae and diatoms.

The method for recovering the sugar solution from the biomass enzyme hydrolyzate of the present invention is to solidify the glycosylated residue microparticles containing lignin as a major component and to convert the glycosylated solution into the macroparticles in a solid-liquid manner. To get a residue. According to Stock's law, the rate at which solid particles settle in a liquid is proportional to the density difference between the solid particles and the medium, and is proportional to the cube of the particle size. Solid-liquid separation is completed within. In addition, the larger particles make it possible to obtain a relatively clear sugar solution even in filtration using a filter cloth. However, even in such solid-liquid separation, some of the sugar solution remains in the precipitate or the filtrate residue, which requires an additional process for recovering the sugar solution.However, in order to easily dismantle the precipitate or the filtrate residue while adding water in the subsequent process, Centrifugation or high pressure press filtration are not preferred. In addition, it is also possible to perform a solid-liquid separation after mixing and stirring a certain amount of water before centrifugation or filtration in order to increase the recovery of sugars.

A method for recovering a sugar solution prepared by saccharification of biomass according to the present invention, comprising: diluting and increasing a sugar solution in a saccharified residue by mixing and stirring a saccharified residue with water after the sugar solution recovery step; And recovering the increased sugar solution that separates the diluted and extended sugar solution from the saccharified residue.

 That is, in the method for recovering the sugar solution from the hydrolyzate of the biomass acid or glycosylation enzyme of the present invention, the solid solution is separated by solid-liquid separation in order to further recover the sugar solution from the saccharified residue containing a part of the sugar solution left in the first solid-liquid separation. The resulting saccharified residue should be mixed with water and homogenized. At this time, the amount of water to be added is added or subtracted according to the target recovery rate of the sugar solution calculated from the water retention rate in the solid content when the solid-liquid separation is performed in a subsequent process.As long as there is no problem in mixing the lumped saccharified residue with water, the method such as stirring and shaking There is no limitation in particular. The new suspension thus prepared still retains a large particle state that can be easily settled or filtered.

In this step, the method for recovering the sugar solution from the hydrolyzate of the biomass acid or glycosylation enzyme of the present invention further recovers the sugar solution by centrifuging or filtering the suspension prepared with fresh water in the above process. When the sugar solution is recovered by this operation, intensive centrifugation or filtration can be used under violent conditions such that the residual amount of sugar solution remains in the residue.For example, in the case of centrifugation, the centrifugation time is increased along with the rotational speed. In the case of filtration, the pressure is increased. However, it is also possible to repeat the above process if you want to further increase the recovery of the sugar solution. This is because the macroparticles produced by the cohesion of the protein are not only relatively durable, but also maintain the adhesion of the denatured protein, so that the particles separated in the process of mixing with water can again aggregate to form the macroparticles.

In the method for recovering the sugar solution prepared by saccharification of the biomass according to the present invention, the diluting increase of the sugar solution may be performed using a batch or continuous dispersion stirrer. That is, in step 3) for efficient solid-liquid separation of the biomass saccharide of the present invention, a slurry may be prepared by adding water to the saccharified residue and stirring it for further recovery of the sugar solution from the saccharified residue discharged after the first solid-liquid separation. Stirrers can be used, and either batch or continuous dispersion agitators can be used.

In the method for recovering the sugar solution prepared by saccharification of the biomass according to the present invention, the step of recovering the extended sugars may be any one of a filter press or a decanter. That is, step 4) for the efficient solid-liquid separation of the biomass enzyme glycosylated compound of the present invention can be seen as a repetition of step 2), when the decanter is used for solid-liquid separation, only by increasing the number of revolutions of the decanter or lengthening the time. Increasing the recovery of sugar solution may differ. In addition, when the filter press is used for the solid-liquid separation, it may be different to reduce the volume of the sugar solution remaining in the saccharified residue by increasing the compressive force.

The method for recovering the sugar solution from the hydrolyzate of the biomass saccharase of the present invention further provides a type of equipment suitable for maximizing the recovery rate of the sugar solution containing the glycosylase and minimizing the amount of water used and an optimal method of use. . In step 1) for efficient solid-liquid separation of the biomass saccharide, a batch or continuous saccharification group that has been saccharified may be used as a reactor for agglomeration of microparticles by adding protein as it is. In other words, by injecting the aqueous solution or suspension prepared in advance in the saccharification device after glycosylation and maintaining the stirring state for a predetermined time, it is possible to induce fine particle aggregation by the protein. In this case, a saccharifier with sufficient spare volume is preferable to further inject water to increase the recovery rate of the sugar solution in the solid-liquid separation of the next step. Alternatively, the sugar particles may be transferred to a batch stirrer capable of stirring after saccharification, and then fine particle aggregation reaction may be performed by protein addition. Also in this case, a stirrer with sufficient spare volume is preferable to further inject water in order to increase the recovery rate of the sugar solution in the solid-liquid separation of the next step. In addition, a continuous reactor capable of inducing fine particle aggregation by stirring while adding a solution of a protein or a suspension during the transfer of the saccharified substance from the saccharifier to the solid-liquid separator may be used. In this case, the recovery rate of the sugar solution in the solid-liquid separation of the next step may be used. A continuous stirrer with sufficient free volume to inject additional water is preferred.

That is, in the first solid-liquid separation of step 2) for the efficient solid-liquid separation of the biomass saccharide of the present invention, a decanter capable of continuously discharging the clear sugar solution and the precipitate may be used. Such a decanter is preferably capable of high speed rotation so as to reduce the power consumption by shortening the operating time. At this time, the high speed rotation speed is preferably 250 × g to 5,000 × g equivalent rotation speed, the residence time of the sugar is preferably within 30 minutes. Considering the method step 3) of the present invention, which further recovers the sugar solution in the precipitate, in the step 2), the decanter discharges the clear sugar solution, but at the same time, by adding water and stirring to discharge the soft precipitate, the slurry can be easily prepared again. It is preferable to operate so that the decanter has a rotational force of 500 × g to 4,000 × g, and a residence time of the sugars is more preferably within 10 minutes. This is because, in most cases, even when solid-liquid separation is performed by a decanter rotating at a high speed, a considerable amount of sugar solution remains in the discharged precipitate, and thus, all of the sugar solution in the precipitate cannot be recovered by only one decanting.

In addition, in the liquid-liquid separation at this stage, a filter press equipped with a filter cloth having fine pores may be used. The filter press can be freely selected from less than 1 micron to several tens of microns in consideration of the turbidity of the sugar solution to be obtained since the size of the particles to be passed varies depending on the pore size of the filter cloth. However, in the present invention, although the solid-liquid separation principle of the biomass enzyme saccharide is to agglomerate microparticles of hydrophobic lignin main ingredient by the addition of a protein having a hydrophobic surface, the average particle diameter of the saccharified residue in the saccharide is increased. It is more effective in increasing the sedimentation rate by separation, whereas the produced macroparticles are not hard and can be cracked again when pressed between the filter cloths so that some fine particles can pass through the filter cloth. However, since the number of particles passing through the filter cloth is significantly smaller, the sugar solution obtained is relatively clear, so that a clear sugar solution can be prepared by further separation such as microfiltration in a subsequent process. In the present invention, when the filter press is used for the solid-liquid separation of the biomass saccharide, the filter cloth to be mounted can not be largely limited because the size of the pore can be freely adjusted, but is preferably 0.1 micron to 50 microns, in order to prepare a clear sugar solution. More preferred are 1 micron to 15 microns.

In the method for recovering a sugar solution prepared by saccharification of biomass according to the present invention, the increased sugar solution may be reused for dilution increase of a slurry in which fine particles are aggregated through an enzyme saccharification step and a fine particle aggregation step. have.

That is, the sugar solution recovered from the increased sugar solution of step 4 may be reused for dilution increase of the slurry in which the fine particles are aggregated in the step of protein addition and fine particle aggregation. For efficient separation of the biomass glycosylated compound of the present invention, it is preferable to use a filter press for the second solid-liquid separation, although the use of a decanter to obtain a clear sugar solution for the first solid-liquid separation is inevitable. It is most preferable to obtain a clear sugar solution while reducing the amount of water used by using a slightly turbid and significantly low sugar concentration obtained from the second solid-liquid separation for the dilution increase of the sugars before the first solid-liquid separation.

Hereinafter, on the basis of the production examples and examples of the present invention will be described in more detail. However, the following Preparation Examples and Examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Preparation Example 1 Protein additive for aggregation of glycated residues containing soy protein as a main ingredient (unmodified)

100 g of soy protein isolated (Soy protein isolated, MP Biomedicals, LLC, France) was added to a 10 liter steamer, and 5 liters of non-ionized water was added and sterilized at 105 ^o C for 20 minutes. A sterilized soy protein solution was added to a plastic container (for 50 liters), and 45 liters of non-ionized water was added to prepare a protein additive for aggregation of glycated residues.

Preparation Example 2 Protein additive for agglutination of glycation residues containing soy protein as a main component (heating denaturation)

1 g of soy protein extract (Soy protein isolated, MP Biomedicals, LLC, France) was added to a 1 liter culture bottle, and 1 liter of water was added, followed by stirring to dissolve. This was sterilized and warm denatured at 121 ^° C. for 20 minutes to prepare protein additives for aggregation of glycated residues.

Preparation Example 3 Protein additive for agglutination of glycation residues containing soybean powder as a main component (heating denaturation)

1 g of soybean flour (Soyma flour, Sigma S-9633, USA) was placed in a 1 liter culture bottle, and 1 liter of water was added thereto, followed by stirring to dissolve. This was sterilized and warm-modified at 121 ^° C. for 20 minutes, and then redispersed for 1 minute with a high-speed rotary stirrer (Goi Bak, Buwon Household Appliances, Korea) to prepare a glycosylated residue protein additive.

Preparation Example 4 Protein additive for agglutination of glycated residues containing bovine serum protein as a main component (heat denatured)

1 g of bovine serum albumin (Sigma A3059, USA) was added to a 1 liter culture bottle, and 1 liter of water was added and stirred to dissolve. This was sterilized and warm denatured at 121 ^° C. for 20 minutes to prepare protein additives for aggregation of glycated residues.

Preparation Example 5 Protein additive for agglutination of glycated residues containing egg protein as a main ingredient (heating degeneration)

1 g of egg protein (egg albumin, Sigma A5503, USA) was added to a 1 liter culture bottle and 1 liter of water was added, followed by stirring to dissolve. This was sterilized and warm-modified at 121 ^° C. for 20 minutes, and then redispersed for 1 minute with a high-speed rotary stirrer (Goi Bak, Buwon Household Appliances, Korea) to prepare a glycosylated residue protein additive.

Example A Recovery of Sugar Solution from Palm Fruit Enzyme Glycosaccharide

Empty wet bunch of oil palm (65% moisture) ground powder (less than 20 mesh, provided by Corindo Group, Indonesia) was injected into a continuous high-pressure reactor (SuPR2G, Advancebio, USA) at 191 ^o C. The hydrothermal treatment was performed for 20 minutes at. Water was added to the pretreatment so that the water content was 10 times that of the raw material drying, followed by injection into a filter press (Taeyoung Filtration, Korea) to separate the liquid. The obtained solid was dispersed and ground by a cutting mill (Korea Powder Machinery Co., Ltd., Korea), and then water was added and mixed. This was injected into a disc mill (laboratory disc mill, manufactured by Andritz, USA) and triturated to prepare an enzyme glycosylation substrate having a water content of 75.6%. A total of 7 liters of saccharification tank weighed in advance was added 1,043 g of the saccharification substrate, 4,006 g of non-ionized water, and 11 ml of the glycosylase Selicctec 3 (Cellic CTec3, manufactured by Novozymes Korea, Seoul). The glycosylated compound was prepared by saccharifying for 72 hours while maintaining a saccharification device at 50 ± 1 ^° C., pH 5.45 ± 0.05, and a stirring speed of 200 rpm. First, after weighing the contents of the saccharification tank, 50 ml of the saccharin was taken and centrifuged, and the supernatant was analyzed for glucose concentration by high performance liquid chromatography (HPLC, Waters, USA). In addition, the residue was washed several times with non-ionized water and lyophilized to calculate the residual ratio of glycated residue.

40 ml of enzyme saccharine was transferred to a Erlenmeyer flask (for 125 ml), a stub was placed, and then placed on a multisterer (Corning Products USA) and stirred at 300 rpm. 4 ml of the glycosylated residue aggregation protein additives of Preparation Examples 1 to 5 were added to the enzyme saccharified, stirred for 5 minutes, and then transferred to a 50 ml falcon tube. The sample was centrifuged at 845 × g for 5 minutes with a control sample containing only 4 ml of water and mixed with glycosylated water (Hanil Science, Korea), and then turbidity was measured using a turbidimeter (HACH 2100AN turbidimeter, USA). Indicated.

	Main ingredient of the additive	Additive manufacturing method	Centrifuge Supernatant Turbidity (NTU)
Comparative Example 1	Saccharide Control (No Additives)	-	Measurement limit exceeded
Example 1	Soy protein extract of Preparation Example 1	No denaturation	71 ± 35
Example 2	Soy Protein Extract of Preparation Example 2	Gaon Degeneration	30.8 ± 0.6
Example 3	Soybean powder of Preparation Example 3	Gaon Degeneration	80.4 ± 7.1
Example 4	Bovine Serum Protein of Preparation Example 4	Gaon Degeneration	40.0 ± 0.6
Example 5	Egg protein of Preparation Example 5	Gaon Degeneration	69.1 ± 1.6

The glucose concentration of the glycosylated product produced by the enzymatic saccharification of the palm fruit and vegetable pretreatment of Example A was 2.6% (weight / weight), the specific gravity was 1.02, and the insoluble residue contained in the sugars was about 2.0%. The sugar solution obtained by centrifuging the saccharides at 845 × g for 5 minutes was very turbid as in Comparative Example 1 of Table 1, and further solid-liquid separation was inevitable. On the other hand, the turbidity of the supernatant obtained by adding 4 ml (4 mg of protein) per 40 g of saccharified residue (0.8 g of saccharified residue) and stirring after adding the protein additive for agglutination of saccharified residue was measured. It was slightly different depending on the type of protein, but it was very low, and all of them produced a clear sugar solution.

Example B Recovery of Sugar Solution from Giant Suspension Enzyme Glycosaccharide Using Decanter and Filter Press

Wet macromolecular crushed water (65% moisture, 20 mesh or less, Korea) was injected into a continuous high pressure reactor (SuPR2G, Advancebio, USA), and hydrothermally treated at 191 ^o C for 20 minutes. Water was added to the pretreatment so that the water content was 10 times that of the raw material drying, followed by injection into a filter press (Taeyoung Filtration, Korea) to separate the liquid. The obtained solid was dispersed and ground by a cutting mill (Korea Powder Machinery Co., Ltd., Korea), and then water was added and mixed. This was injected into a disc mill (laboratory disc mill, manufactured by Andritz, USA) and triturated to prepare an enzyme glycosylation substrate having a water content of 80.7%. The total amount of glycosylation substrate 48,210 g in a total volume of 70 liters of saccharification machine (Hanil Science, Korea) was divided into 12 parts and injected one by one, and the saccharification machine was maintained at 50 ± 1 ^° C., pH 5.0 ± 0.05, and a stirring speed of 200 rpm. To this was added 65.8 ml of glycosylase CelicCec3 (Cellic CTec3, Novozymes Korea, Seoul) every hour. Glycosylated was prepared by adding saccharification substrate and saccharase for a total of 12 hours, and saccharifying for 72 hours from the time of initial substrate addition. A small amount of the saccharide was centrifuged and the supernatant was analyzed for glucose concentration by high-performance liquid chromatography (HPLC, Waters, USA). The residue was washed with water, dried and weighed to calculate the ratio of insoluble glycated residue in the saccharified product. All of the sugars were transferred to a 100-liter mixer (Hanil Science, Korea) to weigh the sugars and calculate the total glucose using this amount and the glucose concentration.

While stirring the glycosylated at 60 rpm per minute, 50 liters of the protein additive for aggregation of glycated residues of Preparation Example 2 was added and further stirred for 5 minutes. The sample was transferred to a decanter (Fine Products, Korea) and programmed to stay at 1,902 × g for 5 minutes and centrifuged. The sugar solution from this process was collected in a 200 liter tank. The solids discharged from the decanter were added to 72.8 liters of non-ionized water while continuously being injected into a small mixer. The disaccharide solution was injected into a filter press (Taeyoung Filtration, Korea) attached with a 5 micron filter cloth and filtered under pressure so that the water content of the saccharified residue discharged by adjusting the pressure of the filter press was about 50%. The sugar solution discharged here was re-injected into the decanter, programmed to stay at 1,902 × g for 5 minutes, and centrifuged, and then the sugar solution discharged was added to the sugar solution obtained from the decanter. A small amount of the sugar solution in the tank was taken to measure the glucose concentration and multiplied by the total amount of the sugar solution to calculate the sugar recovery rate. Table 2 shows the average sugar yield and the turbidity average of the final sugar solution obtained by repeating the above process three times.

Example C Recovery of Sugar Solution from Giant Suspension Enzyme Glycosaccharide Using Decanter

The sugar compound was prepared in the same manner as in Example B. While stirring this glycoside at 60 rpm per minute, 50 liters of the protein additive for aggregation of the saccharified residue of Preparation Example 2 were added and further stirred for 5 minutes. The sample was transferred to a decanter (Fine Products, Korea) and programmed to stay at 1,902 × g for 5 minutes and centrifuged. The sugar solution discharged from this process was collected in a 300 liter tank. The solids discharged from the decanter were added continuously to a small mixer with 74 liters of non-ionized water. The disaccharide solution was solid-liquid separated with the same decanter as above. This operation was repeated once more to recover the sugar solution. A small amount of the sugar solution in the tank was taken to measure the glucose concentration and multiplied by the total amount of the sugar solution to calculate the sugar recovery rate. Table 2 shows the average sugar yield and the turbidity average of the final sugar solution obtained by repeating the above process three times.

division	Example 6 Measurement Results
	Gross weight (g)	Glucose Concentration (%)	Total glucose (g)	importance	% Residual	Yield rate (%)	Turbidity (NTU)
Sugar	50,450	11.1	5,174	1.08	7.6	-	Measurement limit exceeded
Example B Recovery Sugar Solution	165,200	3.08	5,096	1.02	0.0	98.5	21.8
Example C Recovery Sugar Solution	222,000	2.29	5,075	1.02	0.0	98.1	20.2

Table 2 shows that the glycosylated solution has a high specific gravity and a lot of fine particles, so that even after centrifugation for 5 minutes with a force of 1,902 × g, some of the glycosylated residues do not sink and the turbidity is high enough to be impossible to measure. On the other hand, the sugar solution recovered by adding 0.026 g per 2 g of glycosylated residue (2 g per kg of saccharified sugar) to the soy protein extract of Preparation Example 2, which is the protein additive for aggregation of glycated residues of the present invention, was three times the amount. Increasing above and the glucose concentration was lowered to about one third, but it can be seen that the solid solution was effectively separated as a clear solution. In addition, the yield of sugar obtained from the above example was found to reach 98.5%, it can be seen that the sugar solution recovery technique of the present invention can obtain a very high sugar yield even with a simple process.

The sugar solution prepared using the decanter of Example C also had a final volume of four times that of the original sugar solution, but the sugar recovery rate was 98%.

Test Example 1: Confirmation of Enzyme Content of Sugar Solution Recovered from Giant Suspense Enzyme Sulfate

Wet giant pampas grass (65% moisture, 20 mesh or less, Korea) was injected into a continuous high pressure reactor (SuPR2G, Advancebio, USA), and hydrothermally treated at 200 ^° C. for 10 minutes. Prepare the saccharification substrate by adding water to the pretreatment so that the water content is 10 times that of the raw material drying and then injecting it into the filter press (Taeyoung filtration, Korea) to separate the solid and liquid and then injecting it into the cutting mill to lightly grind it. It was. Into a total volume of 7 liter of saccharification machine (Hanil Science, Korea), 4061 g of non-ionized water was added first, and 1190 g of the saccharification substrate was added thereto. The glycosylator was maintained at 50 ± 1 ^° C., pH 5.5 ± 0.05, and agitation speed of 200 rpm. To this was added 24 ml of the glycosylating enzyme Celic Cec3 (Cellic CTec3, manufactured by Novozymes Korea, Seoul). Glycosylated was prepared by saccharification for a total of 144 hours. 200 g each was added while homogenizing the glycosylated solution and placed in four 500 ml centrifuge tubes and two culture bottles, respectively. Two centrifuge tubes containing saccharin were added with 200 ml of the soy protein extract suspension of Preparation Example 2 while stirring with a magnetic stirrer and the stirring was continued for 5 minutes. After centrifugation for 5 minutes at 1,902 × g to obtain a clear supernatant. Two centrifuge tubes containing saccharin were added with 200 ml of non-ionized water, mixed, and centrifuged at 1,902 × g for 90 minutes to obtain a clear supernatant. Two bottles containing the culture per cargo is added to the non-water 200 ml was denatured for 20 minutes at 121 ^o C into the high-temperature sterilizer (autoclave). The contents were transferred to a centrifuge tube and centrifuged at 1,902 × g for 5 minutes to obtain a clear supernatant. 3.17 g of the glycosylation substrate was placed in a 125 ml Erlenmeyer flask, and 8 ml of citric acid buffer (1 M, pH 5.5, Sigma Aldrich, USA), sodium azide (1%, weight / weight, Sigma Aldrich, USA) 1.3 ml, 24 ml of each sugar solution was added, and the acidity was adjusted to pH 5.5. Non-ionized water was added and sealed so that the flask contents weighed 40 g. As the standard enzyme test, Cellic CTec3 as a glycosylase was added to the saccharification system composed of saccharification substrate, citric acid buffer, sodium azide and non-ionized water as described above, and the amount was added in 0.01, 0.02, 0.03, respectively. , 0.04 and 0.05 ml. The sample was placed in a stirrer (orbit shaker, Gaon Scientific, Korea) and glycosylated at 50 ± 1 ^° C. at a stirring speed of 200 rpm for 72 hours. After saccharification, 1 ml of the saccharin was taken and centrifuged (11,000 rpm, 20 minutes), and the glucose concentration in the supernatant was analyzed by high performance liquid chromatography (HPLC, Waters, USA). Table 3 shows the results of the enzyme activity in the sample sugar solution instead of the glycosylation rate drawn using the saccharification rate of the enzyme standard test plot.

division	Glycation rate measurement result (%)
Centrifugation after Carbohydrate Warming	0
Untreated Sugars Centrifugation	11.4 ± 1.1
Centrifugation after protein additive aggregation	15.9 ± 0.1

When the enzyme activity of the sugar solution was measured after different treatments of the sugars, the high-speed centrifugation supernatant showed that the enzyme was contained by converting 11.4% of the glycosylation substrate to glucose under given conditions. On the other hand, in the case of inducing the aggregation of microparticles by denaturing the enzyme containing the saccharin, the enzyme activity of the sugar solution on the newly added glycosylation substrate is not seen at all. In contrast, the sugar solution obtained using the method of the present invention shows a higher enzyme glycosylation rate than the untreated sugar solution, showing that at least it does not inactivate or eliminate the enzyme in the sugar solution.

Claims (10)

1. Biomass sacchalyse preparation step of saccharifying by adding acid or glycosylase to the biomass pretreatment;

   A fine particle aggregation step of preparing a slurry in which fine particles are aggregated by adding and stirring a glycated residue protein additive for biomass saccharification; And

   And a sugar solution recovery step of separating the sugar solution by centrifuging or filtering the slurry in which the fine particles are aggregated.
2. The method according to claim 1,

   The fine particle aggregation step is a method for recovering the sugar solution prepared by saccharification of biomass, characterized in that the addition of the glycosylated residue protein additive for aggregation.
3. The method according to claim 2,

   The protein additive for aggregation of glycated residues has a part of hydrophobic surface in the molecular structure of the protein, so that it is dissolved in water to form a suspension when the acidity (pH) of the aqueous solution is adjusted or heated to form a saccharification of biomass. How to recover a sugar solution.
4. The method according to claim 1,

   The glycosylated residue aggregation protein additive is prepared by glycosylation of biomass, characterized in that it comprises one or more proteins selected from the group consisting of globular protein, fibrous protein or light protein. Recovery method of sugar solution.
5. The method according to claim 4,

   The protein additive for aggregation of glycated residues is soy protein isolated, soy protein concentrate, soy flour, albumin, egg albumin, ovalbumin, bovine serum albumin Serum albumin, globulin (globulin), keratin (keratin), collagen (collagen), fibroin (fibroin), elastin and at least one selected from the group consisting of gluten (gluten) A method for recovering sugar solution prepared by saccharification of mass.
6. The method according to claim 1,

   The sugar solution recovery step is a method for recovering the sugar solution prepared by saccharification of biomass, characterized in that using any one of a centrifuge, filter press or decanter.
7. The method according to claim 1,

   Diluting and increasing the sugar solution in the saccharified residue by mixing and stirring the saccharified residue with water after the sugar solution recovery step; And

   Recovering the sugar solution prepared by saccharification of biomass, characterized in that it further comprises the step of recovering the increased sugar solution for separating the dilute extended sugar solution from the saccharified residue.
8. The method according to claim 7,

   Diluting and increasing the sugar solution is a method of recovering the sugar solution prepared by saccharification of biomass, characterized in that using a batch or continuous dispersion stirrer.
9. The method according to claim 7,

   Recovering the increased sugar solution is a method of recovering the sugar solution prepared by saccharification of biomass, characterized in that using any one of a filter press or a decanter.
10. The method according to claim 7,

    The method of recovering the sugar solution prepared by saccharification of biomass, characterized in that to reuse the increased sugar solution in the dilution increase of the slurry in which the fine particles, which have undergone the biomass glycosylation step and the fine particle aggregation step.

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