JP2010032274A - Separation and quantification method of neutral sugar contained in biomass - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an analyzing method for separating and quantifying five kinds of neutral sugars contained in cellulosic biomass in a high yield with good reproducibility. <P>SOLUTION: The analyzing method includes the process for performing primary hydrolysis solubilizing a sample by the acid treatment of hardly decomposable cellulosic biomass and the secondary hydrolysis of monosaccharide to oligosaccharide, the process for neutralizing an acid treatment solution to add internal standard sugar, the process for perfectly monosaccharifying the remaining oligosaccharide by enzyme, and the sugar measuring process for separating and quantifying the obtained sugar solution. By combining these processes, five kinds of neutral sugars contained in the cellulosic biomass can be separated and quantified in a high yield with good reproducibility. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for measuring the composition of a polysaccharide used as a raw material for bioethanol and biorefinery contained in cellulosic biomass.

  Biomass is attracting attention as a substitute for fossil fuels, reducing carbon dioxide emissions, and preventing global warming. In particular, bioethanol production is rapidly expanding as an alternative to gasoline and other transportation fuels. However, at present, biomass, which is a raw material for bioethanol, is mainly sugar solution and starch such as sugar cane and corn, which is a cause of soaring food and grains. Therefore, utilization of cellulosic biomass is required in order to avoid competition with food. Despite this situation, at present, bioethanol production from cellulosic biomass is extremely difficult. The cause is said to be that hydrolysis of polysaccharides contained in cellulosic biomass is not easy, and lignin and the like have adverse effects such as inhibiting saccharification and alcohol fermentation.

  Cellulosic biomass, unlike carbohydrate and starch biomass, forms more complex cell wall polysaccharides. Among the constituent components, sugars such as acidic sugar are difficult to convert to ethanol, and available sugars are five neutral sugars of glucose, xylose, mannose, arabinose, and galactose. Among these, even if technically possible, the conversion of xylose to ethanol has not reached the industrially feasible range, and research and development for improving the utilization rate of xylose is underway worldwide. Arabinose is also a sugar that is difficult to use at present.

Moreover, all available sugars need to be monosaccharides. In the case of starch, oligosaccharides such as maltose and maltotriose other than glucose are also used in yeast, but cell wall polysaccharides are β-linked polysaccharides, and even disaccharides cannot be used in yeast. From these viewpoints, one of the inventors (Shinichiro Sakamoto) allowed the cellulase enzyme group (accelase) obtained from the culture solution of Aspergillus aculeatus to act on the sugar solution obtained by treating pulp with various cellulases. Thus, the oligosaccharide contained in the enzyme saccharified solution has been successfully saccharified (Non-patent Document 1). As a result, it has been found that a more accurate available sugar amount of a saccharified solution treated with various cellulases can be grasped.

  However, it is difficult to hydrolyze biomass directly to monosaccharides by enzymatic treatment. This is because lignin is not enzymatically decomposed among cellulose, hemicellulose and lignin, which are typical three components constituting biomass, and inhibits the action of polysaccharide hydrolase. The cell wall is composed of a multilayer structure of cellulose, hemicellulose, and lignin, and the polysaccharide hydrolase cannot act on cellulose and hemicellulose unless lignin that is in close contact with cellulose and hemicellulose is removed. Furthermore, it is known that a delignified biomass produces an inhibitor for polysaccharide hydrolase. Therefore, at present, it is extremely difficult to hydrolyze cellulosic biomass directly with a polysaccharide hydrolase without delignification. Further, even delignified biomass has hardly been successfully decomposed to monosaccharides by polysaccharide hydrolase. In sugar analysis of cellulosic biomass, the saccharified solution obtained by enzymatic treatment of delignified pulp contains oligosaccharides. To completely decompose these, the saccharified solution is trifluoroacetic acid (TFA). (Non-patent document 2).

  Currently, the Tappi Standard method (Non-patent Document 3) is known and used as a method for measuring polysaccharide components in wood and pulp. This method is a method in which 72% concentrated sulfuric acid is added to a dried wood flour or pulp, dissolved and subjected to primary hydrolysis, and then subjected to secondary hydrolysis at a temperature of 100 ° C. or higher after lowering the acid concentration. The obtained sugar solution is neutralized, and the constituent sugars are measured by gas chromatography or high performance liquid chromatography. This measurement method is a method developed in Europe and the United States, which is an advanced country of pulp and paper, and is useful for analyzing the sugar composition of biomass mainly made from conifers.

  The reason for this is that hemicellulose contained in conifers has few pentose sugars that are susceptible to hyperlysis. In hydrolysis with sulfuric acid, hemicellulose is more easily hydrolyzed than cellulose, and monosaccharides are generated first. The produced xylose is known to cause excessive decomposition at a temperature of 60 ° C. or higher. Therefore, when cellulose is completely decomposed into monosaccharides, pentoses such as xylose contained in hemicellulose are lost due to excessive decomposition. The less pentose sugar that undergoes hyperlysis, the less sugar loss due to sulfuric acid hydrolysis of conifers. Even in the analysis of sugar composition in conifers, the Tappi Standard method has a drawback that the yield of xylose contained in a small amount is measured low.

  In Japan and temperate areas, hardwoods are more often used as pulp raw materials than conifers. Hardwood contains a lot of hemicellulose, the main component of which is xylose. Furthermore, herbs also contain a large amount of hemicellulose whose main component is xylose. It is difficult to accurately analyze the sugar composition of these hardwoods and herbs. In particular, under the conditions of the Tappi Standard method, it has been pointed out that the xylose constituting hemicellulose is excessively decomposed under the conditions for accurately measuring the amount of glucose constituting cellulose.

Konno, M. Sakamoto, R. Kamaya, Y .: “Quantitative analysis of pulp-derived soluble oligosaccharides by an enzymatic-HPLC method”, J. Ferment. Bioeng., Vol. 82, No. 6, pp. 607-609 (1996) Syverud, K., Moe, S.T., Minja, R.J.A. (1999): "Carbohydrate analysis of pulps using laboratory hydrolysis and High Pressure Liquid Chromatography", 10th International Symposium on Wood and Pulping Chemistry Vol. 2, pp. 232-237 TAPPI Test Method T249 cm-85: “Carbohydrate composition of extractive-free wood and wood pulp by gas-liquid chromatography” in Tappi Test Methods, Atlanta, GA: Technical Association of the Pulp and Paper Industry. Sakamoto, R., Arai, M., Murao, S .: Agric. Biol. Chem., 49,1283 (1985) Shinichiro Sakamoto, Hirotaka Hayashi, Koji Moriyama, Motoo Arai, Sawao Murao “Degradation of cellulosic substances by crude enzymes of Aspergillus aculeatus”: Fermentation Engineering, 60, 333 (1982) Suzuki, M., Sakamoto, R, Aoyagi, T .: Tappi J., 78 (7), 174 (1995)

  An object of the present invention is to accurately analyze the sugar composition of cellulosic biomass containing xylose that is susceptible to hyperlysis. Conventionally, (1) in the acid hydrolysis method, xylose is excessively decomposed or oligosaccharide remains. (2) In the enzyme hydrolysis method, xylose is not excessively decomposed and cannot be applied to untreated biomass. As described above, the conventional methods have merits and demerits, and are not always satisfactory. However, the importance of sugar composition analysis is increasing in promoting the use of cellulosic biomass, and the development of accurate and simple methods has become urgent.

  The present inventors have made various studies in order to develop a saccharification method for completely saccharifying a polysaccharide. First, by utilizing the advantage of sulfuric acid hydrolysis, conditions were set for maximum yield of xylose by combining primary hydrolysis solubilizing biomass and secondary hydrolysis reducing molecular weight. Furthermore, after acid hydrolysis, the acid saccharified solution was neutralized, and then the conditions for treatment with an enzyme solution containing a cellulase enzyme group excellent in monosaccharide production ability were examined. As a result, the present inventors have found a condition under which an oligosaccharide such as cellooligosaccharide remaining undecomposed can be completely decomposed into a monosaccharide while solubilizing the biomass to prevent excessive decomposition of xylose even with untreated biomass. That is, it has been found that treatment conditions that maximize both the yields of glucose and xylose, which are the main constituent sugars, can be obtained. Thereby, the measuring method which analyzes more accurately five neutral sugars of glucose, xylose, mannose, arabinose, and galactose which comprise cellulosic biomass was able to be completed.

  The present invention is constituted by the following sugar composition analysis method for cellulosic biomass.

(1) A method for separating and quantifying five neutral sugars (glucose, xylose, mannose, arabinose, galactose) contained in cellulosic biomass, including the following analytical operation steps (A) to (D).
(A) An acid treatment step in which a certain amount of biomass pulverized and / or ground as a sample is treated with concentrated sulfuric acid, dissolved, diluted, and then hydrolyzed at a high temperature of 100 ° C. or higher.
(B) A neutralization treatment step of neutralizing the obtained acid hydrolyzed solution to pH 3 to 5 and then adding a certain amount of saccharide as an internal standard.
(C) An enzyme treatment step in which an enzyme excellent in monosaccharide production ability is added to an acid saccharified solution containing a neutralized oligosaccharide to convert it into a monosaccharide.
(D) A sugar measurement step of separating and quantifying five neutral sugars by high performance liquid chromatography.

  (2) The method for separating and quantifying neutral sugars contained in the biomass of (1), wherein the biomass as a sample is ground to a particle size of 100 μm or less.

  (3) In step (A), the water content of the sample is adjusted, frozen, concentrated sulfuric acid is added, and the neutral sugar contained in the biomass (1) to (2) is dissolved while grinding. Separation quantification method.

  (4) Separation of neutral sugars contained in biomass of (1) to (3) above, wherein at least one selected from deoxyglucose, inositol, methylglucose and fucose is used as an internal standard in step (B) Quantitative method.

(5) The method for separating and quantifying neutral sugars contained in biomass according to any one of (1) to (4), wherein an enzyme containing at least an enzyme derived from Aspergillus aculeatus is used in step (C).

  (6) In the step (D), the medium contained in the biomass according to any one of (1) to (5) above, wherein ion chromatography for sugar analysis is used for analysis of five hydrolyzed neutral sugars Separation and quantification method of sex sugar.

  (7) Based on the amount of sugar obtained by using the method for separating and quantifying biomass neutral sugar according to any one of (1) to (6) above, the product of the cellulosic biomass in bioethanol or biorefinery A method for determining the yield to sugar.

  The sugar analysis method of the present invention is a method for more accurately measuring five neutral sugars (glucose, xylose, mannose, arabinose, galactose) constituting cellulosic biomass. With the measurement method of the present invention, it is possible to obtain a sugar yield with higher accuracy in the production of bioethanol or biorefinery using cellulosic biomass as a raw material.

  This makes it possible to accurately grasp the sugar composition as an important index for the development of utilization technology of cellulosic biomass, and can be utilized for technological development and improvement of production efficiency. In addition, it can be an extremely useful technique in process management of actual production.

  By this measurement method, five neutral sugars (glucose, xylose, mannose, arabinose, galactose) obtained by hydrolyzing polysaccharides contained in various cellulosic biomass are more accurately separated and quantified with high yield. be able to. This makes it possible to provide sugar composition information useful for the utilization of biomass, and to determine the critical yield of products such as ethanol obtained from various biomass.

  Hereinafter, the present invention will be described in detail. The sugar composition analysis method of the present invention is a measurement method that accurately separates and quantifies five neutral sugars (glucose, xylose, mannose, arabinose, galactose) that are important for the utilization of cellulosic biomass as a sample. is there.

  Cellulose biomass used as a sample includes woody plants and herbs. That is, any biomass containing three components of cellulose, hemicellulose, and lignin can be used as an analysis sample regardless of the type. Of course, measurement is possible even in a sample in which other polysaccharides such as starch are mixed in cellulosic biomass. In that case, starch and cellulose can be separated and quantified.

  The form of the crude sample of cellulosic biomass that has not been pretreated has a great influence on the analysis accuracy. Samples that are cut as finely as possible are preferred. In order to improve the analysis accuracy, it is recommended to use a sample pulverized to 100 μm or less in the case of a dried sample. Furthermore, if possible, a sample ground to a particle size of 50 μm or less is preferable. Even a wet sample can be dissolved by freezing and then adding concentrated sulfuric acid to suppress the temperature rise.

  Grinding is effective as a means of promoting dissolution of the sample with sulfuric acid (primary hydrolysis). In particular, in order to dissolve a sample that is difficult to pulverize, such as a large loss due to pulverization, or a wet sample that cannot be finely divided, it is necessary to add sulfuric acid and then grind by grinding. Of course, even if the dried sample is pulverized, by grinding after adding sulfuric acid, dissolution of the sample is greatly promoted, and the yield of sugar measurement can be improved and the reproduction accuracy can be improved.

  A sample pretreated with cellulosic biomass can be analyzed by any pretreatment. Rather, the pretreatment of biomass is a treatment method for facilitating hydrolysis, and in this sugar analysis method, analysis operations are often facilitated, and analysis accuracy and reproducibility are often improved. The alkali-treated biomass can be dissolved by increasing the amount of sulfuric acid added if necessary. The amount of sulfuric acid used is large relative to the sample, and in many cases there is little need to increase the amount of sulfuric acid added. Moreover, even if the amount of sulfuric acid or alkali increases and the salt concentration after neutralization increases, this analysis method has little influence on the enzyme treatment due to the high sugar analysis sensitivity. Of course, a lower salt concentration is preferred.

  The collection amount of the biomass sample is not limited, but if the amount is large, the analysis operation becomes complicated, and the dry weight is about 100 mg (measured in mg) as a guide. If the sample is larger than this, the amount of secondary hydrolysis with sulfuric acid exceeds 100 mL, and a pressure-resistant glass container having a large capacity is required, and the sample to be ground requires much time for grinding. When the amount of the sample is as small as 10 mg or less (measured in units of 0.1 mg), a problem occurs in analysis accuracy and reproducibility. It is necessary to accurately measure the weight, which may be difficult with wet samples.

  The amount of sulfuric acid added is adjusted so that the sulfuric acid concentration is in the range of 70 to 77%, including the moisture contained in the sample. The concentration of sulfuric acid varies slightly depending on the type of sample, moisture content, storage conditions, and the like. However, when the sulfuric acid concentration is lower than 70%, the sample is not sufficiently dissolved, and accurate measurement becomes difficult. On the other hand, when the sulfuric acid concentration is set to 77% or more, the sugar is excessively decomposed and the saccharified solution is remarkably colored, making accurate measurement difficult. Dissolution of the sample with sulfuric acid is usually performed at room temperature, but can be heated to 40 to 50 ° C. as necessary. However, heating to 60 ° C. or higher is not preferable because excessive decomposition of sugar occurs.

  The conditions for secondary hydrolysis with sulfuric acid differ slightly depending on the type of sample, moisture content, storage conditions, etc., as in primary hydrolysis. The sample dissolved by the primary hydrolysis is transferred to the pressure resistant glass container together with the cleaning liquid. In many cases, optimum treatment can be obtained under the conditions of a sulfuric acid concentration of 4.0 to 4.5%, a heating temperature of 120 ° C. (autoclave pressurization), and a heating time of 10 to 25 minutes. Without being limited to these conditions, the conditions can also be set optimally by selecting the sulfuric acid concentration, the heating temperature, and the heating time.

  The pressure-resistant glass container of the sample after the secondary hydrolysis is cooled to 40 ° C. or less by water cooling, and then the neutralization operation is started. Neutralization is performed with NaOH solution. In the next enzyme treatment step, an acetic acid buffer solution is used as the saccharified solution. Therefore, it is preferable to add a sodium acetate solution to adjust the concentration to 0.1 M before adding NaOH. Subsequently, NaOH solution is added and saccharified liquid is adjusted to pH 4-5.

  Add the sugar solution as the internal standard to the neutralized acid saccharified solution, and measure the total weight in mg. The sugar used for the internal standard is selected from deoxyglucose, inositol, methylglucose, fucose and the like. If necessary, two or more types of internal standards can be used. By adding the internal standard sugar solution at this point, it becomes possible to minimize the influence of measurement variation factors in the operation after this operation.

  The acid saccharified solution that has been neutralized and to which an internal standard has been added contains oligosaccharides such as cellooligosaccharides derived from cellulose. There may also be conditions where xylooligosaccharides derived from hemicellulose remain. Therefore, unless these oligosaccharides are decomposed into monosaccharides, sugar analysis becomes complicated and analysis becomes difficult. Therefore, a cellulase enzyme having an excellent monosaccharide-producing ability is required. There is a need for an enzyme group that not only has a strong cellulase titer, but also has a strong ability to decompose hemicellulase and can decompose all of them into monosaccharides.

Among the cellulase group enzymes, the enzyme group belonging to the genus Aspergillus is well known as an enzyme excellent in monosaccharide production ability. Among them, as the enzyme having the most excellent monosaccharide-producing ability, a crude enzyme (accelase) obtained from a culture solution of A. aculeatus No. F-50 is most recommended. Celluligo-glucosidase that has the strongest activity on cellooligosaccharide without causing a transglycosylation reaction is included in the enzyme group included in the accelerator (Non-patent Document 4). Moreover, 8% sugar solution is obtained from bleached pulp derived from hardwood and alkali pretreated rice straw, and it has been shown that oligosaccharides are not detected by paper chromatography (Non-patent Document 5).

The enzyme group used for the enzyme treatment requires not only cellulose-derived oligosaccharides but also enzymes that hydrolyze hemicellulose-derived oligosaccharides such as xylan to monosaccharides. Therefore, the monosaccharification treatment may be difficult with only a group of cellulase enzymes derived from a single microorganism. Even in such a case, it is also possible to use a combination of cellulase enzymes derived from microorganisms having different origins or single enzymes. For example, when an enzyme group derived from the genus Trichoderma is combined with an accelerator, the saccharification reaction may proceed rapidly or the decomposition limit may increase even if the amount of enzyme added is small.

  The conditions for enzyme treatment vary greatly depending on the type and moisture content of the raw material biomass, the storage state, the presence or absence of pretreatment, the conditions for acid treatment, and the like. Further, the treatment conditions vary depending on the titer of the enzyme to be added and the monosaccharide production ability. As an example, when an accelerator is used, the amount of enzyme added is 1 mg or less, preferably 10 to 50 μg, based on a saccharified solution containing 1 mg of monosaccharide. As other processing conditions, pH 4.5, processing temperature 45 ° C., and processing time 1 to 12 hours are set.

  The saccharified solution that has undergone the enzyme treatment step is appropriately diluted and separated and quantified by high performance liquid chromatography as usual. A method for analyzing a sample sugar solution at intervals of 15 minutes using ion chromatography for sugar analysis set by one of the inventors has been disclosed (Non-Patent Document 6). The measurement conditions vary depending on the type of analyzer and separation column.

  The sugar composition of the biomass obtained by the method of the present invention has a higher xylose content than that of the Tappi Standard method, and can be used for analyzing the sugar composition of various cellulosic biomass. In particular, it exhibits excellent performance in sugar analysis of biomass with high xylose content, such as hardwoods and herbs. Of course, the sugar composition analysis of conifers with low xylose content can also provide more accurate measurements.

  If we can accurately measure the five neutral sugars that make up cellulosic biomass, glucose, xylose, mannose, arabinose and galactose, we can understand the potential for bioethanol and chemicals produced from biomass. Can be accurately evaluated. Also, even in research and development for obtaining products effectively from biomass, if accurate analysis of constituent sugars becomes possible, development of more accurate treatment techniques will be promoted. Moreover, in actual production of bioethanol and the like from biomass, accurate sugar analysis helps process management and helps improve technology to increase productivity more efficiently.

  Thus, the establishment of a technique for accurately measuring the sugar composition of cellulosic biomass is expected to contribute greatly to the development and production of biomass utilization technology.

  EXAMPLES Hereinafter, although an Example is given and the detail of this invention is demonstrated, the technical scope of this invention is not limited by the following Example.

  The rice straw cultivated in Sakai City in 2007 was passed through a φ1.0 mm sieve using a cutter mill (Masuyuki Sangyo, MKCM-5) and an atomizer (Masuyuki Sangyo, MKA-5J). Crushed. Hereinafter, the ground rice straw is referred to as a sample. A completely dry 100 mg sample was collected in a glass mortar (outer diameter 10.5 cm), water (all using pure water) was added to a final weight of 1 g, and the mortar was wrapped in a plastic bag and sealed. This was frozen overnight in a freezer. 3 g of concentrated sulfuric acid was quickly dropped onto the frozen sample and ground with a glass grinder for 5 minutes. Subsequently, the sample was ground for 25 minutes while being kept in a constant temperature bath at 50 ° C. to dissolve the large particles and fibers, and the sample was solubilized. After dissolution, the sample solution was transferred to a 100 mL pressure-resistant glass container, and co-washing was repeated to make the sample solution 80 g including the washing solution. This was treated in an autoclave at 121 ° C. for 10 minutes to carry out secondary hydrolysis with acid. After the heating was completed, the pressure-resistant glass container was quickly taken out, first immersed in warm water at 50 ° C. and cooled, and further cooled to room temperature with tap water. The sample sugar solution subjected to secondary hydrolysis of sulfuric acid was neutralized to pH 4.5 using an appropriate amount of 5N NaOH solution and 5 mL of 1M sodium acetate solution. After neutralization, 1 ml of a 2% deoxyglucose solution was added as an internal standard, the amount added was measured in mg, and the total liquid volume was further measured in mg.

To 150 μL of the sample sugar solution prepared above, 100 μL of 1M acetate buffer (pH 4.5) and 100 μL of enzyme solution (derived from A. aculeatus ) were added, and water was added to a total volume of 1000 μL. This was kept at 45 ° C. overnight to carry out an enzymatic hydrolysis reaction.

  The enzyme hydrolyzed sample was collected in a 1 ml syringe and filtered through a 0.45 μm filter, which was subjected to ion chromatography. Dionex's pumps, controllers, and electrochemical conductivity detectors were used for ion chromatography. The column was CarboPac PA1 manufactured by Dionex, and the flow rate was 1 ml / min and the column temperature was room temperature. Water was used for the mobile phase, and 0.3N sodium hydroxide aqueous solution, 0.1N potassium hydroxide aqueous solution, and 0.25N sodium carbonate aqueous solution were used for the washing liquid. Neutral sugars were separated and eluted in the order of arabinose, galactose, glucose, xylose and mannose. The analysis results were analyzed using PeakNet, a software manufactured by Dionex. The obtained results are shown in Table 1.

Method for calculating sugar content The method for calculating the sugar content from the sugar composition is to calculate the calculated amounts of arabinose, galactose, glucose, xylose and mannose in the treated sample from the analysis results of ion chromatography. The sugar content was calculated by dividing 0.88 by adding 0.90 to the amount of galactose, glucose and mannose multiplied by the sample weight.

Comparative Example 1

Analysis of sugar composition by TAPPI STANDARD method 0.35 g of sample was collected in a conical glass centrifuge tube by absolute drying, 3 ml of 72% sulfuric acid was added thereto, and the mixture was sufficiently stirred using a stir bar. Using a thermostatic bath at 30 ° C., the mixture was held for 1 hour with occasional stirring and dissolved. Wash out the contents thoroughly with 84 ml of water in a beaker. The beaker was covered with a watch glass and subjected to an autoclave and treated at 103 kPa for 1 hour. After cooling, 10 ml of 1% inositol aqueous solution was added and mixed well. Neutralized to pH 4.6 with saturated aqueous barium hydroxide. This was centrifuged to obtain a transparent supernatant. In the TAPPI STANDARD method, monosaccharides in the supernatant are reduced to sugar alcohol, acetylated and subjected to gas chromatography. In Comparative Example 1, the sugar composition was analyzed by ion chromatography in the same manner as in Example 1. In addition, deoxyglucose was used as an internal standard as in Example 1. The obtained results are shown in Table 2.

Comparative Example 2

Sugar composition analysis by enzymatic hydrolysis Add 450 μL of 0.1% suspension of ground rice straw to 100 μL of 1.0 M acetate buffer (pH 4.5), 100 μL of enzyme solution (derived from A. aculeatus ) and water to make 1 ml. After thorough mixing, enzymatic hydrolysis was performed overnight at 45 ° C. The reaction solution was subjected to saccharide composition analysis by ion chromatography in the same manner as in Example 1. In addition, deoxyglucose was used as an internal standard as in Example 1. The obtained results are shown in Table 2.

As shown in Table 1, in the examples using the method of the present invention, the sugar content of the rice straw sample was calculated to be 58.0%. However, in Comparative Example 1, the sugar content is 54.7%, and it is considered that the sugar yield is reduced due to the excessive decomposition of xylose during the operation. In Comparative Example 2, the sugar content is 6.3%, and it is considered that the sample is hardly hydrolyzed.
From the above results, it is difficult to measure an accurate sugar composition when cellulosic biomass is used as a sample in the conventional method, but it is possible to measure a more accurate sugar composition by using the method of the present invention. It becomes.

  A 125 mg sample (externally dry 104 mg) was collected in a glass mortar (outer diameter 10.5 cm), 950 mg of water (all using pure water) was added, wrapped in a plastic bag, and left overnight in a refrigerator freezer. The frozen sample was taken out, 3.00 g of concentrated sulfuric acid was quickly dropped onto the sample, and the sample was ground with a glass pestle. The mortar was held in a thermostatic bath at 50 ° C. and ground while heating. The work of heating and crushing was performed for about 30 minutes, and the solubilization of the sample with a sulfuric acid concentration of 74% was advanced.

  When the powdery rice straw was dissolved, water was added, the sample solution was transferred to a pressure-resistant glass container of 100 mL, and washed carefully with washing water so that no residual liquid remained. The total volume including the wash water was 70 mL, and the sulfuric acid concentration was 4.2%. After the lid of the pressure-resistant glass container was securely closed, secondary hydrolysis was performed by autoclaving. The heating conditions were 121 ° C. and 15 minutes. After the heating was completed, the pressure-resistant glass container was quickly taken out, first immersed in warm water at 50 ° C. and cooled, and further cooled to room temperature with tap water.

  To the sample sugar solution subjected to secondary hydrolysis of sulfuric acid, 5 mL of 1M sodium acetate and an appropriate amount of 5N NaOH were added to adjust to pH 4.5. To this, 1 mL of 2% deoxyglucose was added as an internal standard sugar solution. The amount added was 1,003 mg. The total liquid volume at this time was 90 mL, and the liquid weight was 90,062 mg.

  Next, 70 μL of a sugar solution neutralized and added with an internal standard sugar solution was collected in an Eppendorf tube. The collected weight was 78 mg, and 100 μL of 0.1 M sodium acetate buffer and 100 μL of Aspergillus aculeatus crude enzyme solution (containing 2 mg of crude enzyme) were added to make the total volume 1 mL. This was left still in a 45 degreeC thermostat, and was left for 15 hours.

  The enzyme saccharification reaction completion solution was appropriately diluted and separated and quantified with an ion column chromatograph for sugar analysis (manufactured by DIONEX). The column used was PA-1, and elution was performed with water alone at a rate of 1 mL / min. Neutral sugars were separated and eluted in the order of arabinose, galactose, glucose, xylose and mannose. The internal standard deoxyglucose eluted before arabinose.

  Separately, the five neutral sugars were separated under the same conditions in order to quantify the amount of the eluted sugar, and the conditions under which a standard curve with excellent linearity in the measurement area and the weight of each sugar were set. Furthermore, the conversion coefficient was calculated | required from the linear regression analysis of each saccharide | sugar with respect to the internal standard deoxyglucose. The neutral sugar content contained in the sample rice straw powder was calculated from the conversion factor of each obtained sugar and the measured area. It is calculated as 3.7% arabinose, 0.3% galactose, 46.5% glucose, 21.6% xylose and 1.7% mannose, and the total amount is 73.7%. The sugar content is 65.9%.

  The present invention provides an analytical method for separating and quantifying five types of neutral sugars constituting cellulosic biomass, which is difficult to accurately analyze the sugar composition because of its poor degradability, with high yield. It is. As the use of cellulosic biomass has become increasingly important for protecting the global environment, we have obtained an accurate method for separating and quantifying neutral sugars, which shows the most important applicability. This makes it easier to understand the availability of cellulosic biomass and contributes greatly to the development of technologies that contribute not only to the development of utilization methods but also to the improvement of yields such as bioethanol in actual production. Expected to do.

Claims (7)

A method for separating and quantifying five neutral sugars (glucose, xylose, mannose, arabinose, galactose) contained in cellulosic biomass, including the following analytical operation steps (A) to (D):
(A) An acid treatment step in which a certain amount of pulverized and / or ground biomass as a sample is treated with concentrated sulfuric acid, dissolved, diluted, and then hydrolyzed at a high temperature of 100 ° C. or higher;
(B) A neutralization treatment step of neutralizing the obtained acid hydrolyzate to pH 3 to 5 and then adding a certain amount of sugar as an internal standard;
(C) an enzyme treatment step in which an enzyme having excellent monosaccharide production ability is added to an acid saccharified solution containing a neutralized oligosaccharide to convert it into a monosaccharide;
(D) A sugar measurement step of separating and quantifying five neutral sugars by high performance liquid chromatography.   The method for separating and quantifying neutral sugars contained in biomass according to claim 1, wherein the biomass as a sample is ground to a particle size of 100 µm or less.   The method for separating and quantifying neutral sugars contained in biomass according to claim 1 or 2, wherein in step (A), moisture of the sample is adjusted, frozen, concentrated sulfuric acid is added, and dissolution is performed while grinding.   Separation of neutral sugars contained in biomass according to any one of claims 1 to 3, wherein at least one selected from deoxyglucose, inositol, methylglucose and fucose is used as an internal standard in the step (B). Quantitative method. The method for separating and quantifying neutral sugars contained in biomass according to any one of claims 1 to 4, wherein an enzyme containing at least an enzyme derived from Aspergillus aculeatus is used in step (C).   The method for separating and quantifying neutral sugars contained in biomass according to any one of claims 1 to 6, wherein ion chromatography for sugar analysis is used for analysis of five hydrolyzed neutral sugars in step (D). .   Based on the amount of sugar obtained by using the biomass neutral sugar separation and quantification method according to any one of claims 1 to 6, the yield of sugar in the product of cellulosic biomass in bioethanol or biorefinery is determined. Method.