JP2012100546A

JP2012100546A - Method for producing xylooligosaccharide and acidic xylooligosaccharide

Info

Publication number: JP2012100546A
Application number: JP2010249276A
Authority: JP
Inventors: Kotaro Ishikawa; 稿太郎石川
Original assignee: Oji Paper Co Ltd
Current assignee: New Oji Paper Co Ltd
Priority date: 2010-11-08
Filing date: 2010-11-08
Publication date: 2012-05-31

Abstract

PROBLEM TO BE SOLVED: To provide a method for inexpensively producing high-purity xylooligosaccharide and acidic xylooligosaccharide with reduced coloring.SOLUTION: In the method for producing xyloolgosaccharide and acidic xylooligosaccharide from lignocellulose materials as raw materials, filtrate after treating the lignocellulose materials with hemicellulase is condensed by a reverse osmotic membrane, and after treating the condensed liquid by an ultrafiltration membrane, the filtrate treated by the ultrafiltration membrane is acid-treated. The filtrate treated by the ultrafiltration membrane is continuously returned by a loop to the reverse osmotic membrane.

Description

本発明は、リグノセルロース材料を原料としてキシロオリゴ糖および酸性キシロオリゴ糖を製造する工程で、オリゴ糖に含まれる着色物質を効率的に除去するキシロオリゴ糖および酸性キシロオリゴ糖を製造する方法に関する。また、糖濃度の高いキシロオリゴ糖および酸性キシロオリゴ糖を含む糖液を製造する方法に関する。 The present invention relates to a method for producing a xylooligosaccharide and an acidic xylooligosaccharide that efficiently removes coloring substances contained in the oligosaccharide in a step of producing a xylooligosaccharide and an acidic xylooligosaccharide using a lignocellulose material as a raw material. The present invention also relates to a method for producing a sugar solution containing a high sugar concentration xylooligosaccharide and acidic xylooligosaccharide.

オリゴ糖類は腸内有用細菌の選択的な増殖促進効果によりおなかの調子を良好に保つ機能を有し、特定保健用食品として認定された乳酸菌飲料や菓子類にも利用されている有用な糖類である。食品としての用途だけではなく、畜産飼料に配合されたり、化粧品に乳化剤、保湿成分として配合されて用いられている。
多くのオリゴ糖類の中で、例えば、小麦フスマやコーンコブに由来するキシロオリゴ糖は、他のオリゴ糖と比較し少量で整腸作用を発揮することが知られている。キシロオリゴ糖は、他のオリゴ糖と同様に腸内善玉菌であるビフィズス菌の増殖を選択的に促進し、腸内悪玉菌である大腸菌の増殖を相対的に抑制する作用があることが知られている。大腸菌や腸内腐敗発酵菌は、腸内で増殖しながら発ガン性物質を生産することが報告されており、腸内に存在する大腸菌や腸内腐敗発酵菌の数を減らすことは健康を維持する上で重要である（非特許文献１参照）。 Oligosaccharide is a useful saccharide that has a function to keep the stomach in good condition due to the selective growth promoting effect of useful bacteria in the intestines, and is also used in lactic acid bacteria beverages and confectionery certified as food for specified health use. is there. It is used not only as a food product but also in livestock feeds, and in cosmetics as an emulsifier and a moisturizing ingredient.
Among many oligosaccharides, for example, xylo-oligosaccharides derived from wheat bran and corn cob are known to exert an intestinal regulating action in a small amount as compared with other oligosaccharides. Xylooligosaccharides, like other oligosaccharides, are known to selectively promote the growth of Bifidobacterium, which is a good enterobacteria, and to relatively inhibit the growth of Escherichia coli, which is an enterobacteria. ing. Escherichia coli and intestinal rot-fermenting bacteria have been reported to produce carcinogenic substances while growing in the intestine, and reducing the number of Escherichia coli and intestinal rot-fermenting bacteria present in the intestine maintains health. This is important (see Non-Patent Document 1).

オリゴ糖類を経口摂取した場合、胃酸や消化酵素の酸加水分解によりオリゴ糖の重合度が低下し、最終的には大腸菌やクロストリジウム属等の腐敗性嫌気性菌に資化されることが知られている。しかし、２量体や３量体を主成分とするキシロオリゴ糖組成物は、オリゴ糖の中でも胃酸に対する抵抗性が他のオリゴ糖に比べて比較的高く（特許文献１参照）、経口摂取したオリゴ糖のほとんどが分解されずに腸内に到達する可能性が高い。
現在、平均重合度が５量体程度のキシロオリゴ糖（特許文献２参照）や、平均重合度が１２量体前後の長鎖キシロオリゴ糖が製造可能であることが報告されている（特許文献３参照）。鎖長が長いキシロオリゴ糖は腸内でもキシロース（単糖）まで分解されにくく、鎖長の短いキシロオリゴ糖と比較し強い整腸作用が期待される為、重合度の大きなキシロオリゴ糖の開発が望まれている。 It is known that when oligosaccharides are taken orally, the degree of polymerization of oligosaccharides decreases due to acid hydrolysis of gastric acid and digestive enzymes, and eventually it is assimilated to spoilage anaerobes such as Escherichia coli and Clostridium. ing. However, a xylooligosaccharide composition mainly composed of a dimer or trimer has a relatively high resistance to gastric acid among oligosaccharides compared to other oligosaccharides (see Patent Document 1), and the oligos taken orally. Most of the sugar is likely to reach the intestine without being broken down.
Currently, it has been reported that xylo-oligosaccharides having an average degree of polymerization of about pentamer (see Patent Document 2) and long-chain xylo-oligosaccharides having an average degree of polymerization of around 12-mers can be produced (see Patent Document 3). ). Xylooligosaccharides with a long chain length are difficult to be decomposed into xylose (monosaccharide) even in the intestine, and are expected to have a strong intestinal regulating action compared with a short chain length xylooligosaccharide. ing.

一方、酸性キシロオリゴ糖はキシロオリゴ糖１分子中に少なくとも１つ以上のウロン酸残基を側鎖として有するキシロオリゴ糖である（特許文献４参照）。酸性キシロオリゴ糖には、抗炎症作用（特許文献５参照）、メラニン生成抑制作用（特許文献６参照）、アトピー性皮膚炎改善作用（特許文献７及び８参照）等の機能が認められており、機能性食品だけではなく化粧品等へも利用可能な素材として期待されている。 On the other hand, an acidic xylo-oligosaccharide is a xylo-oligosaccharide having at least one uronic acid residue as a side chain in one molecule of xylo-oligosaccharide (see Patent Document 4). Acid xylo-oligosaccharides have been recognized to have functions such as anti-inflammatory action (see Patent Document 5), melanin production inhibitory action (see Patent Document 6), and atopic dermatitis improving action (see Patent Documents 7 and 8). It is expected as a material that can be used not only for functional foods but also for cosmetics.

キシロオリゴ糖及び酸性キシロオリゴ糖を製造する場合、原料、酵素由来の着色物質、酸処理工程で加熱により生成される着色物質を製造工程において除去することが大きな課題である。酸性キシロオリゴ糖はキシロオリゴ糖の側鎖にウロン酸が付加しており、負電荷を有している。現在、酸性キシロオリゴ糖とキシロオリゴ糖（ウロン酸の付加していないキシロオリゴ糖）を含む糖液をイオン交換樹脂に通液し、酸性キシロオリゴ糖のみを樹脂に吸着させてキシロオリゴ糖と分離させた後、樹脂に吸着している酸性キシロオリゴ糖を塩で溶出させて製造している。特に酸性キシロオリゴ糖については塩による溶出の際に樹脂に吸着している着色物資も酸性キシロオリゴ糖に混合されて溶出されるため、イオン交換樹脂のみでは効率的に着色物質を除去することが困難であった。従って、イオン交換以外に、活性炭処理等の方法を用いて着色物質を除去する必要があった。しかし、活性炭処理では、製造コストの増加、オリゴ糖の収率の低減、処理時間の増大等の問題があった。また、着色物質は、キシロオリゴ糖及び酸性キシロオリゴ糖の生理活性を低減させ、さらに、オリゴ糖の味、溶解性、保存性等の物理的特性にに影響を及ぼす可能性がある。従って、より着色物質や不純物の含量の少ないキシロオリゴ糖及び酸性キシロオリゴ糖を製造する技術の開発が望まれている。 When producing xylo-oligosaccharides and acidic xylo-oligosaccharides, it is a major issue to remove in the production process raw materials, enzyme-derived colored substances, and colored substances produced by heating in the acid treatment process. Acidic xylooligosaccharides have uronic acid added to the side chain of the xylooligosaccharide and have a negative charge. Currently, after passing a sugar solution containing acid xylo-oligosaccharide and xylo-oligosaccharide (xylo-oligosaccharide without added uronic acid) through an ion exchange resin, only the acid xylo-oligosaccharide is adsorbed to the resin and separated from the xylo-oligosaccharide, It is manufactured by eluting the acidic xylooligosaccharide adsorbed on the resin with a salt. Especially for acidic xylo-oligosaccharides, colored substances adsorbed on the resin during elution with salt are also mixed and eluted with acidic xylo-oligosaccharides, so it is difficult to remove colored substances efficiently with ion-exchange resin alone. there were. Therefore, in addition to ion exchange, it has been necessary to remove colored substances using a method such as activated carbon treatment. However, the activated carbon treatment has problems such as an increase in production cost, a reduction in oligosaccharide yield, and an increase in treatment time. In addition, the coloring substance may reduce the physiological activity of xylo-oligosaccharides and acidic xylo-oligosaccharides, and may further affect physical properties such as taste, solubility, and storage stability of the oligosaccharides. Accordingly, it is desired to develop a technique for producing xylooligosaccharides and acidic xylooligosaccharides with less coloring substances and impurities.

また、キシロオリゴ糖及び酸性キシロオリゴ糖を製造する場合、製造工程を効率化し、コストの低減を図ることも重要な課題である。製造工程において、精製前のオリゴ糖溶液の糖濃度を現状より高めることができれば、精製に供するオリゴ糖溶液の処理量が低減できる。これにより、精製工程での負荷が低減でき、製造コストの低下、製造に要する時間の短縮が可能となる。従って、より精製前のオリゴ糖溶液の糖濃度を高める方法の開発が望まれている。 In addition, when producing xylo-oligosaccharides and acidic xylo-oligosaccharides, it is also an important issue to make the production process more efficient and reduce costs. If the sugar concentration of the oligosaccharide solution before purification can be increased from the current level in the production process, the throughput of the oligosaccharide solution used for purification can be reduced. Thereby, the load in the refining process can be reduced, and the production cost can be reduced and the time required for production can be shortened. Therefore, development of a method for increasing the sugar concentration of the oligosaccharide solution before purification is desired.

特許第２５４９６３８号Japanese Patent No. 2549638 特許第１０７８７７８号Japanese Patent No. 10787778 特開２００３−４８９０１JP 2003-48901 A 特許第４０７３６６１号Japanese Patent No. 4073661 特開２００３−２２１３３９JP2003-221339 特許第３７７２７４９号Japanese Patent No. 3772749 特開２００４−２１０６６４JP 2004-210664 A 特許第４２３２４６１号Japanese Patent No. 4232461

金沢ら：大腸細菌叢-とくに胆汁酸代謝と大腸発癌について-.総合臨床、２６、１０４２〜１０５０（１９７７）Kanazawa et al .: Bacterial flora-especially about bile acid metabolism and colorectal carcinogenesis-.

本発明は、リグノセルロース材料を原料としてキシロオリゴ糖および酸性キシロオリゴ糖を製造する工程で、オリゴ糖を含む糖液に含まれる着色を効率的に低減し、また、糖濃度の高い糖液を製造することを課題とする。 The present invention is a process for producing xylooligosaccharides and acidic xylooligosaccharides using a lignocellulosic material as a raw material, efficiently reducing coloring contained in a sugar solution containing oligosaccharides, and producing a sugar solution having a high sugar concentration. This is the issue.

本発明者らは、リグノセルロース材料を原料としてキシロオリゴ糖および酸性キシロオリゴ糖を製造する方法において、リグノセルロース材料をヘミセルラーゼで処理した後のろ液を逆浸透膜で濃縮し、濃縮液を限外ろ過膜で処理後、限外濾過膜で処理したろ液を酸処理することにより糖液の着色を効率的に低減できることを見出した。また、限外濾過膜で処理したろ液をループにより連続的に逆浸透膜に戻すことにより高濃度の糖液の製造ができることを見出した。 In the method for producing xylooligosaccharide and acidic xylooligosaccharide using lignocellulose material as a raw material, the filtrate after concentration of lignocellulose material with hemicellulase is concentrated with a reverse osmosis membrane, and the concentrated solution is limited. It discovered that coloring of a sugar liquid can be efficiently reduced by acid-treating the filtrate processed with the ultrafiltration membrane after processing with a filtration membrane. Moreover, it discovered that a high concentration sugar solution could be manufactured by returning the filtrate processed with the ultrafiltration membrane to the reverse osmosis membrane continuously by the loop.

具体的には、本発明は以下の（１）〜（７）の構成を採用する。
（１）リグノセルロース材料を原料としてキシロオリゴ糖および酸性キシロオリゴ糖を製造する方法において、リグノセルロース材料をヘミセルラーゼで処理した後のろ液を逆浸透膜で濃縮する濃縮工程、得られた濃縮液を限外ろ過膜で処理する脱色工程、限外濾過膜で脱色された糖液を酸で加水分解する酸処理工程、からなる工程を有することを特徴とするキシロオリゴ糖および酸性キシロオリゴ糖の製造方法。
（２）前記（１）項の限外濾過膜で脱色された糖液をループにより連続的に逆浸透膜に戻すことを特徴とするキシロオリゴ糖および酸性キシロオリゴ糖の製造方法。
（３）前記（１）項又は（２）項に記載の限外濾過膜の分画分子量が５０００〜３００００であることを特徴とするキシロオリゴ糖および酸性キシロオリゴ糖の製造方法。
（４）前記（１）項に記載のリグノセルロース材料が、木材の微細化物であることを特徴とするキシロオリゴ糖および酸性キシロオリゴ糖の製造方法。
（５）前記（４）項に記載の木材の微細化物が化学的処理により得られたパルプであることを特徴とするキシロオリゴ糖および酸性キシロオリゴ糖の製造方法。
（６）前記（５）項に記載の化学的処理により得られたパルプが広葉樹由来のクラフトパルプであることを特徴とするキシロオリゴ糖および酸性キシロオリゴ糖の製造方法。
（７）前記（１）項に記載のヘミセルラーゼが、キシラナーゼであることを特徴とするキシロオリゴ糖および酸性キシロオリゴ糖の製造方法。 Specifically, the present invention employs the following configurations (1) to (7).
(1) In a method for producing xylooligosaccharides and acidic xylooligosaccharides using lignocellulose materials as raw materials, a concentration step of concentrating the filtrate after treating lignocellulosic materials with hemicellulase with a reverse osmosis membrane, A method for producing xylooligosaccharides and acidic xylooligosaccharides, comprising a decoloring step for treatment with an ultrafiltration membrane and an acid treatment step for hydrolyzing a sugar solution decolorized with an ultrafiltration membrane with an acid.
(2) A method for producing xylooligosaccharides and acidic xylooligosaccharides, wherein the sugar solution decolorized by the ultrafiltration membrane according to item (1) is continuously returned to a reverse osmosis membrane by a loop.
(3) The method for producing xylooligosaccharides and acidic xylooligosaccharides, wherein the ultrafiltration membrane according to (1) or (2) has a molecular weight cut-off of 5000 to 30000.
(4) The method for producing xylooligosaccharide and acidic xylooligosaccharide, wherein the lignocellulosic material according to item (1) is a refined product of wood.
(5) A method for producing xylooligosaccharides and acidic xylooligosaccharides, characterized in that the refined product of wood described in the above item (4) is a pulp obtained by chemical treatment.
(6) A method for producing xylooligosaccharides and acidic xylooligosaccharides, wherein the pulp obtained by the chemical treatment as described in (5) above is kraft pulp derived from hardwood.
(7) The method for producing a xylo-oligosaccharide and an acidic xylo-oligosaccharide, wherein the hemicellulase described in (1) is a xylanase.

本発明により、キシロオリゴ糖及び酸性キシロオリゴ糖を含む糖液の着色を効率的に低減させることができ、その結果として着色が低減されたキシロオリゴ糖及び酸性キシロオリゴ糖が提供される。さらに、本発明により、精製に供する糖液の糖濃度を効率的に高めることができるため、精製工程での負荷（コスト）を低減でき、また、精製工程に要する時間が短縮できるため生産効率の高いキシロオリゴ糖および酸性キシロオリゴ糖の製造方法が提供される。 According to the present invention, coloring of a sugar solution containing xylo-oligosaccharide and acidic xylo-oligosaccharide can be efficiently reduced, and as a result, xylo-oligosaccharide and acidic xylo-oligosaccharide with reduced coloring are provided. Furthermore, according to the present invention, the sugar concentration of the sugar solution used for purification can be increased efficiently, so that the load (cost) in the purification process can be reduced, and the time required for the purification process can be shortened. Methods for producing high xylo-oligosaccharides and acidic xylo-oligosaccharides are provided.

実施例１、実施例２、実施例３の製造工程フローを示す図The figure which shows the manufacturing process flow of Example 1, Example 2, and Example 3. 比較例１、比較例２、比較例３の製造工程フローを示す図The figure which shows the manufacturing process flow of the comparative example 1, the comparative example 2, and the comparative example 3 実施例４の製造工程フローを示す図The figure which shows the manufacturing process flow of Example 4. 実施例４の糖濃度を示す図The figure which shows the sugar concentration of Example 4

以下、本発明の構成について詳述するが、本発明はこれにより限定されるものではない。本発明のキシロオリゴ及び酸性キシロオリゴ糖を得るための原料として木本性植物、あるいは草本性植物由来のリグノセルロースを用いることができる。木本性植物由来のリグノセルロース原料としては広葉樹、針葉樹の木部や樹皮を用いるのが好ましいが、枝、葉等の他の部位も用いることができる。草本性植物由来のリグノセルロース原料としては、ケナフ、麻、バガス、イネ等の茎、葉等の部位を特に限定なく用いることができる。本発明では、木本性植物の木部や樹皮の部位、あるいは草本性植物の茎、枝葉等の部位を微細化処理した微細化物を用いることができる。微細化処理後、パルプの形状にして用いることが好ましい。用いるパルプとしては、化学パルプ、機械パルプ、脱墨パルプ等、特に限定されないが、広葉樹由来の化学パルプが好ましい。化学パルプを得るための蒸解法としては、クラフト蒸解、ポリサルファイド蒸解、ソーダ蒸解、アルカリサルファイト蒸解等の公知の蒸解法が挙げられ、パルプの品質、パルプを得るためのエネルギー効率等を考慮した場合、クラフト蒸解法を用いることが好ましい。また、クラフト蒸解後、酸素漂白したパルプを用いることがより好ましい。クラフト蒸解後、酸素漂白した広葉樹由来のパルプでは、グルコース、アラビノース等の構成糖がほとんど含まれず、キシロースがほぼ１００％近く含まれるという特徴があるため、用いる原料としてより好ましい。 Hereinafter, although the structure of this invention is explained in full detail, this invention is not limited by this. As raw materials for obtaining the xylo-oligo and acidic xylo-oligosaccharides of the present invention, lignocellulose derived from woody plants or herbaceous plants can be used. As a lignocellulose raw material derived from a woody plant, it is preferable to use hardwood, coniferous wood or bark, but other parts such as branches and leaves can also be used. As a lignocellulose raw material derived from a herbaceous plant, parts such as stems and leaves of kenaf, hemp, bagasse and rice can be used without particular limitation. In the present invention, a refined product obtained by refining a part of a woody plant such as a xylem or bark, or a part of a herbaceous plant such as a stem or a branch, may be used. It is preferable to use in the shape of pulp after the refining treatment. The pulp to be used is not particularly limited, such as chemical pulp, mechanical pulp, deinked pulp, etc., but hardwood-derived chemical pulp is preferable. Examples of cooking methods for obtaining chemical pulp include known cooking methods such as kraft cooking, polysulfide cooking, soda cooking, alkali sulfite cooking, etc., considering the quality of pulp, energy efficiency to obtain pulp, etc. It is preferable to use the kraft cooking method. It is more preferable to use oxygen bleached pulp after kraft cooking. After kraft cooking, oxygen-bleached hardwood-derived pulp is more preferable as a raw material to be used because it contains almost no constituent sugars such as glucose and arabinose and contains almost 100% of xylose.

原料としてパルプを用いる場合、使用するパルプをパルプ製造工程よりキシロオリゴ及び酸性キシロオリゴ糖の糖製造工程へ移送する。移送の手段としては、低濃度のパルプであればポンプによるライン移送、高濃度のパルプであればベルトコンベア等の方法が挙げられ、移送方法は特に限定されない。 When using pulp as a raw material, the pulp to be used is transferred from the pulp production process to the sugar production process of xylooligosaccharide and acidic xylooligosaccharide. Examples of the transfer means include a line transfer by a pump for low-concentration pulp and a belt conveyor method for high-concentration pulp, and the transfer method is not particularly limited.

原料として用いるパルプは、例えば、タンク内で水に懸濁後、ドラムフィルターに移送しドラムフィルターで水で洗浄後、脱水する。この工程でパルプに含まれる微細な懸濁物、リグニンおよび有機酸等の不純物が除かれるため洗浄することが好ましい。パルプの洗浄方法としては、パルプを水で洗浄後、脱水できる装置であれば特に限定されない。また、この洗浄工程で水にｐＨ調整剤や温度調節した洗浄水を添加することにより、洗浄後のパルプのｐＨや温度を調節し、後段でのヘミセルラーゼの至適反応条件を調節することもできる。 Pulp used as a raw material is suspended in water in a tank, transferred to a drum filter, washed with water with a drum filter, and then dehydrated. In this step, impurities such as fine suspension, lignin and organic acid contained in the pulp are removed, so that washing is preferable. The method for washing the pulp is not particularly limited as long as it is an apparatus capable of dewatering after washing the pulp with water. In addition, the pH and temperature of the pulp after washing can be adjusted by adding a pH adjuster or temperature-adjusted washing water to the water in this washing step, and the optimum reaction conditions for hemicellulase in the latter stage can be adjusted. it can.

原料として用いるパルプは、前記の工程の後、ヘミセルラーゼ処理を行う反応装置へポンプで移送する。原料とヘミセルラーゼを混合する方法は、反応槽へ移送する前に混合しても良いし、反応槽内で混合しても良い。高濃度のパルプをヘミセルラーゼで処理する場合、反応槽の形状としては、円筒型が連続的に移送できるため好ましいが、酵素反応が行えるものであれば特に限定されない。高濃度のパルプをヘミセルラーゼで処理する場合、スクリューミキサー等（例えば、トランポスクリュー等）の混合機を用いて反応槽へ移送される前にパルプとキシラナーゼを均一に混和させておくことが好ましい。用いる混合機としては、パルプとヘミセルラーゼを均一に混合できる装置であれば特に限定なく用いることができる。一方、低濃度のパルプをヘミセルラーゼで処理する場合、反応槽の形状は特に限定されないが、例えば、攪拌機を備えた反応槽内でパルプ懸濁液を攪拌しながらオリゴ糖の溶出に最適な滞留時間でヘミセルラーゼ処理を行い、パルプ懸濁液（オリゴ糖を含む）を連続的に回収する方法が挙げられる。これらの反応槽は、ヘミセルラーゼの至適反応温度を維持する為に温度調節用のジャケットや断熱材ケーシングを具備することが好ましい。 The pulp used as a raw material is pumped to the reaction apparatus that performs the hemicellulase treatment after the above-described steps. The method of mixing the raw material and hemicellulase may be mixed before being transferred to the reaction vessel, or may be mixed in the reaction vessel. When treating high-concentration pulp with hemicellulase, the shape of the reaction tank is preferably a cylindrical shape because it can be continuously transferred, but is not particularly limited as long as an enzyme reaction can be performed. When treating high-concentration pulp with hemicellulase, it is preferable that the pulp and xylanase are mixed uniformly before being transferred to the reaction vessel using a mixer such as a screw mixer (eg, tramposcrew). As the mixer to be used, any apparatus can be used without particular limitation as long as it can uniformly mix pulp and hemicellulase. On the other hand, when treating low-concentration pulp with hemicellulase, the shape of the reaction vessel is not particularly limited. For example, the optimum retention for elution of oligosaccharides while stirring the pulp suspension in a reaction vessel equipped with a stirrer A method of performing a hemicellulase treatment over time and continuously recovering a pulp suspension (including oligosaccharides) can be mentioned. These reaction tanks are preferably provided with a temperature adjusting jacket and a heat insulating material casing in order to maintain the optimum reaction temperature of hemicellulase.

本発明で用いるヘミセルラーゼとしては、キシラナーゼ活性を含むものであれば特に限定されない。ヘミセルラーゼとしては、例えば、商品名カルタザイム（クラリアント社製）、パルプザイム（ノボノルディスク社製）、エコパルプ（ローム・エンザイム社製）、スミチーム（新日本化学工業社製）、マルチフェクトキシラナーゼ（ジェネンコア社製）、キシラナーゼコンク（アドバンスド・バイオケミカルス社製）等の市販の酵素製剤や、トリコデルマ（Ｔｒｉｃｈｏｄｅｒｍａ）属、テルモミセス（Ｔｈｅｒｍｏｍｙｃｅｓ）属、オウレオバシヂウム（Ａｕｒｅｏｂａｓｉｄｉｕｍ）属、ストレプトミセス（Ｓｔｒｅｐｔｏｍｙｃｅｓ）属、アスペルギルス（Ａｓｐｅｒｇｉｌｌｕｓ）属、クロストリジウム（Ｃｌｏｓｔｒｉｄｉｕｍ）属、バチルス（Ｂａｃｉｌｌｕｓ）属、テルモトガ（Ｔｈｅｒｍｏｔｏｇａ）属、テルモアスクス（Ｔｈｅｒｍｏａｓｃｕｓ）属、カルドセラム（Ｃａｌｄｏｃｅｌｌｕｍ）属、テルモモノスポラ（Ｔｈｅｒｍｏｍｏｎｏｓｐｏｒａ）属等の微生物により生産されるキシラナーゼが挙げられる。 The hemicellulase used in the present invention is not particularly limited as long as it contains xylanase activity. Examples of hemicellulases include, but are not limited to, the brand names Cartazame (Clariant), Pulpzyme (Novo Nordisk), Eco Pulp (Rohm Enzyme), Sumiteam (New Nippon Chemical Industry), Multifect Xylanase (Genencor) ), Xylanase conch (manufactured by Advanced Biochemicals), and other commercially available enzyme preparations, Trichoderma genus, Thermomyces genus, Aureobasidium genus, Streptomyces genus Aspergillus genus, Clostridium genus, Bacillus genus, Thermotoga genus, Thermoskus Thermoascus) genus Karudoseramu (Caldocellum) genus include xylanases produced by microorganisms such as genus Terumo mono Supora (Thermomonospora).

パルプのヘミセルラーゼ処理工程では、パルプに対するヘミセルラーゼの添加量、反応時間を調節することにより、パルプから溶出されるオリゴ糖の濃度やオリゴ糖の重合度を調節することができる。一般に、ヘミセルラーゼの添加量が多いほど、また反応時間が長いほど反応液中のオリゴ糖濃度は高くなり、且つオリゴ糖の重合度は小さくなる。従って、高重合度のオリゴ糖を含む高濃度の糖液を安定して得るためには、パルプに対して適量のヘミセルラーゼ（低分子まで分解されない程度のヘミセルラーゼ）を添加し、反応後の糖液（パルプを除いたオリゴ糖を含むろ液）の一部を酵素反応槽に戻し再度、酵素反応させることが好ましい。これによりオリゴ糖の重合度を高い状態に維持しながら、時間経過と共に高重合度のオリゴ糖の糖濃度を高めることができる。パルプに対して過剰のヘミセルラーゼを添加したり、反応時間を長くすると、溶出したオリゴ糖が更にヘミセルラーゼで分解され、キシロース、キシロビオースのような鎖長の短いオリゴ糖にまで分解されるため好ましくない。 In the hemicellulase treatment step of pulp, the concentration of oligosaccharide eluted from the pulp and the degree of polymerization of oligosaccharide can be adjusted by adjusting the amount of hemicellulase added to the pulp and the reaction time. In general, the greater the amount of hemicellulase added and the longer the reaction time, the higher the oligosaccharide concentration in the reaction solution and the lower the degree of oligosaccharide polymerization. Therefore, in order to stably obtain a high-concentration sugar solution containing an oligosaccharide having a high degree of polymerization, an appropriate amount of hemicellulase (a hemicellulase that does not decompose to low molecules) is added to the pulp, It is preferable to return a part of the sugar solution (the filtrate containing the oligosaccharide excluding the pulp) to the enzyme reaction tank and react again with the enzyme. As a result, the sugar concentration of the oligosaccharide having a high degree of polymerization can be increased over time while maintaining the degree of polymerization of the oligosaccharide at a high level. If excessive hemicellulase is added to the pulp or the reaction time is lengthened, the eluted oligosaccharide is further decomposed by hemicellulase and is further decomposed to oligosaccharides with a short chain length such as xylose and xylobiose. Absent.

反応槽内でのパルプ濃度、反応時間、キシラナーゼ添加量は任意に設定可能であるが、パルプ濃度は２〜２０質量％が好ましく、７〜１５質量％がさらに好ましい。パルプ濃度が低すぎるとオリゴ糖の濃度を高めることが困難となり、また、処理液の容量が増大するため、反応設備の大型化が必要となるという問題が生じる。一方、パルプ濃度が高すぎるとヘミセルラーゼとパルプの均一な混合やパルプの移送が困難となり、また、糖液の回収量が減少するため好ましくない。パルプに対する適したヘミセルラーゼ添加量及び反応時間は、用いる酵素の種類によって異なる。例えば、マルチフェクトキシラナーゼの場合、反応時間は１０〜２４０分が好ましく、３０〜９０分がより好ましい。また、パルプに対するヘミセルラーゼの添加量は２〜２００（unit/g−乾燥パルプ）が好ましく、１０〜１００（unit/g−乾燥パルプ）がより好ましい。 The pulp concentration, reaction time, and xylanase addition amount in the reaction tank can be arbitrarily set, but the pulp concentration is preferably 2 to 20% by mass, and more preferably 7 to 15% by mass. If the pulp concentration is too low, it is difficult to increase the concentration of the oligosaccharide, and the capacity of the treatment liquid increases, so that there is a problem that the reaction equipment needs to be enlarged. On the other hand, if the pulp concentration is too high, uniform mixing of hemicellulase and pulp and transfer of the pulp become difficult, and the amount of recovered sugar solution decreases, which is not preferable. The appropriate hemicellulase addition amount and reaction time for the pulp vary depending on the type of enzyme used. For example, in the case of multifect xylanase, the reaction time is preferably 10 to 240 minutes, more preferably 30 to 90 minutes. The amount of hemicellulase added to the pulp is preferably 2 to 200 (unit / g-dried pulp), more preferably 10 to 100 (unit / g-dried pulp).

本発明により、パルプをヘミセルラーゼ処理した反応液中に溶出されるオリゴ糖の重合度は、用いる酵素の種類や反応条件によって変動するが、例えば、マルチフェクトキシラナーゼを用いる場合、パルプ濃度１０質量％、反応時間４５分、反応温度５０℃、ｐH６．０、パルプに対する酵素添加量５０（unit/g−乾燥パルプ）の条件では、１〜１５量体の分布を持つ平均重合度が５量体程度のキシロオリゴ糖、および１〜２０量体の分布を持つ平均重合度１０量体程度の酸性キシロオリゴが糖液中に溶出される。 According to the present invention, the degree of polymerization of the oligosaccharide eluted in the reaction solution obtained by treating the pulp with hemicellulase varies depending on the type of enzyme used and the reaction conditions. For example, when multifect xylanase is used, the pulp concentration is 10% by mass. Under the conditions of a reaction time of 45 minutes, a reaction temperature of 50 ° C., a pH of 6.0, and an enzyme addition amount of 50 (unit / g-dried pulp), the average degree of polymerization having a distribution of 1 to 15 mer is about 5 mer. Xylo-oligosaccharides and acidic xylo-oligo having a distribution of 1 to 20-mers and an average degree of polymerization of about 10-mers are eluted in the sugar solution.

ヘミセルラーゼで処理したパルプに含まれるオリゴ糖を回収するための手段は特に限定されないが、例えば、スクリュープレス等の装置を使用してパルプを圧搾してろ液（オリゴ糖含有）を回収しても良いし、ドラムフィルター等の装置を使用してパルプを水で置換洗浄し、オリゴ糖を含有するろ液を回収しても良い。このようにして得られたオリゴ糖を含有するろ液は次の濃縮工程へ移送される。また、前述したようにろ液の一部を酵素反応槽に戻し、再度、ヘミセルラーゼで反応させても良い。 The means for recovering the oligosaccharide contained in the pulp treated with hemicellulase is not particularly limited. For example, even if the pulp is squeezed using an apparatus such as a screw press, the filtrate (containing oligosaccharide) is recovered. Alternatively, the pulp may be replaced with water using an apparatus such as a drum filter and the filtrate containing the oligosaccharide may be recovered. The filtrate containing the oligosaccharide thus obtained is transferred to the next concentration step. Further, as described above, a part of the filtrate may be returned to the enzyme reaction tank and reacted again with hemicellulase.

パルプをヘミセルラーゼで反応させた後のろ液は、次の濃縮工程へ移送する前に前処理により原料由来の残渣を除くことが好ましい。前処理としては、バッグフィルター、フィルタープレス、プレコートフィルター、セラミックフィルター等の精密ろ過処理や凝集沈殿処理が挙げられ、これらの処理を併用しても良い。精密ろ過処理を行う場合は、サブミクロンオーダーの濾過精度を持つフィルターが好ましい。凝集沈殿処理を行う場合は、硫酸バンド、ポリ塩化アルミニウム等の無機系凝集剤や、ポリアクリルアミド系、ポリアミジン系等の高分子凝集剤あるいはキトサン等の天然高分子凝集剤を用いることができる。前処理の方法は、パルプ、懸濁物質等の微細な不溶成分を除去することができる方法であれば、前記の方法に限定されない。 The filtrate after reacting the pulp with hemicellulase preferably removes the residue derived from the raw material by pretreatment before transferring to the next concentration step. Examples of the pretreatment include microfiltration treatment such as bag filter, filter press, precoat filter, ceramic filter, and coagulation precipitation treatment, and these treatments may be used in combination. When performing microfiltration, a filter having submicron order filtration accuracy is preferable. When carrying out the coagulation precipitation treatment, an inorganic coagulant such as a sulfate band or polyaluminum chloride, a polymer coagulant such as polyacrylamide or polyamidine, or a natural polymer coagulant such as chitosan can be used. The pretreatment method is not limited to the above method as long as it can remove fine insoluble components such as pulp and suspended substances.

本発明では、パルプをヘミセルラーゼで反応させた後のろ液中に含まれるオリゴ糖溶液は、後段での精製負荷を低減するために、濃縮により糖液中の濃度を高濃度にすることが望ましい。糖液を濃縮するために逆浸透膜を用いる。逆浸透膜を用いる方法では、キシロース、キシロビオース等の低分子（重合度の小さい糖）や反応後の糖液に含まれる低分子物質（例えば、炭酸ナトリウム、チオ硫酸ナトリウム等の無機物、有機酸、等）が透過液として除去され、高分子（重合度の大きいキシロオリゴ糖）のみが選択的に濃縮されるという利点がある。また、膜濃縮では運転コストが溶媒抽出による方法と比較し安価であり、また、危険性のある溶媒を使用する必要がないため好ましい。
しかし、逆浸透膜の膜エレメントを連続的に運転する場合、オリゴ糖の製造工程で使用する原水中に存在するコロイド性物質や微細な懸濁物質、特に酵素反応後の糖液に不純物として含まれるリグニン等が膜の表面上に付着し、蓄積されて運転時間の経過と共に膜のろ過比抵抗が増大して透過流束が低下するという問題が生じる。特にリグニン等の高分子は透過液として膜外に排出されないためオリゴ糖と共に濃縮されて、結果として時間経過と共に膜の表面に付着し膜の操作圧力を上昇させる。操作圧力が限界値まで上昇すると、連続運転が困難となり、オリゴ糖の生産効率が低下（運転コスト増加、製造時間延長）するため好ましくない。また、精製に供する糖液の糖濃度は、できるだけ高濃度にした方が後段の製造コストが低減できるというメリットがある。しかし、運転開始後、短時間でリグニン等の不純物が膜に付着すると糖濃度が充分に高濃度になる前に膜の操作圧力が限界値に達し糖液の濃度をそれ以上に高濃度にすることができなくなる。従って、膜エレメントの性能劣化を最小限にし、長時間にわたり安定して逆浸透膜装置を運転することが課題であった。この問題を解決するために検討した結果、後述する限外濾過膜で処理した後のろ液をループにより連続的に逆浸透膜に戻すことにより糖濃縮液の糖濃度を高めることが可能となった。 In the present invention, the oligosaccharide solution contained in the filtrate after reacting the pulp with hemicellulase may be concentrated at a high concentration in the sugar solution to reduce the purification load in the subsequent stage. desirable. A reverse osmosis membrane is used to concentrate the sugar solution. In the method using a reverse osmosis membrane, low molecular weight substances such as xylose and xylobiose (sugars with a low degree of polymerization) and low molecular weight substances contained in the sugar solution after the reaction (for example, inorganic substances such as sodium carbonate and sodium thiosulfate, organic acids, Etc.) is removed as a permeate, and only the polymer (xylooligosaccharide having a high degree of polymerization) is selectively concentrated. Membrane concentration is preferable because the operation cost is low compared with the solvent extraction method and there is no need to use a dangerous solvent.
However, when the membrane element of the reverse osmosis membrane is operated continuously, it is included as an impurity in colloidal substances and fine suspended substances present in the raw water used in the oligosaccharide production process, especially in the sugar solution after the enzyme reaction. The lignin or the like deposited on the surface of the membrane accumulates and the filtration specific resistance of the membrane increases with the passage of operating time, resulting in a problem that the permeation flux decreases. In particular, a polymer such as lignin is not discharged out of the membrane as a permeate, and therefore is concentrated together with the oligosaccharide. As a result, it adheres to the surface of the membrane with the passage of time and increases the operating pressure of the membrane. If the operating pressure rises to the limit value, continuous operation becomes difficult, and the oligosaccharide production efficiency decreases (increasing operating costs and extending manufacturing time), which is not preferable. In addition, the sugar concentration of the sugar solution used for purification has an advantage that the production cost in the subsequent stage can be reduced if the concentration is as high as possible. However, if impurities such as lignin adhere to the membrane in a short time after the start of operation, the membrane operating pressure reaches the limit value before the sugar concentration becomes sufficiently high, and the concentration of the sugar solution is increased to a higher level. I can't do that. Therefore, it has been a problem to operate the reverse osmosis membrane device stably for a long time while minimizing the performance deterioration of the membrane element. As a result of studying to solve this problem, it is possible to increase the sugar concentration of the sugar concentrate by continuously returning the filtrate after being treated with an ultrafiltration membrane described later to the reverse osmosis membrane through a loop. It was.

本発明では、逆浸透膜で濃縮した糖液を、次に、限外濾過膜により着色物質や不純物を除去する。逆浸透膜で濃縮中の糖液は、糖液の一部を連続的に限外濾過膜に移送しても良いし、糖濃度が一定の値に達してから限外濾過装置を停止し、濃縮した糖液の全量を限外濾過膜に移送しても良い。
従来は、後述する酸処理の後に限外濾過膜による処理を実施していた。しかし、酸処理前の糖濃縮液には原料や酵素由来の不純物（リグニン、酵素由来のタンパク質等）が混合しており、加熱（酸処理）により、糖液の着色が増大するという問題が生じた。糖液を加熱すると、例えば、アミノ酸や蛋白質の共存下で糖を加熱することによるメイラード反応による糖の褐変化、カラメル物質の増加等、加熱により糖液中に新たな着色物質が増加する。この問題を解決するために検討した結果、後述の酸処理の前に糖濃縮液を限外ろ過膜で処理することにより、糖濃縮液に含まれる不純物をあらかじめ除去することができ、結果として酸処理工程で発生する着色物質の生成を低減することが可能となった。
また、限外濾過膜による処理では、糖濃縮液に元々含まれる原料由来のリグニンや着色物質等の高分子の不純物を除去することもできる。
用いる限外濾過膜の分画分子量は、５０００〜３００００が好ましい。分画分子量が５０００より小さいとオリゴ糖の回収率が低下し、膜への抵抗が増加しオリゴ糖の生産効率が低下するため好ましくない。また、分画分子量が３００００より大きいと着色物質や不純物の除去率が低下するため好ましくない。 In the present invention, the sugar solution concentrated by the reverse osmosis membrane is then subjected to removal of colored substances and impurities by the ultrafiltration membrane. The sugar solution being concentrated in the reverse osmosis membrane may be transferred continuously to the ultrafiltration membrane, or after the sugar concentration reaches a certain value, the ultrafiltration device is stopped, The total amount of the concentrated sugar solution may be transferred to an ultrafiltration membrane.
Conventionally, the treatment with an ultrafiltration membrane has been performed after the acid treatment described later. However, the sugar concentrate before acid treatment contains raw materials and enzyme-derived impurities (lignin, enzyme-derived protein, etc.), and heating (acid treatment) increases the color of the sugar solution. It was. When the sugar liquid is heated, for example, a brown color of the sugar due to the Maillard reaction by heating the sugar in the coexistence of amino acids and proteins, an increase in the caramel substance, and the like, new colored substances increase in the sugar liquid by heating. As a result of studying to solve this problem, the impurities contained in the sugar concentrate can be removed in advance by treating the sugar concentrate with an ultrafiltration membrane before the acid treatment described below. It has become possible to reduce the generation of colored substances generated in the treatment process.
Further, in the treatment with the ultrafiltration membrane, it is also possible to remove high molecular impurities such as lignin derived from raw materials and coloring substances originally contained in the sugar concentrate.
The molecular weight cutoff of the ultrafiltration membrane to be used is preferably 5000 to 30000. If the molecular weight cut off is less than 5000, the oligosaccharide recovery rate is lowered, the resistance to the membrane is increased, and the production efficiency of the oligosaccharide is lowered, which is not preferable. Further, if the molecular weight cut off is larger than 30000, the removal rate of the colored substances and impurities is lowered, which is not preferable.

本発明では、前記の限外濾過膜で処理した後のろ液をループにより連続的に逆浸透膜に戻す（図３参照）。逆浸透膜へ戻す限外濾過膜処理後のろ液の液量はろ液の全量でも良いし、一部でもよい。ろ液の全量を戻す場合は、逆浸透膜内の糖濃度が一定の値に達してから限外濾過装置を停止し、次の酸処理工程へ移送する。一方、ろ液の一部を戻す場合は、逆浸透膜へ戻さない残りのろ液を逆浸透膜から連続的に酸処理工程へ移送する。
前述の逆浸透膜で糖液が濃縮される時にオリゴ糖と共に不純物として含まれるリグニン等の高分子物質も濃度が上昇するが、限外濾過膜で処理した後のろ液をループにより連続的に逆浸透膜に戻すことにより、リグニン等の不純物のみを濃縮中の糖液から除去できる。その結果として、逆浸透膜の操作圧力の上昇を抑制することができ、オリゴ糖の濃度をより高めることができる。 In this invention, the filtrate after processing with the said ultrafiltration membrane is continuously returned to a reverse osmosis membrane by a loop (refer FIG. 3). The amount of the filtrate after the ultrafiltration membrane treatment to be returned to the reverse osmosis membrane may be the whole amount of the filtrate or a part thereof. When returning the total amount of the filtrate, the ultrafiltration apparatus is stopped after the sugar concentration in the reverse osmosis membrane reaches a certain value, and transferred to the next acid treatment step. On the other hand, when returning a part of filtrate, the remaining filtrate which does not return to a reverse osmosis membrane is continuously transferred to an acid treatment process from a reverse osmosis membrane.
When the sugar solution is concentrated in the above reverse osmosis membrane, the concentration of polymer substances such as lignin contained as impurities along with the oligosaccharide also increases, but the filtrate after treatment with the ultrafiltration membrane is continuously passed through the loop. By returning to the reverse osmosis membrane, only impurities such as lignin can be removed from the sugar solution being concentrated. As a result, an increase in the operating pressure of the reverse osmosis membrane can be suppressed, and the concentration of oligosaccharide can be further increased.

前記の限外濾過膜で処理した糖液（図１参照）、あるいは、逆浸透膜で濃縮した糖液（図３参照）は、次に酸処理を行う。糖液中に含まれるキシロオリゴ糖及び酸性キシロオリゴ糖の一部はリグニンと結合して複合体（リグニン−キシロオリゴ糖複合体、リグニン−酸性キシロオリゴ糖複合体）として存在する。酸処理により、複合体からキシロオリゴ糖および酸性キシロオリゴ糖を遊離させることができる。酸処理の方法としては、例えば、糖液に酸を添加してｐＨを５以下に調整し、高温で加熱する方法が挙げられる。ｐＨ調整に用いる酸は、特に限定されないが、例えば、硫酸、塩酸等の鉱酸の他、シュウ酸、酢酸等の有機酸が挙げられる。酸処理のｐＨは２〜５が好ましく、さらにｐＨ３．５〜４．０の範囲がより好ましい。ｐＨが２より低いと糖液に含まれるキシロオリゴ糖及び酸性キシロオリゴ糖からキシロースへの加水分解が促進され、オリゴ糖の回収率が低下するため好ましくない。またｐＨが５より高いと１５０℃以下の温度では複合体からオリゴ糖の遊離が抑制されるため好ましくない。酸処理の温度は、特に限定されないが、１００〜２００℃が好ましく、１０５〜１７０℃がより好ましく、１１０〜１２５℃がさらに好ましい。１００℃より低い温度では、複合体からのオリゴ糖の遊離が抑制されるため好ましくない。また、１７０℃を越えると、キシロオリゴ糖および酸性キシロオリゴ糖からキシロースへの分解が促進されオリゴ糖の収率が低下するため好ましくない。また、酸処理中の圧力は大気圧〜５kg/cm²の範囲であることが好ましい。 The sugar solution treated with the ultrafiltration membrane (see FIG. 1) or the sugar solution concentrated with a reverse osmosis membrane (see FIG. 3) is then subjected to acid treatment. A part of the xylo-oligosaccharide and acidic xylo-oligosaccharide contained in the sugar solution is combined with lignin and exists as a complex (lignin-xylo-oligosaccharide complex, lignin-acidic xylo-oligosaccharide complex). By the acid treatment, xylo-oligosaccharide and acidic xylo-oligosaccharide can be released from the complex. Examples of the acid treatment method include a method of adjusting the pH to 5 or less by adding an acid to a sugar solution and heating at a high temperature. Although the acid used for pH adjustment is not specifically limited, For example, organic acids, such as oxalic acid and an acetic acid other than mineral acids, such as a sulfuric acid and hydrochloric acid, are mentioned. The pH of the acid treatment is preferably 2 to 5, and more preferably in the range of pH 3.5 to 4.0. A pH lower than 2 is not preferable because hydrolysis of xylo-oligosaccharides and acidic xylo-oligosaccharides contained in the sugar solution into xylose is promoted and the oligosaccharide recovery rate is lowered. On the other hand, if the pH is higher than 5, it is not preferable at a temperature of 150 ° C. or lower because oligosaccharide release from the complex is suppressed. Although the temperature of acid treatment is not specifically limited, 100-200 degreeC is preferable, 105-170 degreeC is more preferable, and 110-125 degreeC is further more preferable. A temperature lower than 100 ° C. is not preferable because release of the oligosaccharide from the complex is suppressed. Moreover, when it exceeds 170 degreeC, since decomposition | disassembly to xylose from a xylooligosaccharide and acidic xylooligosaccharide is accelerated | stimulated, the yield of an oligosaccharide falls and it is not preferable. The pressure during the acid treatment is preferably in the range of atmospheric pressure to 5 kg / cm ² .

酸処理後の糖液には、酸処理で生成した着色物質、前記の限外濾過膜で除去されなかった原料由来のリグニン、着色物質等が含まれており、その一部は不溶性残渣として存在している。これらの不溶性残渣を遠心分離、セラミックフィルター等によるフィルターろ過、ろ布等によるろ過で除去することが好ましい。 The sugar solution after acid treatment contains colored substances generated by acid treatment, lignin derived from raw materials not removed by the ultrafiltration membrane, colored substances, etc., some of which exist as insoluble residues is doing. These insoluble residues are preferably removed by centrifugation, filter filtration with a ceramic filter, or filtration with a filter cloth.

酸処理後の糖液中に含まれる着色物質等の不純物の含量をさらに低減するために、活性炭処理を行うことが好ましい。用いる活性炭の種類としては、糖液中の着色物質等の不純物の含量を低減する能力を有するものであれば特に限定されない。 In order to further reduce the content of impurities such as coloring substances contained in the sugar solution after the acid treatment, it is preferable to perform activated carbon treatment. The type of activated carbon used is not particularly limited as long as it has an ability to reduce the content of impurities such as coloring substances in the sugar solution.

前記の操作で着色物質等の不純物を低減した糖液に含まれるキシロオリゴ糖と酸性キシロオリゴ糖をイオン交換樹脂を用いて分離・精製する。分離・精製の方法として、例えば、オリゴ糖を含む糖濃縮液を強カチオン樹脂→弱アニオン樹脂→強カチオン樹脂→弱アニオン樹脂の順に通液する方法を用いることができる。この方法では、酸性キシロオリゴ糖がアニオン樹脂に吸着されるため、キシロオリゴ糖のみをろ液として回収することができる。次に、アニオン樹脂に、例えば、塩化ナトリウム等の塩を通液することにより酸性キシロオリゴ糖を樹脂から溶出させて回収することができる。回収したキシロオリゴ糖及び酸性キシロオリゴ糖を含む溶液を、例えば、エバポレーション等の濃縮装置で濃縮することができる。オリゴ糖を含む溶液をスプレードライで乾燥することにより、キシロオリゴ糖及び酸性キシロオリゴ糖の粉末が得られる。 The xylo-oligosaccharide and the acidic xylo-oligosaccharide contained in the sugar solution in which impurities such as coloring substances are reduced by the above operation are separated and purified using an ion exchange resin. As a method for separation / purification, for example, a method of passing a sugar concentrate containing oligosaccharides in the order of strong cation resin → weak anion resin → strong cation resin → weak anion resin can be used. In this method, since the acidic xylo-oligosaccharide is adsorbed to the anion resin, only the xylo-oligosaccharide can be recovered as a filtrate. Next, acidic xylo-oligosaccharides can be eluted from the resin and recovered by passing a salt such as sodium chloride through the anion resin. The solution containing the recovered xylo-oligosaccharide and acidic xylo-oligosaccharide can be concentrated with a concentration device such as evaporation. By drying the solution containing the oligosaccharide by spray drying, powder of xylo-oligosaccharide and acidic xylo-oligosaccharide can be obtained.

以下に、本発明を実施例により詳細に説明するが、本発明は以下の実施例に限定されるものではない。以下に示す％は、特に断らない限りすべて質量％を示し、対パルプの添加率はパルプの絶乾質量に対する質量の比率を示す。なお、各測定法は以下のとおりである。
〈測定法の概要〉
全糖量の定量
全糖量は検量線をＤ−キシロース（和光純薬工業製）を用いて作製し、フェノール硫酸法（還元糖の定量法；学会出版センター）で定量した。
（２）還元糖量の定量
還元糖量は検量線をＤ−キシロース（和光純薬工業製）を用いて作製し、ソモジ−ネルソン法（還元糖の定量法；学会出版センター）で定量した。
（３）平均重合度の決定法
サンプル糖液を５０℃に保ち１５０００rpmで１５分間遠心分離し不溶物を除去後、上清液の全糖量を還元糖量（共にキシロース換算）を測定した。全糖量を還元糖量で割って平均重合度を求めた。
（４）酵素力価の定義
酵素として用いたキシラナーゼの活性測定にはバーチウッドキシラン（シグマ社製）を基質として用いた。酵素力価の定義はキシラナーゼがキシランを分解することで得られる還元糖の還元力をＤＮＳ法（還元糖の定量法；学会出版センター）を用いて測定し、１分間に１マイクロモルのキシロースに相当する還元力を生成させる酵素量を１unitとした。 EXAMPLES Hereinafter, the present invention will be described in detail with reference to examples, but the present invention is not limited to the following examples. Unless otherwise indicated,% shown below shows all mass%, and the addition rate of a pulp shows the ratio of the mass with respect to the absolute dry mass of a pulp. In addition, each measuring method is as follows.
<Outline of measurement method>
Quantification of total sugar amount The total sugar amount was determined by preparing a calibration curve using D-xylose (manufactured by Wako Pure Chemical Industries, Ltd.) and the phenol sulfate method (quantitative method for reducing sugar; Academic Publishing Center).
(2) Quantification of the amount of reducing sugar The amount of reducing sugar was determined by preparing a calibration curve using D-xylose (manufactured by Wako Pure Chemical Industries, Ltd.) and using the Sommoji-Nelson method (quantitative method for reducing sugar; Society of Science Publishing Center).
(3) Determination of average degree of polymerization The sample sugar solution was kept at 50 ° C. and centrifuged at 15000 rpm for 15 minutes to remove insoluble matters, and then the total sugar amount in the supernatant was measured for reducing sugar amount (both converted to xylose). The average degree of polymerization was determined by dividing the total sugar amount by the reducing sugar amount.
(4) Definition of enzyme titer Birchwood xylan (manufactured by Sigma) was used as a substrate for measuring the activity of xylanase used as an enzyme. Enzyme titer is defined by measuring the reducing power of reducing sugar obtained by decomposing xylan by xylanase using the DNS method (quantitative method for reducing sugar; Academic Publishing Center) to 1 micromole of xylose per minute. The amount of enzyme that generates the corresponding reducing power was 1 unit.

＜実施例１＞
［オリゴ糖濃縮液の製造］
混合広葉樹チップ（国内産広葉樹チップ２０％、ユーカリ材８０％）をクラフト蒸解後、酸素脱リグニンしパルプ(原料)を得た。以降の操作は、連続的に行った。タンクにパルプ及び高温水（６０℃）を添加し、パルプ濃度（最終濃度）を１．６質量％に調製した（容量１０m³）。次に、タンクに濃硫酸を添加、攪拌してｐＨ５．５に調整後、ドラムフィルター（新菱製作所製：φ２０００×６００ SUF）でパルプを脱水洗浄した。脱水後のパルプ（パルプ濃度２０質量％）に温水（５０℃）を添加し、パルプ濃度を１０質量％に調製後、マルチフェクトキシラナーゼ（ジェネンコア社製）を対パルプ５０unit（乾燥パルプ１ｇ当たり）となるように添加して、トランポスクリュー（新菱製作所：220x850 HC-C）で均一に混合した。混合後のパルプを中濃度ポンプ（新菱製作所：２００×RPK）で容積２m³の円筒型反応槽（φ８００mm×４０００mmH）の入口から押し込み、円筒型反応槽内で連続的に酵素反応を行った（反応時間４０分間、温度５０℃）。円筒型反応槽内の出口から排出されたパルプ（酵素反応後のパルプ）をスクリュープレス（新菱製作所：２５０×１０００ SPH−EN）でパルプ濃度が４０質量％になるまで脱水して、キシラナーゼ反応後のろ液を得た。酵素反応開始から６０分以降は、キシラナーゼ反応後のろ液の一部をトランポスクリューに戻し、ドラムフィルターで脱水した２０質量％濃度のパルプを温水（５０℃）の替わりに戻したキシラナーゼ反応後のろ液でパルプ濃度を１０質量％に調整した。酵素反応開始から１５０分後、反応後のろ液中の全糖濃度は０．９２％に上昇し、この時点での糖液に含まれるキシロオリゴ糖の平均重合度は４．７であった。以上のパルプのキシラナーゼ処理を２４時間連続して実施した。酵素反応開始から１５０分以降は、糖濃度約０．９０％の反応ろ液を安定して回収すことができた。最終的に糖濃度０．９０％のキシラナーゼ反応後のろ液を１５ｍ^３得た。得られた反応ろ液を、ミクロンレート１μmのバックフィルター（ＩＳＰフィルターズ製）、次にミクロンレート０．２μmのセラミックフィルター（日本ポール製）でろ過して、清澄なろ液（糖液）を得た。このろ液を逆浸透膜（日東電工製：ＮＴＲ−７４５０、膜質：スルホン化ポリエーテルスルホン系、膜面積：６．５m²−２本）で液温５０℃、流量１４００〜１８００L/hの運転条件で１５倍に濃縮し、全糖濃度１１％の濃縮液を１m³得た。
［オリゴ糖の精製］
図１に示す製造工程で、前記（［オリゴ糖濃縮液の製造］）で得られた濃縮糖液５００Ｌを分画分子量１００００の限外濾過膜（限外濾過装置：オスモニクス製ＵＦ膜モジュール）を用いて、透過流速３５０Ｌ/hで処理した。限外濾過膜を通過したろ液（糖を含む画分）を濃硫酸でｐＨ３．５に調整後、１２１℃で４５分間処理（酸処理）した。ろ液を５０℃まで冷却後、酸処理後で生成した不溶性残渣をミクロンレート０．２μmのセラミックフィルター（日本ポール製）で除去した。この糖液を一部採取して全糖濃度を０．２％に調製（蒸留水で希釈）し、３５０nmの波長における吸光度（着色度）を測定した（表１）。次に、ろ液４８０Lに対して活性炭（三倉化成製：ＰＭ−ＳＸ）１０ｋｇを添加し、５０℃で２時間処理後、糖液を回収した。この糖液を、ＳＶ１．５で強カチオン樹脂（三菱化学製：PK−２１８、３００L）→弱アニオン樹脂（三菱化学製：ＷＡ３０、３００L）→強カチオン樹脂（三菱化学製：ＰＫ−２１８、３００L）→弱アニオン樹脂（三菱化学製：ＷＡ３０、３００L）からなる４床４塔式イオン交換樹脂に通液し、キシロオリゴ糖糖液（糖濃度４．３質量％、５５０L）を得た。得られたキシロオリゴ糖溶液を強カチオン樹脂でｐＨ７に調整後、スプレードライヤー（大川原化工機製：ＯＤＡ−２５型）で処理して、キシロオリゴ糖粉末２３．０ｋｇ（平均重合度５．０）を得た。次に、２塔目及び４塔目の弱アニオン樹脂に５０mM塩化ナトリウム水溶液をＳＶ１．５で通液し、イオン交換樹脂に吸着している酸性キシロオリゴ糖を溶出させ、酸性キシロオリゴ糖溶液（糖濃度２．８質量％、４００Ｌ）を得た。得られた酸性キシロオリゴ糖溶液を、強カチオン樹脂でｐＨ５に調整後、糖濃度２０質量％までエバポール（大川原化工機：CEP-1）で濃縮した。次に、スプレードライヤー（大川原化工機製：ODA-25型）で処理して、酸性キシロオリゴ糖粉末１１．３ｋｇ（平均重合度１１．０）を得た。得られたキシロオリゴ糖及び酸性キシロオリゴ糖を蒸留水で全糖濃度５質量％に調整し、３５０nmの波長における吸光度（着色度）を測定した（表２）。 <Example 1>
[Manufacture of oligosaccharide concentrate]
Mixed hardwood chips (domestic hardwood chips 20%, eucalyptus wood 80%) were kraft cooked and oxygen delignified to obtain pulp (raw material). Subsequent operations were performed continuously. Pulp and hot water (60 ° C.) were added to the tank, and the pulp concentration (final concentration) was adjusted to 1.6 mass% (capacity 10 m ³ ). Next, concentrated sulfuric acid was added to the tank and stirred to adjust the pH to 5.5, and then the pulp was dehydrated and washed with a drum filter (manufactured by Shinryo Seisakusho: φ2000 × 600 SUF). Warm water (50 ° C.) is added to the dehydrated pulp (pulp concentration of 20% by mass), and the pulp concentration is adjusted to 10% by mass. Then, multifect xylanase (Genencor) is added to 50 units (per 1 g of dried pulp) It added so that it might become, and it mixed uniformly with the tramposcrew (Shinryo Seisakusho: 220x850 HC-C). The mixed pulp was pushed from the inlet of a cylindrical reaction tank (φ800 mm × 4000 mmH) having a volume of 2 m ³ with a medium concentration pump (Shinryo Seisakusho: 200 × RPK), and the enzyme reaction was continuously carried out in the cylindrical reaction tank. (Reaction time 40 minutes, temperature 50 ° C.). The pulp discharged from the outlet in the cylindrical reaction tank (the pulp after the enzyme reaction) is dehydrated with a screw press (Shinryo Seisakusho: 250 × 1000 SPH-EN) until the pulp concentration reaches 40% by mass, and the xylanase reaction A later filtrate was obtained. After 60 minutes from the start of the enzyme reaction, a part of the filtrate after the xylanase reaction was returned to the trampo screw, and 20% by mass pulp dehydrated by the drum filter was returned instead of hot water (50 ° C.). The pulp concentration was adjusted to 10% by mass with the filtrate. 150 minutes after the start of the enzyme reaction, the total sugar concentration in the filtrate after the reaction rose to 0.92%, and the average degree of polymerization of the xylo-oligosaccharide contained in the sugar solution at this point was 4.7. The above pulp was subjected to xylanase treatment continuously for 24 hours. After 150 minutes from the start of the enzyme reaction, the reaction filtrate having a sugar concentration of about 0.90% could be stably recovered. Finally, 15 m ³ of a filtrate after xylanase reaction with a sugar concentration of 0.90% was obtained. The obtained reaction filtrate is filtered through a back filter (manufactured by ISP Filters) with a micron rate of 1 μm and then a ceramic filter (manufactured by Nippon Pole) with a micron rate of 0.2 μm to obtain a clear filtrate (sugar solution). It was. This filtrate was operated with a reverse osmosis membrane (manufactured by Nitto Denko: NTR-7450, membrane quality: sulfonated polyethersulfone, membrane area: 6.5 m ² -2) at a liquid temperature of 50 ° C. and a flow rate of 1400-1800 L / h. The mixture was concentrated 15 times under the conditions to obtain 1 m ³ of a concentrated solution having a total sugar concentration of 11%.
[Purification of oligosaccharides]
In the production process shown in FIG. 1, 500 L of concentrated sugar solution obtained in the above ([Manufacturing of oligosaccharide concentrate]) is subjected to ultrafiltration membrane (ultrafiltration device: UF membrane module made by Osmonics) with a molecular weight cut off of 10,000. And processed at a permeation flow rate of 350 L / h. The filtrate (fraction containing sugar) that passed through the ultrafiltration membrane was adjusted to pH 3.5 with concentrated sulfuric acid and then treated (acid treatment) at 121 ° C. for 45 minutes. After cooling the filtrate to 50 ° C., the insoluble residue produced after the acid treatment was removed with a ceramic filter (manufactured by Nippon Pole) having a micron rate of 0.2 μm. A part of this sugar solution was sampled to adjust the total sugar concentration to 0.2% (diluted with distilled water), and the absorbance (coloring degree) at a wavelength of 350 nm was measured (Table 1). Next, 10 kg of activated carbon (manufactured by Mikura Kasei: PM-SX) was added to 480 L of the filtrate, and the sugar solution was recovered after treatment at 50 ° C. for 2 hours. This sugar solution is converted into a strong cation resin (Mitsubishi Chemical: PK-218, 300 L) → weak anion resin (Mitsubishi Chemical: WA30, 300 L) → strong cation resin (Mitsubishi Chemical: PK-218, 300 L) at SV1.5. ) → Passed through a 4-bed 4-tower ion exchange resin made of a weak anion resin (Mitsubishi Chemical: WA30, 300 L) to obtain a xylooligosaccharide sugar solution (sugar concentration 4.3 mass%, 550 L). The obtained xylo-oligosaccharide solution was adjusted to pH 7 with a strong cation resin and then treated with a spray dryer (Okawara Kako Machine: ODA-25 type) to obtain 23.0 kg of xylo-oligosaccharide powder (average polymerization degree 5.0). . Next, 50 mM sodium chloride aqueous solution was passed through the weak anion resin in the second and fourth towers at SV1.5 to elute the acidic xylo-oligosaccharide adsorbed on the ion-exchange resin, and the acidic xylo-oligosaccharide solution (sugar concentration) 2.8% by mass, 400 L) was obtained. The obtained acidic xylo-oligosaccharide solution was adjusted to pH 5 with a strong cationic resin, and then concentrated with Evapor (Okawara Kakoki: CEP-1) to a sugar concentration of 20% by mass. Next, it was treated with a spray dryer (Okawara Chemical Industries, Ltd .: ODA-25 type) to obtain 11.3 kg of acidic xylo-oligosaccharide powder (average degree of polymerization 11.0). The obtained xylo-oligosaccharide and acidic xylo-oligosaccharide were adjusted to a total sugar concentration of 5 mass% with distilled water, and the absorbance (coloring degree) at a wavelength of 350 nm was measured (Table 2).

＜比較例１＞
図２に示す製造工程で、実施例１（［オリゴ糖濃縮液の製造］）で得られた濃縮糖液５００Ｌを、濃硫酸でｐＨ３．５に調整後、１２１℃で４５分間処理（酸処理）した。糖濃縮液を５０℃まで冷却後、酸処理後で生成した不溶性残渣をミクロンレート０．２μmのセラミックフィルター（日本ポール製）で除去した。次に、糖濃縮液を分画分子量１００００の限外濾過膜（限外濾過装置：オスモニクス製ＵＦ膜モジュール）を用いて、透過流速３５０L/hで処理した。この糖液を一部採取して全糖濃度を０．２質量％に調製（蒸留水で希釈）し、３５０nmの波長における吸光度（着色度）を測定した（表１）。次に、限外濾過膜を通過したろ液４８０Lに対して活性炭（三倉化成製：ＰＭ−ＳＸ）１０ｋｇを添加し、５０℃で２時間処理し糖液を回収した。この糖液を、ＳＶ１．５で強カチオン樹脂（三菱化学製：PK−２１８、３００L）→弱アニオン樹脂（三菱化学製：ＷＡ３０、３００L）→強カチオン樹脂（三菱化学製：ＰＫ−２１８、３００L）→弱アニオン樹脂（三菱化学製：ＷＡ３０、３００L）からなる４床４塔式イオン交換樹脂に通液し、キシロオリゴ糖溶液（糖濃度３．９質量％、５５０L）を得た。得られたキシロオリゴ糖溶液を強カチオン樹脂でｐＨ７に調整後、スプレードライヤー（大川原化工機製：ＯＤＡ−２５型）で処理して、キシロオリゴ糖粉末２０．６ｋｇ（平均重合度５．１）を得た。次に、２塔目及び４塔目の弱アニオン樹脂に５０mM塩化ナトリウム水溶液をＳＶ１．５で通液しイオン交換樹脂に吸着している酸性キシロオリゴ糖を溶出させ、酸性キシロオリゴ糖溶液（糖濃度２．４質量％、４００Ｌ）を得た。得られた酸性キシロオリゴ糖溶液を、強カチオン樹脂でｐＨ５に調整後、糖濃度２０質量％までエバポール（大河原化工機：CEP-1）で濃縮した。次に、スプレードライヤー（大川原化工機製：ＯＤＡ−２５型）で処理して、酸性キシロオリゴ糖粉末９．５ｋｇ（平均重合度１１．０）を得た。得られたキシロオリゴ糖及び酸性キシロオリゴ糖を蒸留水で全糖濃度５質量％に調整し、３５０nmの波長における吸光度（着色度）を測定した（表２）。 <Comparative Example 1>
In the production process shown in FIG. 2, 500 L of concentrated sugar solution obtained in Example 1 ([Manufacture of oligosaccharide concentrate]) is adjusted to pH 3.5 with concentrated sulfuric acid and then treated at 121 ° C. for 45 minutes (acid treatment). )did. After the sugar concentrate was cooled to 50 ° C., the insoluble residue produced after the acid treatment was removed with a ceramic filter (manufactured by Nippon Pole) having a micron rate of 0.2 μm. Next, the sugar concentrate was treated at a permeation flow rate of 350 L / h using an ultrafiltration membrane (ultrafiltration device: UF membrane module manufactured by Osmonics) having a molecular weight cut-off of 10,000. A part of this sugar solution was sampled to prepare a total sugar concentration of 0.2% by mass (diluted with distilled water), and the absorbance (coloring degree) at a wavelength of 350 nm was measured (Table 1). Next, 10 kg of activated carbon (manufactured by Mikura Kasei: PM-SX) was added to 480 L of the filtrate that passed through the ultrafiltration membrane, followed by treatment at 50 ° C. for 2 hours to recover the sugar solution. This sugar solution is converted into a strong cation resin (Mitsubishi Chemical: PK-218, 300 L) → weak anion resin (Mitsubishi Chemical: WA30, 300 L) → strong cation resin (Mitsubishi Chemical: PK-218, 300 L) at SV1.5. The solution was passed through a 4-bed 4-tower ion exchange resin made of a weak anion resin (Mitsubishi Chemical: WA30, 300 L) to obtain a xylooligosaccharide solution (sugar concentration: 3.9% by mass, 550 L). The obtained xylo-oligosaccharide solution was adjusted to pH 7 with a strong cation resin and then treated with a spray dryer (Okawara Kako Machine: ODA-25 type) to obtain 20.6 kg of xylo-oligosaccharide powder (average polymerization degree 5.1). . Next, 50 mM sodium chloride aqueous solution is passed through the second and fourth weak anion resins at SV1.5 to elute the acidic xylooligosaccharide adsorbed on the ion exchange resin, and the acidic xylooligosaccharide solution (sugar concentration 2) .4 mass%, 400 L). The obtained acidic xylo-oligosaccharide solution was adjusted to pH 5 with a strong cation resin, and then concentrated with Evapor (Okawara Kakoki: CEP-1) to a sugar concentration of 20% by mass. Next, it processed with the spray dryer (Okawara Chemical Industries make: ODA-25 type | mold), and obtained acid xylo-oligosaccharide powder 9.5kg (average degree of polymerization 11.0). The obtained xylo-oligosaccharide and acidic xylo-oligosaccharide were adjusted to a total sugar concentration of 5 mass% with distilled water, and the absorbance (coloring degree) at a wavelength of 350 nm was measured (Table 2).

分画分子量１００００の限外濾過膜を用いて試験した結果を表１および表２に示す。糖濃縮液を限外濾過した後に酸処理した場合（実施例１）では、糖濃縮液を酸処理後に限外濾過した場合（比較例１）と比較し糖液の着色度（吸光度３５０nm）が減少した（表１）。また、糖濃縮液を限外濾過した後に酸処理した場合（実施例１）では、糖濃縮液を酸処理後に限外濾過した場合（比較例１）と比較し精製後のキシロオリゴ糖液及び酸性キシロオリゴ糖の着色度（吸光度３５０nm）が減少した（表２）。以上の結果から、糖濃縮液を限外濾過した後に酸処理を行うことで、酸処理で生成される着色物質の含量を低減できることが判明した。この結果により、後段の精製工程で脱色を目的として使用する活性炭の量を低減でき、コスト低減が可能となる。 Tables 1 and 2 show the results of testing using an ultrafiltration membrane with a molecular weight cut-off of 10,000. In the case where the sugar concentrate is subjected to acid treatment after ultrafiltration (Example 1), the coloring degree (absorbance 350 nm) of the sugar liquid is higher than in the case where the sugar concentrate is subjected to ultrafiltration after acid treatment (Comparative Example 1). Decreased (Table 1). Further, when the sugar concentrate is subjected to acid treatment after ultrafiltration (Example 1), the purified xylooligosaccharide solution and acidity are compared with the case where the sugar concentrate is ultrafiltered after acid treatment (Comparative Example 1). The coloring degree (absorbance 350 nm) of xylo-oligosaccharide decreased (Table 2). From the above results, it was found that the content of colored substances produced by acid treatment can be reduced by performing acid treatment after ultrafiltration of the sugar concentrate. As a result, the amount of activated carbon used for the purpose of decolorization in the subsequent purification step can be reduced, and the cost can be reduced.

＜実施例２＞
［オリゴ糖の精製］
図１に示す製造工程で実施例１（［オリゴ糖濃縮液の製造］）で得られた濃縮糖液５００Ｌを精製した。分画分子量５０００の限外濾過膜（限外濾過装置：オスモニクス製ＵＦ膜モジュール）を用いた以外は実施例１と同様の方法で実施した。糖液の採取についても実施例１と同様の工程の箇所で実施し、糖液の３５０nmの波長における吸光度（着色度）を測定した（表３）。尚、イオン交換樹脂処理後のキシロオリゴ糖溶液の糖濃度は３．７質量％（５５０L）、酸性キシロオリゴ糖溶液の糖濃度は２．３質量％（４００L）であった。また、精製後、得られたキシロオリゴ糖粉末は１９．１ｋｇ（平均重合度４．９）、酸性キシロオリゴ糖粉末は９．５ｋｇ（平均重合度１０．８）であった。 <Example 2>
[Purification of oligosaccharides]
In the production process shown in FIG. 1, 500 L of concentrated sugar solution obtained in Example 1 ([Manufacturing of oligosaccharide concentrate]) was purified. This was carried out in the same manner as in Example 1 except that an ultrafiltration membrane having a molecular weight cut-off of 5000 (ultrafiltration device: UF membrane module manufactured by Osmonics) was used. The collection of the sugar solution was also carried out at the same step as in Example 1, and the absorbance (coloring degree) of the sugar solution at a wavelength of 350 nm was measured (Table 3). The sugar concentration of the xylo-oligosaccharide solution after the ion exchange resin treatment was 3.7% by mass (550 L), and the sugar concentration of the acidic xylo-oligosaccharide solution was 2.3% by mass (400 L). Further, after purification, the obtained xylo-oligosaccharide powder was 19.1 kg (average polymerization degree 4.9), and the acidic xylo-oligosaccharide powder was 9.5 kg (average polymerization degree 10.8).

＜比較例２＞
図２に示す製造工程で実施例１（［オリゴ糖濃縮液の製造］）で得られた濃縮糖液５００Ｌを精製した。分画分子量５０００の限外濾過膜（限外濾過装置：オスモニクス製ＵＦ膜モジュール）を用いた以外は比較例１と同様の方法で実施した。糖液の採取についても比較例１と同様の工程の箇所で実施し、糖液の３５０nmの波長における吸光度（着色度）を測定した（表３）。尚、イオン交換樹脂処理後のキシロオリゴ糖溶液の糖濃度は３．３質量％（５５０L）、酸性キシロオリゴ糖溶液の糖濃度は２．０質量％（４００L）であった。また、精製後、得られたキシロオリゴ糖粉末は１６．８ｋｇ（平均重合度４．９）、酸性キシロオリゴ糖粉末は８．４ｋｇ（平均重合度１０．９）であった。 <Comparative example 2>
In the production process shown in FIG. 2, 500 L of concentrated sugar solution obtained in Example 1 ([Manufacture of oligosaccharide concentrate]) was purified. This was carried out in the same manner as in Comparative Example 1 except that an ultrafiltration membrane with a molecular weight cut off of 5000 (ultrafiltration device: UF membrane module manufactured by Osmonics) was used. The collection of the sugar solution was also carried out at the same step as in Comparative Example 1, and the absorbance (coloring degree) of the sugar solution at a wavelength of 350 nm was measured (Table 3). The sugar concentration of the xylo-oligosaccharide solution after the ion exchange resin treatment was 3.3% by mass (550 L), and the sugar concentration of the acidic xylo-oligosaccharide solution was 2.0% by mass (400 L). Further, after purification, the obtained xylo-oligosaccharide powder was 16.8 kg (average polymerization degree 4.9), and the acidic xylo-oligosaccharide powder was 8.4 kg (average polymerization degree 10.9).

分画分子量５０００の限外濾過膜を用いて試験した結果を表３および表４に示す。糖濃縮液を限外濾過した後に酸処理した場合（実施例２）では、糖濃縮液を酸処理後に限外濾過した場合（比較例２）と比較し糖液の着色度（吸光度３５０nm）が減少した（表３）。また、糖濃縮液を限外濾過した後に酸処理した場合（実施例２）では、糖濃縮液を酸処理後に限外濾過した場合（比較例２）と比較し精製後のキシロオリゴ糖液及び酸性キシロオリゴ糖の着色度（吸光度３５０nm）が減少した（表４）。 Tables 3 and 4 show the results of testing using an ultrafiltration membrane with a molecular weight cut-off of 5000. In the case where the sugar concentrate is subjected to acid treatment after ultrafiltration (Example 2), the coloring degree (absorbance 350 nm) of the sugar liquid is higher than in the case where the sugar concentrate is subjected to ultrafiltration after acid treatment (Comparative Example 2). Decreased (Table 3). Further, when the sugar concentrate is subjected to acid treatment after ultrafiltration (Example 2), the purified xylooligosaccharide solution and acidity are compared with the case where the sugar concentrate is ultrafiltered after acid treatment (Comparative Example 2). The coloring degree (absorbance 350 nm) of xylo-oligosaccharide decreased (Table 4).

＜実施例３＞
［オリゴ糖の精製］
図１に示す製造工程で実施例１（［オリゴ糖濃縮液の製造］）で得られた濃縮糖液５００Ｌを精製した。分画分子量３００００の限外濾過膜（限外濾過装置：オスモニクス製ＵＦ膜モジュール）を用いた以外は実施例１と同様の方法で実施した。糖液の採取についても実施例１と同様の工程の箇所で実施し、糖液の３５０nmの波長における吸光度（着色度）を測定した（表５）。尚、イオン交換樹脂処理後のキシロオリゴ糖溶液の糖濃度は４．０質量％（５５０L）、酸性キシロオリゴ糖溶液の糖濃度は２．７質量％（４００Ｌ）であった。また、精製後、得られたキシロオリゴ糖粉末は２２．０ｋｇ（平均重合度５．１）、酸性キシロオリゴ糖粉末は１１．０ｋｇ（平均重合度１１．２）であった。 <Example 3>
[Purification of oligosaccharides]
In the production process shown in FIG. 1, 500 L of concentrated sugar solution obtained in Example 1 ([Manufacturing of oligosaccharide concentrate]) was purified. This was carried out in the same manner as in Example 1 except that an ultrafiltration membrane having a molecular weight cut off of 30000 (ultrafiltration device: UF membrane module manufactured by Osmonics) was used. The collection of the sugar solution was also carried out at the same step as in Example 1, and the absorbance (coloring degree) of the sugar solution at a wavelength of 350 nm was measured (Table 5). The sugar concentration of the xylo-oligosaccharide solution after the ion exchange resin treatment was 4.0% by mass (550 L), and the sugar concentration of the acidic xylo-oligosaccharide solution was 2.7% by mass (400 L). Further, after purification, the obtained xylo-oligosaccharide powder was 22.0 kg (average polymerization degree 5.1), and the acidic xylo-oligosaccharide powder was 11.0 kg (average polymerization degree 11.2).

＜比較例３＞
図２に示す製造工程で実施例１（［オリゴ糖濃縮液の製造］）で得られた濃縮糖液５００Ｌを精製した。分画分子量３００００の限外濾過膜（限外濾過装置：オスモニクス製ＵＦ膜モジュール）を用いた以外は比較例１と同様の方法で実施した。糖液の採取についても比較例１と同様の工程の箇所で実施し、糖液の３５０nmの波長における吸光度（着色度）を測定した（表５）。尚、イオン交換樹脂処理後のキシロオリゴ糖溶液の糖濃度は３．９質量％（５５０L）、酸性キシロオリゴ糖溶液の糖濃度は２．５質量％（４００L）であった。また、精製後、得られたキシロオリゴ糖粉末は２１．２ｋｇ（平均重合度５．１）、酸性キシロオリゴ糖粉末は１０．７ｋｇ（平均重合度１１．１）であった。 <Comparative Example 3>
In the production process shown in FIG. 2, 500 L of concentrated sugar solution obtained in Example 1 ([Manufacture of oligosaccharide concentrate]) was purified. This was carried out in the same manner as in Comparative Example 1 except that an ultrafiltration membrane with a molecular weight cut off of 30000 (ultrafiltration device: UF membrane module manufactured by Osmonics) was used. The collection of the sugar solution was also carried out at the same step as in Comparative Example 1, and the absorbance (coloring degree) of the sugar solution at a wavelength of 350 nm was measured (Table 5). The sugar concentration of the xylooligosaccharide solution after the ion exchange resin treatment was 3.9% by mass (550 L), and the sugar concentration of the acidic xylooligosaccharide solution was 2.5% by mass (400 L). Further, after purification, the obtained xylo-oligosaccharide powder was 21.2 kg (average polymerization degree 5.1), and the acidic xylo-oligosaccharide powder was 10.7 kg (average polymerization degree 11.1).

分画分子量３００００の限外濾過膜を用いて試験した結果を表５および表６に示す。糖濃縮液を限外濾過した後に酸処理した場合（実施例３）では、糖濃縮液を酸処理後に限外濾過した場合（比較例３）と比較し糖液の着色度（吸光度３５０nm）が減少した（表５）。また、糖濃縮液を限外濾過した後に酸処理した場合（実施例３）では、糖濃縮液を酸処理後に限外濾過した場合（比較例３）と比較し精製後のキシロオリゴ糖液及び酸性キシロオリゴ糖の着色度（吸光度３５０nm）が減少した（表６）。 Tables 5 and 6 show the results of testing using an ultrafiltration membrane with a molecular weight cut off of 30,000. In the case where the sugar concentrate is subjected to acid treatment after ultrafiltration (Example 3), the coloring degree (absorbance 350 nm) of the sugar liquid is higher than that in the case where the sugar concentrate is subjected to ultrafiltration after acid treatment (Comparative Example 3). Decreased (Table 5). Further, when the sugar concentrate is subjected to acid treatment after ultrafiltration (Example 3), the purified xylooligosaccharide solution and acidity are compared with the case where the sugar concentrate is ultrafiltered after acid treatment (Comparative Example 3). The coloring degree (absorbance 350 nm) of xylo-oligosaccharide decreased (Table 6).

＜実施例４＞
［逆浸透膜と限外濾過膜の間へのループ導入による連続循環の検討］
実施例１（［オリゴ糖濃縮液の製造］）と同様の方法で、パルプを原料として糖濃縮液を製造した。糖濃縮液製造の逆浸透膜（日東電工製：ＮＴＲ−７４５０）で濃縮された糖液を連続的に限外濾過装置（オスモニクス製ＵＦ膜モジュール、分画分子量１００００）を用いて、透過流速３５０L/hで限外ろ過膜による処理を行った。図３に示すように限外濾過膜を通過したろ液（糖を含む画分）を前記の逆浸透膜（日東電工製：ＮＴＲ−７４５０）へ戻し、逆浸透膜→限外濾過膜の工程を連続的に循環させ、糖濃度が一定以上に濃縮されるまで限外濾過を継続した。ループによる連続循環開始後、逆浸透膜入口の操作圧力を経時的に測定した。また、逆浸透膜内の糖液を採取し、糖液の全糖濃度を測定した。 <Example 4>
[Consideration of continuous circulation by introducing a loop between reverse osmosis membrane and ultrafiltration membrane]
In the same manner as in Example 1 ([Production of oligosaccharide concentrate]), a sugar concentrate was produced using pulp as a raw material. A sugar solution concentrated by a reverse osmosis membrane (manufactured by Nitto Denko: NTR-7450) manufactured by a sugar concentrate is continuously used with an ultrafiltration device (UF membrane module made by Osmonics, molecular weight cut off 10,000), and a permeation flow rate of 350 L. Treatment with an ultrafiltration membrane was performed at / h. As shown in FIG. 3, the filtrate (the fraction containing sugar) that has passed through the ultrafiltration membrane is returned to the reverse osmosis membrane (manufactured by Nitto Denko: NTR-7450), and the process of reverse osmosis membrane → ultrafiltration membrane is performed. Was continuously circulated and ultrafiltration was continued until the sugar concentration was concentrated above a certain level. After starting continuous circulation by the loop, the operating pressure at the reverse osmosis membrane inlet was measured over time. In addition, the sugar solution in the reverse osmosis membrane was collected, and the total sugar concentration of the sugar solution was measured.

＜比較例４＞
実施例４記載の方法で、ループによる連続循環を行わない方法を比較例４とした（製造工程フローは図１と同様）。実施例４と同様に、逆浸透膜入口の操作圧力を測定した。また、逆浸透膜内の糖液を採取し、糖液の全糖濃度を測定した（逆浸透膜の運転開始後、同一の時間経過におけるデータを実施例４と比較した）。 <Comparative example 4>
In the method described in Example 4, a method in which continuous circulation by a loop is not performed is referred to as Comparative Example 4 (the manufacturing process flow is the same as in FIG. 1). As in Example 4, the operating pressure at the reverse osmosis membrane inlet was measured. In addition, the sugar solution in the reverse osmosis membrane was collected, and the total sugar concentration of the sugar solution was measured (data in the same time course was compared with Example 4 after the operation of the reverse osmosis membrane was started).

逆浸透膜入口の操作圧力と逆浸透膜内の糖濃度の関係を図４に示す。逆浸透膜と限外濾過膜の間にループをつくり、糖液を連続循環させた場合（実施例４）では、連続循環させない場合（比較例４）と比較し、同じ操作圧力（逆浸透膜）において高濃度の糖液を得ることができた。操作圧力１．９MPaの場合、実施例４では糖濃度１５．２質量％、比較例４では、糖濃度１１．０質量％であった。また、実施例で４は、比較例４と比較し酸処理以降の精製工程に要する時間を約４時間短縮することができた。 FIG. 4 shows the relationship between the operating pressure at the reverse osmosis membrane inlet and the sugar concentration in the reverse osmosis membrane. When a loop is formed between the reverse osmosis membrane and the ultrafiltration membrane and the sugar liquid is continuously circulated (Example 4), the same operating pressure (reverse osmosis membrane is compared with the case where the circulatory solution is not continuously circulated (Comparative Example 4). ), A high concentration sugar solution could be obtained. In the case of operating pressure of 1.9 MPa, the sugar concentration was 15.2% by mass in Example 4, and the sugar concentration was 11.0% by mass in Comparative Example 4. Moreover, in Example 4, compared with Comparative Example 4, the time required for the purification step after the acid treatment could be shortened by about 4 hours.

本発明により、着色が低減された高純度のキシロオリゴ糖および酸性キシロオリゴ糖が効率良く製造できるため、キシロオリゴ糖及び酸性キシロオリゴ糖を安価に製造することができる。 According to the present invention, since high-purity xylo-oligosaccharides and acidic xylo-oligosaccharides with reduced coloring can be efficiently produced, xylo-oligosaccharides and acidic xylo-oligosaccharides can be produced at low cost.

Claims

In the method of producing xylooligosaccharides and acidic xylooligosaccharides from lignocellulosic materials, a concentration step of concentrating the filtrate after treating the lignocellulosic material with hemicellulase with a reverse osmosis membrane, and ultrafiltration of the resulting concentrated solution A method for producing xylooligosaccharides and acidic xylooligosaccharides, comprising: a decoloring step for treatment with a membrane; and an acid treatment step for hydrolyzing a sugar solution decolorized with an ultrafiltration membrane with an acid.

The method for producing xylo-oligosaccharides and acidic xylo-oligosaccharides according to claim 1, wherein all or part of the sugar solution decolorized by the ultrafiltration membrane is continuously returned to the reverse osmosis membrane by a loop.

The method for producing xylooligosaccharides and acidic xylooligosaccharides according to claim 1 or 2, wherein the ultrafiltration membrane has a molecular weight cut-off of 5000 to 30000.

The method for producing xylo-oligosaccharides and acidic xylo-oligosaccharides according to claim 1, wherein the lignocellulosic material is a refined product of wood.

The method for producing xylo-oligosaccharides and acidic xylo-oligosaccharides according to claim 4, wherein the refined product of wood is pulp obtained by chemical treatment.

The method of producing xylo-oligosaccharides and acidic xylo-oligosaccharides according to claim 5, wherein the pulp obtained by chemical treatment is kraft pulp derived from hardwood.

The method for producing a xylo-oligosaccharide and an acidic xylo-oligosaccharide according to claim 1, wherein the hemicellulase is a xylanase.