JP4073661B2 - Method for producing acidic xylooligosaccharide composition - Google Patents
Method for producing acidic xylooligosaccharide composition Download PDFInfo
- Publication number
- JP4073661B2 JP4073661B2 JP2001387322A JP2001387322A JP4073661B2 JP 4073661 B2 JP4073661 B2 JP 4073661B2 JP 2001387322 A JP2001387322 A JP 2001387322A JP 2001387322 A JP2001387322 A JP 2001387322A JP 4073661 B2 JP4073661 B2 JP 4073661B2
- Authority
- JP
- Japan
- Prior art keywords
- xylo
- oligosaccharide
- acidic
- polymerization
- treatment
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
- HEBKCHPVOIAQTA-NGQZWQHPSA-N d-xylitol Chemical compound OC[C@H](O)C(O)[C@H](O)CO HEBKCHPVOIAQTA-NGQZWQHPSA-N 0.000 title claims description 182
- 230000002378 acidificating effect Effects 0.000 title claims description 117
- 239000000203 mixture Substances 0.000 title claims description 64
- 238000004519 manufacturing process Methods 0.000 title claims description 25
- 238000011282 treatment Methods 0.000 claims description 73
- 238000006116 polymerization reaction Methods 0.000 claims description 52
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical compound O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 claims description 49
- 239000002253 acid Substances 0.000 claims description 45
- 238000000034 method Methods 0.000 claims description 42
- 229920005610 lignin Polymers 0.000 claims description 37
- 239000000126 substance Substances 0.000 claims description 33
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 claims description 29
- 238000005903 acid hydrolysis reaction Methods 0.000 claims description 25
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 claims description 25
- 101710121765 Endo-1,4-beta-xylanase Proteins 0.000 claims description 20
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- 229910021645 metal ion Inorganic materials 0.000 claims description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 claims description 2
- 238000005194 fractionation Methods 0.000 claims description 2
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims 3
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- 150000002482 oligosaccharides Chemical class 0.000 description 26
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 21
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- QGGOCWIJGWDKHC-UHFFFAOYSA-N O4-Methyl-D-glucuronsaeure Chemical group OC(=O)C(O)C(OC)C(O)C(O)C=O QGGOCWIJGWDKHC-UHFFFAOYSA-N 0.000 description 11
- 238000010411 cooking Methods 0.000 description 11
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- IAJILQKETJEXLJ-QTBDOELSSA-N aldehydo-D-glucuronic acid Chemical group O=C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C(O)=O IAJILQKETJEXLJ-QTBDOELSSA-N 0.000 description 8
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- LGQKSQQRKHFMLI-SJYYZXOBSA-N (2s,3r,4s,5r)-2-[(3r,4r,5r,6r)-4,5,6-trihydroxyoxan-3-yl]oxyoxane-3,4,5-triol Chemical compound O[C@@H]1[C@@H](O)[C@H](O)CO[C@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O)OC1 LGQKSQQRKHFMLI-SJYYZXOBSA-N 0.000 description 6
- LGQKSQQRKHFMLI-UHFFFAOYSA-N 4-O-beta-D-xylopyranosyl-beta-D-xylopyranose Natural products OC1C(O)C(O)COC1OC1C(O)C(O)C(O)OC1 LGQKSQQRKHFMLI-UHFFFAOYSA-N 0.000 description 6
- SQNRKWHRVIAKLP-UHFFFAOYSA-N D-xylobiose Natural products O=CC(O)C(O)C(CO)OC1OCC(O)C(O)C1O SQNRKWHRVIAKLP-UHFFFAOYSA-N 0.000 description 6
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
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- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 6
- 239000003456 ion exchange resin Substances 0.000 description 6
- 229920003303 ion-exchange polymer Polymers 0.000 description 6
- 125000000969 xylosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)CO1)* 0.000 description 6
- HNSDLXPSAYFUHK-UHFFFAOYSA-N 1,4-bis(2-ethylhexyl) sulfosuccinate Chemical compound CCCCC(CC)COC(=O)CC(S(O)(=O)=O)C(=O)OCC(CC)CCCC HNSDLXPSAYFUHK-UHFFFAOYSA-N 0.000 description 5
- 229920002488 Hemicellulose Polymers 0.000 description 5
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- 238000010586 diagram Methods 0.000 description 5
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- 238000002474 experimental method Methods 0.000 description 5
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- 238000011002 quantification Methods 0.000 description 5
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 4
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 4
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- 150000001450 anions Chemical class 0.000 description 4
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- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
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Description
【０００１】
【発明の属する技術分野】
本発明は、健康食品、食品添加物、薬品・化粧品添加物、農業用生長促進剤などとして利用される酸性キシロオリゴ糖組成物に関する。特に、本発明は、キシロースオリゴマーを主鎖とし、ウロン酸残基を側鎖に持つ酸性キシロオリゴ糖の混合物からなる酸性キシロオリゴ糖組成物とその製造方法に関する。
【０００２】
【従来の技術】
国民の健康に対する関心の高まりから、毎日摂取する食物を通して健康を維持する試みが注目を集めている。特に、食物繊維やオリゴ糖といった多糖やそれらの分解生成物は、それらの消化器官で発揮する生理活性が注目され、近年、熱心に研究されている分野でもある。
食物繊維は、従来から糖質代謝や脂質代謝といった生体の基本的代謝に関わるフードファクターとして重要視されてきた経緯がある。機能性食品のなかにもこの食物繊維の作用をうたったものが少なくない。
【０００３】
食物繊維は、便通性の改善や発ガン物質を吸着することで腸内における発ガン物質の希釈を行うといった機能があり、近年ではヒトのフードファクターとして重要視されている。また、従来の食物繊維の中には血液の高脂血症を改善したり、糖代謝を制御することでヒトの健康を制御できる可能性を有するものも存在している。特に、タマリンド種子ガムやグアーガム、アルギン酸ナトリウムやペクチンといった酸性糖を多く含む食物繊維は血液中の総コレステロールやリン脂質、トリグリセライドの量を低下させ高脂血症を改善すると報告されている。
〔Rotenberg,S. and Jakobsen,P,E.:The effect of dietary pectin on lipid composition of blood, skeletal muscle and internal organs of rats. J.Nutr.,108,1384(1978)〕
【０００４】
しかし、これらの食物繊維は大腸菌の生育を抑え、ビフィズス菌の選択的増殖性を促す働きを持つ糖類ではない。むしろ、小腸の粘膜細胞を物理的にこそぎ落とし、栄養吸収に対して負の影響を与えることも少なくない。〔Fahey Jr.,G.C.:Dietary fiber ; chemistry and nutrition, p.117〜146, Academic Press, New York (1979)〕
【０００５】
一方、オリゴ糖類は腸内有用細菌の選択的な増殖促進効果を通して、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に利用され、チョコレートなどの菓子類にも利用されている有用な糖類である。
また、ヒトの食品用途だけではなく家畜の飼料としての用途もある。さらに、医薬、サニタリー製品の分野でも乳化剤、皮膚の保湿成分としての用途がある。
【０００６】
一般に、特定保健用食品に用いられるオリゴ糖類は、そのほとんどが整腸作用、即ち腸内悪玉菌である大腸菌や腸内腐敗発酵菌であるクロストリジウム属やオイバクテリウム属の菌の数を減らし、相対的に腸内善玉菌といわれるビフィズス菌を増加させる作用を持っている。
いろいろあるオリゴ糖類の中でも、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有名である。キシロオリゴ糖の作用については、他のオリゴ糖と同様に腸内善玉菌のビフィズス菌の選択的増殖を促す一方で、腸内悪玉菌である大腸菌の数を相対的に低下させると言われている。
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知られている〔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)〕ことから、大腸菌や腸内腐敗発酵菌の数を腸内で減らすことは長期にわたる健康を考えた場合に重要である。
【０００７】
近年、オリゴ糖の生理活性の中でも腸管における免疫作用についての研究の進展は特に著しいものがある。特に、キシロオリゴ糖により選択的に増殖したビフィズス菌には腸管免疫の増強作用があることがわかっている。腸管の粘膜細胞下に存在するＢ細胞は、感染防御、食物アレルギーの抑制に関係するＩｇＡ（イムノグロブリンＡ）と呼ばれる物質を生産し、これを腸管内に分泌することで粘膜細胞防御している。ビフィズス菌の細胞壁に由来する成分はマクロファージを介して小腸粘膜細胞下に存在するＢ細胞に作用し、局所免疫をつかさどる成分であるＩｇＡを生産させる方向に働く。
【０００８】
このため、オリゴ糖の摂取により腸管に存在するビフィズス菌をコントロールすることで、外から進入してくる病原菌等の感染を防御したり、アレルギー誘発性の物質が経口でもたらされた時でも中和抗体的に作用するＩｇＡの生産が亢進する可能性が生まれてきた。〔保井久子、加藤紀子、三毛明人、早川和仁、大協真、菅 辰彦.1991.Bifidobacterium breveの経口投与による腸管免疫系（ＩｇＡ抗体生産）の増強.日本農芸化学会誌 65:623〕
【０００９】
整腸作用を期待し、経口でオリゴ糖類を摂取した場合に関しては、胃酸や消化液中の酵素による酸加水分解からくるオリゴ糖の重合度の低下が大きな問題である。オリゴ糖は、酸や酵素による加水分解により徐々に低分子化し、最終的には大腸菌やクロストリジウム属に属する腐敗性嫌気性菌でも資化することが可能な単糖にまで分解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（特許第２５４９６３８号）、それほど分解されずに腸内に届けられる可能性が高い。実際にキシロオリゴ糖をヒトに投与した場合、生体内での整腸効果が確認されている。
【００１０】
現在は、２量体や３量体よりずっと分子量の大きな５量体程度の平均重合度を有するキシロオリゴ糖（特開２００１-２２６４０９号公報）も製造可能である。また、本発明者らは平均重合度が１２量体前後である長鎖キシロオリゴ糖も提案している（特願２００１−２４２９０６号）。鎖長が長いキシロオリゴ糖は腸内でも単糖であるキシロースには変換されにくく、しかも、排泄便量はキシロビオースに比較して増加するというメリットがある。
【００１１】
特に、長鎖キシロオリゴ糖は平均重合度が高く、オリゴ糖と食物繊維の両方の生理活性を有する。つまり、小腸では食物繊維様の作用である抗高脂血症作用を発揮し、小腸を通過して大腸ではビフィズス菌に選択的に資化されることで、整腸作用を発揮するのである。
【００１２】
しかし、これらの優れた生理活性を発揮する長鎖キシロオリゴ糖も１つの問題を有している。その問題とは、溶解性の問題である。中性キシロオリゴ糖は、重合度が大きくなるにつれてその溶解度が低下する。特に、平均重合度が１２量体前後のものは、食物繊維様の作用と整腸作用の両方の作用を併せ持つ有用な糖質であるが、中性キシロオリゴ糖の１２量体は６０℃の水に僅か数％しか溶けない。整腸作用や腸管免疫を発揮させるためには、ある程度水に溶けた状態で腸管に届ける必要があり、長鎖のオリゴ糖の問題点は溶解度にあるといっても過言ではない。また、溶解度が低い点はオリゴ糖の生産効率の上でも大きな問題であり、長鎖であって、なおかつ水溶性が高いキシロオリゴ糖の開発が望まれていた。
【００１３】
食物繊維の中には、生理活性として血液性状の改善をもたらす糖類として酸性糖を含むような多糖類の仲間が存在する。酸性糖を含む多糖類は比較的少ない摂取でも脂質代謝を変化させるので、抗高脂血症の研究実験に非常によく用いられてきた経緯がある。具体的に述べると、酸性糖を含むような多糖や食物繊維は血清中のコレステロールの低下作用を有するものが多く、実験では、コントロール（酸性糖を含まない食餌）を投与されている群に比較して酸性糖を含む食餌を投与されている群の方が約２０〜３０％も血中のコレステロールが低下するようである。〔辻 悦子ほか：栄養学雑誌,３３，２７３（１９７５）、辻 啓介ほか：栄養学雑誌,３５,２２７（１９７７）〕
【００１４】
これらの実験から解かるように、従来より、酸性糖を含む食物繊維は血液性状の改善を期待されて研究を行われてきた経緯がある。特に、ペクチンは酸性糖を含む食物繊維の代表格であり、抗高脂血症の研究だけでなく、血中インスリン上昇抑制作用に関する研究でも良く使われる酸性多糖類である。
〔Jenkins,D.J.A.,Leeds, A.R.,Gassull,M.A.,et al.:Decrease postprandial insulin (and glucose concentrations by guar and pectin. Annual. Internal.Med.,80,20(1977)〕
糖類の吸収抑制は、酸性多糖が胃で水分を吸い膨潤し、そのゲル中に糖分を取り込むため小腸の粘膜細胞が糖質を十分に取り込むことが出来ずに起こると考えられている。
【００１５】
一般に、酸性多糖類は、その糖鎖内にカルボキシル基や硫酸基といった親水性の官能基を持つため水に解けやすく、鎖長が長い割に水に良く溶けるものが多いようである。一方の不溶性の食物繊維も抗高脂血症作用や糖質の吸収抑制作用を持つが、これらは胃から十二指腸へ移動する際、十二指腸の粘膜細胞を傷つける。このため、糖分の吸収は抑制され、血糖は上昇を抑制される。生体の健康度を考えた場合、粘膜細胞を傷つけることは必ずしも得策とはいえないし、むしろ病原菌の感染や貧栄養の一つの原因になり得るようである。このため、抗高脂血症作用や血中インスリン上昇抑制作用といった食物繊維の生理活性も、できる限り「可溶性の食物繊維」で発揮するほうが望ましい。そのため、水溶性が高く、生理活性も豊富な酸性糖を含む多糖やオリゴ糖は、今後ますます機能性食品や医薬品等への応用が可能な産業上重要な糖質であると考えられている。
【００１６】
キシロオリゴ糖は、通常、鎖長が長くなるにつれて溶解度が低下する中性糖である。しかし、キシロオリゴ糖も製造条件が異なると酸性糖を側鎖に持つ形で精製が可能であり、水溶性が高まることが知られている。従来より、植物細胞壁中のヘミセルロース中のキシランには、側鎖としてグルクロン酸残基、もしくは４−０−メチルグルクロン酸残基をもつものが存在することが知られている。トリコデルマ属に属するカビが生産する酵素液で側鎖を有するキシランを処理すると、α−グルクロニダーゼが側鎖を分解除去しつつキシランを分解し、結果的にキシロビオースやキシロトリオースを主成分とするキシロオリゴ糖組成物を作るようである。また、このとき、α-グルクロニダーゼによる消化を免れた酸性糖部分を含む酸性キシロオリゴ糖は２量体もしくは３量体であり、これらのキシロオリゴ糖の非還元末端にグルクロン酸残基もしくは４−０−メチルグルクロン酸残基が存在することが解かっている。〔R.F.H.Dekker,In "Biosythesis and biodegradation of wood components",p.505,Academic Press Inc.(1985)〕
【００１７】
一方、バクテリアが生産するようなキシラナーゼを用いてキシロオリゴ糖を生成させた場合、酵素の基質特異性が真菌類のキシラナーゼと大きく異なるため、生成するキシロオリゴ糖の組成比が２量体であるキシロビオースから５量体までの分布でキシロオリゴ糖を生成することが知られている（特開平１−２５２２８０号公報）。そして、バクテリア由来の酵素は、これらの鎖長のキシロオリゴ糖に酸性糖であるグルクロン酸や４−０−メチルグルクロン酸が非還元末端側のキシロースに１つの側鎖として結合している酸性キシロオリゴ糖を生成する。
【００１８】
広葉樹クラフトパルプを原料とし、これをキシラナーゼ処理して得られた糖液中に存在するキシロオリゴ糖は、リグニンとの複合体を形成して存在することが判明している。特に、キシロオリゴ糖−リグニン複合体は比較的大きなキシロオリゴ糖分子とリグニンが複合体を形成しているため、キシロオリゴ糖部分はキシラナーゼによる消化を逃れることが解かっている。このためキシロオリゴ糖−リグニン複合体を用いて製造されたキシロオリゴ糖は平均分子量が高く、その平均重合度は製造法にもよるが５量体から２０量体まで多岐にわたる。この中性キシロオリゴ糖を主な成分とする長鎖キシロオリゴ糖（特願２００１−２４２９０６号）は可溶性の食物繊維様の生理活性を有するが、一方で溶解度が低い。
【００１９】
【発明が解決しようとする課題】
以上のように、現状では、上市されているキシロオリゴ糖を構成するオリゴ糖は２量体や３量体を主成分とするものがほとんどであり、食物繊維とは異なる物質であると認知されている。
本発明は、鎖長が長く、分解されずに腸まで届くことで整腸作用の効果が高く、また、鎖長が長いために、オリゴ糖の特徴ばかりでなく食物繊維様の作用を有し、かつ、水に対する溶解度が高い酸性キシロオリゴ糖を提供することを課題とするものである。
さらに、本発明は水溶性の長鎖キシロオリゴ糖を大量安価に供給する方法を提供することを課題とするものである。
【００２０】
【課題を解決するための手段】
上記課題を解決するための本発明は、以下の各発明から選択されるものである。
（１）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平均重合度が８〜１５のキシロオリゴ糖混合物からなる酸性キシロオリゴ糖組成物。
【００２１】
（２）前記ウロン酸残基がグルクロン酸残基又は４−０−メチルグルクロン酸残基であることを特徴とする（１）項記載の酸性キシロオリゴ糖組成物。
【００２２】
（３）重合度が５以下の酸性キシロオリゴ糖含有率が５質量％以下であることを特徴とする（１）項又は（２）項に記載の酸性キシロオリゴ糖組成物。
【００２３】
（４）広葉樹クラフトパルプをキシラナーゼで処理してキシロオリゴ糖成分とリグニン成分とが酸加水分解性を有する化学結合により結合されたキシロオリゴ糖−リグニン複合体とキシロオリゴ糖を含有する処理液を得るキシラナーゼ処理工程、
該キシラナーゼ処理工程からのキシロオリゴ糖−リグニン複合体とキシロオリゴ糖を含有する処理液を酸加水分解処理して、酸性キシロオリゴ糖と中性キシロオリゴ糖からなる平均重合度が８〜１５のキシロオリゴ糖成分を含有するキシロオリゴ糖混合物とリグニン様物質とを含有する処理液を得る酸加水分解処理工程、
該酸加水分解処理工程からの処理液から弱アニオン交換樹脂により酸性キシロオリゴ糖成分を吸着して分離する吸着分離工程、
からなる複数工程を有することを特徴とする、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平均重合度が８〜１５の酸性キシロオリゴ糖組成物の製造方法。
【００２８】
（５）前記酸加水分解処理工程は、キシロオリゴ糖−リグニン複合体のキシロオリゴ糖のキシロース分子間のβ−１，４−キシロシド結合を分解しない酸加水分解処理工程であることを特徴とする（４）項記載のキシロオリゴ糖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平均重合度が８〜１５の酸性キシロオリゴ糖組成物の製造方法。
【００２９】
（６）前記キシロオリゴ糖成分とリグニン成分の複合体は、キシロースの２〜２０量体の混合物である酸性キシロオリゴ糖とリグニン様物質の複合体を３０質量％以上含有することを特徴とする（４）項又は（５）項に記載のキシロオリゴ糖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平均重合度が８〜１５の酸性キシロオリゴ糖組成物の製造方法。
【００３０】
（７）キシラン及び／又はヘミセルロースをヘミセルラーゼ処理してキシロオリゴ糖成分とリグニン様成分の複合体を含有する糖溶液を得、該糖溶液を酸加水分解処理し、該加水分解処理液から、重合度が５以下のキシロオリゴ糖含有率が５％以下でかつ平均重合度が８〜１５であり、側鎖としてグルクロン酸又は４−Ｏ−メチルグルクロン酸を少なくとも１つ結合しているキシロオリゴ糖混合物を分離することを特徴とする酸性キシロオリゴ糖組成物の製造方法。
【００３１】
（８）前記酸加水分解処理は、前記ヘミセルラーゼ処理によって得られる糖溶液を濃縮した糖溶液について行われることを特徴とする（７）項記載の酸性キシロオリゴ糖組成物の製造方法。
【００３２】
（９）前記ヘミセルラーゼ処理は、ｐＨ３〜１０、好ましくは５〜９の範囲に調整した糖溶液を、１０℃〜９０℃、好ましくは３０℃〜６０℃の温度で行われる処理であることを特徴とする、（７）項又は（８）項に記載の酸性キシロオリゴ糖組成物の製造方法。
【００３３】
（１０）前記加水分解処理は、ｐＨを３．５、又はそれ以下の値に調整した糖溶液を、１０５℃〜１５０℃、好ましくは１１０℃〜１３０℃の温度で行われる処理であることを特徴とする、（７）項〜（９）項のいずれか１項に記載の酸性キシロオリゴ糖組成物の製造方法。
【００３４】
（１１）前記酸加水分解処理液からの前記酸性キシロオリゴ糖混合物の分離は、前記酸加水分解処理によって得られる糖溶液から、陽イオン交換樹脂カラム−陰イオン交換樹脂カラムを該順序で利用して平均重合度が８〜１５の酸性キシロオリゴ糖混合物を分離する処理であることを特徴とする（７）項〜（１０）項のいずれか１項に記載の酸性キシロオリゴ糖組成物の製造方法。
（１２）前記酸加水分解処理液からの前記酸性キシロオリゴ糖混合物の分離は、前記陰イオン交換樹脂カラムに吸着させた酸性キシロオリゴ糖を、金属イオンの塩化物の濃度勾配を用いて溶出させ、回収する処理を包含する方法であることを特徴とする（１１）項記載の酸性キシロオリゴ糖組成物の製造方法。
【００３５】
【発明の実施の形態】
本発明者らは、今回、使用する酵素がバチルス属に由来する中性好熱キシラナーゼで、かつ原材料が広葉樹化学パルプであるようなリグノセルロース材料を用いた場合、キシロースの２量体から２０量体にわたるキシロオリゴ糖の分布を有するキシロオリゴ糖成分とリグニン成分の複合体を得ることができること、このキシロオリゴ糖成分中には分子量が大きな５量体から２０量体が比較的多く含まれ、さらには、このキシロオリゴ糖成分中のキシロビオースの存在比はキシロオリゴ糖成分中の約１０質量％以下であることを見出した。また、キシロオリゴ糖成分中には酸性糖を側鎖に有する酸性キシロオリゴ糖が存在すること、そして、この酸性キシロオリゴ糖は濃縮された糖液中の全キシロオリゴ糖成分の約３０質量％程度を占めることを見出した。
一般にオリゴ糖は鎖長が長いほど溶解性が落ちるが、この酸性キシロオリゴ糖は酸性糖を側鎖に持つため、主鎖の重合度が６量体以上であっても溶解度は非常に高い。
【００３６】
化学パルプ由来のリグノセルロースを原料として製造された長鎖キシロオリゴ糖成分はコーンコブや綿実殻を原料として製造されたキシロオリゴ糖よりも平均重合度が高いという特徴がある。リグノセルロースを原料とした場合、使用する酵素を調整することで平均重合度が２から平均重合度が５までの任意の比率で新規なキシロオリゴ糖を作ることがある程度は可能である。しかし、今回の新たな知見は、「キシロオリゴ糖−リグニン複合体」を中間体として使用して酸性キシロオリゴ糖を製造し得ることにある。
【００３７】
キシロオリゴ糖−リグニン複合体は、広葉樹、針葉樹クラフトパルプを酵素処理もしくは物理化学的処理した場合に、その処理液中に見出された化合物であり、文献に記載されていない化合物である。この化合物は２量体から２０量体のキシロオリゴ糖鎖にリグニンが結合した物質である。
キシロオリゴ糖に対してリグニンの分子量は圧倒的に大きいため、それが立体的に邪魔になり、キシラナーゼやキシロシダーゼはキシロオリゴ糖−リグニン複合体を分解できない。そのため、酵素処理を行ってもキシロオリゴ糖−リグニン複合体におけるキシロオリゴ糖は分解されずに残り、比較的長鎖のキシロオリゴ糖がリグニンと結合した状態で溶液中に存在する。
しかし、このキシロオリゴ糖−リグニン複合体は、希酸による酸処理でキシロオリゴ糖とリグニンの間を容易に開裂させることができ、この性質を利用することで重合度が５〜２０量体、特に平均重合度が８〜１５のキシロオリゴ糖組成物を容易に製造することができたものである。
【００３８】
以下、本発明をさらに具体的に説明する。
本発明で言う酸性キシロオリゴ糖組成物とは、その酸性キシロオリゴ糖組成物中における比較的重合度の大きなキシロオリゴ糖の含有率が高い組成物であり、そのキシロオリゴ糖１分子中に少なくとも１つのウロン酸を結合しているキシロオリゴ糖からなる組成物のことを言う。具体的には、組成物中の全糖質量に対して、キシロースの６量体以上のキシロオリゴ糖が８０質量％以上であるものであり、かつウロン酸含量が２質量％以上のものをいう。本発明の酸性キシロオリゴ糖組成物の平均重合度を測定した場合、平均重合度は６以上であり、好ましくは８〜１５である。
【００３９】
中でも、酸性キシロオリゴ糖組成物として製造し易く、性能的にも優れているのは、キシロースの６量体〜２０量体を主成分とするものである。ここで主成分とは、全糖質量に対して５０質量％以上であることを言う。
もちろん使用する酵素や酸処理の条件を変更することで平均重合度がさらに高い値の酸性キシロオリゴ糖組成物を自由に設計製造することが可能なことは言うまでもない。
【００４０】
本発明の組成物における長鎖キシロオリゴ糖成分は、リグノセルロース材料をヘミセルラーゼ処理した後に、希酸によりキシロオリゴ糖−リグニン複合体を酸加水分解することによって得られる重合度の大きなキシロオリゴ糖混合物中に存在する。
リグノセルロースとしてのパルプのヘミセルラーゼ処理は、パルプ濃度１〜３０質量％、好ましくは２〜１５質量％の範囲で行われるが、広葉樹クラフトパルプ以外のリグノセルロースを製造原料に用いる場合は、この限りではない。例えば、リグノセルロースに由来するキシランについて、処理時のパルプ濃度を例示すると、小麦フスマ由来キシランでは０．５〜５質量％、好ましくは２質量％前後、コーンコブ由来キシランでは１〜１０質量％、好ましくは５質量％前後、綿実殼由来キシランでは１〜１０質量％、好ましくは５質量％前後、粉砕コーンパイプでは０．２〜５質量％、好ましくは１質量％前後、カラスムギ由来キシランでは０．２〜５質量％、好ましくは１質量％前後などである。
【００４１】
本発明の酸性キシロオリゴ糖組成物を得ることができる原料リグノセルロース物質としては、針葉樹や広葉樹のような木材が好ましく用いられるが、ケナフ、麻、バガス、イネ等の非木本性の植物であってもよく、特に限定されるものではない。本発明に使用されるパルプは、化学パルプ、機械パルプ、脱墨パルプ等何でもよいが、広葉樹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を用いることができるが、パルプ品質、エネルギー効率等を考慮するとクラフト蒸解法が好適に用いられる。
【００４２】
リグノセルロース材料として広葉樹クラフトパルプを用いる場合、まず、アルカリ酸素漂白工程で漂白したパルプをヘミセルラーゼで処理することが望ましいが、蒸解後のパルプや、機械パルプをそのままヘミセルラーゼ処理原料として用いても良い。
【００４３】
クラフト蒸解で得られたパルプ表面には、蒸解工程でパルプ繊維内より溶出されたヘミセルロースが再吸着されていることは周知である。この再吸着されたヘミセルロースは、その９０質量％以上がＤ−キシロースがβ１→４結合することによって構成されたキシランである。
広葉樹クラフトパルプでは、ヘミセルロース中の側鎖であるアラビノースやグルクロン酸はそのほとんどが分解除去されている。また、側鎖の中の４−０−メチルグルクロン酸は側鎖として残存するが、アルカリ条件下でヘキセンウロン酸へと変換される。このヘキセンウロン酸は、酸性条件下で加熱すると容易に分解除去されるのでキシロオリゴ糖の製造にはあまり問題にならない。
【００４４】
化学パルプ表面の再吸着キシランは、通常の植物中の細胞壁内に存在するキシランと違ってパルプの蒸解工程において抽出された際に、その主鎖であるキシランに結合している側鎖の大部分は分解除去されている。そのため、再吸着キシランは、通常の細胞壁中のキシランと比べて側鎖の保有率が非常に低く、キシロース１０個〜２０個に１個程度のグルクロン酸もしくは４−０−メチルグルクロン酸を結合している。また、後述するキシロオリゴ糖−リグニン複合体を形成しているため、再吸着キシランをキシラナーゼで分解、除去するとパルプスラリーにこのキシロオリゴ糖−リグニン複合体が溶出してくるため反応液が茶色になる。この茶色の反応液中には２８０ｎｍの吸収を持つ物質が存在するが、これはリグニンの芳香環に由来する吸収であると考えられている。
【００４５】
再吸着分を含めたキシラン含量は、広葉樹クラフトパルプ絶乾質量の約２０質量％を占める。ヘミセルラーゼ処理においては、酵素が広葉樹クラフトパルプに作用し、再吸着分を含むキシラン全般に作用してこれを低分子化する。例えばバチルス・エスピ−Ｓ−２１１３株のキシラナーゼ（特開平８−２２４０８１号公報参照）を利用する場合、処理反応液中に生じるキシロース及びキシロオリゴ糖の構成糖の割合は、３〜５量体が最も多く、単量体が少ない組成比のオリゴ糖を生成する。
【００４６】
本発明者らは、すでに、広葉樹クラフトパルプのヘミセルラーゼ処理工程より得られる排水中にキシロオリゴ糖とリグニン様物質が結合したキシロオリゴ糖複合体が存在することを見出している。更には、キシロオリゴ糖複合体は比較的重合度の大きなオリゴ糖にリグニン様の物質が結合していることを見出している。このキシロオリゴ糖−リグニン複合体は希酸処理により容易にキシロオリゴ糖とリグニン様物質に分解できるので、重合度の大きなキシロオリゴ糖を大量安価に製造できる。キシロオリゴ糖−リグニン複合体から製造されるキシロオリゴ糖組成物の中で、ウロン酸残基を側鎖に有する酸性キシロオリゴ糖の占める割合は、全糖量に対して約３０質量％にもなる。
【００４７】
現在のところ、大規模な酵素処理工程で利用されている酵素は、そのほとんどがヘミセルラーゼであるが、市販のヘミセルラーゼのいずれも本発明のキシロオリゴ糖の製造方法における酵素処理工程に用いることができる。例えば、商品名カルタザイム（クラリアント社製）、商品名エコパルプ（ローム・エンザイム社製）、商品名スミチーム（新日本化学工業社製）、パルプザイム（ノボノルディクス社製）マルチフェクト７２０（ジェネンコア社）などの市販の酵素製剤や、トリコデルマ属、テルモミセス属、オウレオバシヂウム属、ストレプトミセス属、アスペルギルス属、クロストリジウム属、バチルス属、テルモトガ属、テルモアスクス属、カルドセラム属、テルモモノスポラ属などの微生物により生産されるキシラナーゼを使用することができる。
【００４８】
酵素処理温度は、１０〜９０℃、好ましくは３０〜６０℃の範囲であるが、酵素の至適温度に近い処理温度がより好ましい。一般的な酵素の場合、処理温度が１０℃未満では反応が不十分となる上、そのような温度を得ること自体に多大のコストを要するので適さない。一方、温度が９０℃を超えて高くなると、処理系を密閉化しないと熱ロスが大きくなる上、一般的な酵素の場合、酵素自体が変性し、不活性になるので適さない。処理時の溶液ｐＨは３〜１０、好ましくは５〜９の範囲であるが、酵素の至適ｐＨに近い方がより好ましい。
【００４９】
広葉樹クラフトパルプをアルカリ酸素漂白して得られるパルプを酵素処理して糖液を得る場合、パルプのｐＨがアルカリ側に傾いているため、酵素の至適ｐＨがアルカリ側に近い酵素の方がｐＨを調整する際のコストも低く優位性がある。もし、ｐＨの調整が必要な場合は、任意の酸性溶液又はアルカリ性溶液を添加して調整し、酵素処理を行えばよいことは言うまでもない。
【００５０】
酵素処理により得られた糖液中には、キシロオリゴ糖（２〜２０量体）とキシロオリゴ糖−リグニン複合体が含まれる。酵素処理液中の糖濃度は、バチルス・エスピー２１１３株（独立行政法人 産業技術総合研究所 特許微生物寄託センター寄託菌株ＦＥＲＭ ＢＰ−５２６４）の生産するキシラナーゼを対パルプ絶乾質量当たり１ユニット（１ユニットは、１分間に１マイクロモルのキシロースを遊離させる酵素力）で使用し、１０質量％濃度のパルプスラリー中に添加して処理した場合、約３０００μｇ／ｍｌ（キシロース換算）である。
【００５１】
この糖溶液は、後段の製造工程を考慮に入れた場合、荷電ＮＦ膜やその他の限外ろ過膜、逆浸透膜などの膜分離技術を用いて濃縮したり、エバポレーション等の濃縮作業により糖濃度を上昇させる作業を行うことも可能である。実際に糖液のボリュームを減らすことは、大量の糖液を後段の精製工程で処理する際のハンドリングを容易にする。加えて言うならば、膜濃縮における作業より得られた透過液は、糖濃度が酵素処理液より低く、リグニン等着色性の有機物含量が少ない特徴を持つ。このため、膜濃縮工程より得られる透過液はパルプ製造工程における工業用水として再利用できる。
【００５２】
糖溶液もしくは濃縮処理工程後の糖溶液については、希酸による酸加水分解処理を行ってキシロオリゴ糖−リグニン複合体をキシロオリゴ糖と酸性キシロオリゴ糖、そしてリグニン様物質とに分離する。糖液のｐＨの調整方法としては、糖液に対して鉱酸もしくは有機酸を適宜添加して糖液のｐＨを３．５付近に調整することが一般的であるが、アンバーライト２００Ｃ（商品名、ローム・アンド・ハース社製）といったカチオン交換樹脂で糖液を処理してイオン交換によりｐＨを下げることも可能である。
【００５３】
その後、ｐＨ調整の終わった糖溶液を１０５℃〜１５０℃、好ましくは１１０℃〜１３０℃の範囲で加熱し、酸加水分解の処理を行う。処理時間は１５分以上であるが、好ましくは３０分から６０分である。加熱処理時間を９０分以上に設定するとオリゴ糖の単糖への分解が進み好ましくはない。糖液のｐＨが３．５付近である場合、キシロオリゴ糖複合体とリグニン様物質、キシロオリゴ糖と側鎖の一種であるヘキセンウロン酸は分離除去可能であるが、酸性キシロオリゴ糖自身はほとんど分解することはない。
【００５４】
この処理で、キシロオリゴ糖−リグニン複合体からはリグニン様の有機物が分解除去され、酸性キシロオリゴ糖とキシロオリゴ糖へと変換される。ｐＨ３．５、１２１℃、６０分の処理条件の時のキシロオリゴ糖複合体から酸性キシロオリゴ糖とキシロオリゴ糖への変換効率は約９５％である。このとき、単糖の一部は加水分解が進みフルフラール様物質となり更に縮合して沈殿する。キシロオリゴ糖複合体から切り離されたリグニン様物質も、同様に酸性下で縮合し不溶化して沈殿する。この不溶化した沈殿物は濾紙や珪藻土によるろ過はもちろんのこと、ＵＦ膜やＭＦ膜そしてセラミックフィルター等による分離除去が可能である。
【００５５】
上記のようにしてキシロオリゴ糖−リグニン複合体から得られた酸性キシロオリゴ糖組成物は、比較的鎖長が長い重合度が６〜２０程度のキシロオリゴ糖を高い割合で含んでいる新規な酸性キシロオリゴ糖組成物である。重合度が比較的高い酸性キシロオリゴ糖が得られる理由としては、酵素処理により得られた糖液中のキシロオリゴ糖複合体は２量体から２０量体程度の鎖長の酸性キシロオリゴ糖にリグニン様物質が結合しているため、ヘミセルラーゼによる必要以上の消化を免れていることに起因している。そのような状態から希酸による酸加水分解でリグニン様物質と分離すると、比較的長い鎖長の酸性キシロオリゴ糖が得られる。
【００５６】
酸加水分解して得られた糖液中には、酸性キシロオリゴ糖の他にキシロース、グルコースといった単糖類やリグニン、フラン化合物、フルフラールといった有機物も含まれる。これらの有機物の混合物からキシロオリゴ糖のみを分離、精製する工程としては、イオン交換、分子ふるい、エタノール分画、膜処理などの従来のいかなる精製方法を組み合わせて用いても良い。例えば、活性炭→強カチオン交換樹脂→強アニオン交換樹脂→強カチオン交換樹脂→弱アニオン交換樹脂といった順序でカラムを用いる精製方法では、出発原料である酸処理糖液中の総糖量を１００質量％とした場合、精製酸性キシロオリゴ糖自身の回収率は約３０質量％である。
【００５７】
濃縮糖液を出発原料として酸性キシロオリゴ糖組成物を精製する方法は、次のとおりである。まず、セラミックフィルターを用いて糖液をろ過し比較的分子量の大きな不溶性着色物や希酸処理で得られたリグニン縮合物を除去する。この時糖液の供給側に活性炭を対糖量で数質量％添加してセラミックフィルター処理を行うと、より小さな着色物質などは活性炭に吸着されながらろ過されるので得られたろ過糖液の清澄度が増して後の精製工程が楽になる。
【００５８】
セラミックフィルターでろ過された糖液は、更に分画分子量が２００００以下のＵＦ膜を用いてろ過する。この工程では、更に小さな着色物を除去できる。濃縮側に残された着色物及び重合度が非常に大きなキシロオリゴ糖とリグニンの複合体は廃棄するべきではなく、できる限り前段階のセラミックフィルターを用いた処理における供給側の糖液に戻し、回収率を上げることが好ましい。
【００５９】
セラミックフィルター処理、ＵＦ膜による処理を経て得られた糖液中にはキシロオリゴ糖組成物と酸性キシロオリゴ糖組成物が溶解している。この糖液から酸性キシロオリゴ糖組成物のみを取り出す方法はイオン交換樹脂を用いる方法が適している。糖液を、まずカチオン交換樹脂にて処理し、糖液中の金属イオンを除去する。次いで、強アニオン交換樹脂を用いて糖液中の硫酸イオンなどを除去する。この工程では、硫酸イオンの除去と同時に弱酸である有機酸の一部と着色成分の除去も同時に行っている。
【００６０】
強アニオン交換樹脂で処理された糖液は、もう一度カチオン交換樹脂で処理し、更に金属イオンを除去する。そして、最後に弱アニオン交換樹脂で処理し、着色物質と酸性キシロオリゴ糖を樹脂に吸着させる。中性糖であるキシロオリゴ糖は、このとき弱アニオン交換樹脂には吸着されずそのまま回収され、このときの中性キシロオリゴ糖組成物の回収率は約７０質量％前後に落ち着く。
【００６１】
弱アニオン交換樹脂に吸着された酸性キシロオリゴ糖は、金属塩を溶解した溶出液を用いて弱アニオン交換樹脂から溶出して回収できる。カラムを用いて吸着した酸性キシロオリゴ糖を溶出する場合は、グラジエント操作を行い適正なイオン強度にて樹脂から溶出させることも可能である。また、アルカリを用いて溶出させることも可能である、しかし、このときは溶出画分に着色成分が混入してくる比率が高まるので、後段で着色成分を除去する操作がもう一段必要となる。
【００６２】
金属塩の溶液で酸性キシロオリゴ糖を溶出・回収する際の金属塩は、基本的に何を用いても良い、しかし、回収された酸性キシロオリゴ糖が生理活性を発揮するためには、生物に摂取されることが必須であるため、生体にあまり害を与えないような金属塩が望ましいことはいうまでもない。例えば、塩化ナトリウム、塩化カリウム、塩化カルシウム、塩化マグネシウム、塩化鉄などが挙げられる。その際に、日常では摂取しにくい金属塩を用いて溶出し、酸性キシロオリゴ糖の摂取と同時に、ミネラルとしてこれらの金属塩を同時に摂取できるように酸性キシロオリゴ糖を設計することも可能である。具体的には、近年、日本人はカルシウムやマグネシウムの摂取量が不足がちであるので、カルシウム：マグネシウムのモル比を適当に設定した溶出液を作製し溶出を行い、酸性キシロオリゴ糖の回収することもできる。
【００６３】
精製された酸性キシロオリゴ糖を含む糖溶液をイオンクロマトグラフィー（ダイオネクス社）を用いて分析したところ、２量体ないし２０量体及びそれ以上の酸性キシロオリゴ糖を含む糖液であることが判明した。このときの有機分質量を分析したところ絶乾質量中の全糖量は９５質量％前後であった。また、秤量されたるつぼを用いての灰分の測定では、精製糖液中の灰分は５質量％前後であった。灰分中の金属の酸化物は、溶出に用いた金属塩に由来すると思われる金属がほとんどを占めていた。
【００６４】
前述したように、本発明の新規な酸性キシロオリゴ糖組成物は、リグノセルロース材料を出発原料とし、それをヘミセルラーゼ処理した反応ろ液から分離、精製して得られる重合度の大きな酸性キシロオリゴ糖含有組成物である。また、この重合度の大きな酸性キシロオリゴ糖組成物は、酵素処理や爆砕などの物理化学的手法を用いることで、既知のキシロビオース、キシロトリオース、キシロテトラオース、キシロペンタオースを主鎖の主成分とする酸性キシロオリゴ糖などに容易に変換することもできる。
【００６５】
【実施例】
以下に、実施例及び比較例を挙げて本発明を具体的に説明するが、もちろん、本発明はこれら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％を意味するものであり、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である。なお、各測定法は以下のとおりである。
【００６６】
（１）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）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 発行）にて定量した。
（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）を用いて作製、ソモジ−ネルソン法（「還元糖の定量法」学会出版センター 発行）にて定量した。
【００６７】
（３）ウロン酸量の定量：
ウロン酸は、検量線をＤ−グルクロン酸（和光純薬工業）を用いて作製、カルバゾール硫酸法（「還元糖の定量法」学会出版センター 発行）にて定量した。
（４）平均重合度の決定法：
サンプル糖液を５０℃に保ち、１５０００ｒｐｍにて１５分遠心分離し不溶物を除去し、上清液の全糖量を還元糖量（共にキシロース換算）で割って平均重合度を求めた。
【００６８】
（５）酸性キシロオリゴ糖の定量法
酸性オリゴ糖１分子中のウロン酸残基とキシロース残基の量比は核磁気共鳴装置（以下NMR、日本電子（株）製）用いて行った。サンプルは凍結乾燥、重水素置換を行った後、測定を行った。１Ｈ ＮＭＲ, １３Ｃ ＮＭＲ, ＨＭＱＣ測定によりグリコシド結合をしているキシロース１位（４．４０９ｐｐｍ）とウロン酸１位（５．２２６ｐｐｍ）のプロトンケミカルシフトを決定した。水素の量比に相当する各シグナルの面積を算出しグルクロン酸とキシロースの比率を求めた。また、高速液体クロマトグラフィー質量分析装置（アプライドバイオシステム社製）によって高分解能分子量測定を行いオリゴ糖１分子当たりのウロン酸残基数を求めた。
（６）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、カバキシラン（シグマ社製）を用いた。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」学会出版センター発行）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１ユニットとした。
【００６９】
（７）イオンクロマトグラフによる分析:
キシロオリゴ糖の分析には、イオンクロマトグラフ（ダイオネクス社）を用いた。分析には、糖類の分析に適したカラムとしてＣａｒｂｏ Ｐａｃ ＰＡ−１０（ダイオネクス社）を用いた。
【００７０】
実施例１
<酵素処理工程１>
国内産広葉樹チップ７０％、ユーカリ材３０％からなる混合広葉樹チップを原料として、クラフト蒸解によりカツパー価２０．１、パルプ粘度４１ｃｐｓの工場製の未晒パルプを得た。次いで、酸素脱リグニンを行い、カツパー価９．６、パルプ粘度２５．１ｃｐｓの酸素脱リグニンパルプを得た。
このパルプをパルプ濃度１０％に調整後、希硫酸を加えてｐＨ６．７に調整し、次いでバチルス・エスピーＳ−２１１３株（独立行政法人 産業技術総合研究所 特許微生物寄託センター寄託菌株ＦＥＲＭ ＢＰ−５２６４）一の生産するキシラナーゼを対パルプ１ユニット／ｇとなるように添加し、６０℃で１２０分処理した。処理後、全糖濃度３０００ｍｇ／リッターを含む７６０００リッター（全糖量として２２８ｋｇ）の処理液を得た。続いてＮＦ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、食塩阻止率５０％）を用いて容量比で４０倍に濃縮後、１９００リッターの糖液を回収した。この濃縮液は全糖量を１９０ｋｇ含み、全糖回収率は８３．８％であった。濃縮液中の糖をイオンクロマトグラフィーを用いて分析した結果、中性のキシロオリゴ糖及び溶出時間２４分以降にキシロオリゴ糖複合体のピークが認められた。これを図１に示す。
【００７１】
実施例２
<酵素処理工程２>
酵素としてジェネンコア社のキシラナーゼ（マルチフェクト７２０）を用いてキシロオリゴ糖複合体を生成する実験を行った。酵素処理の実験は実施例１と全く同じパルプ、全く同じ条件で行った。反応生成物をイオンクロマト用カラム（ダイオネクス社:ＣａｒｂｏＰａｃＰＡ−１０）を用いたイオンクロマトグラフィーで分析した結果、実施例１と同じように高濃度のキシロオリゴ糖（２量体〜２０量体）を含むことが判明した。この時、酸性キシロオリゴ糖のピークも一部見られる。これを図２に示す。
【００７２】
実施例３
＜酸加水分解処理工程１＞
酵素処理工程で得られた濃縮糖液１９００リッターに対して硫酸を添加してｐＨを３．５に調整した後、この濃縮糖液を１２１℃にて１時間反応させた。反応生成物をイオンクロマト用カラム（ダイオネクス社：Ｃａｒｂｏ Ｐａｃ ＰＡ−１０）を用いたイオンクロマトグラフィーで分析した結果、高濃度のキシロオリゴ糖（２量体〜２０量体）を含むことが判明した。この時、酸性キシロオリゴ糖のピークも一部見られる。これを図３に示す。
【００７３】
実施例４
＜酸加水分解処理工程２＞
実施例１と全く同様にして得られた濃縮糖液１０００リッターに酢酸を添加してｐＨを３．５に調整し、１２１℃にて１時間反応させた。反応生成物をイオンクロマト用カラム（ダイオネクス社：Ｃａｒｂｏ Ｐａｃ ＰＡ−１０）を用いたイオンクロマトグラフィーで分析した結果、高濃度のキシロオリゴ糖（２量体〜２０量体）を含むことが判明した。この時酸性キシロオリゴ糖のピークも一部見られる。これを図４に示す。
【００７４】
実施例５
＜酸性キシロオリゴ糖の分離・精製工程＞
▲１▼セラミックフィルター処理
酸加水分解処理工程１で調製したキシロオリゴ糖の糖溶液（１００ｍｇ／ｍｌ）１９００リッター、全糖量として１９０ｋｇをろ過面積４．８ｍ２のセラミックフィルターを用いてろ過した。ろ過の条件は液温６０℃にて行い、エアーによる逆洗浄は５分間隔で行った。また、活性炭を対糖３％にて添加してろ過を行った。８時間の連続処理で１９０００リッターを全て処理し、８時間処理での平均Ｆｌｕｘは４０リッター／Ｈｒ／ｍ２であった。
【００７５】
▲２▼イオン交換樹脂による精製
イオン交換には４塔のイオン交換塔（各塔２００リッター容）を使用した。各塔は１２５リッターの樹脂を含むカラムであり、順番に１塔（強カチオンイオン交換樹脂：ＰＫ２１８、三菱化学製）、２塔（強アニオン＋弱アニオン：ＰＡ４０８＋ＷＡ３０＝１：１の比率でミックス、両樹脂とも三菱化学製）、３塔（強カチオンイオン交換樹脂：ＰＫ２１８）、４塔（弱アニオンイオン交換樹脂：ＷＡ３０）である。この４塔のカラムを連続的に用い脱色、脱塩を行った。サンプルはセラミックフィルター処理後の糖液を純水で希釈し（５．０％濃度：６００リッター）使用した。その結果、サンプル６００リッターを通液流速３リッター／ｍｉｎで行い、酸性キシロオリゴ糖を弱アニオンイオン交換樹脂に吸着させた。溶出は７５ｍＭのＮａＣｌ（塩化ナトリウム）水溶液を２００リッター用いて３リッター／ｍｉｎの通液速度で溶出し４．０％濃度の酸性キシロオリゴ糖を１２５リッター、５．０ｋｇの酸性キシロオリゴ糖を回収した。このときの回収率は全糖量に対して３０％であった。この、酸性キシロオリゴ糖は３５％濃度にまで濃縮後、スプレードライヤーで粉体化した。粉体化した酸性キシロオリゴ糖は４．８ｋｇ回収され、回収率は９６．０％であった。この酸性キシロオリゴ糖をイオンクロマトグラフィーにて分析した結果を図５に示す。また、こうして得られた酸性キシロオリゴ糖中のウロン酸の定量を行った結果、ウロン酸：キシロースの質量比は１：１０であった。
【００７６】
実施例６
実施例５と全く同様にして得たセラミックフィルター処理後の糖化（５．０％濃度）６００リッターを４塔のカラムに連続して通液した。通液条件は３リッター／ｍｉｎで行い、弱アニオン交換樹脂に酸性キシロオリゴ糖を吸着させた。溶出には５０ｍＭのＣａＣｌ２（塩化カルシウム）３００リッターを用いて溶出し、溶出液の通液速度は３リッター／ｍｉｎで行った。回収された酸性キシロオリゴ糖は７．５％の濃度で１３４リッターの容量で回収され、回収量は１０ｋｇであり、回収率は３３％であった。
【００７７】
実施例７
＜酸性キシロオリゴ糖の重合度＞
実施例５及び実施例６により精製された酸性キシロオリゴ糖の粉末を超純水に溶解し１％水溶液を作製した。平均重合度は全糖量をフェノール硫酸法で測定し、その後１％水溶液の還元糖量をソモジーネルソン法で測定した。
いずれの測定においても、検量線はＤ−キシロースを用いて作製した。平均重合度は１ｍｌ当たりの全糖量を１ｍｌ当たりの還元糖量で割ることで求めた。その結果、上記方法で作製された塩化カリウムにて溶出された酸性キシロオリゴ糖の平均重合度は１０．２であり、塩化カルシウムにて溶出された酸性キシロオリゴ糖の平均重合度は１１．３であった。
【００７８】
実施例８
実施例５により得られた精製された酸性キシロオリゴ糖１０ｇを１００ｍｌの超純水に溶解し１０％水溶液を作製した。この酸性キシロオリゴ糖水溶液に対して７０％濃度となるようにエタノールを添加した。これを冷蔵庫に一晩放置することで沈殿してくる酸性キシロオリゴ糖と沈殿しない７０％エタノールに可溶の酸性キシロオリゴ糖に分画することが出来た。沈殿と７０％エタノールに可溶性画分とを遠心分離で分離し、おのおの平均重合度を測定したところ７０％可溶性画分は平均重合度が約４．２であり、沈殿した酸性キシロオリゴ糖の再溶解液の平均重合度は１２．８であった。
【００７９】
＜核磁気共鳴装置、質量分析装置での分析＞
上記のうち、７０％エタノール可溶性画分について、エタノールを除去し、純水の溶液として、ＮＭＲ法により重合度及びオリゴ糖１分子中のウロン酸残基とキシロース残基の量比を分析した。キシロース１位（４．４０９ｐｐｍ）とウロン酸１位（５.２２６ｐｐｍ）のプロトンケミカルシフトを決定し、水素の量比に相当する各シグナルの面積を算出した結果、その面積比は５：１であった。従って、ほとんどがキシロースの５量体に４−０−メチルグルクロン酸が一つ結合した酸性キシロオリゴ糖であることが判明した。
【００８０】
更に参考までに、この水溶液を質量分析装置にかけてキシロースと４−０−メチルグルクロン酸のモル比を求めると５：１でありＮＭＲの結果と一致した。
なお、このときの可溶性画分の糖含量はフェノール硫酸法及びカルバゾール硫酸法で測定した場合、初発の１０ｇの酸性キシロオリゴ糖に対して０．４４ｇであった。
これらのことから実施例５により得られた精製された酸性キシロオリゴ糖は重合度が５量体以下のものが４．４％であり、その大部分が５量体であることが判明した。
【００８１】
次に、７０％エタノール沈殿由来の酸性キシロオリゴ糖をエタノールを除去し、純水の溶液とし、ＮＭＲ法により前記と同様に重合度及びオリゴ糖１分子中のウロン酸残基とキシロース残基の量比を分析した。
キシロースと４−０−メチルグルクロン酸のモル比を測定すると１０：１であり、キシロースの１０量体に１つの４−０−メチルグルクロン酸残基が結合した酸性キシロオリゴ糖であった。更に参考までに、この水溶液を質量分析装置にかけてキシロースと４−０−メチルグルクロン酸のモル比を求めると１０：１でありＮＭＲの結果と一致した。
【００８２】
【発明の効果】
本発明により、鎖長が長い酸性キシロオリゴ糖が大量、安価に供給される。この新規酸性キシロオリゴ糖組成物は、酸加水分解、酵素消化などの処理により、容易にキシロビオース、キシロースを主鎖とする酸性キシロオリゴ糖に変換できる。また、中性のキシロオリゴ糖と共に酸性キシロオリゴ糖はビフィズス菌の選択的増殖性があり、機能性食品の材料にも有望である。また、本発明の酸性キシロオリゴ糖組成物は、通常なら溶解度が低い長鎖中性キシロオリゴ糖が主鎖ではあるが、酸性糖鎖を有するために溶解度が非常に高く、製造が容易で、ドリンクや食品への応用が容易な組成物である。
【図面の簡単な説明】
【図１】実施例１のキシロオリゴ糖のイオンクロマトグラフによる分析図。
【図２】実施例２のキシロオリゴ糖のイオンクロマトグラフによる分析図。
【図３】実施例３のキシロオリゴ糖のイオンクロマトグラフによる分析図。
【図４】実施例４のキシロオリゴ糖のイオンクロマトグラフによる分析図。
【図５】実施例５のキシロオリゴ糖のイオンクロマトグラフによる分析図。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an acidic xylo-oligosaccharide composition used as a health food, food additive, medicine / cosmetic additive, agricultural growth promoter and the like. In particular, the present invention relates to an acidic xylo-oligosaccharide composition comprising a mixture of an acidic xylo-oligosaccharide having a xylose oligomer as a main chain and a uronic acid residue as a side chain, and a method for producing the same.
[0002]
[Prior art]
Due to increasing public health concerns, attempts to maintain health through daily food intake are attracting attention. In particular, polysaccharides such as dietary fiber and oligosaccharides, and their degradation products have attracted attention for their physiological activity exhibited in their digestive organs, and are also fields that have been intensively studied in recent years.
Dietary fiber has traditionally been regarded as an important food factor related to basic metabolism in the body, such as carbohydrate metabolism and lipid metabolism. There are many functional foods that claim the action of dietary fiber.
[0003]
Dietary fiber has the function of diluting carcinogens in the intestine by improving the stool and adsorbing carcinogens, and has recently been regarded as an important human food factor. Some conventional dietary fibers have the potential to improve human hyperlipidemia or to control human health by controlling glucose metabolism. In particular, it has been reported that dietary fibers rich in acidic sugars such as tamarind seed gum, guar gum, sodium alginate and pectin reduce the amount of total cholesterol, phospholipids and triglycerides in the blood and improve hyperlipidemia.
(Rotenberg, S. and Jakobsen, P, E .: The effect of dietary pectin on lipid composition of blood, skeletal muscle and internal organs of rats.J. Nutr., 108, 1384 (1978))
[0004]
However, these dietary fibers are not sugars that suppress the growth of E. coli and promote the selective growth of bifidobacteria. Rather, the mucosa cells of the small intestine are physically scraped, often having a negative effect on nutrient absorption. (Fahey Jr., G.C .: Dietary fiber; chemistry and nutrition, p.117-146, Academic Press, New York (1979))
[0005]
Oligosaccharides, on the other hand, have a function to keep the stomach in good condition through the selective growth promotion effect of useful bacteria in the intestines, and are used in lactic acid bacteria beverages that have been certified as foods for specified health use. Is also a useful sugar.
In addition to human food use, it is also used as livestock feed. Furthermore, in the fields of pharmaceuticals and sanitary products, they have applications as emulsifiers and skin moisturizing ingredients.
[0006]
In general, most of the oligosaccharides used in foods for specified health use are intestinal-regulating, that is, reducing the number of bacteria of the genus Clostridium and Eubacterium, which are E. coli, which is an enteric bad bacteria, It has the effect of increasing the number of bifidobacteria, which are said to be relatively good intestinal bacteria.
Among various oligosaccharides, for example, xylooligosaccharides derived from wheat bran and corn cob are famous. As for the action of xylo-oligosaccharides, it is said to promote the selective growth of Bifidobacterium, a good enteric fungus, as well as other oligosaccharides, while relatively reducing the number of E. coli that are enteric bad bacteria. .
It is known that Escherichia coli and intestinal rot-fermenting bacteria produce carcinogens while growing in the intestine [Kanazawa et al .: Colonic bacterial flora-especially bile acid metabolism and colon carcinogenesis-. 1042-1050 (1977)] Therefore, it is important to reduce the number of Escherichia coli and enteric spoilage bacteria in the intestine when considering long-term health.
[0007]
In recent years, among the physiological activities of oligosaccharides, there has been particularly remarkable progress in research on immune effects in the intestinal tract. In particular, it is known that bifidobacteria selectively grown by xylo-oligosaccharide has an intestinal immunity enhancing action. B cells existing under the mucosal cells of the intestine produce a substance called IgA (immunoglobulin A) that is involved in defense of infection and suppression of food allergy, and secrete this into the intestine to protect the mucosal cells. . Components derived from the cell walls of Bifidobacterium act on B cells existing under the small intestinal mucosal cells via macrophages and work to produce IgA, a component that controls local immunity.
[0008]
For this reason, by controlling the bifidobacteria present in the intestinal tract by ingesting oligosaccharides, it is possible to protect against infections such as pathogenic bacteria entering from the outside, or neutralize even when allergenic substances are brought orally There is a possibility that production of IgA acting as an antibody is enhanced. [Hisako Yasui, Noriko Kato, Akito Mige, Kazuhito Hayakawa, Makoto Daikyo, Atsuhiko Tsuji. 1991. Enhancement of the intestinal tract immune system (IgA antibody production) by oral administration of Bifidobacterium breve. Journal of Japanese Society of Agricultural Chemistry 65: 623]
[0009]
In the case of ingesting oligosaccharides orally in anticipation of intestinal regulation, a major problem is the decrease in the degree of polymerization of oligosaccharides resulting from acid hydrolysis by enzymes in stomach acid and digestive fluid. It is known that oligosaccharides are gradually reduced in molecular weight by hydrolysis with acids and enzymes, and finally decomposed into monosaccharides that can be assimilated by spoilage anaerobes belonging to the genus Escherichia coli and Clostridium. It has been.
However, the xylooligosaccharide composition mainly composed of a dimer or a trimer has a relatively high resistance to gastric acid among oligosaccharides compared to other oligosaccharides (Japanese Patent No. 2549638) and is not degraded so much. Likely to be delivered to the intestines. When xylooligosaccharide is actually administered to humans, the intestinal regulating effect in vivo has been confirmed.
[0010]
At present, xylo-oligosaccharides having an average degree of polymerization of about a pentamer having a molecular weight much higher than that of a dimer or trimer (Japanese Patent Laid-Open No. 2001-226409) can be produced. The present inventors have also proposed a long-chain xylo-oligosaccharide having an average degree of polymerization of around 12-mer (Japanese Patent Application No. 2001-242906). Xylooligosaccharides having a long chain length are not easily converted to xylose, which is a monosaccharide even in the intestine, and the amount of excreted stool increases compared to xylobiose.
[0011]
In particular, long-chain xylo-oligosaccharide has a high average degree of polymerization and has physiological activities of both oligosaccharide and dietary fiber. That is, the anti-hyperlipidemic effect, which is a dietary fiber-like effect, is exhibited in the small intestine, and the intestinal regulating effect is exhibited by being selectively assimilated by bifidobacteria in the large intestine.
[0012]
However, long-chain xylo-oligosaccharides that exhibit these excellent physiological activities also have one problem. The problem is a solubility problem. The solubility of neutral xylo-oligosaccharides decreases as the degree of polymerization increases. In particular, those having an average degree of polymerization of around 12-mer are useful carbohydrates that have both dietary fiber-like and intestinal regulating effects, but neutral xylooligosaccharide 12-mer is water at 60 ° C. Only a few percent dissolve. In order to exert intestinal regulation and intestinal immunity, it is necessary to deliver it to the intestinal tract after being dissolved in water to some extent, and it is no exaggeration to say that the problem with long-chain oligosaccharides is solubility. In addition, the low solubility is a big problem in terms of oligosaccharide production efficiency, and it has been desired to develop a xylo-oligosaccharide having a long chain and high water solubility.
[0013]
Among dietary fibers, there is a family of polysaccharides that include acidic sugars as saccharides that improve blood properties as physiological activities. Since polysaccharides including acidic sugars change lipid metabolism even with a relatively low intake, they have been used very often in research experiments for antihyperlipidemia. More specifically, many polysaccharides and dietary fibers that contain acidic sugars have an effect of lowering cholesterol in serum, and compared with the group that received the control (food without acid sugar) in the experiment. Thus, the group in which a diet containing acidic sugar is administered seems to have about 20-30% lower cholesterol in blood. [Reiko Tsuji et al .: Nutrition Journal, 33, 273 (1975), Keisuke Tsuji et al .: Nutrition Journal, 35, 227 (1977)]
[0014]
As can be seen from these experiments, dietary fiber containing acidic sugar has been studied for the improvement of blood properties. In particular, pectin is a representative dietary fiber containing acidic sugars, and is an acidic polysaccharide often used not only in research on antihyperlipidemia but also in research on the inhibitory action on blood insulin elevation.
(Jenkins, D.J.A., Leeds, A.R., Gassull, M.A., et al .: Decrease postprandial insulin (and glucose concentrations by guar and pectin. Annual. Internal. Med., 80, 20 (1977))
Inhibition of saccharide absorption is thought to occur because acidic polysaccharides absorb and swell water in the stomach and take up sugar in the gel, so that mucosal cells in the small intestine cannot take up carbohydrates sufficiently.
[0015]
In general, acidic polysaccharides have a hydrophilic functional group such as a carboxyl group or a sulfate group in their sugar chains, so that they easily dissolve in water, and many of them dissolve well in water for a long chain length. One insoluble dietary fiber also has an antihyperlipidemic action and a carbohydrate absorption inhibitory action, but these damage the duodenal mucosal cells when moving from the stomach to the duodenum. For this reason, absorption of a sugar content is suppressed and an increase in blood glucose is suppressed. In view of the health level of the living body, damaging mucosal cells is not always a good measure, but rather seems to be a cause of pathogen infection and poor nutrition. For this reason, it is desirable that the physiological activity of dietary fiber such as antihyperlipidemic action and blood insulin elevation inhibiting action is also exhibited with “soluble dietary fiber” as much as possible. Therefore, polysaccharides and oligosaccharides, including acidic sugars with high water solubility and abundant physiological activity, are considered to be industrially important carbohydrates that can be applied to functional foods and pharmaceuticals. .
[0016]
Xylooligosaccharides are usually neutral sugars whose solubility decreases with increasing chain length. However, it is known that xylo-oligosaccharides can be purified in the form of having an acidic sugar in the side chain under different production conditions, and the water solubility is increased. Conventionally, it is known that xylan in hemicellulose in a plant cell wall has a glucuronic acid residue or a 4-0-methylglucuronic acid residue as a side chain. When xylan with side chains is treated with an enzyme solution produced by mold belonging to the genus Trichoderma, α-glucuronidase decomposes and removes the side chains, resulting in xylobiose or xylotriose as the main component. It seems to make a sugar composition. At this time, the acidic xylo-oligosaccharide containing an acidic saccharide moiety that has been prevented from being digested by α-glucuronidase is a dimer or trimer, and a glucuronic acid residue or 4-0- It is known that methyl glucuronic acid residues are present. (R.F.H.Dekker, In "Biosythesis and biodegradation of wood components", p.505, Academic Press Inc. (1985))
[0017]
On the other hand, when xylo-oligosaccharides are produced using a xylanase that is produced by bacteria, the composition ratio of the produced xylo-oligosaccharides is dimer from xylobiose because the substrate specificity of the enzyme is greatly different from that of fungal xylanases. It is known to produce xylooligosaccharides with a distribution of up to a pentamer (Japanese Patent Laid-Open No. 1-252280). The enzyme derived from bacteria is an acidic xylooligosaccharide in which glucuronic acid or 4-0-methylglucuronic acid, which is an acidic sugar, is bound to xylose on the non-reducing end as one side chain. Is generated.
[0018]
It has been found that xylo-oligosaccharides present in a sugar solution obtained by treating hardwood kraft pulp with xylanase and forming a complex with lignin are present. In particular, since the xylo-oligosaccharide-lignin complex forms a complex with a relatively large xylo-oligosaccharide molecule and lignin, it has been found that the xylo-oligosaccharide moiety escapes digestion by xylanase. For this reason, the xylo-oligosaccharide produced using the xylo-oligosaccharide-lignin complex has a high average molecular weight, and the average degree of polymerization varies from pentamer to 20-mer depending on the production method. The long-chain xylo-oligosaccharide (Japanese Patent Application No. 2001-242906) mainly composed of this neutral xylo-oligosaccharide has soluble dietary fiber-like physiological activity, but has low solubility.
[0019]
[Problems to be solved by the invention]
As described above, at present, most of the oligosaccharides constituting xylooligosaccharides on the market are mainly composed of dimers and trimers, and are recognized as substances different from dietary fiber. Yes.
In the present invention, the chain length is long and the effect of intestinal regulation is high by reaching the intestine without being decomposed, and since the chain length is long, it has not only the characteristics of oligosaccharide but also the action of dietary fiber. An object of the present invention is to provide an acidic xylo-oligosaccharide having high solubility in water.
Furthermore, an object of the present invention is to provide a method for supplying a water-soluble long-chain xylo-oligosaccharide in a large amount at a low cost.
[0020]
[Means for Solving the Problems]
The present invention for solving the above problems includes the following inventions:Is selected from.
(1) An acidic xylo-oligosaccharide composition comprising a xylo-oligosaccharide mixture having an average degree of polymerization of 8 to 15 and having at least one uronic acid residue as a side chain in one molecule of xylo-oligosaccharide.
[0021]
(2) The acidic xylo-oligosaccharide composition according to (1), wherein the uronic acid residue is a glucuronic acid residue or a 4-0-methylglucuronic acid residue.
[0022]
(3) The content of acidic xylo-oligosaccharide having a degree of polymerization of 5 or less is 5% by mass or less, and the acidic xylo-oligosaccharide composition according to (1) or (2)object.
[0023]
(4)A xylanase treatment step of treating a hardwood kraft pulp with a xylanase to obtain a treatment liquid containing the xylo-oligosaccharide-lignin complex and the xylo-oligosaccharide in which the xylo-oligosaccharide component and the lignin component are bound by a chemical bond having acid hydrolyzability;
A treatment solution containing the xylo-oligosaccharide-lignin complex and xylo-oligosaccharide from the xylanase treatment step is subjected to an acid hydrolysis treatment, and an xylo-oligosaccharide component having an average degree of polymerization of 8 to 15 comprising an acidic xylo-oligosaccharide and a neutral xylo-oligosaccharide is obtained. An acid hydrolysis treatment step for obtaining a treatment liquid containing the xylooligosaccharide mixture and lignin-like substance,
An adsorption separation step of adsorbing and separating acidic xylo-oligosaccharide components from the treatment liquid from the acid hydrolysis treatment step with a weak anion exchange resin;
A method for producing an acidic xylo-oligosaccharide composition having an average degree of polymerization of 8 to 15 having at least one uronic acid residue as a side chain in one molecule of xylo-oligosaccharide, comprising a plurality of steps comprising.
[0028]
(5)in frontThe acid hydrolysis treatment step is performed on the xylooligosaccharide-lignin complex.It is an acid hydrolysis treatment step that does not degrade the β-1,4-xyloside bond between xylose molecules of xylooligosaccharide (4)EntryListedAn average degree of polymerization of 8 to 15 having at least one uronic acid residue as a side chain in one molecule of xylooligosaccharideA method for producing an acidic xylooligosaccharide composition.
[0029]
(6) The complex of the xylooligosaccharide component and the lignin component contains 30% by mass or more of a complex of acidic xylooligosaccharide and lignin-like substance, which is a mixture of 2 to 20-mers of xylose (Item 4) Or (5)In termsDescribedAn average degree of polymerization of 8 to 15 having at least one uronic acid residue as a side chain in one molecule of xylooligosaccharideA method for producing an acidic xylooligosaccharide composition.
[0030]
(7) Xylan and / or hemicellulose is treated with hemicellulase to obtain a sugar solution containing a complex of a xylooligosaccharide component and a lignin-like component, the sugar solution is subjected to an acid hydrolysis treatment, and the degree of polymerization is determined from the hydrolysis treatment solution. A xylooligosaccharide mixture having a xylo-oligosaccharide content of 5 or less and an average polymerization degree of 8 to 15 and having at least one glucuronic acid or 4-O-methylglucuronic acid as a side chain is separated. A method for producing an acidic xylo-oligosaccharide composition.
[0031]
(8) The acid hydrolysis treatment is performed on a sugar solution obtained by concentrating the sugar solution obtained by the hemicellulase treatment (7The method for producing an acidic xylo-oligosaccharide composition as described in the item).
[0032]
(9) The hemicellulase treatment is a treatment in which a sugar solution adjusted to a pH of 3 to 10, preferably 5 to 9, is performed at a temperature of 10 ° C to 90 ° C, preferably 30 ° C to 60 ° C. (7)Or(8The method for producing an acidic xylo-oligosaccharide composition according to item).
[0033]
(10) The hydrolysis treatment is a treatment in which a sugar solution having a pH adjusted to 3.5 or lower is performed at a temperature of 105 ° C. to 150 ° C., preferably 110 ° C. to 130 ° C. (7)-(9The method for producing an acidic xylo-oligosaccharide composition according to any one of items 1).
[0034]
(11) Separation of the acidic xylo-oligosaccharide mixture from the acid hydrolysis treatment liquid is performed by average polymerization from the sugar solution obtained by the acid hydrolysis treatment using a cation exchange resin column-anion exchange resin column in this order. It is a process for separating an acidic xylooligosaccharide mixture having a degree of 8 to 15 (7)-(10The method for producing an acidic xylo-oligosaccharide composition according to any one of items 1).
(12) Separation of the acidic xylo-oligosaccharide mixture from the acid hydrolysis solution is a process of eluting and recovering the acidic xylo-oligosaccharide adsorbed on the anion exchange resin column using a metal ion chloride concentration gradient. Characterized in that the method includes (11The method for producing an acidic xylo-oligosaccharide composition as described in the item).
[0035]
DETAILED DESCRIPTION OF THE INVENTION
When the present invention uses a lignocellulosic material in which the enzyme to be used is a neutral thermophilic xylanase derived from Bacillus and the raw material is hardwood chemical pulp, 20 to 20 amounts of xylose dimer is used. That a complex of xylo-oligosaccharide component and lignin component having a distribution of xylo-oligosaccharide over the body can be obtained, and this xylo-oligosaccharide component contains a relatively large amount of pentamer to 20-mer having a large molecular weight; It has been found that the abundance ratio of xylobiose in the xylooligosaccharide component is about 10% by mass or less in the xylooligosaccharide component. In addition, the xylo-oligosaccharide component contains an acidic xylo-oligosaccharide having an acidic saccharide in the side chain, and the acidic xylo-oligosaccharide accounts for about 30% by mass of the total xylo-oligosaccharide component in the concentrated sugar solution. I found.
In general, oligosaccharides have lower solubility as the chain length is longer. However, since this acidic xylo-oligosaccharide has an acidic sugar in the side chain, the solubility is very high even if the degree of polymerization of the main chain is a hexamer or higher.
[0036]
A long-chain xylo-oligosaccharide component produced using chemical pulp-derived lignocellulose as a raw material is characterized by a higher average degree of polymerization than xylo-oligosaccharide produced using corn cob or cottonseed husk as a raw material. When lignocellulose is used as a raw material, it is possible to some extent to prepare a new xylooligosaccharide at an arbitrary ratio from an average polymerization degree of 2 to an average polymerization degree of 5 by adjusting the enzyme used. However, the new finding of this time is that an acidic xylo-oligosaccharide can be produced using the “xylo-oligosaccharide-lignin complex” as an intermediate.
[0037]
The xylo-oligosaccharide-lignin complex is a compound that is found in a treated solution when a hardwood or softwood kraft pulp is subjected to an enzyme treatment or a physicochemical treatment, and is a compound that is not described in the literature. This compound is a substance in which lignin is bound to a dimer to 20mer xylooligosaccharide chain.
Since the molecular weight of lignin is overwhelmingly larger than that of xylo-oligosaccharide, it is sterically hindered, and xylanase and xylosidase cannot decompose the xylo-oligosaccharide-lignin complex. Therefore, even if the enzyme treatment is performed, the xylooligosaccharide in the xylooligosaccharide-lignin complex remains without being decomposed, and a relatively long-chain xylooligosaccharide exists in the solution in a state of being bound to lignin.
However, this xylo-oligosaccharide-lignin complex can be easily cleaved between xylo-oligosaccharide and lignin by acid treatment with a dilute acid, and by utilizing this property, the degree of polymerization is 5 to 20 mer, especially average A xylo-oligosaccharide composition having a degree of polymerization of 8 to 15 could be easily produced.
[0038]
Hereinafter, the present invention will be described more specifically.
The acidic xylo-oligosaccharide composition referred to in the present invention is a composition having a high content of xylo-oligosaccharide having a relatively large degree of polymerization in the acidic xylo-oligosaccharide composition, and at least one uronic acid in one molecule of the xylo-oligosaccharide. Refers to a composition consisting of xylooligosaccharides linked together. Specifically, the xylose hexamer or higher xylooligosaccharide is 80% by mass or more and the uronic acid content is 2% by mass or more with respect to the total sugar mass in the composition. When the average degree of polymerization of the acidic xylo-oligosaccharide composition of the present invention is measured, the average degree of polymerization is 6 or more, preferably 8-15.
[0039]
Among them, what is easy to produce as an acidic xylooligosaccharide composition and is excellent in performance is mainly composed of a hexamer to 20mer of xylose. Here, the main component means 50% by mass or more based on the total sugar mass.
Of course, it is needless to say that an acidic xylo-oligosaccharide composition having a higher average degree of polymerization can be freely designed and manufactured by changing the enzyme used and the acid treatment conditions.
[0040]
The long-chain xylo-oligosaccharide component in the composition of the present invention is contained in the xylo-oligosaccharide mixture having a high degree of polymerization obtained by subjecting the lignocellulose material to hemicellulase treatment and then acid hydrolysis of the xylo-oligosaccharide-lignin complex with dilute acid. Exists.
The hemicellulase treatment of pulp as lignocellulose is carried out in a pulp concentration range of 1 to 30% by mass, preferably 2 to 15% by mass. However, when lignocellulose other than hardwood kraft pulp is used as a production raw material, this is the limit. is not. For example, with regard to xylan derived from lignocellulose, the pulp concentration at the time of processing is exemplified. For wheat bran derived xylan, 0.5 to 5% by mass, preferably around 2% by mass, and for corn cob derived xylan, 1 to 10% by mass, preferably Is about 5% by weight, about 1 to 10% by weight, preferably about 5% by weight for cottonseed cocoon-derived xylan, about 0.2 to 5% by weight, preferably about 1% by weight for ground corn pipe, and about 0.1% for oat-derived xylan. It is 2-5 mass%, Preferably it is around 1 mass%.
[0041]
As the raw lignocellulosic material from which the acidic xylooligosaccharide composition of the present invention can be obtained, wood such as conifers and hardwoods is preferably used, which is a non-woody plant such as kenaf, hemp, bagasse, rice, etc. There is no particular limitation. The pulp used in the present invention may be any chemical pulp, mechanical pulp, deinked pulp, etc., but hardwood chemical pulp is preferred. As the cooking method for obtaining chemical pulp, known cooking methods such as kraft cooking, polysulfide cooking, soda cooking, alkali sulfite cooking, etc. can be used, but considering the pulp quality, energy efficiency, etc., the kraft cooking method is used. Preferably used.
[0042]
When using hardwood kraft pulp as a lignocellulosic material, it is desirable to first treat the pulp bleached in the alkaline oxygen bleaching step with hemicellulase, but it is also possible to use the pulp after digestion or mechanical pulp as the raw material for hemicellulase treatment. good.
[0043]
It is well known that hemicellulose eluted from the pulp fiber in the cooking process is re-adsorbed on the pulp surface obtained by kraft cooking. 90% by mass or more of the resorbed hemicellulose is xylan constituted by β1- → 4 bonding of D-xylose.
In hardwood kraft pulp, most of arabinose and glucuronic acid, which are side chains in hemicellulose, are decomposed and removed. In addition, 4-0-methylglucuronic acid in the side chain remains as a side chain, but is converted to hexeneuronic acid under alkaline conditions. Since this hexeneuronic acid is easily decomposed and removed when heated under acidic conditions, it is not a problem for the production of xylooligosaccharides.
[0044]
Unlike the xylan present in the cell walls of normal plants, the resorbed xylan on the surface of chemical pulp is the majority of the side chains bound to the main chain, xylan, when extracted in the pulp cooking process. Has been decomposed and removed. Therefore, resorbed xylan has a very low side chain retention rate compared to normal xylan in the cell wall, and binds about 1 glucuronic acid or 4-0-methylglucuronic acid to 10 to 20 xyloses. ing. Further, since the xylooligosaccharide-lignin complex described later is formed, when the resorbed xylan is decomposed and removed by xylanase, the xylooligosaccharide-lignin complex is eluted in the pulp slurry, and the reaction solution becomes brown. In this brown reaction solution, there is a substance having an absorption of 280 nm, which is considered to be an absorption derived from the aromatic ring of lignin.
[0045]
The xylan content including the re-adsorbed component occupies about 20% by mass of the hardwood kraft pulp absolute dry mass. In the hemicellulase treatment, the enzyme acts on hardwood kraft pulp and acts on all xylan containing resorbed components to lower the molecular weight. For example, when the xylanase of the Bacillus espi-S-2113 strain (see JP-A-8-224081) is used, the proportion of xylose and xylooligosaccharide constituent sugars produced in the treatment reaction solution is the most in the 3-5 mer. It produces oligosaccharides with a high composition ratio and a small amount of monomers.
[0046]
The present inventors have already found that a xylo-oligosaccharide complex in which xylo-oligosaccharide and lignin-like substance are bound is present in the wastewater obtained from the hemicellulase treatment process of hardwood kraft pulp. Furthermore, the xylo-oligosaccharide complex has been found to bind a lignin-like substance to an oligosaccharide having a relatively high degree of polymerization. Since this xylo-oligosaccharide-lignin complex can be easily decomposed into a xylo-oligosaccharide and a lignin-like substance by a dilute acid treatment, a xylo-oligosaccharide having a large degree of polymerization can be produced in a large amount at a low cost. In the xylo-oligosaccharide composition produced from the xylo-oligosaccharide-lignin complex, the proportion of the acidic xylo-oligosaccharide having a uronic acid residue in the side chain is about 30% by mass with respect to the total sugar amount.
[0047]
At present, most of the enzymes used in large-scale enzyme treatment steps are hemicellulases, but any commercially available hemicellulase can be used in the enzyme treatment step in the method for producing xylooligosaccharides of the present invention. it can. For example, trade name Cartazame (manufactured by Clariant), trade name Eco Pulp (produced by Rohm Enzyme), trade name Sumiteam (manufactured by Shin Nippon Kagaku Kogyo Co., Ltd.), Pulpzyme (manufactured by Novo Nordics) Multifect 720 (Genencor) Commercially available enzyme preparations and microorganisms such as Trichoderma spp., Thermomyces spp. The xylanase produced can be used.
[0048]
The enzyme treatment temperature is in the range of 10 to 90 ° C., preferably 30 to 60 ° C., but a treatment temperature close to the optimum temperature of the enzyme is more preferred. In the case of a general enzyme, if the treatment temperature is less than 10 ° C., the reaction becomes insufficient, and obtaining such a temperature itself is not suitable because it requires a great deal of cost. On the other hand, if the temperature exceeds 90 ° C., heat loss increases unless the treatment system is sealed, and in the case of a general enzyme, the enzyme itself is denatured and becomes inactive, which is not suitable. The solution pH during the treatment is in the range of 3 to 10, preferably 5 to 9, but is more preferably close to the optimum pH of the enzyme.
[0049]
When a sugar solution is obtained by enzymatic treatment of pulp obtained by bleaching hardwood kraft pulp with alkaline oxygen, the pH of the pulp is inclined to the alkali side, so the enzyme whose pH is closer to the alkali side is the pH. The cost for adjusting the is low and has an advantage. If pH adjustment is necessary, it goes without saying that any acidic solution or alkaline solution may be added to adjust the enzyme treatment.
[0050]
The sugar solution obtained by the enzyme treatment contains xylooligosaccharide (2 to 20 mer) and xylooligosaccharide-lignin complex. The sugar concentration in the enzyme-treated solution is 1 unit (1 unit) of xylanase produced by Bacillus sp. 2113 strain (National Institute of Advanced Industrial Science and Technology, Patent Microorganism Depositary Center deposited strain FERM BP-5264) Is about 3000 μg / ml (in terms of xylose) when used with a pulp slurry having a concentration of 10% by mass, which is used with an enzyme power that releases 1 micromole of xylose per minute.
[0051]
This sugar solution can be concentrated using membrane separation techniques such as charged NF membranes, other ultrafiltration membranes, reverse osmosis membranes, or by concentration work such as evaporation when taking into account the subsequent manufacturing process. It is also possible to work to increase the concentration. Actually reducing the volume of the sugar solution facilitates handling when a large amount of sugar solution is processed in the subsequent purification step. In addition, the permeate obtained from the work in membrane concentration has the characteristics that the sugar concentration is lower than that of the enzyme treatment solution and the content of colored organic substances such as lignin is low. For this reason, the permeate obtained from the membrane concentration step can be reused as industrial water in the pulp production step.
[0052]
The sugar solution or the sugar solution after the concentration treatment step is subjected to an acid hydrolysis treatment with a dilute acid to separate the xylooligosaccharide-lignin complex into xylooligosaccharide, acidic xylooligosaccharide, and lignin-like substance. As a method for adjusting the pH of the sugar solution, it is common to adjust the pH of the sugar solution to around 3.5 by appropriately adding a mineral acid or an organic acid to the sugar solution. It is also possible to treat the sugar solution with a cation exchange resin such as “Rohm & Haas” and lower the pH by ion exchange.
[0053]
Thereafter, the sugar solution whose pH has been adjusted is heated in the range of 105 ° C. to 150 ° C., preferably in the range of 110 ° C. to 130 ° C., to perform acid hydrolysis. The treatment time is 15 minutes or more, preferably 30 to 60 minutes. If the heat treatment time is set to 90 minutes or more, decomposition of oligosaccharides into monosaccharides proceeds, which is not preferable. When the pH of the sugar solution is around 3.5, the xylooligosaccharide complex and lignin-like substance, xylooligosaccharide and hexenuronic acid, which is a side chain, can be separated and removed, but the acidic xylooligosaccharide itself is almost decomposed. There is no.
[0054]
By this treatment, the lignin-like organic substance is decomposed and removed from the xylooligosaccharide-lignin complex, and converted into acidic xylooligosaccharide and xylooligosaccharide. The conversion efficiency from xylo-oligosaccharide complex to acidic xylo-oligosaccharide and xylo-oligosaccharide at pH 3.5, 121 ° C. and 60 minutes is about 95%. At this time, a part of the monosaccharide is hydrolyzed and becomes a furfural-like substance, which is further condensed and precipitated. Similarly, the lignin-like substance cleaved from the xylooligosaccharide complex is condensed, insolubilized and precipitated under acidic conditions. This insolubilized precipitate can be separated and removed by UF membrane, MF membrane, ceramic filter, etc. as well as filtration by filter paper or diatomaceous earth.
[0055]
The acidic xylo-oligosaccharide composition obtained from the xylo-oligosaccharide-lignin complex as described above is a novel acidic xylo-oligosaccharide containing a high proportion of xylo-oligosaccharide having a relatively long chain length and a degree of polymerization of about 6-20. It is a composition. The reason why an acidic xylooligosaccharide having a relatively high degree of polymerization is obtained is that the xylooligosaccharide complex in the sugar solution obtained by the enzyme treatment is converted to an acid xylooligosaccharide having a chain length of about 20 to 20% from a dimer to a lignin-like substance. This is due to the fact that hemicellulase escapes more than necessary. When separated from the lignin-like substance by acid hydrolysis with a dilute acid from such a state, an acidic xylooligosaccharide having a relatively long chain length is obtained.
[0056]
In addition to acidic xylooligosaccharides, the sugar solution obtained by acid hydrolysis includes monosaccharides such as xylose and glucose, and organic substances such as lignin, furan compounds, and furfural. As a step of separating and purifying only xylooligosaccharide from a mixture of these organic substances, any conventional purification method such as ion exchange, molecular sieving, ethanol fractionation, membrane treatment, etc. may be used in combination. For example, in a purification method using a column in the order of activated carbon → strong cation exchange resin → strong anion exchange resin → strong cation exchange resin → weak anion exchange resin, the total amount of sugar in the acid-treated sugar solution as a starting material is 100% by mass. In this case, the recovery rate of the purified acidic xylo-oligosaccharide itself is about 30% by mass.
[0057]
A method for purifying an acidic xylooligosaccharide composition using a concentrated sugar solution as a starting material is as follows. First, the sugar solution is filtered using a ceramic filter to remove insoluble colored substances having a relatively large molecular weight and lignin condensates obtained by dilute acid treatment. At this time, when a ceramic filter treatment is performed by adding activated carbon to the sugar solution supply side in an amount of several percent by mass with respect to the sugar, smaller colored substances are filtered while adsorbed on the activated carbon, so that the obtained filtered sugar solution is clarified. Increasing the degree makes the subsequent purification process easier.
[0058]
The sugar solution filtered through the ceramic filter is further filtered using a UF membrane having a fractional molecular weight of 20000 or less. In this step, even smaller colored matters can be removed. The colored product remaining on the concentration side and the complex of xylo-oligosaccharide and lignin with a very high degree of polymerization should not be discarded, and returned to the supply side sugar solution in the process using the ceramic filter in the previous stage as much as possible and recovered. It is preferable to increase the rate.
[0059]
The xylo-oligosaccharide composition and the acidic xylo-oligosaccharide composition are dissolved in the sugar solution obtained through the ceramic filter treatment and the UF membrane treatment. A method using an ion exchange resin is suitable as a method for taking out only the acidic xylo-oligosaccharide composition from the sugar solution. The sugar solution is first treated with a cation exchange resin to remove metal ions in the sugar solution. Subsequently, sulfate ions and the like in the sugar solution are removed using a strong anion exchange resin. In this step, simultaneously with the removal of sulfate ions, a part of the organic acid, which is a weak acid, and the colored component are simultaneously removed.
[0060]
The sugar solution treated with the strong anion exchange resin is treated once more with the cation exchange resin to further remove metal ions. And finally, it processes with a weak anion exchange resin, and adsorb | sucks a coloring substance and acidic xylo-oligosaccharide to resin. At this time, the xylo-oligosaccharide which is a neutral saccharide is recovered as it is without being adsorbed on the weak anion exchange resin, and the recovery rate of the neutral xylo-oligosaccharide composition at this time is settled to about 70% by mass.
[0061]
The acidic xylooligosaccharide adsorbed on the weak anion exchange resin can be recovered by eluting from the weak anion exchange resin using an eluate in which a metal salt is dissolved. When eluting the adsorbed acidic xylo-oligosaccharide using a column, it is possible to perform elution from the resin with an appropriate ionic strength by performing a gradient operation. It is also possible to elute with an alkali. However, at this time, since the ratio of the colored components mixed into the eluted fraction increases, another operation for removing the colored components at the subsequent stage is required.
[0062]
Any metal salt can be used for elution and recovery of acid xylo-oligosaccharides in a metal salt solution. However, in order for the collected acid xylo-oligosaccharides to exhibit physiological activity, they are ingested by living organisms. Needless to say, a metal salt that does not cause much harm to the living body is desirable. For example, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, iron chloride and the like can be mentioned. At that time, it is also possible to design the acidic xylo-oligosaccharide so that it can be eluted using a metal salt that is difficult to ingest daily, and at the same time as the intake of the acidic xylo-oligosaccharide, these metal salts can be taken simultaneously as minerals. Specifically, in recent years, Japanese people tend to be deficient in calcium and magnesium intake, so an eluate with an appropriate calcium: magnesium molar ratio is prepared and eluted to recover acidic xylooligosaccharides. You can also.
[0063]
The sugar solution containing the purified acidic xylo-oligosaccharide was analyzed using ion chromatography (Dionex Co., Ltd.), and it was found that it was a saccharide solution containing dimer to 20-mer and higher acidic xylo-oligosaccharide. When the organic content mass at this time was analyzed, the total sugar amount in the absolutely dry mass was around 95% by mass. Moreover, in the measurement of the ash content using the weighed crucible, the ash content in the refined sugar solution was around 5% by mass. Most of the metal oxides in the ash accounted for the metal that seems to be derived from the metal salt used for elution.
[0064]
As described above, the novel acidic xylo-oligosaccharide composition of the present invention contains a lignocellulosic material as a starting material, and contains an acidic xylo-oligosaccharide having a high degree of polymerization obtained by separation and purification from a reaction filtrate treated with hemicellulase. It is a composition. In addition, this acidic xylo-oligosaccharide composition having a high degree of polymerization is obtained by using known xylobiose, xylotriose, xylotetraose, and xylopentaose as main components by using physicochemical methods such as enzyme treatment and explosion. It can be easily converted into acidic xylo-oligosaccharides.
[0065]
【Example】
EXAMPLES The present invention will be specifically described below with reference to examples and comparative examples, but the present invention is of course not limited to these examples. Unless otherwise specified, all percentages shown below mean mass%, and the addition ratio of pulp is the ratio of mass to the absolute dry mass of pulp. In addition, each measuring method is as follows.
[0066]
(1) Quantification of total sugar content:
The total sugar amount was quantified by preparing a calibration curve using D-xylose (Wako Pure Chemical Industries) and the phenol-sulfuric acid method (published by the “Reducing Sugar Quantification Method” Society of Publishing Press Center).
(2) Quantification of reducing sugar content:
The amount of reducing sugar was prepared by using a calibration curve using D-xylose (Wako Pure Chemical Industries) and quantified by the Sommoji-Nelson method (published by the "Reducing Sugar Quantification Method" Society Publishing Center).
[0067]
(3) Determination of the amount of uronic acid:
The uronic acid was prepared using D-glucuronic acid (Wako Pure Chemical Industries) with a calibration curve, and quantified by the carbazole sulfate method (published by the “Reducing Sugar Determination Method” published by the Japan Society for the Pressing of the Society).
(4) Method for determining the average degree of polymerization:
The sample sugar solution was kept at 50 ° C., centrifuged at 15000 rpm for 15 minutes to remove insoluble matters, and the total amount of sugar in the supernatant was divided by the amount of reducing sugar (both converted to xylose) to determine the average degree of polymerization.
[0068]
(5) Quantitative determination of acidic xylooligosaccharides
The quantitative ratio of uronic acid residues to xylose residues in one molecule of acidic oligosaccharide was determined using a nuclear magnetic resonance apparatus (hereinafter referred to as NMR, manufactured by JEOL Ltd.). The sample was measured after freeze-drying and deuterium substitution.1H NMR,13The proton chemical shift between xylose 1 position (4.409 ppm) and uronic acid 1 position (5.226 ppm) having glycosidic bonds was determined by C NMR and HMQC measurements. The area of each signal corresponding to the amount ratio of hydrogen was calculated to determine the ratio of glucuronic acid and xylose. In addition, high-resolution molecular weight measurement was performed using a high performance liquid chromatography mass spectrometer (Applied Biosystems) to determine the number of uronic acid residues per oligosaccharide molecule.
(6) Definition of enzyme titer:
For measuring the activity of the xylanase used as the enzyme, Kabukilan (manufactured by Sigma) was used. The enzyme titer is defined by measuring the reducing power of reducing sugar obtained by xylanase degrading xylan using the DNS method (published by the “Reducing Sugar Determination Method” Society of Science Publishing Center) and measuring 1 micromole per minute. Generate reducing power equivalent to xylose
The amount of enzyme used was 1 unit.
[0069]
(7) Analysis by ion chromatography:
An ion chromatograph (Dionex) was used for analysis of xylooligosaccharides. For the analysis, Carbo Pac PA-10 (Dionex) was used as a column suitable for the analysis of saccharides.
[0070]
Example 1
<Enzyme treatment process 1>
Using unmixed hardwood chips consisting of 70% domestic hardwood chips and 30% eucalyptus wood as raw materials, unbleached pulp made by the factory with a kappa number of 20.1 and a pulp viscosity of 41 cps was obtained by kraft cooking. Subsequently, oxygen delignification was performed to obtain an oxygen delignified pulp having a Cupper number of 9.6 and a pulp viscosity of 25.1 cps.
After adjusting this pulp to a pulp concentration of 10%, dilute sulfuric acid was added to adjust to pH 6.7, and then Bacillus sp. S-2113 strain (Independent Administrative Institution National Institute of Advanced Industrial Science and Technology, Patent Microorganism Depositary Center deposited strain FERM BP-5264) ) One xylanase produced was added to 1 unit / g of pulp and treated at 60 ° C. for 120 minutes. After the treatment, a treatment liquid of 76000 liters (total sugar amount 228 kg) containing a total sugar concentration of 3000 mg / liter was obtained. Subsequently, the mixture was concentrated 40 times by volume using an NF membrane (Nitto Denko: NTR-7450, membrane quality: sulfonated polyethersulfone, salt rejection 50%), and 1900 liters of sugar solution was recovered. This concentrate contained 190 kg of total sugar, and the total sugar recovery rate was 83.8%. As a result of analyzing the sugar in the concentrated solution using ion chromatography, neutral xylo-oligosaccharide and a peak of xylo-oligosaccharide complex were observed after 24 minutes elution time. This is shown in FIG.
[0071]
Example 2
<Enzyme treatment process 2>
Experiments were carried out to generate xylooligosaccharide conjugates using Genencor xylanase (Multifect 720) as the enzyme. The enzyme treatment experiment was performed under exactly the same conditions as in Example 1. The reaction product was analyzed by ion chromatography using an ion chromatography column (Dionex: CarboPacPA-10). It has been found. At this time, some acidic xylo-oligosaccharide peaks are also observed. This is shown in FIG.
[0072]
Example 3
<Acid hydrolysis treatment step 1>
Sulfuric acid was added to the 1900 liter concentrated sugar solution obtained in the enzyme treatment step to adjust the pH to 3.5, and then this concentrated sugar solution was reacted at 121 ° C. for 1 hour. As a result of analyzing the reaction product by ion chromatography using a column for ion chromatography (Dionex: Carbo Pac PA-10), it was found to contain a high concentration of xylooligosaccharides (dimer to 20mer). At this time, some acidic xylo-oligosaccharide peaks are also observed. This is shown in FIG.
[0073]
Example 4
<Acid hydrolysis treatment step 2>
Acetic acid was added to 1000 liters of concentrated sugar solution obtained in exactly the same manner as in Example 1 to adjust the pH to 3.5, and the mixture was reacted at 121 ° C. for 1 hour. As a result of analyzing the reaction product by ion chromatography using a column for ion chromatography (Dionex: Carbo Pac PA-10), it was found to contain a high concentration of xylooligosaccharides (dimer to 20mer). At this time, some acidic xylo-oligosaccharide peaks are also observed. This is shown in FIG.
[0074]
Example 5
<Separation and purification process of acidic xylooligosaccharides>
(1) Ceramic filter treatment
1900 liters of xylo-oligosaccharide solution (100 mg / ml) prepared in acid hydrolysis treatment step 1, 190 kg as total sugar amount, filtration area 4.8m2It filtered using the ceramic filter. Filtration conditions were performed at a liquid temperature of 60 ° C., and backwashing with air was performed at intervals of 5 minutes. Moreover, it filtered by adding activated carbon to sugar 3%. All 19000 liters are processed in 8 hours of continuous processing, and the average flux over 8 hours is 40 liters / Hr / m.2Met.
[0075]
(2) Purification by ion exchange resin
For ion exchange, four ion exchange towers (each
[0076]
Example 6
600 liters of saccharified (5.0% concentration) saccharified (5.0% concentration) obtained in exactly the same manner as in Example 5 was continuously passed through four columns of columns. The liquid passing condition was 3 liters / min, and the acidic xylo-oligosaccharide was adsorbed on the weak anion exchange resin. 50 mM CaCl for elution2(Calcium chloride) Elution was performed using 300 liters, and the elution rate was 3 liters / min. The recovered acidic xylo-oligosaccharide was recovered in a volume of 134 liters at a concentration of 7.5%, the recovery amount was 10 kg, and the recovery rate was 33%.
[0077]
Example 7
<Degree of polymerization of acidic xylo-oligosaccharide>
The acid xylo-oligosaccharide powder purified in Example 5 and Example 6 was dissolved in ultrapure water to prepare a 1% aqueous solution. The average degree of polymerization was determined by measuring the total sugar amount by the phenol-sulfuric acid method, and then measuring the reducing sugar amount of the 1% aqueous solution by the Somogene Nelson method.
In any measurement, a calibration curve was prepared using D-xylose. The average degree of polymerization was determined by dividing the total amount of sugar per ml by the amount of reducing sugar per ml. As a result, the average degree of polymerization of acidic xylo-oligosaccharides eluted with potassium chloride prepared by the above method was 10.2, and the average degree of polymerization of acidic xylo-oligosaccharides eluted with calcium chloride was 11.3. It was.
[0078]
Example 8
10 g of the purified acidic xylo-oligosaccharide obtained in Example 5 was dissolved in 100 ml of ultrapure water to prepare a 10% aqueous solution. Ethanol was added to a concentration of 70% with respect to this acidic xylooligosaccharide aqueous solution. This was allowed to fractionate into acidic xylo-oligosaccharides that precipitated in 70% ethanol that did not precipitate and acid xylo-oligosaccharides that precipitated when left in the refrigerator overnight. The precipitate and the fraction soluble in 70% ethanol were separated by centrifugation, and the average degree of polymerization was measured. The average degree of polymerization of the 70% soluble fraction was about 4.2, and the precipitated acidic xylooligosaccharide was redissolved. The average degree of polymerization of the liquid was 12.8.
[0079]
<Analysis with nuclear magnetic resonance apparatus and mass spectrometer>
Among the above, 70% ethanol soluble fraction was subjected to ethanol removal and analyzed as a pure water solution by NMR method for the degree of polymerization and the quantitative ratio of uronic acid residue and xylose residue in one oligosaccharide molecule. The proton chemical shift between xylose 1-position (4.409 ppm) and uronic acid 1-position (5.226 ppm) was determined, and the area of each signal corresponding to the amount ratio of hydrogen was calculated. As a result, the area ratio was 5: 1. there were. Therefore, it was found that most of them were acidic xylooligosaccharides in which one 4-0-methylglucuronic acid was bonded to a xylose pentamer.
[0080]
For further reference, the molar ratio of xylose and 4-0-methylglucuronic acid was determined by applying this aqueous solution to a mass spectrometer, which was 5: 1, which was consistent with the NMR results.
The sugar content of the soluble fraction at this time was 0.44 g with respect to the first 10 g of acidic xylo-oligosaccharide as measured by the phenol sulfate method and the carbazole sulfate method.
From these facts, it was found that the purified acidic xylo-oligosaccharide obtained in Example 5 had a degree of polymerization of 4.4% or less, and the majority was a pentamer.
[0081]
Next, the ethanol is removed from the acidic xylooligosaccharide derived from 70% ethanol precipitation to obtain a pure water solution, and the degree of polymerization and the amount of uronic acid residue and xylose residue in one oligosaccharide molecule are determined by NMR method as described above. The ratio was analyzed.
The molar ratio of xylose and 4-0-methylglucuronic acid was measured to be 10: 1, and it was an acidic xylooligosaccharide in which one 4-0-methylglucuronic acid residue was bound to a xylose decamer. For further reference, the molar ratio of xylose and 4-0-methylglucuronic acid was determined by applying this aqueous solution to a mass spectrometer, which was 10: 1, which was consistent with the NMR results.
[0082]
【The invention's effect】
According to the present invention, a large amount of acidic xylo-oligosaccharide having a long chain length is supplied at low cost. This novel acidic xylo-oligosaccharide composition can be easily converted into acidic xylo-oligosaccharide having xylobiose and xylose as the main chain by treatments such as acid hydrolysis and enzymatic digestion. In addition, neutral xylo-oligosaccharides and acidic xylo-oligosaccharides have selective growth of bifidobacteria and are promising for functional food materials. The acidic xylo-oligosaccharide composition of the present invention is usually a long-chain neutral xylo-oligosaccharide having a low solubility, but it has an acidic glycan, so it has a very high solubility and is easy to produce, It is a composition that can be easily applied to food.
[Brief description of the drawings]
FIG. 1 is an analysis diagram of the xylo-oligosaccharide of Example 1 by ion chromatography.
2 is an analysis diagram of the xylo-oligosaccharide of Example 2 by ion chromatography. FIG.
FIG. 3 is an analysis diagram of the xylooligosaccharide of Example 3 by ion chromatography.
4 is an ion chromatographic analysis diagram of the xylooligosaccharide of Example 4. FIG.
5 is an analysis diagram of the xylooligosaccharide of Example 5 by ion chromatography. FIG.
Claims (5)
該キシラナーゼ処理工程からのキシロオリゴ糖−リグニン複合体とキシロオリゴ糖を含有する処理液を酸加水分解処理して、酸性キシロオリゴ糖と中性キシロオリゴ糖からなる平均重合度が８〜１５のキシロオリゴ糖成分を含有するキシロオリゴ糖混合物とリグニン様物質とを含有する処理液を得る酸加水分解処理工程、
該酸加水分解処理工程からの処理液から弱アニオン交換樹脂により酸性キシロオリゴ糖成分を吸着して分離する吸着分離工程、
からなる複数工程を有することを特徴とする、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平均重合度が８〜１５の酸性キシロオリゴ糖組成物の製造方法。 A xylanase treatment step of treating a hardwood kraft pulp with a xylanase to obtain a treatment liquid containing the xylo-oligosaccharide-lignin complex and the xylo-oligosaccharide in which the xylo-oligosaccharide component and the lignin component are bound by a chemical bond having acid hydrolyzability;
A treatment solution containing the xylo-oligosaccharide-lignin complex and xylo-oligosaccharide from the xylanase treatment step is subjected to an acid hydrolysis treatment, and an xylo-oligosaccharide component having an average degree of polymerization of 8 to 15 comprising an acidic xylo-oligosaccharide and a neutral xylo-oligosaccharide is obtained. An acid hydrolysis treatment step for obtaining a treatment liquid containing the xylooligosaccharide mixture and lignin-like substance,
An adsorption separation step of adsorbing and separating acidic xylo-oligosaccharide components from the treatment liquid from the acid hydrolysis treatment step with a weak anion exchange resin;
A method for producing an acidic xylo-oligosaccharide composition having an average degree of polymerization of 8 to 15 having at least one uronic acid residue as a side chain in one molecule of xylo-oligosaccharide, comprising a plurality of steps consisting of:
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| JP4792845B2 (en) * | 2005-07-12 | 2011-10-12 | 王子製紙株式会社 | Stomatitis improving agent |
| ES2542504T3 (en) * | 2006-05-30 | 2015-08-06 | Nutrition Sciences N.V./S.A. | Tri- and tetraoligosaccharides for use as agglutination agents for enteric pathogens |
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| MX2020002288A (en) | 2016-08-31 | 2020-07-14 | Oji Holdings Corp | Production method for acidic xylooligosaccharide, and acidic xylooligosaccharide. |
| JP6225321B1 (en) | 2016-08-31 | 2017-11-08 | 王子ホールディングス株式会社 | Method for producing polysulfate pentosan |
| JP6281659B1 (en) | 2017-02-28 | 2018-02-21 | 王子ホールディングス株式会社 | Polysulfate pentosan, pharmaceutical composition and anticoagulant |
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| KR102678054B1 (en) | 2017-09-12 | 2024-06-24 | 오지 홀딩스 가부시키가이샤 | Method for producing polypentosan sulfate and polypentosan sulfate |
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