JP2017197468A - Method for producing chitin oligomer, n-acetylglucosamine, and 1-o-alkyl-n-acetylglucosamine - Google Patents
Method for producing chitin oligomer, n-acetylglucosamine, and 1-o-alkyl-n-acetylglucosamine Download PDFInfo
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- 229920002101 Chitin Polymers 0.000 title claims abstract description 141
- 229950006780 n-acetylglucosamine Drugs 0.000 title claims abstract description 61
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 title claims abstract description 48
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 48
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 title claims abstract description 40
- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 title claims abstract description 40
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- 230000000694 effects Effects 0.000 claims description 11
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- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 description 1
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- DUKURNFHYQXCJG-UHFFFAOYSA-N Lewis A pentasaccharide Natural products OC1C(O)C(O)C(C)OC1OC1C(OC2C(C(O)C(O)C(CO)O2)O)C(NC(C)=O)C(OC2C(C(OC3C(OC(O)C(O)C3O)CO)OC(CO)C2O)O)OC1CO DUKURNFHYQXCJG-UHFFFAOYSA-N 0.000 description 1
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- MSWZFWKMSRAUBD-UHFFFAOYSA-N beta-D-galactosamine Natural products NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 description 1
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Abstract
Description
本発明は、酸触媒を用いたキチンの加水分解反応により、キチン含有バイオマスからキチンオリゴマー、Ｎ−アセチルグルコサミン（ＮＡＧ）及び１−Ｏ−アルキル−Ｎ−アセチルグルコサミンを製造する方法に関する。 The present invention relates to a method for producing chitin oligomers, N-acetylglucosamine (NAG) and 1-O-alkyl-N-acetylglucosamine from chitin-containing biomass by a hydrolysis reaction of chitin using an acid catalyst.
キチンは、ＮＡＧがβ−１，４−グリコシド結合した含窒素多糖高分子であり、エビ、カニなどの甲殻類、昆虫類、キノコなどの真菌に含まれる、自然界に豊富に存在するバイオマスである。 Chitin is a nitrogen-containing polysaccharide polymer in which NAG is β-1,4-glycosidically bound, and is a biomass that exists in abundance in nature, including crustaceans such as shrimp and crabs, insects, and fungi such as mushrooms. .
キチンの加水分解生成物や加アルコール分解生成物などの加溶媒分解生成物には、様々な効果効能を有し各種機能素材として利用できるものがある。
例えば、加水分解生成物であるＮＡＧが２〜７個程度重合したキチンオリゴマーは、ＮＡＧ取得のための前駆体として有用な成分であることに加え、抗腫瘍、免疫賦活、抗菌の作用（Trend Food Sci. Technol., 1999, 10, 37-51；非特許文献１）や、ビフィズス菌増殖による腸内環境調整作用、植物生体防御機構活性化作用であるエリシター活性が報告されており、医薬用素材、機能性食品、農業資材としても注目されている。
Some solvolysis products such as chitin hydrolysis products and alcoholysis decomposition products have various effects and effects and can be used as various functional materials.
For example, a chitin oligomer obtained by polymerizing about 2 to 7 NAGs as hydrolysis products is a useful component as a precursor for obtaining NAG, as well as antitumor, immunostimulatory and antibacterial actions (Trend Food Sci. Technol., 1999, 10, 37-51; Non-patent document 1), and the elicitor activity, which is an action to adjust the intestinal environment by the growth of bifidobacteria and the action to activate the plant defense mechanism, has been reported. It is also attracting attention as a functional food and agricultural material.
また、加水分解生成物の単量体であるＮＡＧは、体内に取り込まれることによりヒアルロン酸などのムコ多糖類の生合成を促進して、ひざ・関節痛の改善や美肌・保湿などの効果が認められる成分であり、同様の効果を示すグルコサミンより苦みがなく、利用効率が高いことから、近年、機能性食品、医薬関連原料、美容関連原料としての利用が期待されている。 In addition, NAG, which is a monomer of the hydrolysis product, promotes biosynthesis of mucopolysaccharides such as hyaluronic acid by being taken into the body, and has effects such as improvement of knee and joint pain, skin beautification and moisturizing. In recent years, it is expected to be used as a functional food, a pharmaceutical-related raw material, and a beauty-related raw material because it is a recognized ingredient, has less bitterness than glucosamine, which exhibits the same effect, and has high utilization efficiency.
さらに、メタノールの加アルコール分解生成物であり、ＮＡＧ誘導体である１−Ｏ−メチル−Ｎ−アセチルグルコサミン（以下、ＭｅＮＡＧと略記することがある。）は、血球凝集を阻害することによるインフルエンザ及びがんの抑制効果を有していることから医薬品原料として注目されており（Arch. Biochem. Biophys., 1987, 259, 79-88；非特許文献２、J. Biol. Chem., 1989,264, 173-177；非特許文献３）、さらに有機触媒（J. Org. Chem., 2012, 6390-6406；非特許文献４）、リガンド（J. Org. Chem., 1997, 62, 6012-6028；非特許文献５）、ゲル化剤（Tetrahedron, 2010, 66, 962-5971；非特許文献６）などへの用途展開の可能性が示されている物質である。 Furthermore, 1-O-methyl-N-acetylglucosamine (hereinafter, may be abbreviated as MeNAG), which is an alcohol decomposition product of methanol and is a NAG derivative, is caused by influenza and (Arch. Biochem. Biophys., 1987, 259, 79-88; Non-Patent Document 2, J. Biol. Chem., 1989, 264, 173-177; Non-Patent Document 3), further an organic catalyst (J. Org. Chem., 2012, 6390-6406; Non-Patent Document 4), a ligand (J. Org. Chem., 1997, 62, 6012-6028; Non-patent document 5), a substance that has been shown to have potential for use in gelling agents (Tetrahedron, 2010, 66, 962-5971; non-patent document 6).
酸を用いる、キチンからのキチンオリゴマーやＮＡＧの製造方法としては、キチンに濃塩酸を用いて４０℃前後の加熱条件で３〜４時間の反応を行い部分加水分解してオリゴマーを生成させ、引き続き後工程を行う方法が開発されている（特公平５−３３０３７号公報；特許文献１、特開２００９−１６７１４０号公報；特許文献２、特許５４２６０９９号公報；特許文献３）。特許文献１及び２では、後工程に酵素反応を導入し、特許文献３では後工程に冷却晶析を導入してＮＡＧの生成している。しかしながら、これらの方法は、酸触媒のモル数に対する、基質であるキチン中のＮ−アセチルグルコサミン単位（Ｃ8Ｈ13ＮО5）のモル数の比（以下、Ｓ／Ｃ比と略記する。）が０．０８〜０．１４であって、基質に対して大量の濃塩酸が使用されている上、製造されるオリゴマーやＮＡＧのキチンに対する収率が低いという問題がある。また別の酸では、硫酸を用いて０〜５０℃で５分〜２時間の処理をする方法も開発されている（特開２００２−８８０９３；特許文献４）が、この方法もＳ／Ｃは０．０５〜０．５であり大量の硫酸を使用している。
このように現在開発されている酸を用いる製造方法は、いずれも危険な強酸を大量に用いるため、環境負荷とコストがかかるという問題がある。
As a method for producing chitin oligomers or NAG from chitin using an acid, chitin is subjected to a reaction for 3 to 4 hours under concentrated heating conditions using concentrated hydrochloric acid under a heating condition of about 40 ° C. to generate oligomers by partial hydrolysis. A method for performing a post-process has been developed (Japanese Patent Publication No. 5-33037; Patent Document 1, Japanese Patent Application Laid-Open No. 2009-167140; Patent Document 2, Japanese Patent No. 5426099; Patent Document 3). In Patent Documents 1 and 2, an enzyme reaction is introduced into the subsequent process, and in Patent Document 3, cooling crystallization is introduced into the subsequent process to generate NAG. However, in these methods, the ratio of the number of moles of N-acetylglucosamine units (C 8 H 13 NO 5 ) in chitin as a substrate to the number of moles of acid catalyst (hereinafter abbreviated as S / C ratio). 0.08 to 0.14, and a large amount of concentrated hydrochloric acid is used with respect to the substrate, and the yield of the produced oligomer or NAG with respect to chitin is low. As another acid, a method of treating with sulfuric acid at 0 to 50 ° C. for 5 minutes to 2 hours has been developed (Japanese Patent Laid-Open No. 2002-88093; Patent Document 4). 0.05-0.5 and a large amount of sulfuric acid is used.
As described above, all of the production methods using acids that are currently developed have a problem of high environmental burden and cost because they use a large amount of dangerous strong acids.
一方、酸を用いず、キチンに対してキチン分解酵素生産能を有する微生物を利用する方法が提案されている（特開２００４−４１０３５号公報；特許文献５、特開２００８−２５３２５２号公報；特許文献６）。しかし、これらの方法は、４〜５日の長時間を要し、キチンの分解効率も低く、生産性が低いという問題を抱えている。 On the other hand, there has been proposed a method using a microorganism having an ability to produce a chitin degrading enzyme with respect to chitin without using an acid (Japanese Patent Application Laid-Open No. 2004-41035; Patent Document 5, Japanese Patent Application Laid-Open No. 2008-253252; Patent Reference 6). However, these methods require a long time of 4 to 5 days, have low chitin decomposition efficiency, and have low productivity.
また、ＭｅＮＡＧは、特許文献１〜３に記載の通り、塩酸でキチンを部分加水分解して作成したキチンオリゴマーに、メタノールを加えて加熱する加メタノール分解法により製造されているが、この場合も大量の酸を使用する問題がある。 In addition, as described in Patent Documents 1 to 3, MeNAG is produced by a methanol decomposition method in which methanol is added to a chitin oligomer prepared by partial hydrolysis of hydrochloric acid with hydrochloric acid and heated. There is a problem of using a large amount of acid.
以上のことから、生産性が高く効率的で、酸の使用量が少なく環境負荷が低いキチンオリゴマー、Ｎ−アセチルグルコサミン及び１−Ｏ−アルキル−Ｎ−アセチルグルコサミンを製造する方法の確立が望まれている。 In view of the above, establishment of a method for producing chitin oligomers, N-acetylglucosamine and 1-O-alkyl-N-acetylglucosamine that is highly productive and efficient, uses less acid and has a low environmental impact is desired. ing.
本発明の目的は、従来法よりも酸触媒を少ない量用いたキチン含有バイオマスの加水分解反応によりキチンオリゴマー、Ｎ−アセチルグルコサミン及び１−Ｏ−アルキル−Ｎ−アセチルグルコサミンを製造する方法を提供することにある。 The object of the present invention is to provide a method for producing chitin oligomers, N-acetylglucosamine and 1-O-alkyl-N-acetylglucosamine by hydrolysis reaction of chitin-containing biomass using a smaller amount of acid catalyst than conventional methods. There is.
本発明者らは、酸触媒を用いたキチン含有バイオマスの加水分解反応において、従来法に比べて少量の酸触媒と水の存在下でキチン含有バイオマスを物理的に粉砕することにより部分加水分解してキチンオリゴマーを製造できることを見出した。また、このキチンオリゴマーに水を加えて加熱し、加水分解することによりＮ−アセチルグルコサミンを製造できること、さらに、キチンオリゴマーにアルコールを加えて加熱し、加アルコール分解することにより、１−Ｏ−アルキル−Ｎ−アセチルグルコサミンを製造できることを見出し、本発明を完成するに至った。 In the hydrolysis reaction of chitin-containing biomass using an acid catalyst, the present inventors have partially hydrolyzed chitin-containing biomass by physically pulverizing the chitin-containing biomass in the presence of a small amount of acid catalyst and water compared to the conventional method. And found that a chitin oligomer can be produced. In addition, N-acetylglucosamine can be produced by adding water to the chitin oligomer and heating and hydrolyzing it, and further adding 1-alcohol to the chitin oligomer and heating it, followed by alcoholysis, thereby producing 1-O-alkyl. It has been found that -N-acetylglucosamine can be produced, and the present invention has been completed.
すなわち、本発明は以下の［１］〜［９］のキチンオリゴマーの製造方法、［１０］〜［１１］のＮ−アセチルグルコサミンの製造方法、及び［１２］〜［１５］の１−Ｏ−アルキル−Ｎ−アセチルグルコサミンの製造方法に関する。
［１］ キチン含有バイオマスを、硫酸、硝酸、塩酸及び過塩素酸から選択される酸触媒及び水の共存下で粉砕装置を用い粉砕しつつ部分加水分解することを特徴とするキチンオリゴマーの製造方法。
［２］ 前記酸触媒が硫酸である前項１に記載のキチンオリゴマーの製造方法。
［３］ 前記酸触媒のモル数（Ｃ）に対するキチン中のＮ−アセチルグルコサミン単位（Ｃ8Ｈ13ＮО5）のモル数（Ｓ）の比（Ｓ／Ｃ）が０．２〜２０である前項１または２に記載のキチンオリゴマーの製造方法。
［４］ 予め酸触媒を含浸させたキチン含有バイオマスを水の存在下で粉砕する前項１〜３のいずれかに記載のキチンオリゴマーの製造方法。
［５］ 酸触媒を溶解した溶媒とキチン含有バイオマスを混合し、その後溶媒を除去して酸触媒をキチン含有バイオマスに含浸させ、得られた酸触媒を含浸させたキチン含有バイオマスを水の存在下で粉砕する前項４に記載のキチンオリゴマーの製造方法。
［６］ 前記溶媒が、キチン含有バイオマスを変性させず、酸触媒活性を阻害せず、加熱あるいは蒸留で除去できるものである前項５に記載のキチンオリゴマーの製造方法。
［７］ 前記溶媒が、水、ジエチルエーテル、ヘキサン、及びベンゼンから選択される前項６に記載のキチンオリゴマーの製造方法。
［８］ 粉砕装置がボールミルである前項１〜７のいずれかに記載のキチンオリゴマーの製造方法。
［９］ ボールミルが、遊星ボールミル、転動ボールミルのいずれかである前項８に記載のキチンオリゴマーの製造方法。
［１０］ 前項１〜９のいずれかに記載の方法により得られたキチンオリゴマーに水を加えて加熱し加水分解することを特徴とするＮ−アセチルグルコサミンの製造方法。
［１１］ 加熱温度が１００〜２６０℃である前項１０に記載のＮ−アセチルグルコサミンの製造方法。
［１２］ 前項１〜９のいずれかに記載の方法により得られたキチンオリゴマーにアルコールを加えて加アルコール分解することを特徴とする１−Ｏ−アルキル−Ｎ−アセチルグルコサミンの製造方法。
［１３］ アルコールが１価アルコールである前項１２に記載の１−Ｏ−アルキル−Ｎ−アセチルグルコサミンの製造方法。
［１４］ 前記アルコールがメタノールであり、前記１−Ｏ−アルキル−Ｎ−アセチルグルコサミンが１−Ｏ−メチル−Ｎ−アセチルグルコサミンである前項１２または１３に記載の製造方法。
［１５］ 加熱温度が１２０〜２８０℃である前項１２〜１４のいずれかに記載の１−Ｏ−アルキル−Ｎ−アセチルグルコサミンの製造方法。
That is, the present invention provides a method for producing the following chitin oligomers [1] to [9], a method for producing N-acetylglucosamine [10] to [11], and 1-O— of [12] to [15]. The present invention relates to a method for producing alkyl-N-acetylglucosamine.
[1] A method for producing a chitin oligomer, characterized in that chitin-containing biomass is partially hydrolyzed while being pulverized using a pulverizer in the presence of an acid catalyst selected from sulfuric acid, nitric acid, hydrochloric acid and perchloric acid and water. .
[2] The method for producing a chitin oligomer according to item 1 above, wherein the acid catalyst is sulfuric acid.
[3] The ratio (S / C) of the number of moles (S) of N-acetylglucosamine units (C 8 H 13 NO 5 ) in chitin to the number of moles (C) of the acid catalyst is 0.2-20. 3. A method for producing a chitin oligomer according to item 1 or 2.
[4] The method for producing a chitin oligomer according to any one of items 1 to 3, wherein chitin-containing biomass impregnated with an acid catalyst in advance is pulverized in the presence of water.
[5] Mixing the solvent in which the acid catalyst is dissolved and the chitin-containing biomass, then removing the solvent and impregnating the chitin-containing biomass with the acid catalyst, and the resulting chitin-containing biomass impregnated with the acid catalyst in the presence of water 5. The method for producing a chitin oligomer according to item 4 above, wherein the chitin oligomer is pulverized by the method.
[6] The method for producing a chitin oligomer according to item 5 above, wherein the solvent does not denature the chitin-containing biomass and does not inhibit the acid catalyst activity and can be removed by heating or distillation.
[7] The method for producing a chitin oligomer according to [6], wherein the solvent is selected from water, diethyl ether, hexane, and benzene.
[8] The method for producing a chitin oligomer according to any one of 1 to 7 above, wherein the pulverizer is a ball mill.
[9] The method for producing a chitin oligomer as described in 8 above, wherein the ball mill is either a planetary ball mill or a rolling ball mill.
[10] A method for producing N-acetylglucosamine, wherein water is added to the chitin oligomer obtained by the method according to any one of 1 to 9 above and heated to hydrolyze.
[11] The method for producing N-acetylglucosamine according to item 10 above, wherein the heating temperature is 100 to 260 ° C.
[12] A method for producing 1-O-alkyl-N-acetylglucosamine, wherein an alcohol is added to the chitin oligomer obtained by the method according to any one of 1 to 9 above to cause alcoholysis.
[13] The process for producing 1-O-alkyl-N-acetylglucosamine according to item 12, wherein the alcohol is a monohydric alcohol.
[14] The production method according to item 12 or 13, wherein the alcohol is methanol and the 1-O-alkyl-N-acetylglucosamine is 1-O-methyl-N-acetylglucosamine.
[15] The method for producing 1-O-alkyl-N-acetylglucosamine according to any one of 12 to 14 above, wherein the heating temperature is 120 to 280 ° C.
本発明によれば、キチン含有バイオマスから、酸触媒を用いてキチンオリゴマー、Ｎ−アセチルグルコサミン及び１−Ｏ−アルキル−Ｎ−アセチルグルコサミンを環境負荷を低減し、低コストで製造することができる。 According to the present invention, chitin oligomers, N-acetylglucosamine and 1-O-alkyl-N-acetylglucosamine can be produced from chitin-containing biomass at low cost using an acid catalyst.
以下、本発明方法の実施形態について説明する。なお、以下に説明する実施形態は本発明の代表的な例を示したものであり、本発明はそれらに限定されるものではない。 Hereinafter, embodiments of the method of the present invention will be described. In addition, embodiment described below shows the typical example of this invention, and this invention is not limited to them.
キチン含有バイオマス（固体基質）：
バイオマスとは一般的には「再生可能な生物由来の有機性資源で化石資源を除いたもの」を指すが、本発明で使用する「キチン含有バイオマス」（以下、固体基質ということがある。）は、例えば、エビ、カニなどの甲殻類、節足動物、昆虫類、イカ、貝、沖アミなどの殻や表皮、キノコなどの菌類の細胞壁などの主にキチンを含むバイオマスである。
Chitin-containing biomass (solid substrate):
Biomass generally refers to “renewable biological organic resources excluding fossil resources”, but “chitin-containing biomass” used in the present invention (hereinafter sometimes referred to as solid substrate). Is, for example, biomass mainly containing chitin such as shellfish such as shrimp and crab, arthropods, insects, squid, shellfish, shells such as offshore mites, and fungal cell walls such as mushrooms.
キチン含有バイオマスは、精製処理してあるものでも、精製処理してないものでも用いることができる。精製処理してあるものとしては、アルカリによるタンパク質の溶解、酸によるカルシウムの溶解などの処理をした後に、中和、固液分離、水洗などの処理をしてタンパク質やカルシウムなどの不純物の除去を行い、少なくともキチンを含有するものが挙げられる。さらに、工業的に調製したキチンなどでもよい。
キチン含有バイオマスは、不純物として原料由来のタンパク質、リン酸、鉄、銅、亜鉛、モリブデン、珪素、アルミニウム、カルシウム、マグネシウム、カリウム、ナトリウムなどを含有してもかまわない。
The chitin-containing biomass can be used whether it has been purified or not. For those that have undergone purification treatment, after processing such as protein dissolution with alkali and calcium dissolution with acid, neutralization, solid-liquid separation, washing with water, etc. are performed to remove impurities such as protein and calcium. And at least containing chitin. Furthermore, industrially prepared chitin may be used.
Chitin-containing biomass may contain raw material-derived proteins, phosphoric acid, iron, copper, zinc, molybdenum, silicon, aluminum, calcium, magnesium, potassium, sodium, and the like as impurities.
キチン含有バイオマスの形態は、乾体でも湿体でもかまわず、結晶性でも非結晶性でもかまわない。キチン含有バイオマスは反応に先立ち粗粉砕することが望ましい。粗粉砕により触媒との接触性が増加して、加水分解反応が促進される。従って、キチン含有バイオマスの形状・大きさは、粉砕するのに適していることが好ましい。そのような形状・大きさとしては、例えば粒径が２０〜１０００μｍの粉体状が挙げられる。 The form of chitin-containing biomass may be dry or wet, and may be crystalline or non-crystalline. The chitin-containing biomass is desirably coarsely pulverized prior to the reaction. Coarse pulverization increases the contact with the catalyst and promotes the hydrolysis reaction. Therefore, the shape and size of the chitin-containing biomass is preferably suitable for pulverization. Examples of such a shape and size include a powder form having a particle size of 20 to 1000 μm.
粗粉砕処理は、例えば、シュレッダー、ジョークラッシャー、ジャイレトリクラッシャー、コーンクラッシャー、ハンマークラッシャー、ロールクラッシャー、及びロールミルなどの粗粉砕機、スタンプミル、エッジランナ、切断・せん断ミル、ロッドミル、自生粉砕機及びローラミルなどの中粉砕機を用いて実施することができる。原料の処理時間は、処理後原料が均一に微粉化されるのであれば限定されるものではない。 Coarse pulverization processing includes, for example, shredders, jaw crushers, gyratory crushers, cone crushers, hammer crushers, roll crushers, and roll mills, etc. It can be carried out using a medium pulverizer. The processing time of a raw material will not be limited if the raw material after a process is pulverized uniformly.
酸触媒：
本発明において用いる酸触媒は、キチンを加水分解できる触媒であれば特に限定されるものではなく、例えば、主成分であるキチンの主鎖を形成しているβ−１，４グリコシド結合を加水分解する活性を有する触媒が好ましい。
Acid catalyst:
The acid catalyst used in the present invention is not particularly limited as long as it is a catalyst capable of hydrolyzing chitin. For example, β-1,4 glycosidic bond forming the main chain of chitin which is a main component is hydrolyzed. A catalyst having the activity of
酸触媒としては、硫酸、塩酸、硝酸、過塩素酸などの強酸を用いることができる。また、これらを併用することも可能である。酸触媒によるキチン含有バイオマスの加水分解速度を高めることができることから、硫酸、塩酸、硝酸がより好ましく、硫酸が最も好ましい。 As the acid catalyst, a strong acid such as sulfuric acid, hydrochloric acid, nitric acid or perchloric acid can be used. These can also be used in combination. Sulfuric acid, hydrochloric acid, and nitric acid are more preferable, and sulfuric acid is most preferable because the hydrolysis rate of chitin-containing biomass by an acid catalyst can be increased.
本発明は、図３のスキームに示すように、固体基質中のキチンを部分加水分解してキチンオリゴマーを製造する方法（方法１）、方法１で製造したキチンオリゴマーを水中で加水分解してＮ−アセチルグルコサミンを製造する方法（方法２）、及び方法１で製造したキチンオリゴマーをアルコール溶媒中で加アルコール分解して１−Ｏ−アルキル−Ｎ−アセチルグルコサミンを製造する方法（方法３）からなる。以下、各方法を説明する。 In the present invention, as shown in the scheme of FIG. 3, a chitin oligomer in a solid substrate is partially hydrolyzed to produce a chitin oligomer (Method 1), and the chitin oligomer produced in Method 1 is hydrolyzed in water to form N -A method for producing acetylglucosamine (Method 2) and a method for producing 1-O-alkyl-N-acetylglucosamine by hydrolyzing the chitin oligomer produced in Method 1 in an alcohol solvent (Method 3). . Each method will be described below.
方法１（部分加水分解反応）：
固体基質の部分加水分解は、基質に酸触媒を含浸させた後、粉砕による機械応力を掛けることにより行う。なお部分加水分解とは、キチンの解重合生成物の大部分が水溶性のオリゴマー単位に留まり、モノマー単位まで達する解重合が少ない加水分解反応のことを言う。
Method 1 (partial hydrolysis reaction):
Partial hydrolysis of a solid substrate is performed by impregnating the substrate with an acid catalyst and then applying mechanical stress due to grinding. The partial hydrolysis refers to a hydrolysis reaction in which most of the depolymerized product of chitin remains in the water-soluble oligomer unit and there is little depolymerization reaching the monomer unit.
キチンは、ＮＡＧのポリマー主鎖を形成するβ−１，４グリコシド結合と、Ｃ２位に側鎖を形成しているアセトアミド基（−ＮＨＣＯＣＨ3）のアミド結合の２つの加水分解対象となる結合を有するが、本発明の目的化合物はいずれも、側鎖のアセトアミド基を有するものであるため、主鎖のβ−１，４グリコシド結合が選択的に加水分解されることが重要である。 Chitin is composed of two bonds to be hydrolyzed: a β-1,4 glycosidic bond that forms the polymer main chain of NAG, and an amide bond of the acetamide group (—NHCOCH 3 ) that forms a side chain at the C2 position. However, since all of the target compounds of the present invention have side chain acetamide groups, it is important that the β-1,4 glycosidic bond of the main chain is selectively hydrolyzed.
酸触媒（Ｃ）と固体基質（Ｓ）の比率は、特に限定されるものではないが、反応時の部分加水分解効率、反応後の基質残渣低減の観点から、Ｓ／Ｃのモル比が０．２〜２０で行うことができ、０．５〜１５が好ましく、１〜１０がより好ましい。 The ratio of the acid catalyst (C) to the solid substrate (S) is not particularly limited, but the molar ratio of S / C is 0 from the viewpoint of partial hydrolysis efficiency during the reaction and reduction of the substrate residue after the reaction. 2 to 20, preferably 0.5 to 15, and more preferably 1 to 10.
固体基質への酸触媒の含浸方法は、それぞれを直接混合して行うことができる。また酸触媒を溶解した溶媒と基質を混合したのち、溶媒を蒸留や加熱することにより除去することもできる。使用する溶媒は、基質を変性せず、酸触媒活性を阻害も失活もせず、不揮発性でない（すなわち、加熱・蒸留で除去できる）ものであれば特に限定されない。例えば、水、ジエチルエーテル、ヘキサン、ベンゼンなどが適当である。 The method of impregnating the acid catalyst into the solid substrate can be carried out by directly mixing each of them. Further, after the solvent in which the acid catalyst is dissolved and the substrate are mixed, the solvent can be removed by distillation or heating. The solvent used is not particularly limited as long as it does not denature the substrate, does not inhibit or deactivate the acid catalyst activity, and is not nonvolatile (that is, can be removed by heating and distillation). For example, water, diethyl ether, hexane, benzene and the like are suitable.
部分加水分解時の水分は、酸触媒を含浸した固体基質に物理吸着した水分が１〜３質量％程度あれば十分に部分加水分解を賄えるので、通常は水を添加しなくても問題ないが、水を添加することもできる。
酸触媒を含浸した固体基質を粉砕することにより、部分加水分解を実現することができる。ここでの粉砕の効果としては、固体基質が微粉化されることによる、含浸した酸触媒のより均一な拡散、比表面積増大に伴う物理応力の伝達効率の向上、キチンのアモルファス化による固体基質の加水分解性の向上が考えられる。
The water at the time of partial hydrolysis can sufficiently cover partial hydrolysis if the water physically adsorbed on the solid substrate impregnated with the acid catalyst is about 1 to 3% by mass, so there is usually no problem even if water is not added. Water can also be added.
Partial hydrolysis can be achieved by grinding a solid substrate impregnated with an acid catalyst. The effect of the pulverization here is that the solid substrate is pulverized more uniformly, the impregnated acid catalyst is more uniformly diffused, the transmission efficiency of physical stress accompanying the increase in specific surface area is improved, and the solid substrate is made amorphous by chitin amorphization. An improvement in hydrolyzability can be considered.
粉砕処理に用いる装置としては、ポットミル、チューブミル、コニカルミルなどの転動ボールミル、旋回流型ジェットミル、衝突タイプジェットミル、流動層型ジェットミル、湿式タイプジェットミルなどのジェット粉砕機、らいかい機（擂潰機）、オングミルなどのせん断ミル、乳鉢、石うすなどのコロイドミル、ハンマーミル、ケージミル、ピンミル、ディスインテグレータ、スクリーンミル、ターボ型ミル、遠心分級ミルなどの衝撃式粉砕機、さらには自転及び公転の運動を採用した種類の粉砕機である遊星ボールミルなどが挙げられる。
本発明の目的である、主鎖のグリコシド結合の選択的な加水分解の実現の観点からは、用いる粉砕装置は、固体基質に圧縮力が強く加わり、主鎖の両方向に引っ張り応力が加えられるボールミルが好ましく、遊星ボールミル、転動ボールミルがより好ましく、遊星ボールミルが最も好ましい。
Equipment used for pulverization treatment includes rolling mills such as pot mills, tube mills and conical mills, swirling flow jet mills, impingement type jet mills, fluidized bed jet mills, wet type jet mills and other jet crushers, and large machines (Crusher), shearing mills such as ong mills, colloidal mills such as mortars and stones, hammer mills, cage mills, pin mills, disintegrators, screen mills, turbo mills, centrifugal classifiers, and other impact-type crushers, and even rotation And a planetary ball mill which is a kind of pulverizer adopting a revolving motion.
From the viewpoint of realizing selective hydrolysis of the main chain glycosidic bond, which is the object of the present invention, the grinding device used is a ball mill in which a compressive force is strongly applied to the solid substrate and tensile stress is applied in both directions of the main chain. Are preferred, planetary ball mills and rolling ball mills are more preferred, and planetary ball mills are most preferred.
粉砕処理の温度は、側鎖のアセトアミド基を脱離させずに、主鎖のβ−１，４グリコシド結合を選択的に加水分解できれば、特に限定されるものではないが、常温〜６０℃が好ましい。
粉砕処理の時間は、固体基質の部分加水分解が進み、水溶性になるのであれば、特に限定されるものではない。処理終点を見極めるために、経時的に取得したサンプルの水溶性を確認することが好ましい。
The temperature of the pulverization treatment is not particularly limited as long as the β-1,4 glycoside bond of the main chain can be selectively hydrolyzed without removing the side chain acetamide group. preferable.
The time for the pulverization treatment is not particularly limited as long as partial hydrolysis of the solid substrate proceeds and becomes water-soluble. In order to determine the treatment end point, it is preferable to confirm the water solubility of the sample obtained over time.
部分加水分解（方法１）で得られた反応生成物は、引き続き、後述の加水分解を行う方法２や加アルコール分解を行う方法３の原料として使用できる他、中和や脱塩などの精製処理を行いキチンオリゴマーとして使用することもできる。 The reaction product obtained by partial hydrolysis (Method 1) can be used as a raw material for Method 2 for subsequent hydrolysis and Method 3 for alcoholysis, as well as purification treatment such as neutralization and desalting. It can also be used as a chitin oligomer.
方法２（加水分解反応）及び方法３（加アルコール分解反応）：
方法２の加水分解反応、及び方法３の加アルコール分解反応では、いずれも方法１の部分加水分解で得られた反応生成物を原料として用いることができる。実際には、原料を溶媒（水またはアルコール）に溶解した後、加熱して反応を行う。方法２では溶媒に水を使用し、キチンオリゴマーからＮＡＧが製造される。方法３では溶媒にアルコールを使用し、キチンオリゴマーからＮＡＧ誘導体である１−Ｏ−アルキル−Ｎ−アセチルグルコサミンが製造される。
方法３で使用する溶媒には、メタノール、エタノール、１−プロパノール、２−プロパノール、１−ブタノール、２−ブタノール、２メチル−１プロパノール、２−メチル−２−プロパノールなどの１価アルコールが好ましい。原料を溶解させて反応の効率や均一性を得るという観点から、溶媒は、メタノール、エタノールがより好ましく、メタノールが最も好ましい。
Method 2 (hydrolysis reaction) and method 3 (alcohol decomposition reaction):
In both the hydrolysis reaction in Method 2 and the alcoholysis reaction in Method 3, the reaction product obtained by partial hydrolysis in Method 1 can be used as a raw material. In practice, the raw material is dissolved in a solvent (water or alcohol) and then heated to carry out the reaction. In Method 2, NAG is produced from chitin oligomers using water as a solvent. In Method 3, alcohol is used as a solvent, and 1-O-alkyl-N-acetylglucosamine, which is a NAG derivative, is produced from a chitin oligomer.
The solvent used in Method 3 is preferably a monohydric alcohol such as methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 2methyl-1propanol, or 2-methyl-2-propanol. From the viewpoint of obtaining the efficiency and uniformity of the reaction by dissolving the raw materials, the solvent is more preferably methanol or ethanol, and most preferably methanol.
反応は、通常は、原料と溶媒の存在下、常圧で密閉した容器中で行う。例えば、溶媒が水の場合、水蒸気分圧が０．１ＭＰａ以上の加圧状態となる温度で行う。加圧状態となる加熱温度は、方法２の加水分解反応は、好ましくは１００〜２６０℃、より好ましくは１３０〜２３０℃、さらに好ましくは１５０〜２１０℃である。また、方法３の加アルコール分解反応は、好ましくは１２０〜２８０℃、より好ましくは１５０〜２５０℃、さらに好ましくは１７０〜２３０℃である。なお、加熱温度は反応時の溶液の温度である。当該範囲とすることで、目的生成物の製造収率を高めることができる。本発明の製造方法における反応は、通常はオートクレーブなどの密閉容器内で実施されるため、加熱開始時は常圧であっても、上記温度に反応系が加熱されると加圧状態となる。 The reaction is usually carried out in a sealed vessel at normal pressure in the presence of raw materials and a solvent. For example, when the solvent is water, it is performed at a temperature at which the water vapor partial pressure is in a pressurized state of 0.1 MPa or more. The heating temperature at which the pressure is applied is preferably 100 to 260 ° C, more preferably 130 to 230 ° C, and still more preferably 150 to 210 ° C in the hydrolysis reaction of Method 2. In addition, the alcoholysis reaction of Method 3 is preferably 120 to 280 ° C, more preferably 150 to 250 ° C, and further preferably 170 to 230 ° C. The heating temperature is the temperature of the solution during the reaction. By setting it as the said range, the manufacture yield of a target product can be raised. The reaction in the production method of the present invention is usually carried out in a closed container such as an autoclave. Therefore, even when the heating is started at normal pressure, the reaction system is heated to the above temperature.
さらに、反応前または反応中に密閉容器内を加圧し、反応することもできる。加圧する圧力は、例えば０．１〜３０ＭＰａ、好ましくは１〜２０ＭＰａ、さらに好ましくは２〜１０ＭＰａである。 Further, the reaction can be carried out by pressurizing the inside of the sealed container before or during the reaction. The pressure to pressurize is, for example, 0.1 to 30 MPa, preferably 1 to 20 MPa, and more preferably 2 to 10 MPa.
反応における加熱では、室温から反応温度に到達する時間は、好ましくは５〜６０分、より好ましくは５〜３０分、さらに好ましくは５〜２０分である。反応温度までに到達すると同時に、加熱を止めて冷却することが好ましい。このようにすることで、目的生成物の製造収率を高めることができる。 In the heating in the reaction, the time to reach the reaction temperature from room temperature is preferably 5 to 60 minutes, more preferably 5 to 30 minutes, and further preferably 5 to 20 minutes. At the same time as reaching the reaction temperature, the heating is preferably stopped and cooled. By doing in this way, the production yield of the target product can be increased.
分解に用いる溶媒の量（原料１質量部に対する溶媒の質量比）は、少なくともバイオマスのキチンオリゴマーを全量加溶媒分解できる量であるが、反応混合物の流動性や撹拌性などを考慮して、好ましくは１〜５００質量部、より好ましくは２〜３５０質量部、さらに好ましくは２〜２００質量部である。当該範囲とすることにより、分解反応において高い生成物収率と生成物濃度を両立できる。 The amount of the solvent used for the decomposition (mass ratio of the solvent with respect to 1 part by mass of the raw material) is an amount capable of solvolytic decomposition of at least the chitin oligomer of the biomass, but preferably in consideration of the fluidity and agitation of the reaction mixture. Is 1 to 500 parts by mass, more preferably 2 to 350 parts by mass, and still more preferably 2 to 200 parts by mass. By setting it as the said range, a high product yield and product concentration can be made compatible in a decomposition reaction.
反応は反応混合物を撹拌しながら行うことが好ましい。反応形式は、バッチ式または連続式などのいずれでもよい。 The reaction is preferably carried out while stirring the reaction mixture. The reaction format may be either batch type or continuous type.
以下、実施例及び比較例を挙げて本発明をさらに説明するが、本発明はこれらの記載により何ら限定を受けるものではない。 Hereinafter, although an example and a comparative example are given and the present invention is further explained, the present invention is not limited at all by these statements.
［使用原料］
カニ殻を乾燥、粉砕し、高温の希水酸化ナトリウム水溶液と室温の希塩酸水溶液にそれぞれ数時間浸漬することにより、タンパク質、炭酸カルシウムを除去して精製したキチンを用いた。
[Raw materials]
The chitin purified by removing protein and calcium carbonate by drying and crushing the crab shell and immersing it in a dilute aqueous sodium hydroxide solution and a dilute aqueous hydrochloric acid solution at room temperature for several hours, respectively.
［キチンの部分加水分解反応（方法１）］
比較例１：未処理キチン
精製したキチン(平均粒径７３μｍ)をそのまま用いた。このサンプルを未処理キチンとする。
[Partial hydrolysis reaction of chitin (method 1)]
Comparative Example 1: Untreated chitin Purified chitin (average particle size 73 μm) was used as it was. This sample is untreated chitin.
比較例２：硫酸含浸キチン
未処理キチン（平均粒径７３μｍ）５．０ｇ（ＮＡＧ単位として２４．６ｍｍｏｌ）を硫酸０．３ｇ（３．０４ｍｍｏｌ、Ｓ／Ｃ比８．１）を含むジエチルエーテル１５ｍＬに分散させた後、ジエチルエーテルを乾燥して取得した粉末を６時間静置した。このサンプルを硫酸含浸キチンとする。
Comparative Example 2: Sulfur impregnated chitin 15 mL of diethyl ether containing 5.0 g of untreated chitin (average particle size 73 μm) (24.6 mmol as NAG unit) and 0.3 g of sulfuric acid (3.04 mmol, S / C ratio 8.1) Then, the powder obtained by drying diethyl ether was allowed to stand for 6 hours. This sample is designated as sulfuric acid-impregnated chitin.
比較例３：キチン粉砕物
未処理キチン（平均粒径７３μｍ）５．０ｇを直径５ｍｍのアルミナボール１００ｇと共に容量２５０ｍＬのアルミナポットに入れた。このポットを遊星ボールミル（フリッチュ社製、ＰＵＬＶＥＲＩＳＥＴＴＥ（登録商標）６）にセットして５００ｒｐｍで１０分間処理した後に１０分間のインターバルを置くサイクルを１２時間行った。このサンプルをキチン粉砕物（平均粒径４２μｍ）とする。
Comparative Example 3: Ground chitin 5.0 g of untreated chitin (average particle size 73 μm) was placed in an alumina pot with a capacity of 250 mL together with 100 g of alumina balls having a diameter of 5 mm. This pot was set on a planetary ball mill (Purverisete (registered trademark) 6 manufactured by Fritsch), treated at 500 rpm for 10 minutes, and then subjected to a cycle having an interval of 10 minutes for 12 hours. Let this sample be a chitin ground material (average particle diameter of 42 micrometers).
比較例４：「キチン粉砕物＋硫酸」
比較例３に記載のキチン粉砕物（平均粒径４２μｍ）５．０ｇ（ＮＡＧ単位として２４．６ｍｍｏｌ）を硫酸０．３ｇ（３．０４ｍｍｏｌ、Ｓ／Ｃ比８．１）を含むジエチルエーテル１５ｍＬに分散させた後、ジエチルエーテルを乾燥して取得した粉末を６時間静置した。このサンプルを「キチン粉砕物＋硫酸」とする。
Comparative Example 4: “chitin pulverized product + sulfuric acid”
To 15 mL of diethyl ether containing 5.0 g (3.04 mmol, S / C ratio 8.1) of sulfuric acid 5.0 g (24.6 mmol as NAG unit) of the chitin pulverized product described in Comparative Example 3 (average particle size 42 μm) After dispersion, the powder obtained by drying diethyl ether was allowed to stand for 6 hours. This sample is referred to as “chitin pulverized product + sulfuric acid”.
実施例１：硫酸含浸キチン粉砕物
未処理キチン（平均粒径７３μｍ）５．０ｇ（ＮＡＧ単位として２４．６ｍｍｏｌ）を硫酸０．３ｇ（３．０４ｍｍｏｌ、Ｓ／Ｃ比８．１）を含むジエチルエーテル１５ｍＬに分散させた後、ジエチルエーテルを乾燥して取得した４．９ｇの粉末を、直径５ｍｍのアルミナボール１００ｇと共に容量２５０ｍＬのアルミナポットに入れた。このポットを遊星ボールミル（フリッチュ社製、ＰＵＬＶＥＲＩＳＥＴＴＥ６）にセットして５００ｒｐｍで１０分間処理した後に１０分間のインターバルを置くサイクルを１２時間行った。従って、インターバルを除いた粉砕処理の時間は６時間である。その結果、１．５質量％の物理吸着水を含有する粉末を得た。この手順により得た粉末サンプルを硫酸含浸キチン粉砕物とする。
Example 1: Sulfur-impregnated chitin pulverized product Untreated chitin (average particle size 73 μm) 5.0 g (24.6 mmol as a NAG unit) and sulfuric acid 0.3 g (3.04 mmol, S / C ratio 8.1) diethyl After dispersing in 15 mL of ether, 4.9 g of powder obtained by drying diethyl ether was placed in an alumina pot having a capacity of 250 mL together with 100 g of alumina balls having a diameter of 5 mm. This pot was set on a planetary ball mill (Purverisete 6 manufactured by Fritsch) and treated at 500 rpm for 10 minutes, and then a cycle with an interval of 10 minutes was performed for 12 hours. Therefore, the pulverization time excluding the interval is 6 hours. As a result, a powder containing 1.5% by mass of physically adsorbed water was obtained. The powder sample obtained by this procedure is used as a sulfuric acid-impregnated chitin pulverized product.
［キチンの部分加水分解反応サンプルの溶解度測定］
実施例１及び比較例１〜４の各サンプル１００ｍｇを秤量して、蒸留水５０ｍＬに添加し振とうした後、１０分間の超音波処理を行い、可溶分を溶解させた。一連の処理は温度２５℃で行い、得られた懸濁液は、０．１μｍのポリテトラフルオロエチレン（ＰＴＦＥ）のフィルターでろ過して、固体残渣はさらに５ｍＬの蒸留水でろ過洗浄し、１１０℃のオーブンに入れて一晩乾燥した後、質量を測定し、以下の計算式で溶解度を算出した。
100 mg of each sample of Example 1 and Comparative Examples 1 to 4 was weighed, added to 50 mL of distilled water and shaken, and then subjected to ultrasonic treatment for 10 minutes to dissolve soluble components. A series of treatments were performed at a temperature of 25 ° C., and the resulting suspension was filtered through a 0.1 μm polytetrafluoroethylene (PTFE) filter, and the solid residue was further filtered and washed with 5 mL of distilled water. After putting in an oven at 0 ° C. and drying overnight, the mass was measured and the solubility was calculated by the following formula.
溶解度の測定結果を表１に示す。酸存在下でボールミル粉砕した実施例１の硫酸含浸キチン粉砕物サンプルは１００％の溶解度を示したのに対し、未処理のキチン（比較例１）は０％で全く溶解性を示さなかった。また処理を施したものにおいても、酸添加のみ行った硫酸含浸キチンサンプル（比較例２）は３．７％、ボールミル粉砕のみ行ったキチン粉砕物サンプル（比較例３）は５．２％、ボールミル粉砕の後に酸添加した「キチン粉砕物＋硫酸」サンプル（比較例４）は７．８％であり、いずれも大幅に低い溶解度しか示されなかった。
これらの結果から、酸触媒の添加、粉砕による機械的応力付与のそれぞれの処理によるキチンを加水分解し可溶化する効果は限定的であるが、２つの処理を同時に実施することにより格段の相乗効果が得られ、キチン可溶化を大幅に促進することが確認された。
The solubility measurement results are shown in Table 1. The sulfuric acid-impregnated chitin pulverized sample of Example 1 ball milled in the presence of acid showed a solubility of 100%, while untreated chitin (Comparative Example 1) showed no solubility at 0%. Also in the treated sample, the sulfuric acid-impregnated chitin sample (Comparative Example 2) subjected to acid addition alone was 3.7%, and the chitin pulverized sample sample (Comparative Example 3) subjected only to ball milling was 5.2%. The “chitin pulverized product + sulfuric acid” sample (Comparative Example 4) added with acid after pulverization was 7.8%, and all showed significantly low solubility.
From these results, the effect of hydrolyzing and solubilizing chitin by each treatment of adding an acid catalyst and applying mechanical stress by pulverization is limited, but a remarkable synergistic effect by carrying out two treatments simultaneously It was confirmed that chitin solubilization was greatly promoted.
［キチンの部分加水分解反応サンプルの水懸濁液ろ液の酢酸測定］
溶解率１００％であった硫酸含浸キチン粉砕物サンプル（実施例１）の水懸濁液ろ液を、高速液体クロマトグラフ（装置：島津製作所製ＬＣ−１０ＡＴＶＰ、カラム：Ｐｈｅｎｏｍｅｎｅｘ（登録商標） Ｓｙｎｅｒｇｉ ４μｍ Ｈｙｄｒｏ−ＲＰ ８０Å φ４．６×２５０ｍｍ、移動相：４０ｍＭリン酸カリウム緩衝液、ｐＨ２．９、０．８ｍＬ／分、３０℃、検出：示差屈折率）により酢酸を定量分析した。その結果、酢酸は検出されなかった。
これにより、酸触媒共存下のボールミル粉砕によるキチンの部分加水分解では、キチンを構成するＮＡＧユニットのＣ２位にあるアセトアミド基（−ＮＨＣＯＣＨ3）のアミド結合は保持され、ＮＡＧユニット同士を繋ぐグリコシド結合が選択的に切断されることが示唆された。これは、少量の酸触媒が共存し加水分解しやすい系において、ボールミル粉砕による機械的応力がキチンを押しつぶして重合の両方向に引き伸ばす作用、すなわち主鎖を形成するグリコシド結合を引っ張る作用を及ぼすことにより、従来の酸添加のみで行う加水分解では得られなかったグリコシド結合の選択的な加水分解を加速させる相乗効果が得られるものと推測される（図１参照）。
[Measurement of acetic acid in aqueous suspension filtrate of partial hydrolysis reaction sample of chitin]
An aqueous suspension filtrate of a sulfuric acid-impregnated chitin pulverized sample (Example 1) having a dissolution rate of 100% was subjected to a high performance liquid chromatograph (equipment: LC-10ATVP, manufactured by Shimadzu Corporation, column: Phenomenex (registered trademark) Synergi 4 μm. Acetic acid was quantitatively analyzed by Hydro-RP 80 φ4.6 × 250 mm, mobile phase: 40 mM potassium phosphate buffer, pH 2.9, 0.8 mL / min, 30 ° C., detection: differential refractive index). As a result, acetic acid was not detected.
As a result, in the partial hydrolysis of chitin by ball milling in the presence of an acid catalyst, the amide bond of the acetamide group (—NHCOCH 3 ) at the C2 position of the NAG unit constituting the chitin is retained, and the glycoside bond connecting the NAG units. Was suggested to be selectively cleaved. This is because the mechanical stress due to ball milling crushes chitin and stretches it in both directions of polymerization, that is, pulls the glycosidic bond that forms the main chain, in a system that is easily hydrolyzed with a small amount of acid catalyst. It is presumed that a synergistic effect that accelerates the selective hydrolysis of glycosidic bonds, which was not obtained by hydrolysis performed only by conventional acid addition, is obtained (see FIG. 1).
［キチンの部分加水分解反応サンプルの水懸濁液ろ液のＨＰＬＣ分析］
実施例１の水懸濁液ろ液を、ＨＰＬＣ（高速液体クロマトグラフ）（装置：島津製作所製ＬＣ−１０ＡＴＶＰ、カラム：Ｐｈｅｎｏｍｅｎｅｘ Ｒｅｚｅｘ ＲＰＭ−Ｍｏｎｏｓａｃｃｈａｒｉｄｅ Ｐｂ＋＋ φ７．８×３００ｍｍ、移動相：水、０．６ｍＬ／分、７０℃、検出：示差屈折率）で分析し、ＮＡＧ及びオリゴ糖（重合度２〜８）の収率を以下の計算式により算出した。
The aqueous suspension filtrate of Example 1 was subjected to HPLC (high performance liquid chromatograph) (equipment: LC-10ATVP manufactured by Shimadzu Corporation), column: Phenomenex Rezex RPM-Monosaccharide Pb ++ φ7.8 × 300 mm, mobile phase: water, 0. Analysis was performed at 6 mL / min, 70 ° C., detection: differential refractive index), and the yields of NAG and oligosaccharide (degree of polymerization 2 to 8) were calculated by the following formula.
［方法２（加水分解反応）及び方法３（加メタノール分解反応）］
実施例２〜５及び比較例５〜８：
表２に記載したＳ／Ｃ比の各サンプルをキチンとして４０６ｍｇ分（ＮＡＧ単位として２ｍｍｏｌ）と、水またはメタノール４０ｍＬを、高圧反応器（内容積１００ｍＬ，オーエムラボテック（株）製オートクレーブ，材質：ハステロイ（登録商標）Ｃ２２製）に入れた後、６００ｒｐｍで撹拌しながら室温から表２に記載した反応温度まで約１６分加熱した。反応温度に到達した時点で加熱を止め、反応器を風冷し、冷却後、反応液を遠心分離装置により液体と固体に分離して上清サンプルの分析を行った。反応温度１９０℃の場合の温度プロファイルは図２に示す通りである。
[Method 2 (hydrolysis reaction) and method 3 (methanol addition reaction)]
Examples 2-5 and Comparative Examples 5-8:
Each sample of S / C ratio described in Table 2 is 406 mg as chitin (2 mmol as NAG unit), 40 mL of water or methanol, a high pressure reactor (internal volume 100 mL, autoclave manufactured by OM Labtech Co., Ltd., material: Hastelloy (Registered Trademark) manufactured by C22) and then heated from room temperature to the reaction temperatures shown in Table 2 with stirring at 600 rpm for about 16 minutes. When the reaction temperature was reached, heating was stopped, the reactor was cooled with air, and after cooling, the reaction solution was separated into a liquid and a solid using a centrifuge and the supernatant sample was analyzed. The temperature profile when the reaction temperature is 190 ° C. is as shown in FIG.
固形分を除去した液相サンプルの生成物は、ＨＰＬＣ（装置：島津製作所製ＬＣ−１０ＡＴＶＰ、カラム：Ｐｈｅｎｏｍｅｎｅｘ Ｒｅｚｅｘ ＲＰＭ−Ｍｏｎｏｓａｃｃｈａｒｉｄｅ Ｐｂ＋＋ φ７．８×３００ｍｍ、移動相：水、０．６ｍＬ／分、７０℃、及びカラム：Ｓｈｏｄｅｘ（登録商標） ＳＵＧＡＲ ＳＨ−１０１１ φ８×３００ｍｍ、移動相：水、０．５ｍＬ／分、５０℃、検出：示差屈折率）によりＮＡＧ及びＭｅＮＡＧを定量分析した。 The product of the liquid phase sample from which the solid content was removed was HPLC (apparatus: LC-10ATVP manufactured by Shimadzu Corporation, column: Phenomenex Rezex RPM-Monosaccharide Pb ++ φ7.8 × 300 mm, mobile phase: water, 0.6 mL / min, 70 NAG and MeNAG were quantitatively analyzed by C. and column: Shodex (registered trademark) SUGAR SH-1011 φ8 × 300 mm, mobile phase: water, 0.5 mL / min, 50 ° C., detection: differential refractive index.
以下に生成物の収率の計算式を示す。
分析結果を表２に示す。方法２の加水分解反応では、Ｓ／Ｃ比８．１の硫酸含浸キチン粉砕物サンプル、反応温度１９０℃（実施例２）のＮＡＧ収率は３２％、Ｓ／Ｃ比２の硫酸含浸キチン粉砕物サンプル、反応温度１７０℃（実施例３）のＮＡＧ収率は５３％となり、硫酸濃度を高くし、反応温度を下げた条件で収率が向上した。 The analysis results are shown in Table 2. In the hydrolysis reaction of Method 2, a sulfuric acid-impregnated chitin pulverized sample with an S / C ratio of 8.1, an NAG yield of a reaction temperature of 190 ° C. (Example 2) is 32%, and an sulfuric acid-impregnated chitin pulverization with an S / C ratio of 2 is performed. The NAG yield of the product sample and the reaction temperature of 170 ° C. (Example 3) was 53%, and the yield was improved under the condition that the sulfuric acid concentration was increased and the reaction temperature was lowered.
方法３のメタノールを溶媒に使用した加メタノール分解反応では、Ｓ／Ｃ比８．１の硫酸含浸キチン粉砕物サンプル、反応温度１９０℃（実施例４）のＭｅＮＡＧ収率は６８％、Ｓ／Ｃ比４．１の硫酸含浸キチン粉砕物サンプル、反応温度１９０℃（実施例５）のＮＡＧ収率は７０％となり、１９０℃での加水分解反応のＮＡＧ収率より高い値を示した。メタノール中でのＭｅＮＡＧの１９０℃の安定性試験では残存率が９４％で高い安定性を示したことから、１９０℃においても加メタノール分解反応では生成したＭｅＮＡＧはほとんど分解せず高い収率が得られたものと推測される。ＭｅＮＡＧはＣ１にメトキシ基が結合しＮＡＧにはあるヘミアセタール基を含有していない構造であることが熱安定性を示す理由と考えられる。 In the methanol decomposition reaction using methanol of Method 3 as a solvent, a sample of sulfuric acid-impregnated chitin having an S / C ratio of 8.1, a MeNAG yield of 68% at a reaction temperature of 190 ° C. (Example 4), S / C The sample of sulfuric acid-impregnated chitin pulverized with a ratio of 4.1 and the reaction temperature of 190 ° C. (Example 5) had a NAG yield of 70%, which was higher than the NAG yield of the hydrolysis reaction at 190 ° C. In the 190 ° C stability test of MeNAG in methanol, the residual rate was 94%, indicating a high stability. Even at 190 ° C, the MeNAG produced in the methanol decomposition reaction hardly decomposed and a high yield was obtained. It is presumed that It is considered that MeNAG has a structure in which a methoxy group is bonded to C1 and NAG does not contain a certain hemiacetal group, and exhibits thermal stability.
加メタノール分解反応は、１９０℃で未処理キチンサンプル（比較例５）、硫酸含浸キチンサンプル（比較例６）、キチン粉砕物サンプル（比較例７）、「キチン粉砕物＋硫酸」サンプル（比較例８）についても行った。それらのＭｅＮＡＧ収率は、比較例５と７が０％、比較例６が４％、比較例８が１６％といずれも低く、高収率を得るためには、酸触媒共存下での機械応力付与が必要であることが示された。 The methanol decomposition reaction was carried out at 190 ° C. in an untreated chitin sample (Comparative Example 5), a sulfuric acid-impregnated chitin sample (Comparative Example 6), a chitin pulverized sample (Comparative Example 7), and a “chitin pulverized product + sulfuric acid” sample (Comparative Example). We also performed 8). The yields of MeNAG were as low as 0% in Comparative Examples 5 and 7, 4% in Comparative Example 6, and 16% in Comparative Example 8, and in order to obtain high yields, the machine in the presence of an acid catalyst was used. It was shown that stressing was necessary.
本発明の方法によれば、キチンから、酸触媒を用いてキチンオリゴマー、Ｎ−アセチルグルコサミン及び１−Ｏ−アルキルＮ−アセチルグルコサミンを既存の製造方法よりも少ない酸触媒の使用量で、効率的に製造でき、医薬、化粧品、食品、飼料分野に有用な高機能素材を提供することができる。 According to the method of the present invention, the chitin oligomer, N-acetylglucosamine and 1-O-alkyl N-acetylglucosamine can be efficiently produced from chitin using an acid catalyst with a smaller amount of acid catalyst than in the existing production method. It is possible to provide a highly functional material useful in the fields of medicine, cosmetics, food and feed.
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| JP2013018852A (en) * | 2011-07-11 | 2013-01-31 | Earth Technica:Kk | Method and apparatus for producing chitin ground product |
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